U.S. patent application number 14/135876 was filed with the patent office on 2014-07-24 for signal-sensor polynucleotides.
This patent application is currently assigned to MODERNA THERAPEUTICS, INC.. The applicant listed for this patent is MODERNA THERAPEUTICS, INC.. Invention is credited to Tirtha Chakraborty, Antonin de Fougerolles, Stephen G. Hoge.
Application Number | 20140206852 14/135876 |
Document ID | / |
Family ID | 49448268 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140206852 |
Kind Code |
A1 |
Hoge; Stephen G. ; et
al. |
July 24, 2014 |
SIGNAL-SENSOR POLYNUCLEOTIDES
Abstract
The invention relates to compositions and methods for the
preparation, manufacture and therapeutic use of signal-sensor
polynucleotides, primary transcripts and mmRNA molecules.
Inventors: |
Hoge; Stephen G.; (New York,
NY) ; de Fougerolles; Antonin; (Waterloo, BE)
; Chakraborty; Tirtha; (Medford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MODERNA THERAPEUTICS, INC. |
CAMBRIDGE |
MA |
US |
|
|
Assignee: |
MODERNA THERAPEUTICS, INC.
CAMBRIDGE
MA
|
Family ID: |
49448268 |
Appl. No.: |
14/135876 |
Filed: |
December 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14041011 |
Sep 30, 2013 |
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14135876 |
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61754159 |
Jan 18, 2013 |
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61781097 |
Mar 14, 2013 |
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61829334 |
May 31, 2013 |
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61839893 |
Jun 27, 2013 |
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61842733 |
Jul 3, 2013 |
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61857304 |
Jul 23, 2013 |
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Current U.S.
Class: |
536/23.5 |
Current CPC
Class: |
A61P 35/02 20180101;
A61K 31/711 20130101; A61K 48/0058 20130101; A61K 31/7105 20130101;
C12N 15/85 20130101; C12N 2310/141 20130101; C12N 15/113 20130101;
A61P 35/00 20180101; A61K 38/00 20130101; C07K 14/47 20130101 |
Class at
Publication: |
536/23.5 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Claims
1. An isolated synthetic signal-sensor polynucleotide, wherein said
isolated synthetic signal-sensor polynucleotide comprises an mRNA
which encodes an oncology-related polypeptide of interest and a
sensor sequence comprising SEQ ID NO: 3741.
2. The isolated synthetic signal-sensor polynucleotide of claim 1,
wherein the open reading frame of the mRNA is codon optimized.
3. The isolated synthetic signal-sensor polynucleotide of claim 1,
wherein the isolated synthetic signal-sensor polynucleotide
comprises at least one chemical modification, wherein the chemical
modification is 1-methylpseudouridine.
4. The isolated synthetic signal-sensor polynucleotide of claim 3,
further comprising the chemical modification 5-methylcytidine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/041,011, filed Sep. 30, 2013, entitled,
Signal-Sensor Polynucleotides for the Alteration of Cellular
Phenotypes which claims priority to U.S. Provisional Application
No. 61,753,661, filed Jan. 17, 2013, entitled Signal-Sensor
Polynucleotides for the Alternation of Cellular Phenotypes and
Microenvironments; U.S. Provisional Application No. 61/754,159,
filed Jan. 18, 2013, entitled Signal-Sensor Polynucleotides for the
Alternation of Cellular Phenotypes and Microenvironments; U.S.
Provisional Application No. 61/781,097, filed Mar. 14, 2013,
entitled Signal-Sensor Polynucleotides for the Alternation of
Cellular Phenotypes and Microenvironments; U.S. Provisional
Application No. 61/829,334, filed May 31, 2013, entitled
Signal-Sensor Polynucleotides for the Alternation of Cellular
Phenotypes and Microenvironments; U.S. Provisional Application No.
61/839,893, filed Jun. 27, 2013, entitled Signal-Sensor
Polynucleotides for the Alternation of Cellular Phenotypes and
Microenvironments; U.S. Provisional Application No. 61/842,733,
filed Jul. 3, 2013, entitled Signal-Sensor Polynucleotides for the
Alternation of Cellular Phenotypes and Microenvironments; and U.S.
Provisional Application No. 61/857,304, filed Jul. 23, 2013,
entitled Signal-Sensor Polynucleotides for the Alternation of
Cellular Phenotypes and Microenvironments; the contents of each of
which is herein incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence listing file, entitled
M37USCON.txt, was created on Dec. 16, 2013 and is 9,748,522 bytes
in size. The information in electronic format of the Sequence
Listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to compositions, methods, processes,
kits and devices for the design, preparation, manufacture and/or
formulation of signal-sensor polynucleotides, primary constructs
and mRNA molecules for the alteration of cellular phenotypes and
micro environments.
BACKGROUND OF THE INVENTION
[0004] Cancer is a disease characterized by uncontrolled cell
division and growth within the body. In the United States, roughly
a third of all women and half of all men will experience cancer in
their lifetime. Polypeptides are involved in every aspect of the
disease including cancer cell biology (carcinogenesis, cell cycle
suppression, DNA repair and angiogenesis), treatment
(immunotherapy, hormone manipulation, enzymatic inhibition),
diagnosis and determination of cancer type (molecular markers for
breast, prostate, colon and cervical cancer for example). With the
host of undesired consequences brought about by standard treatments
such as chemotherapy and radiotherapy used today, genetic therapy
for the manipulation of disease-related peptides and their
functions provides a more targeted approach to disease diagnosis,
treatment and management.
[0005] To this end, it has been previously shown that certain
modified mRNA sequences have the potential as therapeutics with
benefits beyond just evading, avoiding or diminishing the immune
response. Such studies are detailed in published co-pending
International Publication No WO2012019168 filed Aug. 5, 201,
International Publication No WO2012045082 filed Oct. 3, 2011,
International Publication No WO2012045075 filed Oct. 3, 2011,
International Publication No WO2013052523 filed Oct. 3, 2012, and
International Publication No WO2013090648 filed Dec. 14, 2012 the
contents of which are incorporated herein by reference in their
entirety.
[0006] The use of modified polynucleotides in the fields of
antibodies, viruses, veterinary applications and a variety of in
vivo settings have been explored and are disclosed in, for example,
co-pending and co-owned U.S. Provisional Patent Application No.
61/618,862, filed Apr. 2, 2012, entitled Modified Polynucleotides
for the Production of Biologics; U.S. Provisional Patent
Application No. 61/681,645, filed Aug. 10, 2012, entitled Modified
Polynucleotides for the Production of Biologics; U.S. Provisional
Patent Application No. 61/737,130, filed Dec. 14, 2012, entitled
Modified Polynucleotides for the Production of Biologics; U.S.
Provisional Patent Application No. 61/618,866, filed Apr. 2, 2012,
entitled Modified Polynucleotides for the Production of Antibodies;
U.S. Provisional Patent Application No. 61/681,647, filed Aug. 10,
2012, entitled Modified Polynucleotides for the Production of
Antibodies; U.S. Provisional Patent Application No. 61/737,134,
filed Dec. 14, 2012, entitled Modified Polynucleotides for the
Production of Antibodies; U.S. Provisional Patent Application No.
61/618,868, filed Apr. 2, 2012, entitled Modified Polynucleotides
for the Production of Vaccines; U.S. Provisional Patent Application
No. 61/681,648, filed Aug. 10, 2012, entitled Modified
Polynucleotides for the Production of Vaccines; U.S. Provisional
Patent Application No. 61/737,135, filed Dec. 14, 2012, entitled
Modified Polynucleotides for the Production of Vaccines; U.S.
Provisional Patent Application No. 61/618,870, filed Apr. 2, 2012,
entitled Modified Polynucleotides for the Production of Therapeutic
Proteins and Peptides; U.S. Provisional Patent Application No.
61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotides
for the Production of Therapeutic Proteins and Peptides; U.S.
Provisional Patent Application No. 61/737,139, filed Dec. 14, 2012,
Modified Polynucleotides for the Production of Therapeutic Proteins
and Peptides; U.S. Provisional Patent Application No. 61/618,873,
filed Apr. 2, 2012, entitled Modified Polynucleotides for the
Production of Secreted Proteins; U.S. Provisional Patent
Application No. 61/681,650, filed Aug. 10, 2012, entitled Modified
Polynucleotides for the Production of Secreted Proteins; U.S.
Provisional Patent Application No. 61/737,147, filed Dec. 14, 2012,
entitled Modified Polynucleotides for the Production of Secreted
Proteins; U.S. Provisional Patent Application No. 61/618,878, filed
Apr. 2, 2012, entitled Modified Polynucleotides for the Production
of Plasma Membrane Proteins; U.S. Provisional Patent Application
No. 61/681,654, filed Aug. 10, 2012, entitled Modified
Polynucleotides for the Production of Plasma Membrane Proteins;
U.S. Provisional Patent Application No. 61/737,152, filed Dec. 14,
2012, entitled Modified Polynucleotides for the Production of
Plasma Membrane Proteins; U.S. Provisional Patent Application No.
61/618,885, filed Apr. 2, 2012, entitled Modified Polynucleotides
for the Production of Cytoplasmic and Cytoskeletal Proteins; U.S.
Provisional Patent Application No. 61/681,658, filed Aug. 10, 2012,
entitled Modified Polynucleotides for the Production of Cytoplasmic
and Cytoskeletal Proteins; U.S. Provisional Patent Application No.
61/737,155, filed Dec. 14, 2012, entitled Modified Polynucleotides
for the Production of Cytoplasmic and Cytoskeletal Proteins; U.S.
Provisional Patent Application No. 61/618,896, filed Apr. 2, 2012,
entitled Modified Polynucleotides for the Production of
Intracellular Membrane Bound Proteins; U.S. Provisional Patent
Application No. 61/668,157, filed Jul. 5, 2012, entitled Modified
Polynucleotides for the Production of Intracellular Membrane Bound
Proteins; U.S. Provisional Patent Application No. 61/681,661, filed
Aug. 10, 2012, entitled Modified Polynucleotides for the Production
of Intracellular Membrane Bound Proteins; U.S. Provisional Patent
Application No. 61/737,160, filed Dec. 14, 2012, entitled Modified
Polynucleotides for the Production of Intracellular Membrane Bound
Proteins; U.S. Provisional Patent Application No. 61/618,911, filed
Apr. 2, 2012, entitled Modified Polynucleotides for the Production
of Nuclear Proteins; U.S. Provisional Patent Application No.
61/681,667, filed Aug. 10, 2012, entitled Modified Polynucleotides
for the Production of Nuclear Proteins; U.S. Provisional Patent
Application No. 61/737,168, filed Dec. 14, 2012, entitled Modified
Polynucleotides for the Production of Nuclear Proteins; U.S.
Provisional Patent Application No. 61/618,922, filed Apr. 2, 2012,
entitled Modified Polynucleotides for the Production of Proteins;
U.S. Provisional Patent Application No. 61/681,675, filed Aug. 10,
2012, entitled Modified Polynucleotides for the Production of
Proteins; U.S. Provisional Patent Application No. 61/737,174, filed
Dec. 14, 2012, entitled Modified Polynucleotides for the Production
of Proteins; U.S. Provisional Patent Application No. 61/618,935,
filed Apr. 2, 2012, entitled Modified Polynucleotides for the
Production of Proteins Associated with Human Disease; U.S.
Provisional Patent Application No. 61/681,687, filed Aug. 10, 2012,
entitled Modified Polynucleotides for the Production of Proteins
Associated with Human Disease; U.S. Provisional Patent Application
No. 61/737,184, filed Dec. 14, 2012, entitled Modified
Polynucleotides for the Production of Proteins Associated with
Human Disease; U.S. Provisional Patent Application No. 61/618,945,
filed Apr. 2, 2012, entitled Modified Polynucleotides for the
Production of Proteins Associated with Human Disease; U.S.
Provisional Patent Application No. 61/681,696, filed Aug. 10, 2012,
entitled Modified Polynucleotides for the Production of Proteins
Associated with Human Disease; U.S. Provisional Patent Application
No. 61/737,191, filed Dec. 14, 2012, entitled Modified
Polynucleotides for the Production of Proteins Associated with
Human Disease; U.S. Provisional Patent Application No. 61/618,953,
filed Apr. 2, 2012, entitled Modified Polynucleotides for the
Production of Proteins Associated with Human Disease; U.S.
Provisional Patent Application No. 61/681,704, filed Aug. 10, 2012,
entitled Modified Polynucleotides for the Production of Proteins
Associated with Human Disease; U.S. Provisional Patent Application
No. 61/737,203, filed Dec. 14, 2012, entitled Modified
Polynucleotides for the Production of Proteins Associated with
Human Disease; U.S. Provisional Patent Application No. 61/681,720,
filed Aug. 10, 2012, entitled Modified Polynucleotides for the
Production of Cosmetic Proteins and Peptides; U.S. Provisional
Patent Application No. 61/737,213, filed Dec. 14, 2012, entitled
Modified Polynucleotides for the Production of Cosmetic Proteins
and Peptides; U.S. Provisional Patent Application No. 61/681,742,
filed Aug. 10, 2012, entitled Modified Polynucleotides for the
Production of Oncology-Related Proteins and Peptides; International
Application No PCT/US2013/030062, filed Mar. 9, 2013, entitled
Modified Polynucleotides for the Production of Biologics and
Proteins Associated with Human Disease; U.S. patent application
Ser. No. 13/791,922, filed Mar. 9, 2013, entitled Modified
Polynucleotides for the Production of Biologics and Proteins
Associated with Human Disease; International Application No
PCT/US2013/030063, filed Mar. 9, 2013, entitled Modified
Polynucleotides; International Application No. PCT/US2013/030064,
entitled Modified Polynucleotides for the Production of Secreted
Proteins; U.S. patent application Ser. No. 13/791,921, filed Mar.
9, 2013, entitled Modified Polynucleotides for the Production of
Secreted Proteins; International Application No PCT/US2013/030059,
filed Mar. 9, 2013, entitled Modified Polynucleotides for the
Production of Membrane Proteins; International Application No.
PCT/US2013/030066, filed Mar. 9, 2013, entitled Modified
Polynucleotides for the Production of Cytoplasmic and Cytoskeletal
Proteins; International Application No. PCT/US2013/030067, filed
Mar. 9, 2013, entitled Modified Polynucleotides for the Production
of Nuclear Proteins; International Application No.
PCT/US2013/030060, filed Mar. 9, 2013, entitled Modified
Polynucleotides for the Production of Proteins; International
Application No. PCT/US2013/030061, filed Mar. 9, 2013, entitled
Modified Polynucleotides for the Production of Proteins Associated
with Human Disease; U.S. patent application Ser. No. 13/791,910,
filed Mar. 9, 2013, entitled Modified Polynucleotides for the
Production of Proteins Associated with Human Disease; International
Application No. PCT/US2013/030068, filed Mar. 9, 2013, entitled
Modified Polynucleotides for the Production of Cosmetic Proteins
and Peptides; and International Application No. PCT/US2013/030070,
filed Mar. 9, 2013, entitled Modified Polynucleotides for the
Production of Oncology-Related Proteins and Peptides; International
Patent Application No. PCT/US2013/031821, filed Mar. 15, 2013,
entitled In Vivo Production of Proteins; the contents of each of
which are herein incorporated by reference in their entireties.
[0007] Formulations and delivery of modified polynucleotides are
described in, for example, co-pending and co-owned International
Publication No WO2013090648, filed Dec. 14, 2012, entitled Modified
Nucleoside, Nucleotide, Nucleic Acid Compositions and US
Publication No US20130156849, filed Dec. 14, 2012, entitled
Modified Nucleoside, Nucleotide, Nucleic Acid Compositions; the
contents of each of which are herein incorporated by reference in
their entireties.
[0008] The next generation of therapeutics must also address the
complex cellular microenvironment of the cancer and have the
capacity for cell, tissue, organ or patient stratification, whether
structurally or functionally.
[0009] The present invention addresses this need by providing
nucleic acid based compounds or polynucleotide-encoding nucleic
acid-based compounds (e.g., signal-sensor polynucleotides) which
encode a polypeptide of interest and which have structural and/or
chemical features that allow for greater selectivity, profiling or
stratification along defineable disease characteristics or
metrics.
SUMMARY OF THE INVENTION
[0010] Described herein are compositions, methods, processes, kits
and devices for the design, preparation, manufacture and/or
formulation of signal-sensor polynucleotide molecules encoding at
least one oncology-related polypeptide of interest. Such
signal-sensor polynucleotides may be chemically modified mRNA
(mmRNA) molecules.
[0011] The present invention provides an isolated signal-sensor
polynucleotide comprising a region encoding an oncology-related
polypeptide of interest that functions, when translated, to send a
death or survival signal. Such death or survival signals include
those which (i) alter (increase or decrease) the expression of one
or more proteins, nucleic acids, or non-coding nucleic acids, (ii)
alter the binding properties of biomolecules within the cell,
and/or (iii) perturb the cellular microenvironment in a
therapeutically beneficial way.
[0012] Optionally, the signal-sensor polynucleotide may also encode
in a flanking region, one or more sensor sequences. Such sensor
sequences function to "sense" the cell, tissue or organ
microenvironment and confer upon the signal-sensor polynucleotide
an altered expression or half life profile (increased or decreased)
depending on the interactions of the sensor sequence with the cell,
tissue or organ microenvironment.
[0013] In one aspect, provided herein are signal-sensor
polynucleotide comprising, a first region of linked nucleosides, a
first flanking region located 5' relative to said first region and
a second flanking region located 3' relative to said first region.
The first region may encode an oncology-related polypeptide of
interest such as, but not limited to, SEQ ID NOs: 1321-2487,
6611-6616 and 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516 and
7517 and the first flanking region may include a sequence of linked
nucleosides such as, but not limited to, the native 5' untranslated
region (UTR) of any of the nucleic acids that encode any of SEQ ID
NOs: 1321-2487, 6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514,
7516, 7517, SEQ ID NO: 1-4 and functional variants thereof. The
first region may comprise at least an open reading frame of a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 2488-2496, 6617-6621, 7348-7354, 7362-7489, 7491, 7494, 7506,
7511 and 7513.
[0014] The second flanking region may include a sequence of linked
nucleosides such as, but not limited to, the native 3' UTR of any
of the nucleic acids that encode any of SEQ ID NOs: 1321-2487,
6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516, 7517, SEQ
ID NO: 5-21 and functional variants thereof, and one or more sensor
sequences located such as, but not limited to, SEQ ID NOs:
3529-4549, SEQ ID NOs: 5571-6591 and functional variants thereof.
The signal-sensor polynucleotide may also include a 3' tailing
sequence of linked nucleosides.
[0015] In another aspect, provided herein is a signal-sensor
polynucleotide which comprises an mRNA encoding an oncology-related
polypeptide of interest and one or more sensor sequences such as,
but not limited to, SEQ ID NOs: 3529-4549, SEQ ID NOs: 5571-6591
and functional variants thereof. The oncology-related polypeptide
of interest may be, but is not limited to, SEQ ID NOs: 1321-2487,
6611-6616, 7355-7361, 7490, 7492, 7493, 7512, 7514, 7516 and 7517.
The mRNA may include at least one open reading frame of a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
2488-2496, 6617-6621, 7348-7354, 7362-7489, 7491, 7494, 7506, 7511
and 7513.
[0016] The signal-sensor polynucleotides may comprise one, two,
three or more than three stop codons. In one aspect, the
signal-sensor polynucleotides comprise two stop codons. As a
non-limiting example, the first stop codon is "TGA" and the second
stop codon is selected from the group consisting of "TAA," "TGA"
and "TAG." In another aspect, signal-sensor polynucleotides
comprise three stop codons.
[0017] The signal-sensor polynucleotides may have a 3' tailing
sequence of linked nucleosides such as, but not limited to, a
poly-A tail of at least 140 nucleotides, a triple helix, and a poly
A-G quartet.
[0018] The signal-sensor polynucleotides may have a 5' cap such as,
but not limited to, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,
2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,
2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
[0019] In one aspect, the signal-sensor polynucleotides may include
at least one chemical modification such as, but not limited to,
modifications located on one or more of a nucleoside and/or the
backbone of the nucleotides. In one embodiment, the signal-sensor
polynucleotides comprise a pseudouridine analog such as, but not
limited to, 1-carboxymethyl-pseudouridine,
1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine,
1-taurinomethyl-4-thio-pseudouridine, 1-methyl-pseudouridine
(m.sup.1.psi.), 1-methyl-4-thio-pseudouridine
(m.sup.1s.sup.4.psi.), 4-thio-1-methyl-pseudouridine,
3-methyl-pseudouridine (m.sup.3.psi.),
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,
4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,
1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp.sup.3.psi.),
and 2'-O-methyl-pseudouridine (.psi.m). In another embodiment, the
signal-sensor polynucleotides comprise the pseudouridine analog
1-methylpseudouridine. In yet another embodiment, the signal-sensor
polynucleotides comprise the pseudouridine analog
1-methylpseudouridine and the modified nucleoside
5-methylcytidine.
[0020] In another aspect, the signal-sensor polynucleotides may
include at least two chemical modifications such as, but not
limited to, modifications located on one or more of a nucleoside
and/or the backbone of the nucleotides. As a non-limiting example,
the signal-sensor polynucleotide comprises the chemical
modifications 1-methylpseudouridine and 5-methylcytidine.
[0021] The signal-sensor polynucleotides may comprise at least one
translation enhancer element (TEE) such as, but not limited to,
TEE-001-TEE-705.
[0022] In one aspect, the signal-sensor polynucleotide encodes a
factor modulating the affinity between HIF subunits and/or
HIF-dependent gene expression such as, but not limited to, SEQ ID
NO: 6611-6616.
[0023] The signal-sensor polynucleotides may be purified and/or
formulated.
[0024] Employing the signal-sensor polynucleotides, the present
invention provides a method of treating a disease, disorder and/or
condition in a subject in need thereof by increasing the level of
an oncology-related polypeptide of interest comprising
administering to said subject an isolated signal-sensor
polynucleotide encoding said oncology-related polypeptide. The
disease, disorder and/or condition may include, but is not limited
to, adrenal cortical cancer, advanced cancer, anal cancer, aplastic
anemia, bileduct cancer, bladder cancer, bone cancer, bone
metastasis, brain tumors, brain cancer, breast cancer, childhood
cancer, cancer of unknown primary origin, Castleman disease,
cervical cancer, colon/rectal cancer, endometrial cancer, esophagus
cancer, Ewing family of tumors, eye cancer, gallbladder cancer,
gastrointestinal carcinoid tumors, gastrointestinal stromal tumors,
gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma,
renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute
lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic
leukemia, chronic myeloid leukemia, chronic myelomonocytic
leukemia, liver cancer, non-small cell lung cancer, small cell lung
cancer, lung carcinoid tumor, lymphoma of the skin, malignant
mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal
cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal
cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile
cancer, pituitary tumors, prostate cancer, retinoblastoma,
rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft
tissue, basal and squamous cell skin cancer, melanoma, small
intestine cancer, stomach cancer, testicular cancer, throat cancer,
thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer,
vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and
secondary cancers caused by cancer treatment.
[0025] The present invention provides a method of reducing,
eliminating, or preventing tumor growth in a subject in need
thereof by increasing the level of an oncology-related polypeptide
of interest comprising administering to said subject an isolated
signal-sensor polynucleotide encoding said oncology-related
polypeptide. The tumor growth may be associated with or results
from a disease, disorder and/or condition such as, but not limited
to, adrenal cortical cancer, advanced cancer, anal cancer, aplastic
anemia, bileduct cancer, bladder cancer, bone cancer, bone
metastasis, brain tumors, brain cancer, breast cancer, childhood
cancer, cancer of unknown primary origin, Castleman disease,
cervical cancer, colon/rectal cancer, endometrial cancer, esophagus
cancer, Ewing family of tumors, eye cancer, gallbladder cancer,
gastrointestinal carcinoid tumors, gastrointestinal stromal tumors,
gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma,
renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute
lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic
leukemia, chronic myeloid leukemia, chronic myelomonocytic
leukemia, liver cancer, non-small cell lung cancer, small cell lung
cancer, lung carcinoid tumor, lymphoma of the skin, malignant
mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal
cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal
cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile
cancer, pituitary tumors, prostate cancer, retinoblastoma,
rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft
tissue, basal and squamous cell skin cancer, melanoma, small
intestine cancer, stomach cancer, testicular cancer, throat cancer,
thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer,
vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and
secondary cancers caused by cancer treatment.
[0026] The present invention provides a method of reducing and/or
ameriorating at least one symptom of cancer in a subject in need
thereof by increasing the level of a polypeptide of interest
comprising administering to said subject an isolated signal-sensor
polynucleotide encoding said oncology-related polypeptide.
Non-limiting examples of symptoms include weakness, aches and
pains, fever, fatigue, weight loss, blood clots, increased blood
calcium levels, low white blood cell count, short of breath,
dizziness, headaches, hyperpigmentation, jaundice, erthema,
pruritis, excessive hair growth, change in bowel habits, change in
bladder function, long-lasting sores, white patches inside the
mouth, white spots on the tongue, unusual bleeding or discharge,
thickening or lump on parts of the body, indigestion, trouble
swallowing, changes in warts or moles, change in new skin and
nagging cough and hoarseness.
[0027] The present invention provides a method of preferentially
inducing cell death in cancer cells in a tissue or organ comprising
contacting the tissue or organ with a signal-sensor polynucleotide
encoding an oncology-related polypeptide whose expression triggers
apoptosis or cell death and at least one microRNA binding site of a
microRNA where the expression of the microRNA in the cancer cell is
lower than the expression of the mircroRNA in normal non-cancerous
cells.
[0028] The signal-sensor polynucleotide may be administered at a
total daily dose of between 0.001 ug and 150 ug. Administration of
a signal-sensor polynucleotide may be by injection, topical
administration, ophthalmic administration or intranasal
administration. In one aspect, administration may be by injection
such as, but not limited to, intradermal, subcutaneous and
intramuscular. In another aspect, administration may be topical
such as, but not limited to, using creams, lotions, ointments,
gels, sprays, solutions and the like.
[0029] The details of various embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The foregoing and other objects, features and advantages
will be apparent from the following description of particular
embodiments of the invention, as illustrated in the accompanying
drawings in which like reference characters refer to the same parts
throughout the different views. The drawings are not necessarily to
scale, emphasis instead being placed upon illustrating the
principles of various embodiments of the invention.
[0031] FIG. 1 is a schematic of a primary construct of the present
invention.
[0032] FIG. 2 is an expanded schematic of the second flanking
region of a primary construct of the present invention illustrating
the signal-sensor elements of the polynucleotide.
[0033] FIG. 3 is a gel profile of Apoptosis-Inducing Factor short
(AIFsh) protein from AIFsh modified mRNA in mammals. FIG. 3A shows
the expected size of AIFsh. FIG. 3B shows the expected size of
AIFsh.
[0034] FIG. 4 is a gel profile of Siah E3 ubiquitin protein ligase
1 (SIAH1) protein from SIAH1 modified mRNA in mammals. FIG. 4A
shows the expected size of SIAH1. FIG. 4B shows the expected size
of SIAH1.
[0035] FIG. 5 is a gel profile of constitutively active (C.A.)
caspase 3 (also known as reverse caspase 3 (Rev-Caspase 3)) protein
from C.A. caspase 3 modified mRNA in mammals. FIG. 5A shows the
expected size of C.A. caspase 3. FIG. 5B shows the expected size of
C.A. caspase 3.
[0036] FIG. 6 is a gel profile of Granulysin protein from
granulysin modified mRNA in mammals. FIG. 6A shows the expected
size of granulysin. FIG. 6B shows the expected size of
granulysin.
[0037] FIG. 7 is a western blot of C.A. caspase 3 and C.A. caspase
6. FIG. 7A shows protein from C.A. caspase 3 modified mRNA fully
modified with 5-methylcytidine and 1-methylpseudouridine or fully
modified with 1-methylpseudouridine. FIG. 7B shows protein from
C.A. caspase 6 modified mRNA fully modified with 5-methylcytidine
and 1-methylpseudouridine or fully modified with
1-methylpseudouridine.
DETAILED DESCRIPTION
[0038] It is of great interest in the fields of therapeutics,
diagnostics, reagents and for biological assays to be able to
deliver a nucleic acid, e.g., a ribonucleic acid (RNA) inside a
cell, whether in vitro, in vivo, in situ or ex vivo, such as to
cause intracellular translation of the nucleic acid and production
of an encoded polypeptide of interest. Of particular importance is
the delivery and function of a non-integrative polynucleotide.
[0039] Described herein are compositions (including pharmaceutical
compositions) and methods for the design, preparation, manufacture
and/or formulation of polynucleotides encoding one or more
polypeptides of interest. Also provided are systems, processes,
devices and kits for the selection, design and/or utilization of
the polynucleotides encoding the polypeptides of interest described
herein.
[0040] To this end, polypeptides of the present invention are
encoded by a new class of polynucleotide therapeutics, termed
"signal-sensor polynucleotides" which are particularly useful in
the stratification, profiling and/or personalization of the
polynucleotide therapeutice (e.g., mRNA) and which are tailored to
a particular cell type, disease or cell microenvironment or
biological profile.
[0041] It is known that cancers exhibit diverse gene expression
patterns, physicochemical environments and metastatic or motility
behaviors and according to Hanahan and Weinberg (Cell, 2011,
144:646-674) there are six hallmarks of cancer. These include
sustaining a proliferative signaling, evading growth suppressors,
resisting cell death, enabling replicative immortality, inducing
angiogenesis, and activating invasion and metastasis. These
hallmarks or functions of cancer allow the cancer to survive,
proliferate and disseminate and each arises at different times and
in different patterns depending on the cancer type.
[0042] The development of cancer therapeutics which to selectively
target the cancer cells while sparing normal cells dominates
ongoing efforts in every area of oncology. The polynucleotides of
the present invention represent such therapeutics; having the
ability to selectively stabilize or destabilize cell systems,
signal proliferation (survival) or death, trigger the cell cycle or
senescence and/or activate or avoid the immune response depending
on the cell type, e.g., cancer or normal cell.
[0043] According to the present invention, signal-sensor
polynucleotide therapeutics may be used to destabilize the survival
advantages or hallmarks of a cancer cell (hence they would be
cytotoxic). In one embodiment diagnostic efforts would include the
profiling of the cancer (although this would not be required a
priori) including including metabolic state (hypoxic, acidotic),
apoptotic vs. survival gene profiles, cell cycle vs. senescent
stage, immune status, and stromal factors present.
[0044] In one embodiment the signal-sensor polynucleotide disrupts
the transcriptome of the cancer cell. The disruption may affect one
or more signaling or expression events. For example the encoded
oncology-related polypeptide may act upstream of a transcription
factor known to induce or enhance the expression of genes
associated with a cancer. Delivery of the signal-sensor
polynucleotide encoding the oncology-related polypeptide which
inhibits such a transcription factor (either by binding or
sequestration or degradation) would thereby alter the transcriptome
of the cancer cell and have a therapeutic benefit. One such
transcription factor is HIF-1alpha. A signal-sensor polynucleotide
encoding a protein which is capable of binding HIF-1alpha or whose
expression results in lower HIF-1alpha, would effectively turn down
HIF-1alpha regulated genes, e.g., VEGFA or SLC2A1, and destabilize
the cancer.
[0045] In one embodiment, the profile of the cancer may be
evaluated before the signal-sensor polynucleotide is selected. Such
profiling data would inform the selection of which oncology-related
polypeptide to be delivered. The profile of gene expression,
categorized by hallmark class such as apoptosis, replicative
capacity or metabolic signature would allow dynamic instability
scoring for a polypeptide and an optimization of therapeutic window
for the signal-sensor polynucleotide. As used herein, a "dynamic
instability index" refers to a dose of signal-sensor polynuclotide
sufficient to induce 50% increase of the oncology-related target
protein in vitro in a cancer cell as compared to a normal matched
cell.
[0046] Profiling may also be done within hallmark classes such as
the distinction between caspase-dependent and caspase independent
gene expression for the apoptosis class. Alternatively, profiling
could be conducted across classes such as gene profiling of
apoptosis, senescence (replicative capacity), and metabolic
classes.
[0047] In one embodiment, the signal-sensor polynucleotides
described herein may be used to reduce the expression and/or amount
of a polypeptide in a cell. As a non-limiting example, MYC
inhibitor A, MYC inhibitor B, MYC inhibitor C or MYC inhibitor D
may be used on Hep3B cells in order to determine the potentcy of
MYC inhibitor A, MYC inhibitor B, MYC inhibitor C or MYC inhibitor
D at various concentrations (see e.g., Example 55).
[0048] In one embodiment, the signal-sensor polynucleotides
described hereim may direct either cytotoxic or cytoprotective
therapeutic benefit to specific cells, e.g., normal vs.
cancerous.
[0049] In one embodiment signal-sensor polynucleotides would not
only encode an oncology-related polypeptide but also a sensor
sequence. Sensor sequences include, for example, microRNA binding
sites, transcription factor binding sites, artificial binding sites
engineered to act as pseudo-receptors for endogenous nucleic acid
binding molecules. A "sensor region" is a region of linked
nucleosides of the signal-sensor polynucleotide comprising at least
one sensor sequence. The signal-sensor polynucleotides of the
present invention may have one or more sensor regions.
[0050] In one embodiment, one or more sensor regions may be located
in the first flanking region. As a non-limiting example, the sensor
region in the first flanking region may comprise at least one
sensor sequence. The sensor sequence may be, but is not limited to,
mir-122, mir-142-3p, mir-142-5p, mir-146, fragments or variants
thereof. As another non-limiting example, the sensor region in the
first flanking region may comprise at least one sensor sequence
such as a mir-122 sequence. The mir-122 sequence may be, but is not
limited to, a mir-122 binding site, mir-122 seed sequence, mir-122
binding site without the seed sequence or a combination thereof
[0051] In another embodiment, one or more sensor regions may be
located in the second flanking region. As a non-limiting example,
the sensor region in the second flanking region may include a
sensor sequence such as mir-122, mir-142-3p, mir-142-5p, mir-146,
fragments or variants thereof. As another non-limiting example, the
sensor region in the second flanking region may include three
sensor sequences. The sensor sequences may be, but are not limited
to, mir-122 sequences such as mir-122 binding sites, mir-122 seed
sequences, mir-122 binding sites without the seed sequence or a
combination thereof. As yet another non-limiting example, the
sensor region in the second flanking region is located in the 3'UTR
and the sensor region may include a sensor sequence which is a
mir-122 sequence. The mir-122 sequence may be, but is not limited
to, a mir-122 binding site, mir-122 seed sequence, mir-122 binding
site without the seed sequence or a combination thereof
[0052] In one embodiment, two or more sensor regions may be located
in the same region of the signal-sensor polynucleotide such as, but
not limited to, a first region first region of linked nucleotides,
the first flanking region and/or the second flanking region. As a
non-limiting example, the two or more sensor regions are located in
the second flanking region. As yet another non-limiting example,
three sensor regions are located in the 3' UTR in the second
flanking region. The three sensor regions may include, mir-122
binding sites, mir-122 seed sequences, mir-122 binding sites
without the seed sequence or a combination thereof
[0053] In another embodiment, two or more sensor regions may be
located in different regions of the signal-sensor polynucleotide
such as, but not limited to, the first region of linked
nucleotides, the first flanking region and/or the second flanking
region. As a non-limiting example, a first sensor region is located
in the first flanking region and a second sensor region is located
in the second flanking region. The sensor regions may comprise the
same sensor sequence or different sensor sequences.
[0054] In one embodiment, a start codon is located within a sensor
region.
[0055] In one embodiment, a sensor region may comprise two or more
sensor sequences. The sensor sequences may be the same or
different.
[0056] In one embodiment, the sensor region may comprise two or
more sensor sequence which are different from each other but they
may be based on the same mir binding site. As a non-limiting
example, the sensor region may include at least one miR binding
site sequence and at least one mir binding site sequence with the
seed removed. As another non-limiting example, the sensor region
may include at least one miR binding site sequence and at least one
miR seed sequence. As yet another non-limiting example, the sensor
region may include at least one miR binding site sequence with the
seed removed and at least one miR seed sequence.
[0057] In another embodiment, the sensor region may comprise two or
more sensor sequences which are in a pattern such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice,
or more than three times. In these patterns, each letter, A, B, or
C represent a different miR sequence.
[0058] In yet another embodiment, the signal-sensor polynucleotide
may include two or more sensor regions with each sensor region
having one or more sensor sequences. As a non-limiting example, the
sensor sequences may be in a pattern such as ABABAB or AABBAABBAABB
or ABCABCABC or variants thereof repeated once, twice, or more than
three times in each of the sensor regions. As another non-limiting
example, the sensor sequences may be in a pattern such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice,
or more than three times across the entire signal-sensor
polynucleotide. In these patterns, each letter, A, B, or C
represent a different miR sequence. As a non-limiting example, the
first sensor region may have sensor sequences in the pattern ABA
and the second sensor region may have sensor sequences in the
pattern BAB so the overall pattern of the sensor sequences in the
signal-sensor polynucleotide is ABABAB. As another non-limiting
example, the first sensor region may have sensor sequences AA, the
second sensor region may have sensor sequences BB, the third sensor
region may have sensor sequences AA and the fourth sensor region
may have sensor seuqences BB so the overall pattern of the sensor
sequences in the signal-sensor polynucleotide is AABBAABB.
[0059] The sensor sequences in the signal-sensor polynucleotides of
the present invention may include one or more regulatory sequences
in the 3-UTR and/or 5'UTR of natural mRNAs, which regulate mRNA
stability and translation in different tissues and cells. Such
cis-regulatory elements may include, but are not limited to,
Cis-RNP (Ribonucleoprotein)/RBP (RNA binding protein) regulatory
elements, AU-rich element AUE, structured stem-loop, constitutive
decay elements (CDEs), GC-richness and other structured mRNA motifs
(Parker B J et al., Genome Research, 2011, 21, 1929-1943, which is
herein incorporated by reference in its entirety.). For example,
CDEs are a class of regulatory motifs that mediate mRNA degradation
through their interaction with Roquin proteins. In particular, CDEs
are found in many mRNAs that encode regulators of development and
inflammation to limit cytokine production in macrophage (Leppek K
et al., Cell, 2013, 153, 869-881, which is herein incorporated by
reference in its entirety.).
[0060] In one embodiment, a particular CDE can be introduced to the
signal-sensor polynucleotide when the degradation of polypeptides
in a cell or tissue is desired. A particular CDE can also be
removed from the signal-sensor polynucleotide in order to maintain
a more stable mRNA in a cell or tissue for sustaining protein
expression.
[0061] In one embodiment, microRNA (miRNA) profiling of the cancer
cells or tissues may be conducted to determine the presence or
absence of miRNA in the cells or tissues to determine the
appropriate microRNA to use as sensor sequences in the signal
sensor polynucleotides.
[0062] MicroRNA gene regulation may be influenced by the sequence
surrounding the microRNA such as, but not limited to, the species
of the surrounding sequence, the type of sequence (e.g.,
heterologous, homologous and artificial), regulatory elements in
the surrounding sequence and/or structural elements in the
surrounding sequence. The microRNA may be influenced by the 5'UTR
and/or the 3'UTR. As a non-limiting example, a non-human 3'UTR may
increase the regulatory effect of the microRNA sequence on the
expression of a polypeptide of interest compared to a human 3'UTR
of the same sequence type.
[0063] Other regulatory elements and/or structural elements of the
5'-UTR can influence microRNA mediated gene regulation. One such
example is a structured IRES (Internal Ribosome Entry Site) in the
5'UTR, which is necessary for the binding of translational
elongation factors to initiate protein translation. EIF4A2 binding
to this secondarily structured element in the 5'UTR is necessary
for microRNA mediated gene expression (Meijer H A et al., Science,
2013, 340, 82-85, herein incorporated by reference in its
entirety). The sensor-signal polynucleotide can further be modified
to include this structured 5'-UTR in order to enhance microRNA
mediated gene regulation.
[0064] At least one microRNA site can be engineered into the 3' UTR
of the signal-sensor polynucleotides of the present invention. In
this context, at least two, at least three, at least four, at least
five, at least six, at least seven, at least eight, at least nine,
at least ten or more microRNA sites may be engineered into the 3'
UTR of the signal-sensor polynucleotides of the present invention.
In one embodiment, the microRNA sites incorporated into the
signal-sensor polynucleotides may be the same or may be different
microRNA sites. In another embodiment, the microRNA sites
incorporated into the signal-sensor polynucleotides may target the
same or different tissues in the body. As a non-limiting example,
through the introduction of tissue-, cell-type-, or
disease-specific microRNA binding sites in the 3' UTR of a
signal-sensor polynucleotide, the degree of expression in specific
cell types (e.g. hepatocytes, myeloid cells, endothelial cells,
cancer cells, etc.) can be reduced.
[0065] In one embodiment, a microRNA site can be engineered near
the 5' terminus of the 3'UTR, about halfway between the 5' terminus
and 3' terminus of the 3'UTR and/or near the 3' terminus of the
3'UTR. As a non-limiting example, a microRNA site may be engineered
near the 5' terminus of the 3'UTR and about halfway between the 5'
terminus and 3' terminus of the 3'UTR. As another non-limiting
example, a microRNA site may be engineered near the 3' terminus of
the 3'UTR and about halfway between the 5' terminus and 3' terminus
of the 3'UTR. As yet another non-limiting example, a microRNA site
may be engineered near the 5' terminus of the 3'UTR and near the 3'
terminus of the 3'UTR.
[0066] In another embodiment, a 3'UTR can comprise 4 microRNA
sites. The microRNA sites may be complete microRNA binding sites,
microRNA seed sequences and/or microRNA binding site sequences
without the seed sequence.
[0067] In one embodiment, a signal-sensor polynucleotide may be
engineered to include microRNA sites which are expressed in
different tissues of a subject. As a non-limiting example, a
signal-sensor polynucleotide of the present invention may be
engineered to include miR-192 and miR-122 to regulate expression of
the signal-sensor polynucleotide in the liver and kidneys of a
subject. In another embodiment, a signal-sensor polynucleotide may
be engineered to include more than one microRNA sites for the same
tissue. For example a signal-sensor polynucleotide of the present
invention may be engineered to include miR-17-92 and miR-126 to
regulate expression of the signal-sensor polynucleotide in
endothelial cells of a subject.
[0068] In one embodiment, the therapeutic window and or
differential expression associated with the oncology-related
polypeptide encoded by the signal-sensor polynucleotide of the
invention may be altered. For example, signal-sensor
polynucleotides may be designed whereby a death signal is more
highly expressed in cancer cells (or a survival signal in a normal
cell) by virtue of the miRNA signature of those cells. Where a
cancer cell expresses a lower level of a particular miRNA, the
signal-sensor polynucleotide encoding the binding site for that
miRNA (or miRNAs) would be more highly expressed. Hence, the
oncology-related polypeptide encoded by the signal-sensor
polynucleotide is selected as a protein which triggers or induces
cell death. Neigboring noncancer cells, harboring a higher
expression of the same miRNA would be less affected by the encoded
death signal as the signal-sensor polynucleotide would be expressed
at a lower level due to the affects of the miRNA binding to the
binding site or "sensor" encoded in the 3'UTR. Conversely, cell
survival or cytoprotective signals may be delivered to tissues
containing cancer and non cancerous cells where a miRNA has a
higher expression in the cancer cells--the result being a lower
survival signal to the cancer cell and a larger survival signature
to the normal cell. Multiple signal-sensor polynucleotides may be
designed and administered having different signals according to the
previous paradigm.
[0069] In one embodiment, the expression of a signal-sensor
polynucleotide may be controlled by incorporating at least one
sensor sequence in the signal-sensor polynucleotide and formulating
the signal-sensor polynucleotide. As a non-limiting example, a
polynucleotide may be targeted to an orthotopic tumor by having a
polynucleotide incorporating a miR-122 binding site and formulated
in a lipid nanoparticle comprising the cationic lipid DLin-KC2-DMA
(see e.g., the experiments described in Example 56A and 56B).
[0070] Through an understanding of the expression patterns of
microRNA in different cell types, signal-sensor polynucleotides can
be engineered for more targeted expression in specific cell types
or only under specific biological conditions. Through introduction
of tissue-specific microRNA binding sites, signal-sensor
polynucleotides could be designed that would be optimal for protein
expression in a tissue or in the context of a biological condition
such as cancer.
[0071] Transfection experiments can be conducted in relevant cell
lines, using engineered signal-sensor polynucleotides and protein
production can be assayed at various time points post-transfection.
For example, cells can be transfected with different microRNA
binding site-engineering nucleic acids or signal-sensor
polynucleotides and by using an ELISA kit to the relevant protein
and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, 72 hr
and 7 days post-transfection. In vivo experiments can also be
conducted using microRNA-binding site-engineered molecules to
examine changes in tissue-specific expression of formulated
signal-sensor polynucleotides.
[0072] In one embodiment, the signal-sensor polynucleotides of the
invention may include at least one microRNA in order to dampen the
antigen presentation by antigen presenting cells. The microRNA may
be the complete microRNA sequence, the microRNA seed sequence, the
microRNA sequence without the seed or a combination thereof. As a
non-limiting example, the microRNA incorporated into the
signal-sensor polynucleotide may be specific to the hematopoietic
system. As another non-limiting example, the microRNA incorporated
into the signal-sensor polynucleotides of the invention to dampen
antigen presentation is miR-142-3p.
[0073] In one embodiment, the signal-sensor polynucleotides of the
invention may include at least one microRNA in order to dampen
expression of the encoded polypeptide in a cell of interest. As a
non-limiting example, the signal-sensor polynucleotides of the
invention may include at least one miR-122 binding site in order to
dampen expression of an encoded polypeptide of interest in the
liver. As another non-limiting example, the signal-sensor
polynucleotides of the invention may include at least one
miR-142-3p binding site, miR-142-3p seed sequence, miR-142-3p
binding site without the seed, miR-142-5p binding site, miR-142-5p
seed sequence, miR-142-5p binding site without the seed, miR-146
binding site, miR-146 seed sequence and/or miR-146 binding site
without the seed sequence (see e.g., the experiment outlined in
Example 47 and Example 60).
[0074] According to the present invention, the signal-sensor
polynucleotides described herein may be modified as to avoid the
deficiencies of other polypeptide-encoding molecules of the art.
Hence, in this embodiment the signal-sensor polynucleotides are
referred to as modified signal-sensor polynucleotides or primary
constructs, modified mRNA or mmRNA.
[0075] Provided herein, in part, are signal-sensor polynucleotide
polynucleotides, primary constructs and/or mmRNA encoding
oncology-related polypeptides of interest which have been designed
to improve one or more of the stability and/or clearance in
tissues, receptor uptake and/or kinetics, cellular access by the
compositions, engagement with translational machinery, mRNA
half-life, translation efficiency, immune evasion, protein
production capacity, secretion efficiency (when applicable),
accessibility to circulation, protein half-life and/or modulation
of a cell's status, function and/or activity.
I. Compositions of the Invention
[0076] The present invention provides nucleic acid molecules,
specifically signal-sensor polynucleotides, primary constructs
and/or mmRNA which encode one or more oncology-related polypeptides
of interest. Specifically the invention contemplates signal-sensor
polynucleotides which are useful in cancer or cancer related
diseases, disorders. As used herein, "signal-sensor
polynucleotides" are nucleic acid transcripts which encode one or
more oncology-related polypeptides of interest that, when
translated, delivers a "signal" to the cell (cancer or
noncancerous) which results in the therapeutic benefit to the
organism of either being detrimental to the cancer cell or
beneficial to normal cells or both detrimental to cancer cells and
advantageous to normal cells. The signal-sensor polynucleotides may
optionally further comprise a sequence (translatable or not) which
"senses" the microenvironment of the polynucleotide and alters (a)
the function or phenotypic outcome associated with the peptide or
protein which is translated, (b) the expression level of the
signal-sensor polynucleotide, and/or both.
[0077] The term "nucleic acid," in its broadest sense, includes any
compound and/or substance that comprise a polymer of nucleotides.
These polymers are often referred to as polynucleotides. Exemplary
nucleic acids or polynucleotides of the invention include, but are
not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids
(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),
peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including
LNA having a .beta.-D-ribo configuration, .alpha.-LNA having an
.alpha.-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA
having a 2'-amino functionalization, and 2'-amino-.alpha.-LNA
having a 2'-amino functionalization) or hybrids thereof
[0078] In preferred embodiments, the signal-sensor polynucleotide
or nucleic acid molecule is a messenger RNA (mRNA). As used herein,
the term "messenger RNA" (mRNA) refers to any polynucleotide which
encodes a polypeptide of interest and which is capable of being
translated to produce the encoded polypeptide of interest in vitro,
in vivo, in situ or ex vivo. Signal-sensor polynucleotides of the
invention may be mRNA or any nucleic acid molecule and may or may
not be chemically modified.
[0079] Traditionally, the basic components of an mRNA molecule
include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a
poly-A tail. Building on this wild type modular structure, the
present invention expands the scope of functionality of traditional
mRNA molecules by providing signal-sensor polynucleotides or
primary RNA constructs which maintain a modular organization, but
which comprise one or more structural and/or chemical modifications
or alterations which impart useful properties to the polynucleotide
including, in some embodiments, the lack of a substantial induction
of the innate immune response of a cell into which the
signal-sensor polynucleotide is introduced. As such, modified mRNA
molecules of the present invention, which may be synthetic, are
termed "mmRNA." As used herein, a "structural" feature or
modification is one in which two or more linked nucleotides are
inserted, deleted, duplicated, inverted or randomized in a
signal-sensor polynucleotide polynucleotide, primary construct or
mmRNA without significant chemical modification to the nucleotides
themselves. Because chemical bonds will necessarily be broken and
reformed to effect a structural modification, structural
modifications are of a chemical nature and hence are chemical
modifications. However, structural modifications will result in a
different sequence of nucleotides. For example, the polynucleotide
"ATCG" may be chemically modified to "AT-5meC-G". The same
polynucleotide may be structurally modified from "ATCG" to
"ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting
in a structural modification to the polynucleotide.
Signal-Sensor Polynucleotide, Primary Construct or mmRNA
Architecture
[0080] The signal-sensor polynucleotides of the present invention
are distinguished from wild type mRNA in their functional and/or
structural design features which serve to, as evidenced herein,
overcome existing problems of effective polypeptide production
using nucleic acid-based therapeutics.
[0081] FIG. 1 shows a representative signal-sensor primary
construct 100 of the present invention. As used herein, the term
"primary construct" or "primary mRNA construct" refers to a
signal-sensor polynucleotide transcript which encodes one or more
polypeptides of interest and which retains sufficient structural
and/or chemical features to allow the polypeptide of interest
encoded therein to be translated. Signal-sensor primary constructs
may be polynucleotides of the invention. When structurally or
chemically modified, the signal-sensor primary construct may be
referred to as a mmRNA.
[0082] Returning to FIG. 1, the primary construct 100 here contains
a first region of linked nucleotides 102 that is flanked by a first
flanking region 104 and a second flaking region 106. As used
herein, the "first region" may be referred to as a "coding region"
or "region encoding" or simply the "first region." This first
region may include, but is not limited to, the encoded
oncology-related polypeptide of interest. The oncology-related
polypeptide of interest may comprise at its 5' terminus one or more
signal peptide sequences encoded by a signal peptide sequence
region 103. The flanking region 104 may comprise a region of linked
nucleotides comprising one or more complete or incomplete 5' UTRs
sequences. The flanking region 104 may also comprise a 5' terminal
cap 108. The second flanking region 106 may comprise a region of
linked nucleotides comprising one or more complete or incomplete 3'
UTRs. The flanking region 106 may also comprise a 3' tailing
sequence 110 and a 3'UTR 120.
[0083] Bridging the 5' terminus of the first region 102 and the
first flanking region 104 is a first operational region 105.
Traditionally this operational region comprises a start codon. The
operational region may alternatively comprise any translation
initiation sequence or signal including a start codon.
[0084] Bridging the 3' terminus of the first region 102 and the
second flanking region 106 is a second operational region 107.
Traditionally this operational region comprises a stop codon. The
operational region may alternatively comprise any translation
initiation sequence or signal including a stop codon. According to
the present invention, multiple serial stop codons may also be
used. In one embodiment, the operation region of the present
invention may comprise two stop codons. The first stop codon may be
"TGA" and the second stop codon may be selected from the group
consisting of "TAA," "TGA" and "TAG." The operation region may
further comprise three stop codons. The third stop codon may be
selected from the group consisting of "TAA," "TGA" and "TAG."
[0085] Turning to FIG. 2, the 3'UTR 120 of the second flanking
region 106 may comprise one or more sensor sequences 130. A region
comprising at least one sensor sequence is referred to as a "sensor
region." These sensor sequences as discussed herein operate as
pseudo-receptors (or binding sites) for ligands of the local
microenvironment of the primary construct or signal-sensor
polynucleotide. For example, microRNA binding sites or miRNA seeds
may be used as sensors such that they function as pseudoreceptors
for any microRNAs present in the environment of the
polynucleotide.
[0086] Generally, the shortest length of the first region of the
signal-sensor primary construct of the present invention can be the
length of a nucleic acid sequence that is sufficient to encode for
a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a
hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a
decapeptide. In another embodiment, the length may be sufficient to
encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25,
10-25, or 10-20 amino acids. The length may be sufficient to encode
for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30
amino acids, or a peptide that is no longer than 40 amino acids,
e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10
amino acids. Examples of dipeptides that the polynucleotide
sequences can encode or include, but are not limited to, carnosine
and anserine.
[0087] Generally, the length of the first region encoding the
oncology-related polypeptide of interest of the present invention
is greater than about 30 nucleotides in length (e.g., at least or
greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800,
1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000,
9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,
80,000, 90,000 or up to and including 100,000 nucleotides). As used
herein, the "first region" may be referred to as a "coding region"
or "region encoding" or simply the "first region."
[0088] In some embodiments, the signal-sensor polynucleotide
polynucleotide, primary construct, or mmRNA includes from about 30
to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100,
from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500,
from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to
10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000,
from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to
1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000,
from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from
100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to
1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000,
from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500
to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to
1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to
5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to
25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to
100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to
7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to
50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to
3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to
10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to
70,000, and from 2,000 to 100,000).
[0089] According to the present invention, the first and second
flanking regions may range independently from 15-1,000 nucleotides
in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90,
100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,
700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60,
70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450,
500, 600, 700, 800, 900, and 1,000 nucleotides).
[0090] According to the present invention, the tailing sequence may
range from absent to 500 nucleotides in length (e.g., at least 60,
70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or
500 nucleotides). Where the tailing region is a polyA tail, the
length may be determined in units of or as a function of polyA
binding protein binding. In this embodiment, the polyA tail is long
enough to bind at least 4 monomers of polyA binding protein. PolyA
binding protein monomers bind to stretches of approximately 38
nucleotides. As such, it has been observed that polyA tails of
about 80 nucleotides and 160 nucleotides are functional.
[0091] According to the present invention, the capping region may
comprise a single cap or a series of nucleotides forming the cap.
In this embodiment the capping region may be from 1 to 10, e.g.
2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides
in length. In some embodiments, the cap is absent.
[0092] According to the present invention, the first and second
operational regions may range from 3 to 40, e.g., 5-30, 10-20, 15,
or at least 4, or 30 or fewer nucleotides in length and may
comprise, in addition to a start and/or stop codon, one or more
signal and/or restriction sequences.
Cyclic Signal-Sensor Polynucleotides
[0093] According to the present invention, a signal-sensor primary
construct or mmRNA may be cyclized, or concatemerized, to generate
a translation competent molecule to assist interactions between
poly-A binding proteins and 5'-end binding proteins. The mechanism
of cyclization or concatemerization may occur through at least 3
different routes: 1) chemical, 2) enzymatic, and 3) ribozyme
catalyzed. The newly formed 5'-/3'-linkage may be intramolecular or
intermolecular.
[0094] In the first route, the 5'-end and the 3'-end of the nucleic
acid may contain chemically reactive groups that, when close
together, form a new covalent linkage between the 5'-end and the
3'-end of the molecule. The 5'-end may contain an NHS-ester
reactive group and the 3'-end may contain a 3'-amino-terminated
nucleotide such that in an organic solvent the 3'-amino-terminated
nucleotide on the 3'-end of a synthetic mRNA molecule will undergo
a nucleophilic attack on the 5'-NHS-ester moiety forming a new
5'-/3'-amide bond.
[0095] In the second route, T4 RNA ligase may be used to
enzymatically link a 5'-phosphorylated nucleic acid molecule to the
3'-hydroxyl group of a nucleic acid forming a new phosphorodiester
linkage. In an example reaction, 1 .mu.g of a nucleic acid molecule
is incubated at 37.degree. C. for 1 hour with 1-10 units of T4 RNA
ligase (New England Biolabs, Ipswich, Mass.) according to the
manufacturer's protocol. The ligation reaction may occur in the
presence of a split oligonucleotide capable of base-pairing with
both the 5'- and 3'-region in juxtaposition to assist the enzymatic
ligation reaction.
[0096] In the third route, either the 5'- or 3'-end of the cDNA
template encodes a ligase ribozyme sequence such that during in
vitro transcription, the resultant nucleic acid molecule can
contain an active ribozyme sequence capable of ligating the 5'-end
of a nucleic acid molecule to the 3'-end of a nucleic acid
molecule. The ligase ribozyme may be derived from the Group I
Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or
may be selected by SELEX (systematic evolution of ligands by
exponential enrichment). The ribozyme ligase reaction may take 1 to
24 hours at temperatures between 0 and 37.degree. C.
Signal-Sensor Polynucleotide Multimers
[0097] According to the present invention, multiple distinct
signal-sensor polynucleotides, primary constructs or mmRNA may be
linked together through the 3'-end using nucleotides which are
modified at the 3'-terminus. Chemical conjugation may be used to
control the stoichiometry of delivery into cells. For example, the
glyoxylate cycle enzymes, isocitrate lyase and malate synthase, may
be supplied into HepG2 cells at a 1:1 ratio to alter cellular fatty
acid metabolism. This ratio may be controlled by chemically linking
signal-sensor polynucleotides, primary constructs or mmRNA using a
3'-azido terminated nucleotide on one signal-sensor polynucleotide,
primary construct or mmRNA species and a C5-ethynyl or
alkynyl-containing nucleotide on the opposite signal-sensor
polynucleotide, primary construct or mmRNA species. The modified
nucleotide is added post-transcriptionally using terminal
transferase (New England Biolabs, Ipswich, Mass.) according to the
manufacturer's protocol. After the addition of the 3'-modified
nucleotide, the two signal-sensor polynucleotide, primary construct
or mmRNA species may be combined in an aqueous solution, in the
presence or absence of copper, to form a new covalent linkage via a
click chemistry mechanism as described in the literature.
[0098] In another example, more than two signal-sensor
polynucleotides may be linked together using a functionalized
linker molecule. For example, a functionalized saccharide molecule
may be chemically modified to contain multiple chemical reactive
groups (SH--, NH.sub.2--, N.sub.3, etc. . . . ) to react with the
cognate moiety on a 3'-functionalized signal-sensor polynucleotide
molecule (i.e., a 3'-maleimide ester, 3'-NHS-ester, alkynyl). The
number of reactive groups on the modified saccharide can be
controlled in a stoichiometric fashion to directly control the
stoichiometric ratio of conjugated signal-sensor polynucleotide,
primary construct or mmRNA.
Signal-Sensor Polynucleotide Conjugates and Combinations
[0099] In order to further enhance oncology-related protein
production, signal-sensor polynucleotide primary constructs or
mmRNA of the present invention can be designed to be conjugated to
other polynucleotides, oncology-related polypeptides, dyes,
intercalating agents (e.g. acridines), cross-linkers (e.g.
psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin),
polycyclic aromatic hydrocarbons (e.g., phenazine,
dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating
agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG,
[MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers,
enzymes, haptens (e.g. biotin), transport/absorption facilitators
(e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases,
proteins, e.g., glycoproteins, or peptides, e.g., molecules having
a specific affinity for a co-ligand, or antibodies e.g., an
antibody, that binds to a specified cell type such as a cancer
cell, endothelial cell, or bone cell, hormones and hormone
receptors, non-peptidic species, such as lipids, lectins,
carbohydrates, vitamins, cofactors, or a drug.
[0100] Conjugation may result in increased stability and/or half
life and may be particularly useful in targeting the signal-sensor
polynucleotides, primary constructs or mmRNA to specific sites in
the cell, tissue or organism.
[0101] According to the present invention, the signal-sensor
polynucleotide mmRNA or primary constructs may be administered
with, or further encode one or more of RNAi agents, siRNAs, shRNAs,
miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic
DNA, tRNA, RNAs that induce triple helix formation, aptamers or
vectors, and the like.
[0102] In one embodiment, the signal-sensor polynucleotides
described herein may be conjugated with a moiety to target various
cancer cells such as, but not limited to, the moieties described in
US Patent Application No. US20130216561, the contents of which are
herein incorporated by reference in its entirety. The linkage
between the signal-sensor polynucleotides and the cancer targeting
moiety may be an acid cleavable linkage that can increase the
efficacy of the conjugate such as, but not limited to, the linkages
described in US Patent Application No. US20130216561, the contents
of which are herein incorporated by reference in its entirety.
Bifunctional Signal-Sensor Polynucleotide
[0103] In one embodiment of the invention are bifunctional
signal-sensor polynucleotides (e.g., bifunctional primary
constructs or bifunctional mmRNA). As the name implies,
bifunctional signal-sensor polynucleotides are those having or
capable of at least two functions. These molecules may also by
convention be referred to as multi-functional.
[0104] The multiple functionalities of bifunctional signal-sensor
polynucleotides may be encoded by the RNA (the function may not
manifest until the encoded product is translated) or may be a
property of the polynucleotide itself. It may be structural or
chemical. Bifunctional modified signal-sensor polynucleotides may
comprise a function that is covalently or electrostatically
associated with the polynucleotides. Further, the two functions may
be provided in the context of a complex of a signal-sensor
polynucleotide and another molecule.
[0105] Bifunctional signal-sensor polynucleotides may encode
oncology-related peptides which are anti-proliferative. These
peptides may be linear, cyclic, constrained or random coil. They
may function as aptamers, signaling molecules, ligands or mimics or
mimetics thereof. Anti-proliferative peptides may, as translated,
be from 3 to 50 amino acids in length. They may be 5-40, 10-30, or
approximately 15 amino acids long. They may be single chain,
multichain or branched and may form complexes, aggregates or any
multi-unit structure once translated.
Noncoding Signal-Sensor Polynucleotides
[0106] As described herein, provided are signal-sensor
polynucleotides and primary constructs having sequences that are
partially or substantially not translatable, e.g., having a
noncoding region. Such noncoding region may be the "first region"
of the signal-sensor primary construct. Alternatively, the
noncoding region may be a region other than the first region. Such
molecules are generally not translated, but can exert an effect on
protein production by one or more of binding to and sequestering
one or more translational machinery components such as a ribosomal
protein or a transfer RNA (tRNA), thereby effectively reducing
protein expression in the cell or modulating one or more pathways
or cascades in a cell which in turn alters protein levels. The
signal-sensor polynucleotide and/or primary construct may contain
or encode one or more long noncoding RNA (1ncRNA, or lincRNA) or
portion thereof, a small nucleolar RNA (sno-RNA), micro RNA
(miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA
(piRNA).
Auxotrophic Signal-Sensor Polynucleotides
[0107] In one embodiment, the signal-sensor polynucleotides of the
present invention may be auxotrophic. As used herein, the term
"auxotrophic" refers to signal-sensor polynucleotides that comprise
at least one feature that triggers, facilitates or induces the
degradation or inactivation of the itself in response to spatial or
temporal cues such that oncology-related protein expression is
substantially prevented or reduced. Such spatial or temporal cues
include the location of the signal-sensor polynucleotide to be
translated such as a particular tissue or organ or cellular
environment. Also contemplated are cues involving temperature, pH,
ionic strength, moisture content, and the like.
[0108] In one embodiment, the feature is located in a terminal
region of the signal-sensor polynucleotides of the present
invention. As a non-limiting example, the auxotrophic mRNA may
contain a miR binding site in the terminal region which binds to a
miR expressed in a selected tissue so that the expression of the
auxotrophic mRNA is substantially prevented or reduced in the
selected tissue. To this end and for example, an auxotrophic mRNA
containing a miR-122 binding site will not produce protein if
localized to the liver since miR-122 is expressed in the liver and
binding of the miR would effectuate destruction of the auxotrophic
mRNA. As a non-limiting example, HEK293 cells do not express
miR-122 so there would be little to no downregulation of a
signal-sensor polynucleotide having a miR-122 sequence in HEK293
but for hepatocytes which do expression miR-122 there would be a
downregulation of a signal-sensor polynucleotide having a miR-122
sequence in hepatocytes (see e.g., the study outlined Example 19).
As another non-limiting example, the miR-122 level can be measured
in HeLa cells, primary human hepatocytes and primary rat
hepatocytes prior to administration with a signal-sensor
polynucleotide encoding having at least one miR-122 binding site,
miR-122 binding site without the seed sequence or a miR-122 binding
site After administration the expression of the signal-sensor
polynucleotide can be measured to determine the dampening effect of
the miR-122 in the signal-sensor polynucleotide (see e.g., the
studies outlined in Examples 41, 42, 43 57, 58 and 59). As yet
another non-limiting example, the effectiveness of the miR-122
binding site, miR-122 seed or the miR-122 binding site without the
seed in different 3'UTRs may be evaluated in order to determine the
proper UTR for the desired outcome such as, but not limited to, the
highest dampening effect (see e.g., the study outlined in Example
46).
[0109] In one embodiment, the degradation or inactivation of
auxotrophic mRNA may comprise a feature responsive to a change in
pH. As a non-limiting example, the auxotrophic mRNA may be
triggered in an environment having a pH of between pH 4.5 to 8.0
such as at a pH of 5.0 to 6.0 or a pH of 6.0 to 6.5. The change in
pH may be a change of 0.1 unit, 0.2 units, 0.3 units, 0.4 units,
0.5 units, 0.6 units, 0.7 units, 0.8 units, 0.9 units, 1.0 units,
1.1 units, 1.2 units, 1.3 units, 1.4 units, 1.5 units, 1.6 units,
1.7 units, 1.8 units, 1.9 units, 2.0 units, 2.1 units, 2.2 units,
2.3 units, 2.4 units, 2.5 units, 2.6 units, 2.7 units, 2.8 units,
2.9 units, 3.0 units, 3.1 units, 3.2 units, 3.3 units, 3.4 units,
3.5 units, 3.6 units, 3.7 units, 3.8 units, 3.9 units, 4.0 units or
more.
[0110] In another embodiment, the degradation or inactivation of
auxotrophic mRNA may be triggered or induced by changes in
temperature. As a non-limiting example, a change of temperature
from room temperature to body temperature. The change of
temperature may be less than 1.degree. C., less than 5.degree. C.,
less than 10.degree. C., less than 15.degree. C., less than
20.degree. C., less than 25.degree. C. or more than 25.degree.
C.
[0111] In yet another embodiment, the degradation or inactivation
of auxotrophic mRNA may be triggered or induced by a change in the
levels of ions in the subject. The ions may be cations or anions
such as, but not limited to, sodium ions, potassium ions, chloride
ions, calcium ions, magnesium ions and/or phosphate ions.
Oncology-Related Polypeptides of Interest
[0112] According to the present invention, the signal-sensor
primary construct is designed to encode one or more
oncology-related polypeptides of interest or fragments thereof. An
oncology-related polypeptide of interest may include, but is not
limited to, whole polypeptides, a plurality of polypeptides or
fragments of polypeptides, which independently may be encoded by
one or more nucleic acids, a plurality of nucleic acids, fragments
of nucleic acids or variants of any of the aforementioned. As used
herein, the term "oncology-related polypeptides of interest" refers
to any polypeptide which is selected to be encoded in the
signal-sensor primary construct of the present invention. As used
herein, "polypeptide" means a polymer of amino acid residues
(natural or unnatural) linked together most often by peptide bonds.
The term, as used herein, refers to proteins, polypeptides, and
peptides of any size, structure, or function. In some instances the
polypeptide encoded is smaller than about 50 amino acids and the
polypeptide is then termed a peptide. If the polypeptide is a
peptide, it will be at least about 2, 3, 4, or at least 5 amino
acid residues long. Thus, polypeptides include gene products,
naturally occurring polypeptides, synthetic polypeptides, homologs,
orthologs, paralogs, fragments and other equivalents, variants, and
analogs of the foregoing. A polypeptide may be a single molecule or
may be a multi-molecular complex such as a dimer, trimer or
tetramer. They may also comprise single chain or multichain
polypeptides such as antibodies or insulin and may be associated or
linked. Most commonly disulfide linkages are found in multichain
polypeptides. The term polypeptide may also apply to amino acid
polymers in which one or more amino acid residues are an artificial
chemical analogue of a corresponding naturally occurring amino
acid.
[0113] The term "polypeptide variant" refers to molecules which
differ in their amino acid sequence from a native or reference
sequence. The amino acid sequence variants may possess
substitutions, deletions, and/or insertions at certain positions
within the amino acid sequence, as compared to a native or
reference sequence. Ordinarily, variants will possess at least
about 50% identity (homology) to a native or reference sequence,
and preferably, they will be at least about 80%, more preferably at
least about 90% identical (homologous) to a native or reference
sequence.
[0114] In some embodiments "variant mimics" are provided. As used
herein, the term "variant mimic" is one which contains one or more
amino acids which would mimic an activated sequence. For example,
glutamate may serve as a mimic for phosphoro-threonine and/or
phosphoro-serine. Alternatively, variant mimics may result in
deactivation or in an inactivated product containing the mimic,
e.g., phenylalanine may act as an inactivating substitution for
tyrosine; or alanine may act as an inactivating substitution for
serine.
[0115] "Homology" as it applies to amino acid sequences is defined
as the percentage of residues in the candidate amino acid sequence
that are identical with the residues in the amino acid sequence of
a second sequence after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent homology.
Methods and computer programs for the alignment are well known in
the art. It is understood that homology depends on a calculation of
percent identity but may differ in value due to gaps and penalties
introduced in the calculation.
[0116] By "homologs" as it applies to polypeptide sequences means
the corresponding sequence of other species having substantial
identity to a second sequence of a second species.
[0117] "Analogs" is meant to include polypeptide variants which
differ by one or more amino acid alterations, e.g., substitutions,
additions or deletions of amino acid residues that still maintain
one or more of the properties of the parent or starting
polypeptide.
[0118] The present invention contemplates several types of
compositions which are polypeptide based including variants and
derivatives. These include substitutional, insertional, deletion
and covalent variants and derivatives. The term "derivative" is
used synonymously with the term "variant" but generally refers to a
molecule that has been modified and/or changed in any way relative
to a reference molecule or starting molecule.
[0119] As such, signal-sensor polynucleotides encoding
oncology-related polypeptides containing substitutions, insertions
and/or additions, deletions and covalent modifications with respect
to reference sequences, in particular the oncology-related
polypeptide sequences disclosed herein, are included within the
scope of this invention. For example, sequence tags or amino acids,
such as one or more lysines, can be added to the peptide sequences
of the invention (e.g., at the N-terminal or C-terminal ends).
Sequence tags can be used for peptide purification or localization.
Lysines can be used to increase peptide solubility or to allow for
biotinylation. Alternatively, amino acid residues located at the
carboxy and amino terminal regions of the amino acid sequence of a
peptide or protein may optionally be deleted providing for
truncated sequences. Certain amino acids (e.g., C-terminal or
N-terminal residues) may alternatively be deleted depending on the
use of the sequence, as for example, expression of the sequence as
part of a larger sequence which is soluble, or linked to a solid
support.
[0120] "Substitutional variants" when referring to polypeptides are
those that have at least one amino acid residue in a native or
starting sequence removed and a different amino acid inserted in
its place at the same position. The substitutions may be single,
where only one amino acid in the molecule has been substituted, or
they may be multiple, where two or more amino acids have been
substituted in the same molecule.
[0121] As used herein the term "conservative amino acid
substitution" refers to the substitution of an amino acid that is
normally present in the sequence with a different amino acid of
similar size, charge, or polarity. Examples of conservative
substitutions include the substitution of a non-polar (hydrophobic)
residue such as isoleucine, valine and leucine for another
non-polar residue. Likewise, examples of conservative substitutions
include the substitution of one polar (hydrophilic) residue for
another such as between arginine and lysine, between glutamine and
asparagine, and between glycine and serine. Additionally, the
substitution of a basic residue such as lysine, arginine or
histidine for another, or the substitution of one acidic residue
such as aspartic acid or glutamic acid for another acidic residue
are additional examples of conservative substitutions. Examples of
non-conservative substitutions include the substitution of a
non-polar (hydrophobic) amino acid residue such as isoleucine,
valine, leucine, alanine, methionine for a polar (hydrophilic)
residue such as cysteine, glutamine, glutamic acid or lysine and/or
a polar residue for a non-polar residue.
[0122] "Insertional variants" when referring to polypeptides are
those with one or more amino acids inserted immediately adjacent to
an amino acid at a particular position in a native or starting
sequence. "Immediately adjacent" to an amino acid means connected
to either the alpha-carboxy or alpha-amino functional group of the
amino acid.
[0123] "Deletional variants" when referring to polypeptides are
those with one or more amino acids in the native or starting amino
acid sequence removed. Ordinarily, deletional variants will have
one or more amino acids deleted in a particular region of the
molecule.
[0124] "Covalent derivatives" when referring to polypeptides
include modifications of a native or starting protein with an
organic proteinaceous or non-proteinaceous derivatizing agent,
and/or post-translational modifications. Covalent modifications are
traditionally introduced by reacting targeted amino acid residues
of the protein with an organic derivatizing agent that is capable
of reacting with selected side-chains or terminal residues, or by
harnessing mechanisms of post-translational modifications that
function in selected recombinant host cells. The resultant covalent
derivatives are useful in programs directed at identifying residues
important for biological activity, for immunoassays, or for the
preparation of anti-protein antibodies for immunoaffinity
purification of the recombinant glycoprotein. Such modifications
are within the ordinary skill in the art and are performed without
undue experimentation.
[0125] Certain post-translational modifications are the result of
the action of recombinant host cells on the expressed
oncology-related polypeptide. Glutaminyl and asparaginyl residues
are frequently post-translationally deamidated to the corresponding
glutamyl and aspartyl residues. Alternatively, these residues are
deamidated under mildly acidic conditions. Either form of these
residues may be present in the oncology-related polypeptides
produced in accordance with the present invention.
[0126] Other post-translational modifications include hydroxylation
of proline and lysine, phosphorylation of hydroxyl groups of seryl
or threonyl residues, methylation of the alpha-amino groups of
lysine, arginine, and histidine side chains (T. E. Creighton,
Proteins: Structure and Molecular Properties, W.H. Freeman &
Co., San Francisco, pp. 79-86 (1983)).
[0127] "Features" when referring to polypeptides are defined as
distinct amino acid sequence-based components of a molecule.
Features of the polypeptides encoded by the mmRNA of the present
invention include surface manifestations, local conformational
shape, folds, loops, half-loops, domains, half-domains, sites,
termini or any combination thereof
[0128] As used herein when referring to polypeptides the term
"surface manifestation" refers to a polypeptide based component of
a protein appearing on an outermost surface.
[0129] As used herein when referring to polypeptides the term
"local conformational shape" means a polypeptide based structural
manifestation of a protein which is located within a definable
space of the protein.
[0130] As used herein when referring to polypeptides the term
"fold" refers to the resultant conformation of an amino acid
sequence upon energy minimization. A fold may occur at the
secondary or tertiary level of the folding process. Examples of
secondary level folds include beta sheets and alpha helices.
Examples of tertiary folds include domains and regions formed due
to aggregation or separation of energetic forces. Regions formed in
this way include hydrophobic and hydrophilic pockets, and the
like.
[0131] As used herein the term "turn" as it relates to protein
conformation means a bend which alters the direction of the
backbone of a peptide or polypeptide and may involve one, two,
three or more amino acid residues.
[0132] As used herein when referring to polypeptides the term
"loop" refers to a structural feature of a polypeptide which may
serve to reverse the direction of the backbone of a peptide or
polypeptide. Where the loop is found in a polypeptide and only
alters the direction of the backbone, it may comprise four or more
amino acid residues. Oliva et al. have identified at least 5
classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).
Loops may be open or closed. Closed loops or "cyclic" loops may
comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the
bridging moieties. Such bridging moieties may comprise a
cysteine-cysteine bridge (Cys-Cys) typical in polypeptides having
disulfide bridges or alternatively bridging moieties may be
non-protein based such as the dibromozylyl agents used herein.
[0133] As used herein when referring to polypeptides the term
"half-loop" refers to a portion of an identified loop having at
least half the number of amino acid resides as the loop from which
it is derived. It is understood that loops may not always contain
an even number of amino acid residues. Therefore, in those cases
where a loop contains or is identified to comprise an odd number of
amino acids, a half-loop of the odd-numbered loop will comprise the
whole number portion or next whole number portion of the loop
(number of amino acids of the loop/2+/-0.5 amino acids). For
example, a loop identified as a 7 amino acid loop could produce
half-loops of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3
or 4).
[0134] As used herein when referring to polypeptides the term
"domain" refers to a motif of a polypeptide having one or more
identifiable structural or functional characteristics or properties
(e.g., binding capacity, serving as a site for protein-protein
interactions).
[0135] As used herein when referring to polypeptides the term
"half-domain" means a portion of an identified domain having at
least half the number of amino acid resides as the domain from
which it is derived. It is understood that domains may not always
contain an even number of amino acid residues. Therefore, in those
cases where a domain contains or is identified to comprise an odd
number of amino acids, a half-domain of the odd-numbered domain
will comprise the whole number portion or next whole number portion
of the domain (number of amino acids of the domain/2+/-0.5 amino
acids). For example, a domain identified as a 7 amino acid domain
could produce half-domains of 3 amino acids or 4 amino acids
(7/2=3.5+/-0.5 being 3 or 4). It is also understood that
sub-domains may be identified within domains or half-domains, these
subdomains possessing less than all of the structural or functional
properties identified in the domains or half domains from which
they were derived. It is also understood that the amino acids that
comprise any of the domain types herein need not be contiguous
along the backbone of the polypeptide (i.e., nonadjacent amino
acids may fold structurally to produce a domain, half-domain or
subdomain).
[0136] As used herein when referring to polypeptides the terms
"site" as it pertains to amino acid based embodiments is used
synonymously with "amino acid residue" and "amino acid side chain."
A site represents a position within a peptide or polypeptide that
may be modified, manipulated, altered, derivatized or varied within
the polypeptide based molecules of the present invention.
[0137] As used herein the terms "termini" or "terminus" when
referring to polypeptides refers to an extremity of a peptide or
polypeptide. Such extremity is not limited only to the first or
final site of the peptide or polypeptide but may include additional
amino acids in the terminal regions. The polypeptide based
molecules of the present invention may be characterized as having
both an N-terminus (terminated by an amino acid with a free amino
group (NH2)) and a C-terminus (terminated by an amino acid with a
free carboxyl group (COOH)). Proteins of the invention are in some
cases made up of multiple polypeptide chains brought together by
disulfide bonds or by non-covalent forces (multimers, oligomers).
These sorts of proteins will have multiple N- and C-termini.
Alternatively, the termini of the polypeptides may be modified such
that they begin or end, as the case may be, with a non-polypeptide
based moiety such as an organic conjugate.
[0138] Once any of the features have been identified or defined as
a desired component of a polypeptide to be encoded by the
signal-sensor primary construct or mmRNA of the invention, any of
several manipulations and/or modifications of these features may be
performed by moving, swapping, inverting, deleting, randomizing or
duplicating. Furthermore, it is understood that manipulation of
features may result in the same outcome as a modification to the
molecules of the invention. For example, a manipulation which
involved deleting a domain would result in the alteration of the
length of a molecule just as modification of a nucleic acid to
encode less than a full length molecule would.
[0139] Modifications and manipulations can be accomplished by
methods known in the art such as, but not limited to, site directed
mutagenesis. The resulting modified molecules may then be tested
for activity using in vitro or in vivo assays such as those
described herein or any other suitable screening assay known in the
art.
[0140] According to the present invention, the oncology-related
polypeptides may comprise a consensus sequence which is discovered
through rounds of experimentation. As used herein a "consensus"
sequence is a single sequence which represents a collective
population of sequences allowing for variability at one or more
sites.
[0141] As recognized by those skilled in the art, protein
fragments, functional protein domains, and homologous proteins are
also considered to be within the scope of oncology-related
polypeptides of interest of this invention. For example, provided
herein is any protein fragment (meaning an oncology-related
polypeptide sequence at least one amino acid residue shorter than a
reference oncology-related polypeptide sequence but otherwise
identical) of a reference oncology-related protein 10, 20, 30, 40,
50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length.
In another example, any oncology-related protein that includes a
stretch of about 20, about 30, about 40, about 50, or about 100
amino acids which are about 40%, about 50%, about 60%, about 70%,
about 80%, about 90%, about 95%, or about 100% identical to any of
the sequences described herein can be utilized in accordance with
the invention. In certain embodiments, a polypeptide to be utilized
in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9,
10, or more mutations as shown in any of the sequences provided or
referenced herein.
Encoded Oncology-Related Polypeptides
[0142] The signal-sensor primary constructs or mmRNA of the present
invention may be designed to encode oncology-related polypeptides
of interest such as oncology-related peptides and proteins.
[0143] In one embodiment, signal-sensor primary constructs or mmRNA
of the present invention may encode variant polypeptides which have
a certain identity with a reference oncology-related polypeptide
sequence. As used herein, a "reference oncology-related polypeptide
sequence" refers to a starting oncology-related polypeptide
sequence. Reference sequences may be wild type sequences or any
sequence to which reference is made in the design of another
sequence. A "reference polypeptide sequence" may, e.g., be any one
of the protein sequence listed in Table 6.
[0144] The term "identity" as known in the art, refers to a
relationship between the sequences of two or more peptides, as
determined by comparing the sequences. In the art, identity also
means the degree of sequence relatedness between peptides, as
determined by the number of matches between strings of two or more
amino acid residues. Identity measures the percent of identical
matches between the smaller of two or more sequences with gap
alignments (if any) addressed by a particular mathematical model or
computer program (i.e., "algorithms"). Identity of related peptides
can be readily calculated by known methods. Such methods include,
but are not limited to, those described in Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York, 1991; and
Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).
[0145] In some embodiments, the polypeptide variant may have the
same or a similar activity as the reference oncology-related
polypeptide. Alternatively, the variant may have an altered
activity (e.g., increased or decreased) relative to a reference
oncology-related polypeptide. Generally, variants of a particular
signal-sensor polynucleotide or oncology-related polypeptide of the
invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% but less than 100% sequence identity to that particular
reference signal-sensor polynucleotide or oncology-related
polypeptide as determined by sequence alignment programs and
parameters described herein and known to those skilled in the art.
Such tools for alignment include those of the BLAST suite (Stephen
F. Altschul, Thomas L. Madden, Alejandro A. Schiffer, Jinghui
Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997),
"Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs", Nucleic Acids Res. 25:3389-3402.) Other tools are
described herein, specifically in the definition of "identity."
[0146] Default parameters in the BLAST algorithm include, for
example, an expect threshold of 10, Word size of 28, Match/Mismatch
Scores 1, -2, Gap costs Linear. Any filter can be applied as well
as a selection for species specific repeats, e.g., Homo
sapiens.
[0147] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may be used to treat a disease,
disorder and/or condition in a subject.
[0148] In one embodiment, the polynucleotides, primary constructs
and/or mmRNA may be used to reduce, eliminate or prevent tumor
growth in a subject.
[0149] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may be used to recude and/or
ameliorate at least one symptom of cancer in a subject. A symptom
of cancer may include, but is not limited to, weakness, aches and
pains, fever, fatigue, weight loss, blood clots, increased blood
calcium levels, low white blood cell count, short of breath,
dizziness, headaches, hyperpigmentation, jaundice, erthema,
pruritis, excessive hair growth, change in bowel habits, change in
bladder function, long-lasting sores, white patches inside the
mouth, white spots on the tongue, unusual bleeding or discharge,
thickening or lump on parts of the body, indigestion, trouble
swallowing, changes in warts or moles, change in new skin and
nagging cough or hoarseness. Further, the signal-sensor
polynucleotides, primary constructs and/or mmRNA may reduce a
side-effect associated with cancer such as, but not limited to,
chemo brain, peripheral neuropathy, fatigue, depression, nausea,
vomiting, pain, anemia, lymphedema, infections, sexual side
effects, reduced fertility or infertility, ostomics, insomnia and
hair loss.
Oncology-Related Proteins or Oncology-Related Peptides
[0150] The signal-sensor primary constructs or mmRNA disclosed
herein, may encode one or more validated or "in testing"
oncology-related proteins or oncology-related peptides.
[0151] According to the present invention, one or more
oncology-related proteins or oncology-related peptides currently
being marketed or in development may be encoded by the
oncology-related signal-sensor polynucleotide, primary constructs
or mmRNA of the present invention. While not wishing to be bound by
theory, it is believed that incorporation into the signal-sensor
primary constructs or mmRNA of the invention will result in
improved therapeutic efficacy due at least in part to the
specificity, purity and selectivity of the construct designs.
[0152] The signal-sensor polynucleotides, primary constructs and/or
mmRNA may alter a biological and/or physiolocial process and/or
compound such as, but not limited to, the cell cycle, the DNA
damage response (e.g., DNA damage repair), apoptosis, angiogenesis,
cell motility, the epithelial to mesenchymal transition in
epithelial cells, the phosphatidyl inositol 3 (PI3) kinase/Akt
cellular signaling pathway, telomerase activity and/or expression,
tumor metastasis, tumorigenesis, cathepsins, cell senescence,
receptor tyrosine kinase signaling, metabolism and drug metabolism,
G protein signaling, growth factors and receptors, heat shock
proteins, histone deacetylases, hormone receptors, hypoxia, poly
ADP-ribose polymerases, protein kinases, RAS signaling,
topisomerases, transcription factors and tumor suppressor activity
in cancerous, precancerous and/or other cells.
[0153] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may used to express a polypeptide
in cells or tissues for the purpose of replacing the protein
produced from a deleted or mutated gene.
[0154] Further, the polynucleotides, primary constructs or mmRNA of
the invention may be used to treat cancer which has been caused by
carcinogens of natural and/or synthetic origin. In another
embodiment, the use of the polynucleotides, primary constructs
and/or mmRNA may be used to treat cancer caused by other organisms
and/or cancers caused by viral infection.
Sensors in the Flanking Regions: Untranslated Regions (UTRs)
[0155] Untranslated regions (UTRs) of a gene are transcribed but
not translated. The 5'UTR starts at the transcription start site
and continues to the start codon but does not include the start
codon; whereas, the 3'UTR starts immediately following the stop
codon and continues until the transcriptional termination signal.
There is growing body of evidence about the regulatory roles played
by the UTRs in terms of stability of the nucleic acid molecule and
translation. The regulatory features of a UTR can be incorporated
into the signal-sensor polynucleotides, primary constructs and/or
mmRNA of the present invention to enhance the stability of the
molecule. The specific features can also be incorporated to ensure
controlled down-regulation of the transcript in case they are
misdirected to undesired organs sites. The untranslated regions may
be incorporated into a vector system which can produce mRNA and/or
be delivered to a cell, tissue and/or organism to produce a
polypeptide of interest.
[0156] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA of the present may comprise at
least one terminal modification. Non-limiting examples of terminal
modifications are described in U.S. Provisional Patent Application
No. 61/729,933, filed Nov. 26, 2012, entitled Terminally Optimized
Modified RNAs, U.S. Provisional Patent application No. 61/737,224,
filed Dec. 14, 2012, entitled Terminally Optimized RNAs, U.S.
Provisional Patent Application No. 61/758,921, filed Jan. 31, 2013,
entitled Differential Targeting Using RNA Constructs, U.S.
Provisional Patent Application No. 61/781,139, filed Mar. 14, 2013,
entitled Differential Targeting Using RNA Constructs, U.S.
Provisional Patent Application No. 61/829,359, filed May 31, 2013,
entitled Differential Targeting Using RNA Constructs, U.S.
Provisional Patent Application No. 61/839,903, filed Jun. 27, 2013,
entitled Differential Targeting Using RNA Constructs, U.S.
Provisional Patent Application No. 61/842,709, filed Jul. 3, 2013,
entitled Differential Targeting Using RNA Constructs, and U.S.
Provisional Patent Application No. 61/857,436, filed Jul. 23, 2013,
entitled Differential Targeting Using RNA Constructs, the contents
of each of which are herein incorporated by reference in their
entireties. These terminal modifications include, but are not
limited to, 5' caps, microRNA binding sites in the terminal region,
chain terminating nucleosides, translation enhancer elements in the
terminal region and tailing sequences including a polyA-G quartet
and stem loop sequences.
5' UTR and Translation Initiation
[0157] Natural 5'UTRs bear features which play roles in for
translation initiation. They harbor signatures like Kozak sequences
which are commonly known to be involved in the process by which the
ribosome initiates translation of many genes. Kozak sequences have
the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or
guanine) three bases upstream of the start codon (AUG), which is
followed by another `G`. 5'UTR also have been known to form
secondary structures which are involved in elongation factor
binding. For example, one of the secondary 5'-UTR structures is the
structured IRES for eIF4A2 elongation factor binding, which is
necessary for the microRNA mediated gene repression at 3'-UTR.
[0158] 5'UTR secondary structures involved in elongation factor
binding can interact with other RNA binding molecules in the 5'UTR
or 3'UTR to regulate gene expression. For example, the elongation
factor EIF4A2 binding to a secondarily structured element in the
5'UTR is necessary for microRNA mediated repression (Meijer H A et
al., Science, 2013, 340, 82-85, herein incorporated by reference in
its entirety). The different secondary structures in the 5'UTR can
be incorporated into the flanking region to either stabilize or
selectively destalized mRNAs in specific tissues or cells.
[0159] By engineering the features typically found in abundantly
expressed genes of specific target organs, one can enhance the
stability and oncology-related protein production of the
signal-sensor polynucleotides, primary constructs or mmRNA of the
invention. For example, introduction of 5' UTR of liver-expressed
mRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E,
transferrin, alpha fetoprotein, erythropoietin, or Factor VIII,
could be used to enhance expression of a nucleic acid molecule,
such as a mmRNA, in hepatic cell lines or liver. Likewise, use of
5' UTR from other tissue-specific mRNA to improve expression in
that tissue is possible--for muscle (MyoD, Myosin, Myoglobin,
Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for
myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1,
i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36,
GLUT4, ACRP30, adiponectin) and for lung epithelial cells
(SP-A/B/C/D).
[0160] Other non-UTR sequences may be incorporated into the 5' (or
3' UTR) UTRs. For example, introns or portions of introns sequences
may be incorporated into the flanking regions of the signal-sensor
polynucleotides, primary constructs or mmRNA of the invention.
Incorporation of intronic sequences may increase protein production
as well as mRNA levels.
Translation Enhancer Elements (TEEs)
[0161] In one embodiment, the 5'UTR of the signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA may include at least one translational enhancer
polynucleotide, translation enhancer element, translational
enhancer elements (collectively referred to as "TEE"s). As a
non-limiting example, the TEE may be located between the
transcription promoter and the start codon. The signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA with at least one TEE in the 5'UTR may include a cap at the
5'UTR. Further, at least one TEE may be located in the 5'UTR of
signal-sensor polynucleotides, primary constructs, modified nucleic
acids and/or mmRNA undergoing cap-dependent or cap-independent
translation.
[0162] The term "translational enhancer element" or "translation
enhancer element" (herein collectively referred to as "TEE") refers
to sequences that increase the amount of polypeptide or protein
produced from an mRNA.
[0163] In one embodiment, TEEs are conserved elements in the UTR
which can promote translational activity of a nucleic acid such as,
but not limited to, cap-dependent or cap-independent translation.
The conservation of these sequences has been previously shown by
Panek et al (Nucleic Acids Research, 2013, 1-10; herein
incorporated by reference in its entirety) across 14 species
including humans.
[0164] In one embodiment, the TEE may be any of the TEEs listed in
Table 35 in Example 45, including portion and/or fragments thereof.
The TEE sequence may include at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 99% or more than 99% of
the TEE sequences disclosed in Table 35 and/or the TEE sequence may
include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a
5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10
nucleotide fragment of the TEE sequences disclosed in Table 35.
[0165] In one non-limiting example, the TEEs known may be in the
5'-leader of the Gtx homeodomain protein (Chappell et al., Proc.
Natl. Acad. Sci. USA 101:9590-9594, 2004, herein incorporated by
reference in their entirety).
[0166] In another non-limiting example, TEEs are disclosed as SEQ
ID NOs: 1-35 in US Patent Publication No. US20090226470, SEQ ID
NOs: 1-35 in US Patent Publication US20130177581, SEQ ID NOs: 1-35
in International Patent Publication No. WO2009075886, SEQ ID NOs:
1-5, and 7-645 in International Patent Publication No.
WO2012009644, SEQ ID NO: 1 in International Patent Publication No.
WO1999024595, SEQ ID NO: 1 in U.S. Pat. No. 6,310,197, and SEQ ID
NO: 1 in U.S. Pat. No. 6,849,405, each of which is herein
incorporated by reference in its entirety.
[0167] In yet another non-limiting example, the TEE may be an
internal ribosome entry site (IRES), HCV-IRES or an IRES element
such as, but not limited to, those described in U.S. Pat. No.
7,468,275, US Patent Publication Nos. US20070048776 and
US20110124100 and International Patent Publication Nos.
WO2007025008 and WO2001055369, each of which is herein incorporated
by reference in its entirety. The IRES elements may include, but
are not limited to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt,
Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci. USA
101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005) and
in US Patent Publication Nos. US20070048776 and US20110124100 and
International Patent Publication No. WO2007025008, each of which is
herein incorporated by reference in its entirety.
[0168] "Translational enhancer polynucleotides" or "translation
enhancer polynucleotide sequences" are polynucleotides which
include one or more of the specific TEE exemplified herein and/or
disclosed in the art (see e.g., U.S. Pat. No. 6,310,197, U.S. Pat.
No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395,
US20090226470, US20070048776, US20110124100, US20090093049,
US20130177581, WO2009075886, WO2007025008, WO2012009644,
WO2001055371 WO1999024595, and EP2610341A1 and EP2610340A1; each of
which is herein incorporated by reference in its entirety) or their
variants, homologs or functional derivatives. One or multiple
copies of a specific TEE can be present in the signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA. The TEEs in the translational enhancer polynucleotides can
be organized in one or more sequence segments. A sequence segment
can harbor one or more of the specific TEEs exemplified herein,
with each TEE being present in one or more copies. When multiple
sequence segments are present in a translational enhancer
polynucleotide, they can be homogenous or heterogeneous. Thus, the
multiple sequence segments in a translational enhancer
polynucleotide can harbor identical or different types of the
specific TEEs exemplified herein, identical or different number of
copies of each of the specific TEEs, and/or identical or different
organization of the TEEs within each sequence segment.
[0169] In one embodiment, the signal-sensor polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA may include
at least one TEE that is described in International Patent
Publication No. WO1999024595, WO2012009644, WO2009075886,
WO2007025008, WO1999024595, European Patent Publication No.
EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.
6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395, US
Patent Publication No. US20090226470, US20110124100, US20070048776,
US20090093049 and US20130177581, each of which is herein
incorporated by reference in its entirety. The TEE may be located
in the 5'UTR of the signal-sensor polynucleotides, primary
constructs, modified nucleic acids and/or mmRNA.
[0170] In another embodiment, the signal-sensor polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA may include
at least one TEE that has at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95% or at least 99% identity with the
TEEs described in US Patent Publication Nos. US20090226470,
US20070048776, US20130177581 and US20110124100, International
Patent Publication No. WO1999024595, WO2012009644, WO2009075886 and
WO2007025008, European Patent Publication No. EP2610341A1 and
EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S.
Pat. No. 7,456,273, U.S. Pat. No. 7,183,395, each of which is
herein incorporated by reference in its entirety.
[0171] In one embodiment, the 5'UTR of the signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA may include at least 1, at least 2, at least 3, at least 4,
at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18 at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 30, at least 35, at least 40, at least 45, at
least 50, at least 55 or more than 60 TEE sequences. The TEE
sequences in the 5'UTR of the signal-sensor polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA of the
present invention may be the same or different TEE sequences. The
TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or
ABCABCABC or variants thereof repeated once, twice, or more than
three times. In these patterns, each letter, A, B, or C represent a
different TEE sequence at the nucleotide level.
[0172] In one embodiment, the 5'UTR may include a spacer to
separate two TEE sequences. As a non-limiting example, the spacer
may be a 15 nucleotide spacer and/or other spacers known in the
art. As another non-limiting example, the 5'UTR may include a TEE
sequence-spacer module repeated at least once, at least twice, at
least 3 times, at least 4 times, at least 5 times, at least 6
times, at least 7 times, at least 8 times and at least 9 times or
more than 9 times in the 5'UTR.
[0173] In another embodiment, the spacer separating two TEE
sequences may include other sequences known in the art which may
regulate the translation of the signal-sensor polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA of the
present invention such as, but not limited to, miR sequences
described herein (e.g., miR binding sites and miR seeds). As a
non-limiting example, each spacer used to separate two TEE
sequences may include a different miR sequence or component of a
miR sequence (e.g., miR seed sequence).
[0174] In one embodiment, the TEE in the 5'UTR of the signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention may include at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 99% or more
than 99% of the TEE sequences disclosed in US Patent Publication
Nos. US20090226470, US20070048776, US20130177581 and US20110124100,
International Patent Publication No. WO1999024595, WO2012009644,
WO2009075886 and WO2007025008, European Patent Publication No.
EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.
6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395. In
another embodiment, the TEE in the 5'UTR of the signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention may include a 5-30 nucleotide
fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a
5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE
sequences disclosed in US Patent Publication Nos. US20090226470,
US20070048776, US20130177581 and US20110124100, International
Patent Publication No. WO1999024595, WO2012009644, WO2009075886 and
WO2007025008, European Patent Publication No. EP2610341A1 and
EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S.
Pat. No. 7,456,273, U.S. Pat. No. 7,183,395; each of which are
herein incorporated by reference in their entirety.
[0175] In one embodiment, the TEE in the 5'UTR of the signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA of the present invention may include at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 99% or more
than 99% of the TEE sequences disclosed in Chappell et al. (Proc.
Natl. Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al. (PNAS
102:6273-6278, 2005), in Supplemental Table 1 and in Supplemental
Table 2 disclosed by Wellensiek et al (Genome-wide profiling of
human cap-independent translation-enhancing elements, Nature
Methods, 2013; DOI:10.1038/NMETH.2522); each of which is herein
incorporated by reference in its entirety. In another embodiment,
the TEE in the 5'UTR of the signal-sensor polynucleotides, primary
constructs, modified nucleic acids and/or mmRNA of the present
invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide
fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a
5-10 nucleotide fragment of the TEE sequences disclosed in Chappell
et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004) and Zhou et
al. (PNAS 102:6273-6278, 2005), in Supplemental Table 1 and in
Supplemental Table 2 disclosed by Wellensiek et al (Genome-wide
profiling of human cap-independent translation-enhancing elements,
Nature Methods, 2013; DOI:10.1038/NMETH.2522); each of which is
herein incorporated by reference in its entirety.
[0176] In one embodiment, the TEE used in the 5'UTR of the
signal-sensor polynucleotides, primary constructs, modified nucleic
acids and/or mmRNA of the present invention is an IRES sequence
such as, but not limited to, those described in U.S. Pat. No.
7,468,275 and International Patent Publication No. WO2001055369,
each of which is herein incorporated by reference in its
entirety.
[0177] In one embodiment, the TEEs used in the 5'UTR of the
signal-sensor polynucleotides, primary constructs, modified nucleic
acids and/or mmRNA of the present invention may be identified by
the methods described in US Patent Publication No. US20070048776
and US20110124100 and International Patent Publication Nos.
WO2007025008 and WO2012009644, each of which is herein incorporated
by reference in its entirety.
[0178] In another embodiment, the TEEs used in the 5'UTR of the
signal-sensor polynucleotides, primary constructs, modified nucleic
acids and/or mmRNA of the present invention may be a transcription
regulatory element described in U.S. Pat. No. 7,456,273 and U.S.
Pat. No. 7,183,395, US Patent Publication No. US20090093049, and
International Publication No. WO2001055371, each of which is herein
incorporated by reference in their entirety. The transcription
regulatory elements may be identified by methods known in the art,
such as, but not limited to, the methods described in U.S. Pat. No.
7,456,273 and U.S. Pat. No. 7,183,395, US Patent Publication No.
US20090093049, and International Publication No. WO2001055371, each
of which is herein incorporated by reference in their entirety.
[0179] In yet another embodiment, the TEE used in the 5'UTR of the
signal-sensor polynucleotides, primary constructs, modified nucleic
acids and/or mmRNA of the present invention is an oligonucleotide
or portion thereof as described in U.S. Pat. No. 7,456,273 and U.S.
Pat. No. 7,183,395, US Patent Publication No. US20090093049, and
International Publication No. WO2001055371, each of which is herein
incorporated by reference in their entirety.
[0180] The 5' UTR comprising at least one TEE described herein may
be incorporated in a monocistronic sequence such as, but not
limited to, a vector system or a nucleic acid vector. As a
non-limiting example, the vector systems and nucleic acid vectors
may include those described in U.S. Pat. Nos. 7,456,273 and
7,183,395, US Patent Publication No. US20070048776, US20090093049
and US20110124100 and International Patent Publication Nos.
WO2007025008 and WO2001055371, each of which is herein incorporated
by reference in its entirety.
[0181] In one embodiment, the TEEs described herein may be located
in the 5'UTR and/or the 3'UTR of the signal-sensor polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA. The TEEs
located in the 3'UTR may be the same and/or different than the TEEs
located in and/or described for incorporation in the 5'UTR.
[0182] In one embodiment, the 3'UTR of the signal-sensor
polynucleotides, primary constructs, modified nucleic acids and/or
mmRNA may include at least 1, at least 2, at least 3, at least 4,
at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18 at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 30, at least 35, at least 40, at least 45, at
least 50, at least 55 or more than 60 TEE sequences. The TEE
sequences in the 3'UTR of the signal-sensor polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA of the
present invention may be the same or different TEE sequences. The
TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or
ABCABCABC or variants thereof repeated once, twice, or more than
three times. In these patterns, each letter, A, B, or C represent a
different TEE sequence at the nucleotide level.
[0183] In one embodiment, the 3'UTR may include a spacer to
separate two TEE sequences. As a non-limiting example, the spacer
may be a 15 nucleotide spacer and/or other spacers known in the
art. As another non-limiting example, the 3'UTR may include a TEE
sequence-spacer module repeated at least once, at least twice, at
least 3 times, at least 4 times, at least 5 times, at least 6
times, at least 7 times, at least 8 times and at least 9 times or
more than 9 times in the 3'UTR.
[0184] In another embodiment, the spacer separating two TEE
sequences may include other sequences known in the art which may
regulate the translation of the signal-sensor polynucleotides,
primary constructs, modified nucleic acids and/or mmRNA of the
present invention such as, but not limited to, miR sequences
described herein (e.g., miR binding sites and miR seeds). As a
non-limiting example, each spacer used to separate two TEE
sequences may include a different miR sequence or component of a
miR sequence (e.g., miR seed sequence).
[0185] In one embodiment, the incorporation of a miR sequence
and/or a TEE sequence changes the shape of the stem loop region
which may increase and/or descrease translation. (see e.g, Kedde et
al. A Pumilio-induced RNA structure switch in p27-3'UTR controls
miR-221 and miR-22 accessibility. Nature Cell Biology. 2010, herein
incorporated by reference in its entirety).
[0186] In one embodiment, the 5'UTR may comprise at least one
microRNA sequence. The microRNA sequence may be, but is not limited
to, a 19 or 22 nucleotide sequence and/or a microRNA sequence
without the seed.
[0187] In one embodiment the microRNA sequence in the 5'UTR may be
used to stabilize the nucleic acid and/or mRNA described
herein.
[0188] In another embodiment, a microRNA sequence in the 5'UTR may
be used to decrease the accessibility of the site of translation
initiation such as, but not limited to a start codon. Matsuda et al
(PLoS One. 2010 11(5):e15057; herein incorporated by reference in
its entirety) used antisense locked nucleic acid (LNA)
oligonucleotides and exon-junctino complexes (EJCs) around a start
codon (-4 to +37 where the A of the AUG codons is +1) in order to
decrease the accessibility to the first start codon (AUG). Matsuda
showed that altering the sequence around the start codon with an
LNA or EJC the efficiency, length and structural stability of the
nucleic acid or mRNA is affected. The signal-sensor polynucleotides
of the present invention may comprise a microRNA sequence, instead
of the LNA or EJC sequence described by Matsuda et al, near the
site of translation initiation in order to decrease the
accessibility to the site of translation initiation. The site of
translation initiation may be prior to, after or within the
microRNA sequence. As a non-limiting example, the site of
translation initiation may be located within a microRNA sequence
such as a seed sequence or binding site. As another non-limiting
example, the site of translation initiation may be located within a
miR-122 sequence such as the seed sequence or the mir-122 binding
site.
[0189] In one embodiment, the nucleic acids or mRNA of the present
invention comprises at least one microRNA sequence in a region of
the nucleic acid or mRNA which may interact with a RNA binding
protein.
RNA Motifs for RNA Binding Proteins (RBPs)
[0190] RNA binding proteins (RBPs) can regulate numerous aspects of
co- and post-transcription gene expression such as, but not limited
to, RNA splicing, localization, translation, turnover,
polyadenylation, capping, modification, export and localization.
RNA-binding domains (RBDs), such as, but not limited to, RNA
recognition motif (RR) and hnRNP K-homology (KH) domains, typically
regulate the sequence association between RBPs and their RNA
targets (Ray et al. Nature 2013. 499:172-177; herein incorporated
by reference in its entirety). In one embodiment, the canonical
RBDs can bind short RNA sequences. In another embodiment, the
canonical RBDs can recognize structure RNAs.
[0191] In one embodiment, the nucleic acids and/or mRNA may
comprise at least one RNA-binding motif such as, but not limited to
a RNA-binding domain (RBD).
[0192] In one embodiment, the RBD may be any of the RBDs, fragments
or variants thereof descried by Ray et al. (Nature 2013.
499:172-177; herein incorporated by reference in its entirety).
[0193] In one embodiment, the nucleic acids or mRNA of the present
invention may comprise a sequence for at least one RNA-binding
domain (RBDs). When the nucleic acids or mRNA of the present
invention comprise more than one RBD, the RBDs do not need to be
from the same species or even the same structural class.
[0194] In one embodiment, at least one flanking region (e.g., the
5'UTR and/or the 3'UTR) may comprise at least one RBD. In another
embodiment, the first flanking region and the second flanking
region may both comprise at least one RBD. The RBD may be the same
or each of the RBDs may have at least 60% sequence identity to the
other RBD. As a non-limiting example, at least on RBD may be
located before, after and/or within the 3'UTR of the nucleic acid
or mRNA of the present invention. As another non-limiting example,
at least one RBD may be located before or within the first 300
nucleosides of the 3'UTR.
[0195] In another embodiment, the nucleic acids and/or mRNA of the
present invention may comprise at least one RBD in the first region
of linked nucleosides. The RBD may be located before, after or
within a coding region (e.g., the ORF).
[0196] In yet another embodiment, the first region of linked
nucleosides and/or at least one flanking region may comprise at
least on RBD. As a non-limiting example, the first region of linked
nucleosides may comprise a RBD related to splicing factors and at
least one flanking region may comprise a RBD for stability and/or
translation factors.
[0197] In one embodiment, the nucleic acids and/or mRNA of the
present invention may comprise at least one RBD located in a coding
and/or non-coding region of the nucleic acids and/or mRNA.
[0198] In one embodiment, at least one RBD may be incorporated into
at least one flanking region to increase the stability of the
nucleic acid and/or mRNA of the present invention.
[0199] In one embodiment, a microRNA sequence in a RNA binding
protein motif may be used to decrease the accessibility of the site
of translation initiation such as, but not limited to a start
codon. The signal-sensor polynucleotdies of the present invention
may comprise a microRNA sequence, instead of the LNA or EJC
sequence described by Matsuda et al, near the site of translation
initiation in order to decrease the accessibility to the site of
translation initiation. The site of translation initiation may be
prior to, after or within the microRNA sequence. As a non-limiting
example, the site of translation initiation may be located within a
microRNA sequence such as a seed sequence or binding site. As
another non-limiting example, the site of translation initiation
may be located within a miR-122 sequence such as the seed sequence
or the mir-122 binding site.
[0200] In another embodiment, an antisense locked nucleic acid
(LNA) oligonucleotides and exon-junctino complexes (EJCs) may be
used in the RNA binding protein motif. The LNA and EJCs may be used
around a start codon (-4 to +37 where the A of the AUG codons is
+1) in order to decrease the accessibility to the first start codon
(AUG).
3' UTR and the AU Rich Elements
[0201] 3'UTRs are known to have stretches of Adenosines and
Uridines embedded in them. These AU rich signatures are
particularly prevalent in genes with high rates of turnover. Based
on their sequence features and functional properties, the AU rich
elements (AREs) can be separated into three classes (Chen et al,
1995): Class I AREs contain several dispersed copies of an AUUUA
motif within U-rich regions. C-Myc and MyoD contain class I AREs.
Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A)
nonamers. Molecules containing this type of AREs include GM-CSF and
TNF-a. Class III ARES are less well defined. These U rich regions
do not contain an AUUUA motif c-Jun and Myogenin are two
well-studied examples of this class. Most proteins binding to the
AREs are known to destabilize the messenger, whereas members of the
ELAV family, most notably HuR, have been documented to increase the
stability of mRNA. HuR binds to AREs of all the three classes.
Engineering the HuR specific binding sites into the 3' UTR of
nucleic acid molecules will lead to HuR binding and thus,
stabilization of the message in vivo.
[0202] Introduction, removal or modification of 3' UTR AU rich
elements (AREs) can be used to modulate the stability of
signal-sensor polynucleotides, primary constructs or mmRNA of the
invention. When engineering specific polynucleotides, primary
constructs or mmRNA, one or more copies of an ARE can be introduced
to make polynucleotides, primary constructs or mmRNA of the
invention less stable and thereby curtail translation and decrease
production of the resultant protein. Likewise, AREs can be
identified and removed or mutated to increase the intracellular
stability and thus increase translation and production of the
resultant protein. Transfection experiments can be conducted in
relevant cell lines, using signal-sensor polynucleotides, primary
constructs or mmRNA of the invention and protein production can be
assayed at various time points post-transfection. For example,
cells can be transfected with different ARE-engineering molecules
and by using an ELISA kit to the relevant protein and assaying
protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and 7 days
post-transfection.
3' UTR and Triple Helices
[0203] In one embodiment, signal-sequence polynucleotides of the
present invention may include a triple helix on the 3' end of the
signal-sequence polynucleotides. The 3' end of the nucleic acids of
the present invention may include a triple helix alone or in
combination with a Poly-A tail.
[0204] In one embodiment, the signal-sequence polynucleotides of
the present invention may comprise at least a first and a second
U-rich region, a conserved stem loop region between the first and
second region and an A-rich region. The first and second U-rich
region and the A-rich region may associate to form a triple helix
on the 3' end of the nucleic acid. This triple helix may stabilize
the nucleic acid, enhance the translational efficiency of the
nucleic acid and/or protect the 3' end from degradation. Exemplary
triple helices include, but are not limited to, the triple helix
sequence of metastasis-associated lung adenocarcinoma transcript 1
(MALAT1), MEN-.beta. and polyadenylated nuclear (PAN) RNA (See
Wilusz et al., Genes & Development 2012 26:2392-2407; herein
incorporated by reference in its entirety). In one embodiment, the
3' end of the modified nucleic acids, enhanced modified RNA or
ribonucleic acids of the present invention comprises a first U-rich
region comprising TTTTTCTTTT (SEQ ID NO: 1), a second U-rich region
comprising TTTTGCTTTTT (SEQ ID NO: 2) or TTTTGCTTTT (SEQ ID NO: 3),
an A-rich region comprising AAAAAGCAAAA (SEQ ID NO: 4). In another
embodiment, the 3' end of the nucleic acids of the present
invention comprises a triple helix formation structure comprising a
first U-rich region, a conserved region, a second U-rich region and
an A-rich region.
[0205] In one embodiment, the triple helix may be formed from the
cleavage of a MALAT1 sequence prior to the cloverleaf structure.
While not meaning to be bound by theory, MALAT1 is a long
non-coding RNA which, when cleaved, forms a triple helix and a
tRNA-like cloverleaf structure. The MALAT1 transcript then
localizes to nuclear speckles and the tRNA-like cloverleaf
localizes to the cytoplasm (Wilusz et al. Cell 2008 135(5):
919-932; herein incorporated by reference in its entirety).
[0206] As a non-limiting example, the terminal end of the nucleic
acid of the present invention comprising the MALAT1 sequence can
then form a triple helix structure, after RNaseP cleavage from the
cloverleaf structure, which stabilizes the nucleic acid (Peart et
al. Non-mRNA 3' end formation: how the other half lives; WIREs RNA
2013; herein incorporated by reference in its entirety).
[0207] In one embodiment, the signal-sequence polynucleotides
described herein comprise a MALAT1 sequence. In another embodiment,
the signal-sequence polynucleotides may be polyadenylated. In yet
another embodiment, the signal-sequence polynucleotides is not
polyadenylated but has an increased resistance to degradation
compared to unmodified nucleic acids or mRNA.
[0208] In one embodiment, the signal-sequence polynucleotides of
the present invention may comprise a MALAT1 sequence in the second
flanking region (e.g., the 3'UTR). As a non-limiting example, the
MALAT1 sequence may be human or mouse.
[0209] In another embodiment, the cloverleaf structure of the
MALAT1 sequence may also undergo processing by RNaseZ and CCA
adding enzyme to form a tRNA-like structure called mascRNA
(MALAT1-associated small cytoplasmic RNA). As a non-limiting
example, the mascRNA may encode a protein or a fragment thereof
and/or may comprise a microRNA sequence. The mascRNA may comprise
at least one chemical modification described herein.
Stem Loop
[0210] In one embodiment, the nucleic acids of the present
invention may include a stem loop such as, but not limited to, a
histone stem loop. The stem loop may be a nucleotide sequence that
is about 25 or about 26 nucleotides in length such as, but not
limited to, SEQ ID NOs: 7-17 as described in International Patent
Publication No. WO2013103659, herein incorporated by reference in
its entirety. The histone stem loop may be located 3' relative to
the coding region (e.g., at the 3' terminus of the coding region).
As a non-limiting example, the stem loop may be located at the 3'
end of a nucleic acid described herein.
[0211] In one embodiment, the stem loop may be located in the
second terminal region. As a non-limiting example, the stem loop
may be located within an untranslated region (e.g., 3'UTR) in the
second terminal region.
[0212] In one embodiment, the nucleic acid such as, but not limited
to mRNA, which comprises the histone stem loop may be stabilized by
the addition of at least one chain terminating nucleoside. Not
wishing to be bound by theory, the addition of at least one chain
terminating nucleoside may slow the degradation of a nucleic acid
and thus can increase the half-life of the nucleic acid.
[0213] In one embodiment, the chain terminating nucleoside may be,
but is not limited to, those described in International Patent
Publication No. WO2013103659, herein incorporated by reference in
its entirety. In another embodiment, the chain terminating
nucleosides which may be used with the present invention includes,
but is not limited to, 3'-deoxyadenosine (cordycepin),
3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine,
3'-deoxythymine, 2',3'-dideoxynucleosides, such as
2',3'-dideoxyadenosine, 2',3'-dideoxyuridine,
2',3'-dideoxycytosine, 2',3'-dideoxyguanosine,
2',3'-dideoxythymine, a 2'-deoxynucleoside, or a
--O-methylnucleoside.
[0214] In another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprises the histone stem loop may be
stabilized by a modification to the 3' region of the nucleic acid
that can prevent and/or inhibit the addition of oligio(U) (see
e.g., International Patent Publication No. WO2013103659, herein
incorporated by reference in its entirety).
[0215] In yet another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprises the histone stem loop may be
stabilized by the addition of an oligonucleotide that terminates in
a 3'-deoxynucleoside, 2',3'-dideoxynucleoside
3''-0-methylnucleosides, 3'-0-ethylnucleosides, 3'-arabinosides,
and other modified nucleosides known in the art and/or described
herein.
[0216] In one embodiment, the nucleic acids of the present
invention may include a histone stem loop, a polyA tail sequence
and/or a 5' cap structure. The histone stem loop may be before
and/or after the polyA tail sequence. The nucleic acids comprising
the histone stem loop and a polyA tail sequence may include a chain
terminating nucleoside described herein.
[0217] In another embodiment, the nucleic acids of the present
invention may include a histone stem loop and a 5' cap structure.
The 5' cap structure may include, but is not limited to, those
described herein and/or known in the art.
[0218] In one embodiment, the conserved stem loop region may
comprise a miR sequence described herein. As a non-limiting
example, the stem loop region may comprise the seed sequence of a
miR sequence described herein. In another non-limiting example, the
stem loop region may comprise a miR-122 seed sequence.
[0219] In another embodiment, the conserved stem loop region may
comprise a miR sequence described herein and may also include a TEE
sequence.
[0220] In one embodiment, the incorporation of a miR sequence
and/or a TEE sequence changes the shape of the stem loop region
which may increase and/or descrease translation. (see e.g, Kedde et
al. A Pumilio-induced RNA structure switch in p27-3'UTR controls
miR-221 and miR-22 accessibility. Nature Cell Biology. 2010, herein
incorporated by reference in its entirety).
5' Capping
[0221] The 5' cap structure of an mRNA is involved in nuclear
export, increasing mRNA stability and binds the mRNA Cap Binding
Protein (CBP), which is responsibile for mRNA stability in the cell
and translation competency through the association of CBP with
poly(A) binding protein to form the mature cyclic mRNA species. The
cap further assists the removal of 5' proximal introns removal
during mRNA splicing.
[0222] Endogenous mRNA molecules may be 5'-end capped generating a
5'-ppp-5'-triphosphate linkage between a terminal guanosine cap
residue and the 5'-terminal transcribed sense nucleotide of the
mRNA molecule. This 5'-guanylate cap may then be methylated to
generate an N7-methyl-guanylate residue. The ribose sugars of the
terminal and/or anteterminal transcribed nucleotides of the 5' end
of the mRNA may optionally also be 2'-O-methylated. 5'-decapping
through hydrolysis and cleavage of the guanylate cap structure may
target a nucleic acid molecule, such as an mRNA molecule, for
degradation.
[0223] Modifications to the signal-sensor polynucleotides, primary
constructs, and mmRNA of the present invention may generate a
non-hydrolyzable cap structure preventing decapping and thus
increasing mRNA half-life. Because cap structure hydrolysis
requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified
nucleotides may be used during the capping reaction. For example, a
Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.)
may be used with .alpha.-thio-guanosine nucleotides according to
the manufacturer's instructions to create a phosphorothioate
linkage in the 5'-ppp-5' cap. Additional modified guanosine
nucleotides may be used such as .alpha.-methyl-phosphonate and
seleno-phosphate nucleotides.
[0224] Additional modifications include, but are not limited to,
2'-O-methylation of the ribose sugars of 5'-terminal and/or
5'-anteterminal nucleotides of the mRNA (as mentioned above) on the
2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap
structures can be used to generate the 5'-cap of a nucleic acid
molecule, such as an mRNA molecule.
[0225] Cap analogs, which herein are also referred to as synthetic
cap analogs, chemical caps, chemical cap analogs, or structural or
functional cap analogs, differ from natural (i.e. endogenous,
wild-type or physiological) 5'-caps in their chemical structure,
while retaining cap function. Cap analogs may be chemically (i.e.
non-enzymatically) or enzymatically synthesized and/linked to a
nucleic acid molecule.
[0226] For example, the Anti-Reverse Cap Analog (ARCA) cap contains
two guanines linked by a 5'-5'-triphosphate group, wherein one
guanine contains an N7 methyl group as well as a 3'-O-methyl group
(i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine
(m.sup.7G-3'mppp-G; which may equivaliently be designated 3'
O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other, unmodified,
guanine becomes linked to the 5'-terminal nucleotide of the capped
nucleic acid molecule (e.g. an mRNA or mmRNA). The N7- and
3'-O-methlyated guanine provides the terminal moiety of the capped
nucleic acid molecule (e.g. mRNA or mmRNA).
[0227] Another exemplary cap is mCAP, which is similar to ARCA but
has a 2'-O-methyl group on guanosine (i.e.,
N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine,
m.sup.7Gm-ppp-G).
[0228] While cap analogs allow for the concomitant capping of a
nucleic acid molecule in an in vitro transcription reaction, up to
20% of transcripts remain uncapped. This, as well as the structural
differences of a cap analog from an endogenous 5'-cap structures of
nucleic acids produced by the endogenous, cellular transcription
machinery, may lead to reduced translational competency and reduced
cellular stability.
[0229] Signal-sensor polynucleotides, primary constructs and mmRNA
of the invention may also be capped post-transcriptionally, using
enzymes, in order to generate more authentic 5'-cap structures. As
used herein, the phrase "more authentic" refers to a feature that
closely mirrors or mimics, either structurally or functionally, an
endogenous or wild type feature. That is, a "more authentic"
feature is better representative of an endogenous, wild-type,
natural or physiological cellular function and/or structure as
compared to synthetic features or analogs, etc., of the prior art,
or which outperforms the corresponding endogenous, wild-type,
natural or physiological feature in one or more respects.
Non-limiting examples of more authentic 5' cap structures of the
present invention are those which, among other things, have
enhanced binding of cap binding proteins, increased half life,
reduced susceptibility to 5' endonucleases and/or reduced 5'
decapping, as compared to synthetic 5' cap structures known in the
art (or to a wild-type, natural or physiological 5' cap structure).
For example, recombinant Vaccinia Virus Capping Enzyme and
recombinant 2'-O-methyltransferase enzyme can create a canonical
5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an
mRNA and a guanine cap nucleotide wherein the cap guanine contains
an N7 methylation and the 5'-terminal nucleotide of the mRNA
contains a 2'-O-methyl. Such a structure is termed the Cap1
structure. This cap results in a higher translational-competency
and cellular stability and a reduced activation of cellular
pro-inflammatory cytokines, as compared, e.g., to other 5' cap
analog structures known in the art. Cap structures include
7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and
7mG(5')-ppp(5')NlmpN2mp (cap 2).
[0230] Because the signal-sensor polynucleotides, primary
constructs or mmRNA may be capped post-transcriptionally, and
because this process is more efficient, nearly 100% of the
signal-sensor polynucleotides, primary constructs or mmRNA may be
capped. This is in contrast to -80% when a cap analog is linked to
an mRNA in the course of an in vitro transcription reaction.
[0231] According to the present invention, 5' terminal caps may
include endogenous caps or cap analogs. According to the present
invention, a 5' terminal cap may comprise a guanine analog. Useful
guanine analogs include inosine, N1-methyl-guanosine,
2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,
2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
Viral Sequences
[0232] Additional viral sequences such as, but not limited to, the
translation enhancer sequence of the barley yellow dwarf virus
(BYDV-PAV) can be engineered and inserted in the 3' UTR of the
signal-sensor polynucleotides, primary constructs or mmRNA of the
invention and can stimulate the translation of the construct in
vitro and in vivo. Transfection experiments can be conducted in
relevant cell lines at and protein production can be assayed by
ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7
post-transfection.
IRES Sequences
[0233] Further, provided are signal-sensor polynucleotides, primary
constructs or mmRNA which may contain an internal ribosome entry
site (IRES). First identified as a feature Picorna virus RNA, IRES
plays an important role in initiating protein synthesis in absence
of the 5' cap structure. An IRES may act as the sole ribosome
binding site, or may serve as one of multiple ribosome binding
sites of an mRNA. signal-sensor polynucleotides, primary constructs
or mmRNA containing more than one functional ribosome binding site
may encode several oncology-related peptides or oncology-related
polypeptides that are translated independently by the ribosomes
("multicistronic nucleic acid molecules"). When signal-sensor
polynucleotides, primary constructs or mmRNA are provided with an
IRES, further optionally provided is a second translatable region.
Examples of IRES sequences that can be used according to the
invention include without limitation, those from picornaviruses
(e.g. FMDV), pest viruses (CFFV), polio viruses (PV),
encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses
(FMDV), hepatitis C viruses (HCV), classical swine fever viruses
(CSFV), murine leukemia virus (MLV), simian immune deficiency
viruses (SIV) or cricket paralysis viruses (CrPV).
Poly-A Tails
[0234] During RNA processing, a long chain of adenine nucleotides
(poly-A tail) may be added to a polynucleotide such as an mRNA
molecule in order to increase stability. Immediately after
transcription, the 3' end of the transcript may be cleaved to free
a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine
nucleotides to the RNA. The process, called polyadenylation, adds a
poly-A tail that can be between 100 and 250 residues long.
[0235] It has been discovered that unique poly-A tail lengths
provide certain advantages to the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention.
[0236] Generally, the length of a poly-A tail of the present
invention is greater than 30 nucleotides in length. In another
embodiment, the poly-A tail is greater than 35 nucleotides in
length (e.g., at least or greater than about 35, 40, 45, 50, 55,
60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400,
450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400,
1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000
nucleotides). In some embodiments, the signal-sensor
polynucleotides, primary construct, or mmRNA includes from about 30
to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100,
from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000,
from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to
100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to
1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from
50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000,
from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100
to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500,
from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000
to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to
3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to
3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to
3,000).
[0237] In one embodiment, the poly-A tail is designed relative to
the length of the overall signal-sensor polynucleotides, primary
constructs or mmRNA. This design may be based on the length of the
coding region, the length of a particular feature or region (such
as the first or flanking regions), or based on the length of the
ultimate product expressed from the polynucleotides, primary
constructs or mmRNA.
[0238] In this context the poly-A tail may be 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100% greater in length than the signal-sensor
polynucleotides, primary constructs or mmRNA or feature thereof.
The poly-A tail may also be designed as a fraction of
polynucleotides, primary constructs or mmRNA to which it belongs.
In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70,
80, or 90% or more of the total length of the construct or the
total length of the construct minus the poly-A tail.
[0239] In one embodiment, engineered binding sites and/or
conjugation of signal-sensor polynucleotides, primary constructs or
mmRNA for Poly-A binding protein may be used to enhance expression.
The engineered binding sites may be sensor sequences which can
operate as binding sites for ligands of the local microenvironment
of the nucleic acids and/or mRNA. As a non-limiting example, the
nucleic acids and/or mRNA may comprise at least one engineered
binding site to alter the binding affinity of Poly-A binding
protein (PABP) and analogs thereof. The incorporation of at least
one engineered binding site may increase the binding affinity of
the PABP and analogs thereof
[0240] Additionally, multiple distinct signal-sensor
polynucleotides, primary constructs or mmRNA may be linked together
to the PABP (Poly-A binding protein) through the 3'-end using
modified nucleotides at the 3'-terminus of the poly-A tail.
Transfection experiments can be conducted in relevant cell lines
and protein production can be assayed by ELISA at 12 hr, 24 hr, 48
hr, 72 hr and day 7 post-transfection. As a non-limiting example,
the transfection experiments may be used to evaluate the effect on
PABP or analogs thereof binding affinity as a result of the
addition of at least one engineered binding site.
[0241] In one embodiment, the signal-sensor polynucleotides and
primary constructs of the present invention are designed to include
a polyA-G Quartet. The G-quartet is a cyclic hydrogen bonded array
of four guanine nucleotides that can be formed by G-rich sequences
in both DNA and RNA. In this embodiment, the G-quartet is
incorporated at the end of the poly-A tail. The resultant mmRNA
construct is assayed for stability, protein production and other
parameters including half-life at various time points. It has been
discovered that the polyA-G quartet results in protein production
equivalent to at least 75% of that seen using a poly-A tail of 120
nucleotides alone.
[0242] In one embodiment, the nucleic acids or mRNA of the present
invention may comprise a polyA tail and may be stabilized by the
addition of a chain terminating nucleoside. The nucleic acids
and/or mRNA with a polyA tail may further comprise a 5' cap
structure.
[0243] In another embodiment, the nucleic acids or mRNA of the
present invention may comprise a polyA-G Quartet. The nucleic acids
and/or mRNA with a polyA-G Quartet may further comprise a 5' cap
structure.
[0244] In one embodiment, the chain terminating nucleoside which
may be used to stabilize the nucleic acid or mRNA comprising a
polyA tail or polyA-G Quartet may be, but is not limited to, those
described in International Patent Publication No. WO2013103659,
herein incorporated by reference in its entirety. In another
embodiment, the chain terminating nucleosides which may be used
with the present invention includes, but is not limited to,
3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine,
3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, such
as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine,
2',3'-dideoxycytosine, 2',3'-dideoxyguanosine,
2',3'-dideoxythymine, a 2'-deoxynucleoside, or
a-O-methylnucleoside.
[0245] In another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprise a polyA tail or a polyA-G Quartet
may be stabilized by a modification to the 3' region of the nucleic
acid that can prevent and/or inhibit the addition of oligio(U) (see
e.g., International Patent Publication No. WO2013103659, herein
incorporated by reference in its entirety).
[0246] In yet another embodiment, the nucleic acid such as, but not
limited to mRNA, which comprise a polyA tail or a polyA-G Quartet
may be stabilized by the addition of an oligonucleotide that
terminates in a 3'-deoxynucleoside, 2',3'-dideoxynucleoside
methylnucleosides, 3'-0-ethylnucleosides, 3'-arabinosides, and
other modified nucleosides known in the art and/or described
herein.
Quantification
[0247] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be
quantified in exosomes derived from one or more bodily fluid. As
used herein "bodily fluids" include peripheral blood, serum,
plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva,
bone marrow, synovial fluid, aqueous humor, amniotic fluid,
cerumen, breast milk, broncheoalveolar lavage fluid, semen,
prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat,
fecal matter, hair, tears, cyst fluid, pleural and peritoneal
fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial
fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal
secretion, stool water, pancreatic juice, lavage fluids from sinus
cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and
umbilical cord blood. Alternatively, exosomes may be retrieved from
an organ selected from the group consisting of lung, heart,
pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin,
colon, breast, prostate, brain, esophagus, liver, and placenta.
[0248] In the quantification method, a sample of not more than 2 mL
is obtained from the subject and the exosomes isolated by size
exclusion chromatography, density gradient centrifugation,
differential centrifugation, nanomembrane ultrafiltration,
immunoabsorbent capture, affinity purification, microfluidic
separation, or combinations thereof. In the analysis, the level or
concentration of signal-sensor polynucleotides, primary construct
or mmRNA may be an expression level, presence, absence, truncation
or alteration of the administered construct. It is advantageous to
correlate the level with one or more clinical phenotypes or with an
assay for a human disease biomarker. The assay may be performed
using construct specific probes, cytometry, qRT-PCR, real-time PCR,
PCR, flow cytometry, electrophoresis, mass spectrometry, or
combinations thereof while the exosomes may be isolated using
immunohistochemical methods such as enzyme linked immunosorbent
assay (ELISA) methods. Exosomes may also be isolated by size
exclusion chromatography, density gradient centrifugation,
differential centrifugation, nanomembrane ultrafiltration,
immunoabsorbent capture, affinity purification, microfluidic
separation, or combinations thereof.
[0249] These methods afford the investigator the ability to
monitor, in real time, the level of signal-sensor polynucleotides,
primary constructs or mmRNA remaining or delivered. This is
possible because the polynucleotides, primary constructs or mmRNA
of the present invention differ from the endogenous forms due to
the structural and/or chemical modifications.
II. Design and Synthesis of Signal-Sensor Polynucleotides
[0250] Signal-sensor polynucleotides, primary constructs or mmRNA
for use in accordance with the invention may be prepared according
to any available technique including, but not limited to chemical
synthesis, enzymatic synthesis, which is generally termed in vitro
transcription (IVT) or enzymatic or chemical cleavage of a longer
precursor, etc. Methods of synthesizing RNAs are known in the art
(see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a
practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL
Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:
methods and applications, Methods in Molecular Biology, v. 288
(Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are
incorporated herein by reference).
[0251] The process of design and synthesis of the signal-sensor
primary constructs of the invention generally includes the steps of
gene construction, mRNA production (either with or without
modifications) and purification. In the enzymatic synthesis method,
a target signal-sensor polynucleotide sequence encoding the
oncology-related polypeptide of interest is first selected for
incorporation into a vector which will be amplified to produce a
cDNA template. Optionally, the target signal-sensor polynucleotide
sequence and/or any flanking sequences may be codon optimized. The
cDNA template is then used to produce mRNA through in vitro
transcription (IVT). After production, the mRNA may undergo
purification and clean-up processes. The steps of which are
provided in more detail below.
Gene Construction
[0252] The step of gene construction may include, but is not
limited to gene synthesis, vector amplification, plasmid
purification, plasmid linearization and clean-up, and cDNA template
synthesis and clean-up.
Gene Synthesis
[0253] Once an oncology-related polypeptide of interest, or target,
is selected for production, a signal-sensor primary construct is
designed. Within the primary construct, a first region of linked
nucleosides encoding the polypeptide of interest may be constructed
using an open reading frame (ORF) of a selected nucleic acid (DNA
or RNA) transcript. The ORF may comprise the wild type ORF, an
isoform, variant or a fragment thereof. As used herein, an "open
reading frame" or "ORF" is meant to refer to a nucleic acid
sequence (DNA or RNA) which is capable of encoding an
oncology-related polypeptide of interest. ORFs often begin with the
start codon, ATG and end with a nonsense or termination codon or
signal.
[0254] Further, the nucleotide sequence of the first region may be
codon optimized. Codon optimization methods are known in the art
and may be useful in efforts to achieve one or more of several
goals. These goals include to match codon frequencies in target and
host organisms to ensure proper folding, bias GC content to
increase mRNA stability or reduce secondary structures, minimize
tandem repeat codons or base runs that may impair gene construction
or expression, customize transcriptional and translational control
regions, insert or remove protein trafficking sequences, remove/add
post translation modification sites in encoded protein (e.g.
glycosylation sites), add, remove or shuffle protein domains,
insert or delete restriction sites, modify ribosome binding sites
and mRNA degradation sites, to adjust translational rates to allow
the various domains of the protein to fold properly, or to reduce
or eliminate problem secondary structures within the mRNA. Codon
optimization tools, algorithms and services are known in the art,
non-limiting examples include services from GeneArt (Life
Technologies) and/or DNA2.0 (Menlo Park Calif.). In one embodiment,
the ORF sequence is optimized using optimization algorithms. Codon
options for each amino acid are given in Table 1.
TABLE-US-00001 TABLE 1 Codon Options Single Letter Amino Acid Code
Codon Options Isoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA,
CTG, TTA, TTG Valine V GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC
Methionine M ATG Cysteine C TGT, TGC Alanine A GCT, GCC, GCA, GCG
Glycine G GGT, GGC, GGA, GGG Proline P CCT, CCC, CCA, CCG Threonine
T ACT, ACC, ACA, ACG Serine S TCT, TCC, TCA, TCG, AGT, AGC Tyrosine
Y TAT, TAC Tryptophan W TGG Glutamine Q CAA, CAG Asparagine N AAT,
AAC Histidine H CAT, CAC Glutamic acid E GAA, GAG Aspartic acid D
GAT, GAC Lysine K AAA, AAG Arginine R CGT, CGC, CGA, CGG, AGA, AGG
Selenocysteine Sec UGA in mRNA in presence of Selenocystein
insertion element (SECIS) Stop codons Stop TAA, TAG, TGA
[0255] In one embodiment, after a nucleotide sequence has been
codon optimized it may be further evaluated for regions containing
restriction sites. At least one nucleotide within the restriction
site regions may be replaced with another nucleotide in order to
remove the restriction site from the sequence but the replacement
of nucleotides does alter the amino acid sequence which is encoded
by the codon optimized nucleotide sequence.
[0256] Features, which may be considered beneficial in some
embodiments of the present invention, may be encoded by the
signal-sensor primary construct and may flank the ORF as a first or
second flanking region. The flanking regions may be incorporated
into the signal-sensor primary construct before and/or after
optimization of the ORF. It is not required that a signal-sensor
primary construct contain both a 5' and 3' flanking region.
Examples of such features include, but are not limited to,
untranslated regions (UTRs), Kozak sequences, an oligo(dT)
sequence, and detectable tags and may include multiple cloning
sites which may have XbaI recognition.
[0257] In some embodiments, a 5' UTR and/or a 3' UTR may be
provided as flanking regions. Multiple 5' or 3' UTRs may be
included in the flanking regions and may be the same or of
different sequences. Any portion of the flanking regions, including
none, may be codon optimized and any may independently contain one
or more different structural or chemical modifications, before
and/or after codon optimization. Combinations of features may be
included in the first and second flanking regions and may be
contained within other features. For example, the ORF may be
flanked by a 5' UTR which may contain a strong Kozak translational
initiation signal and/or a 3' UTR which may include an oligo(dT)
sequence for templated addition of a poly-A tail.
[0258] Tables 2 and 3 provide a listing of exemplary UTRs which may
be utilized in the signal-sensor primary construct of the present
invention as flanking regions. Shown in Table 2 is a representative
listing of a 5'-untranslated region of the invention. Variants of
5' UTRs may be utilized wherein one or more nucleotides are added
or removed to the termini, including A, T, C or G.
TABLE-US-00002 TABLE 2 5'-Untranslated Regions 5' UTR Name/ SEQ ID
Identifier Description Sequence NO. Native Wild type UTR See wild
type sequence -- 5UTR-001 Synthetic UTR
GGGAAATAAGAGAGAAAAGAAGAGTAAGA 1 AGAAATATAAGAGCCACC 5UTR-002
Upstream UTR GGGAGATCAGAGAGAAAAGAAGAGTAAGA 2 AGAAATATAAGAGCCACC
5UTR-003 Upstream UTR GGAATAAAAGTCTCAACACAACATATACAA 3
AACAAACGAATCTCAAGCAATCAAGCATTC TACTTCTATTGCAGCAATTTAAATCATTTCT
TTTAAAGCAAAAGCAATTTTCTGAAAATTT TCACCATTTACGAACGATAGCAAC 5UTR-004
Upstream UTR GGGAGACAAGCUUGGCAUUCCGGUACUGU 4 UGGUAAAGCCACC
[0259] Shown in Table 3 is a representative listing of
3'-untranslated regions of the invention. Variants of 3' UTRs may
be utilized wherein one or more nucleotides are added or removed to
the termini, including A, T, C or G.
TABLE-US-00003 TABLE 3 3'-Untranslated Regions SEQ 3' UTR Name/ ID
Identifier Description Sequence NO. 3UTR-001 Creatine
GCGCCTGCCCACCTGCCACCGACTGCTGGAACC 5 Kinase
CAGCCAGTGGGAGGGCCTGGCCCACCAGAGTCC
TGCTCCCTCACTCCTCGCCCCGCCCCCTGTCCCA
GAGTCCCACCTGGGGGCTCTCTCCACCCTTCTCA
GAGTTCCAGTTTCAACCAGAGTTCCAACCAATG
GGCTCCATCCTCTGGATTCTGGCCAATGAAATAT
CTCCCTGGCAGGGTCCTCTTCTTTTCCCAGAGCT
CCACCCCAACCAGGAGCTCTAGTTAATGGAGAG
CTCCCAGCACACTCGGAGCTTGTGCTTTGTCTCC
ACGCAAAGCGATAAATAAAAGCATTGGTGGCCT
TTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTA GA 3UTR-002 Myoglobin
GCCCCTGCCGCTCCCACCCCCACCCATCTGGGCC 6
CCGGGTTCAAGAGAGAGCGGGGTCTGATCTCGT
GTAGCCATATAGAGTTTGCTTCTGAGTGTCTGCT
TTGTTTAGTAGAGGTGGGCAGGAGGAGCTGAGG
GGCTGGGGCTGGGGTGTTGAAGTTGGCTTTGCAT
GCCCAGCGATGCGCCTCCCTGTGGGATGTCATCA
CCCTGGGAACCGGGAGTGGCCCTTGGCTCACTG
TGTTCTGCATGGTTTGGATCTGAATTAATTGTCC
TTTCTTCTAAATCCCAACCGAACTTCTTCCAACC
TCCAAACTGGCTGTAACCCCAAATCCAAGCCATT
AACTACACCTGACAGTAGCAATTGTCTGATTAAT
CACTGGCCCCTTGAAGACAGCAGAATGTCCCTTT
GCAATGAGGAGGAGATCTGGGCTGGGCGGGCCA
GCTGGGGAAGCATTTGACTATCTGGAACTTGTGT
GTGCCTCCTCAGGTATGGCAGTGACTCACCTGGT
TTTAATAAAACAACCTGCAACATCTCATGGTCTT TGAATAAAGCCTGAGTAGGAAGTCTAGA
3UTR-003 .alpha.-actin ACACACTCCACCTCCAGCACGCGACTTCTCAGG 7
ACGACGAATCTTCTCAATGGGGGGGCGGCTGAG
CTCCAGCCACCCCGCAGTCACTTTCTTTGTAACA
ACTTCCGTTGCTGCCATCGTAAACTGACACAGTG
TTTATAACGTGTACATACATTAACTTATTACCTC
ATTTTGTTATTTTTCGAAACAAAGCCCTGTGGAA
GAAAATGGAAAACTTGAAGAAGCATTAAAGTCA
TTCTGTTAAGCTGCGTAAATGGTCTTTGAATAAA GCCTGAGTAGGAAGTCTAGA 3UTR-004
Albumin CATCACATTTAAAAGCATCTCAGCCTACCATGAG 8
AATAAGAGAAAGAAAATGAAGATCAAAAGCTT
ATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCC
AACACCCTGTCTAAAAAACATAAATTTCTTTAAT
CATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAA
AAAATGGAAAGAATCTAATAGAGTGGTACAGCA
CTGTTATTTTTCAAAGATGTGTTGCTATCCTGAA
AATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCT
CTCTTATTCCACTTCGGTAGAGGATTTCTAGTTT
CTTGTGGGCTAATTAAATAAATCATTAATACTCT
TCTAATGGTCTTTGAATAAAGCCTGAGTAGGAA GTCTAGA 3UTR-005 .alpha.-globin
GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGC 9
CCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTC
TTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCT CGAGCATGCATCTAGA 3UTR-006 G-CSF
GCCAAGCCCTCCCCATCCCATGTATTTATCTCTA 10
TTTAATATTTATGTCTATTTAAGCCTCATATTTAA
AGACAGGGAAGAGCAGAACGGAGCCCCAGGCC
TCTGTGTCCTTCCCTGCATTTCTGAGTTTCATTCT
CCTGCCTGTAGCAGTGAGAAAAAGCTCCTGTCCT
CCCATCCCCTGGACTGGGAGGTAGATAGGTAAA
TACCAAGTATTTATTACTATGACTGCTCCCCAGC
CCTGGCTCTGCAATGGGCACTGGGATGAGCCGC
TGTGAGCCCCTGGTCCTGAGGGTCCCCACCTGGG
ACCCTTGAGAGTATCAGGTCTCCCACGTGGGAG
ACAAGAAATCCCTGTTTAATATTTAAACAGCAGT
GTTCCCCATCTGGGTCCTTGCACCCCTCACTCTG
GCCTCAGCCGACTGCACAGCGGCCCCTGCATCC CCTTGGCTGTGAGGCCCCTGGACAAGCAGAGGT
GGCCAGAGCTGGGAGGCATGGCCCTGGGGTCCC
ACGAATTTGCTGGGGAATCTCGTTTTTCTTCTTA
AGACTTTTGGGACATGGTTTGACTCCCGAACATC
ACCGACGCGTCTCCTGTTTTTCTGGGTGGCCTCG
GGACACCTGCCCTGCCCCCACGAGGGTCAGGAC
TGTGACTCTTTTTAGGGCCAGGCAGGTGCCTGGA
CATTTGCCTTGCTGGACGGGGACTGGGGATGTG GGAGGGAGCAGACAGGAGGAATCATGTCAGGC
CTGTGTGTGAAAGGAAGCTCCACTGTCACCCTCC
ACCTCTTCACCCCCCACTCACCAGTGTCCCCTCC
ACTGTCACATTGTAACTGAACTTCAGGATAATAA
AGTGTTTGCCTCCATGGTCTTTGAATAAAGCCTG
AGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 3UTR-007 Col1a2;
ACTCAATCTAAATTAAAAAAGAAAGAAATTTGA 11 collagen,
AAAAACTTTCTCTTTGCCATTTCTTCTTCTTCTTT type I, alpha 2
TTTAACTGAAAGCTGAATCCTTCCATTTCTTCTG
CACATCTACTTGCTTAAATTGTGGGCAAAAGAG AAAAAGAAGGATTGATCAGAGCATTGTGCAATA
CAGTTTCATTAACTCCTTCCCCCGCTCCCCCAAA
AATTTGAATTTTTTTTTCAACACTCTTACACCTGT
TATGGAAAATGTCAACCTTTGTAAGAAAACCAA AATAAAAATTGAAAAATAAAAACCATAAACATT
TGCACCACTTGTGGCTTTTGAATATCTTCCACAG
AGGGAAGTTTAAAACCCAAACTTCCAAAGGTTT
AAACTACCTCAAAACACTTTCCCATGAGTGTGAT
CCACATTGTTAGGTGCTGACCTAGACAGAGATG
AACTGAGGTCCTTGTTTTGTTTTGTTCATAATAC
AAAGGTGCTAATTAATAGTATTTCAGATACTTGA
AGAATGTTGATGGTGCTAGAAGAATTTGAGAAG
AAATACTCCTGTATTGAGTTGTATCGTGTGGTGT
ATTTTTTAAAAAATTTGATTTAGCATTCATATTTT
CCATCTTATTCCCAATTAAAAGTATGCAGATTAT
TTGCCCAAATCTTCTTCAGATTCAGCATTTGTTCT
TTGCCAGTCTCATTTTCATCTTCTTCCATGGTTCC
ACAGAAGCTTTGTTTCTTGGGCAAGCAGAAAAA
TTAAATTGTACCTATTTTGTATATGTGAGATGTT
TAAATAAATTGTGAAAAAAATGAAATAAAGCAT GTTTGGTTTTCCAAAAGAACATAT 3UTR-008
Col6a2; CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTCGT 12 collagen,
GAGCCCACCCCGTCCATGGTGCTAAGCGGGCCC type VI,
GGGTCCCACACGGCCAGCACCGCTGCTCACTCG alpha 2
GACGACCCCCTGGGCCTGCACCTCTCCAGCTCCT
CCCACGGGGTCCCCGTAGCCCCGGCCCCCGCCC AGCCCCAGGTCTCCCCAGGCCCTCCGCAGGCTG
CCCGGCCTCCCTCCCCCTGCAGCCATCCCAAGGC
TCCTGACCTACCTGGCCCCTGAGCTCTGGAGCAA GCCCTGACCCAATAAAGGCTTTGAACCCAT
3UTR-009 RPN1; GGGGCTAGAGCCCTCTCCGCACAGCGTGGAGAC 13 ribophorin I
GGGGCAAGGAGGGGGGTTATTAGGATTGGTGGT
TTTGTTTTGCTTTGTTTAAAGCCGTGGGAAAATG
GCACAACTTTACCTCTGTGGGAGATGCAACACT GAGAGCCAAGGGGTGGGAGTTGGGATAATTTTT
ATATAAAAGAAGTTTTTCCACTTTGAATTGCTAA
AAGTGGCATTTTTCCTATGTGCAGTCACTCCTCT
CATTTCTAAAATAGGGACGTGGCCAGGCACGGT
GGCTCATGCCTGTAATCCCAGCACTTTGGGAGGC
CGAGGCAGGCGGCTCACGAGGTCAGGAGATCGA
GACTATCCTGGCTAACACGGTAAAACCCTGTCTC
TACTAAAAGTACAAAAAATTAGCTGGGCGTGGT GGTGGGCACCTGTAGTCCCAGCTACTCGGGAGG
CTGAGGCAGGAGAAAGGCATGAATCCAAGAGG CAGAGCTTGCAGTGAGCTGAGATCACGCCATTG
CACTCCAGCCTGGGCAACAGTGTTAAGACTCTGT
CTCAAATATAAATAAATAAATAAATAAATAAAT AAATAAATAAAAATAAAGCGAGATGTTGCCCTC
AAA 3UTR-010 LRP1; low GGCCCTGCCCCGTCGGACTGCCCCCAGAAAGCC 14 density
TCCTGCCCCCTGCCAGTGAAGTCCTTCAGTGAGC lipoprotein
CCCTCCCCAGCCAGCCCTTCCCTGGCCCCGCCGG receptor-
ATGTATAAATGTAAAAATGAAGGAATTACATTT related
TATATGTGAGCGAGCAAGCCGGCAAGCGAGCAC protein 1
AGTATTATTTCTCCATCCCCTCCCTGCCTGCTCCT
TGGCACCCCCATGCTGCCTTCAGGGAGACAGGC AGGGAGGGCTTGGGGCTGCACCTCCTACCCTCC
CACCAGAACGCACCCCACTGGGAGAGCTGGTGG
TGCAGCCTTCCCCTCCCTGTATAAGACACTTTGC
CAAGGCTCTCCCCTCTCGCCCCATCCCTGCTTGC
CCGCTCCCACAGCTTCCTGAGGGCTAATTCTGGG
AAGGGAGAGTTCTTTGCTGCCCCTGTCTGGAAG ACGTGGCTCTGGGTGAGGTAGGCGGGAAAGGAT
GGAGTGTTTTAGTTCTTGGGGGAGGCCACCCCA AACCCCAGCCCCAACTCCAGGGGCACCTATGAG
ATGGCCATGCTCAACCCCCCTCCCAGACAGGCC
CTCCCTGTCTCCAGGGCCCCCACCGAGGTTCCCA
GGGCTGGAGACTTCCTCTGGTAAACATTCCTCCA
GCCTCCCCTCCCCTGGGGACGCCAAGGAGGTGG GCCACACCCAGGAAGGGAAAGCGGGCAGCCCC
GTTTTGGGGACGTGAACGTTTTAATAATTTTTGC
TGAATTCCTTTACAACTAAATAACACAGATATTG TTATAAATAAAATTGT 3UTR-011 Nnt1;
ATATTAAGGATCAAGCTGTTAGCTAATAATGCC 15 cardiotrophin-
ACCTCTGCAGTTTTGGGAACAGGCAAATAAAGT like
ATCAGTATACATGGTGATGTACATCTGTAGCAA cytokine
AGCTCTTGGAGAAAATGAAGACTGAAGAAAGCA factor 1
AAGCAAAAACTGTATAGAGAGATTTTTCAAAAG CAGTAATCCCTCAATTTTAAAAAAGGATTGAAA
ATTCTAAATGTCTTTCTGTGCATATTTTTTGTGTT
AGGAATCAAAAGTATTTTATAAAAGGAGAAAGA
ACAGCCTCATTTTAGATGTAGTCCTGTTGGATTT
TTTATGCCTCCTCAGTAACCAGAAATGTTTTAAA
AAACTAAGTGTTTAGGATTTCAAGACAACATTAT
ACATGGCTCTGAAATATCTGACACAATGTAAAC
ATTGCAGGCACCTGCATTTTATGTTTTTTTTTTCA
ACAAATGTGACTAATTTGAAACTTTTATGAACTT
CTGAGCTGTCCCCTTGCAATTCAACCGCAGTTTG
AATTAATCATATCAAATCAGTTTTAATTTTTTAA
ATTGTACTTCAGAGTCTATATTTCAAGGGCACAT
TTTCTCACTACTATTTTAATACATTAAAGGACTA
AATAATCTTTCAGAGATGCTGGAAACAAATCAT
TTGCTTTATATGTTTCATTAGAATACCAATGAAA
CATACAACTTGAAAATTAGTAATAGTATTTTTGA
AGATCCCATTTCTAATTGGAGATCTCTTTAATTT
CGATCAACTTATAATGTGTAGTACTATATTAAGT
GCACTTGAGTGGAATTCAACATTTGACTAATAA AATGAGTTCATCATGTTGGCAAGTGATGTGGCA
ATTATCTCTGGTGACAAAAGAGTAAAATCAAAT
ATTTCTGCCTGTTACAAATATCAAGGAAGACCTG
CTACTATGAAATAGATGACATTAATCTGTCTTCA
CTGTTTATAATACGGATGGATTTTTTTTCAAATC
AGTGTGTGTTTTGAGGTCTTATGTAATTGATGAC
ATTTGAGAGAAATGGTGGCTTTTTTTAGCTACCT
CTTTGTTCATTTAAGCACCAGTAAAGATCATGTC
TTTTTATAGAAGTGTAGATTTTCTTTGTGACTTTG
CTATCGTGCCTAAAGCTCTAAATATAGGTGAATG
TGTGATGAATACTCAGATTATTTGTCTCTCTATA
TAATTAGTTTGGTACTAAGTTTCTCAAAAAATTA
TTAACACATGAAAGACAATCTCTAAACCAGAAA
AAGAAGTAGTACAAATTTTGTTACTGTAATGCTC
GCGTTTAGTGAGTTTAAAACACACAGTATCTTTT
GGTTTTATAATCAGTTTCTATTTTGCTGTGCCTGA
GATTAAGATCTGTGTATGTGTGTGTGTGTGTGTG
TGCGTTTGTGTGTTAAAGCAGAAAAGACTTTTTT
AAAAGTTTTAAGTGATAAATGCAATTTGTTAATT
GATCTTAGATCACTAGTAAACTCAGGGCTGAATT
ATACCATGTATATTCTATTAGAAGAAAGTAAAC
ACCATCTTTATTCCTGCCCTTTTTCTTCTCTCAAA
GTAGTTGTAGTTATATCTAGAAAGAAGCAATTTT
GATTTCTTGAAAAGGTAGTTCCTGCACTCAGTTT
AAACTAAAAATAATCATACTTGGATTTTATTTAT
TTTTGTCATAGTAAAAATTTTAATTTATATATATT
TTTATTTAGTATTATCTTATTCTTTGCTATTTGCC
AATCCTTTGTCATCAATTGTGTTAAATGAATTGA
AAATTCATGCCCTGTTCATTTTATTTTACTTTATT
GGTTAGGATATTTAAAGGATTTTTGTATATATAA
TTTCTTAAATTAATATTCCAAAAGGTTAGTGGAC
TTAGATTATAAATTATGGCAAAAATCTAAAAAC
AACAAAAATGATTTTTATACATTCTATTTCATTA
TTCCTCTTTTTCCAATAAGTCATACAATTGGTAG
ATATGACTTATTTTATTTTTGTATTATTCACTATA
TCTTTATGATATTTAAGTATAAATAATTAAAAAA
ATTTATTGTACCTTATAGTCTGTCACCAAAAAAA
AAAAATTATCTGTAGGTAGTGAAATGCTAATGTT
GATTTGTCTTTAAGGGCTTGTTAACTATCCTTTAT
TTTCTCATTTGTCTTAAATTAGGAGTTTGTGTTTA
AATTACTCATCTAAGCAAAAAATGTATATAAAT CCCATTACTGGGTATATACCCAAAGGATTATAA
ATCATGCTGCTATAAAGACACATGCACACGTAT
GTTTATTGCAGCACTATTCACAATAGCAAAGACT
TGGAACCAACCCAAATGTCCATCAATGATAGAC TTGATTAAGAAAATGTGCACATATACACCATGG
AATACTATGCAGCCATAAAAAAGGATGAGTTCA TGTCCTTTGTAGGGACATGGATAAAGCTGGAAA
CCATCATTCTGAGCAAACTATTGCAAGGACAGA AAACCAAACACTGCATGTTCTCACTCATAGGTG
GGAATTGAACAATGAGAACACTTGGACACAAGG
TGGGGAACACCACACACCAGGGCCTGTCATGGG GTGGGGGGAGTGGGGAGGGATAGCATTAGGAG
ATATACCTAATGTAAATGATGAGTTAATGGGTG CAGCACACCAACATGGCACATGTATACATATGT
AGCAAACCTGCACGTTGTGCACATGTACCCTAG AACTTAAAGTATAATTAAAAAAAAAAAGAAAAC
AGAAGCTATTTATAAAGAAGTTATTTGCTGAAAT
AAATGTGATCTTTCCCATTAAAAAAATAAAGAA
ATTTTGGGGTAAAAAAACACAATATATTGTATTC
TTGAAAAATTCTAAGAGAGTGGATGTGAAGTGT TCTCACCACAAAAGTGATAACTAATTGAGGTAA
TGCACATATTAATTAGAAAGATTTTGTCATTCCA
CAATGTATATATACTTAAAAATATGTTATACACA
ATAAATACATACATTAAAAAATAAGTAAATGTA 3UTR-012 Col6a1;
CCCACCCTGCACGCCGGCACCAAACCCTGTCCTC 16 collagen,
CCACCCCTCCCCACTCATCACTAAACAGAGTAA type VI,
AATGTGATGCGAATTTTCCCGACCAACCTGATTC alpha 1
GCTAGATTTTTTTTAAGGAAAAGCTTGGAAAGCC
AGGACACAACGCTGCTGCCTGCTTTGTGCAGGG
TCCTCCGGGGCTCAGCCCTGAGTTGGCATCACCT
GCGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTA GTGTCACCTGCACAGGGCCCTCTGAGGCTCAGC
CCTGAGCTGGCGTCACCTGTGCAGGGCCCTCTGG
GGCTCAGCCCTGAGCTGGCCTCACCTGGGTTCCC
CACCCCGGGCTCTCCTGCCCTGCCCTCCTGCCCG
CCCTCCCTCCTGCCTGCGCAGCTCCTTCCCTAGG
CACCTCTGTGCTGCATCCCACCAGCCTGAGCAAG
ACGCCCTCTCGGGGCCTGTGCCGCACTAGCCTCC
CTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCA
ATCCTCACCTAACAGTTACTTTACAATTAAACTC
AAAGCAAGCTCTTCTCCTCAGCTTGGGGCAGCC
ATTGGCCTCTGTCTCGTTTTGGGAAACCAAGGTC
AGGAGGCCGTTGCAGACATAAATCTCGGCGACT
CGGCCCCGTCTCCTGAGGGTCCTGCTGGTGACCG
GCCTGGACCTTGGCCCTACAGCCCTGGAGGCCG CTGCTGACCAGCACTGACCCCGACCTCAGAGAG
TACTCGCAGGGGCGCTGGCTGCACTCAAGACCC
TCGAGATTAACGGTGCTAACCCCGTCTGCTCCTC
CCTCCCGCAGAGACTGGGGCCTGGACTGGACAT GAGAGCCCCTTGGTGCCACAGAGGGCTGTGTCT
TACTAGAAACAACGCAAACCTCTCCTTCCTCAGA
ATAGTGATGTGTTCGACGTTTTATCAAAGGCCCC
CTTTCTATGTTCATGTTAGTTTTGCTCCTTCTGTG
TTTTTTTCTGAACCATATCCATGTTGCTGACTTTT CCAAATAAAGGTTTTCACTCCTCTC
3UTR-013 Calr; AGAGGCCTGCCTCCAGGGCTGGACTGAGGCCTG 17 calreticulin
AGCGCTCCTGCCGCAGAGCTGGCCGCGCCAAAT
AATGTCTCTGTGAGACTCGAGAACTTTCATTTTT
TTCCAGGCTGGTTCGGATTTGGGGTGGATTTTGG
TTTTGTTCCCCTCCTCCACTCTCCCCCACCCCCTC
CCCGCCCTTTTTTTTTTTTTTTTTTAAACTGGTAT
TTTATCTTTGATTCTCCTTCAGCCCTCACCCCTGG
TTCTCATCTTTCTTGATCAACATCTTTTCTTGCCT
CTGTCCCCTTCTCTCATCTCTTAGCTCCCCTCCAA
CCTGGGGGGCAGTGGTGTGGAGAAGCCACAGGC
CTGAGATTTCATCTGCTCTCCTTCCTGGAGCCCA
GAGGAGGGCAGCAGAAGGGGGTGGTGTCTCCAA CCCCCCAGCACTGAGGAAGAACGGGGCTCTTCT
CATTTCACCCCTCCCTTTCTCCCCTGCCCCCAGG
ACTGGGCCACTTCTGGGTGGGGCAGTGGGTCCC AGATTGGCTCACACTGAGAATGTAAGAACTACA
AACAAAATTTCTATTAAATTAAATTTTGTGTCTCC 3UTR-014 Colla1;
CTCCCTCCATCCCAACCTGGCTCCCTCCCACCCA 18 collagen,
ACCAACTTTCCCCCCAACCCGGAAACAGACAAG type I, alpha 1
CAACCCAAACTGAACCCCCTCAAAAGCCAAAAA ATGGGAGACAATTTCACATGGACTTTGGAAAAT
ATTTTTTTCCTTTGCATTCATCTCTCAAACTTAGT
TTTTATCTTTGACCAACCGAACATGACCAAAAAC
CAAAAGTGCATTCAACCTTACCAAAAAAAAAAA AAAAAAAAGAATAAATAAATAACTTTTTAAAAA
AGGAAGCTTGGTCCACTTGCTTGAAGACCCATG
CGGGGGTAAGTCCCTTTCTGCCCGTTGGGCTTAT
GAAACCCCAATGCTGCCCTTTCTGCTCCTTTCTC
CACACCCCCCTTGGGGCCTCCCCTCCACTCCTTC
CCAAATCTGTCTCCCCAGAAGACACAGGAAACA
ATGTATTGTCTGCCCAGCAATCAAAGGCAATGCT
CAAACACCCAAGTGGCCCCCACCCTCAGCCCGC TCCTGCCCGCCCAGCACCCCCAGGCCCTGGGGG
ACCTGGGGTTCTCAGACTGCCAAAGAAGCCTTG
CCATCTGGCGCTCCCATGGCTCTTGCAACATCTC
CCCTTCGTTTTTGAGGGGGTCATGCCGGGGGAGC
CACCAGCCCCTCACTGGGTTCGGAGGAGAGTCA GGAAGGGCCACGACAAAGCAGAAACATCGGATT
TGGGGAACGCGTGTCAATCCCTTGTGCCGCAGG
GCTGGGCGGGAGAGACTGTTCTGTTCCTTGTGTA
ACTGTGTTGCTGAAAGACTACCTCGTTCTTGTCT
TGATGTGTCACCGGGGCAACTGCCTGGGGGCGG GGATGGGGGCAGGGTGGAAGCGGCTCCCCATTT
TATACCAAAGGTGCTACATCTATGTGATGGGTG GGGTGGGGAGGGAATCACTGGTGCTATAGAAAT
TGAGATGCCCCCCCAGGCCAGCAAATGTTCCTTT
TTGTTCAAAGTCTATTTTTATTCCTTGATATTTTT
CTTTTTTTTTTTTTTTTTTTGTGGATGGGGACTTG
TGAATTTTTCTAAAGGTGCTATTTAACATGGGAG
GAGAGCGTGTGCGGCTCCAGCCCAGCCCGCTGC
TCACTTTCCACCCTCTCTCCACCTGCCTCTGGCTT
CTCAGGCCTCTGCTCTCCGACCTCTCTCCTCTGA
AACCCTCCTCCACAGCTGCAGCCCATCCTCCCGG
CTCCCTCCTAGTCTGTCCTGCGTCCTCTGTCCCCG
GGTTTCAGAGACAACTTCCCAAAGCACAAAGCA GTTTTTCCCCCTAGGGGTGGGAGGAAGCAAAAG
ACTCTGTACCTATTTTGTATGTGTATAATAATTT
GAGATGTTTTTAATTATTTTGATTGCTGGAATAA
AGCATGTGGAAATGACCCAAACATAATCCGCAG
TGGCCTCCTAATTTCCTTCTTTGGAGTTGGGGGA
GGGGTAGACATGGGGAAGGGGCTTTGGGGTGAT
GGGCTTGCCTTCCATTCCTGCCCTTTCCCTCCCCA
CTATTCTCTTCTAGATCCCTCCATAACCCCACTC
CCCTTTCTCTCACCCTTCTTATACCGCAAACCTTT
CTACTTCCTCTTTCATTTTCTATTCTTGCAATTTC
CTTGCACCTTTTCCAAATCCTCTTCTCCCCTGCAA
TACCATACAGGCAATCCACGTGCACAACACACA
CACACACTCTTCACATCTGGGGTTGTCCAAACCT
CATACCCACTCCCCTTCAAGCCCATCCACTCTCC
ACCCCCTGGATGCCCTGCACTTGGTGGCGGTGG GATGCTCATGGATACTGGGAGGGTGAGGGGAGT
GGAACCCGTGAGGAGGACCTGGGGGCCTCTCCT
TGAACTGACATGAAGGGTCATCTGGCCTCTGCTC
CCTTCTCACCCACGCTGACCTCCTGCCGAAGGAG
CAACGCAACAGGAGAGGGGTCTGCTGAGCCTGG CGAGGGTCTGGGAGGGACCAGGAGGAAGGCGT
GCTCCCTGCTCGCTGTCCTGGCCCTGGGGGAGTG AGGGAGACAGACACCTGGGAGAGCTGTGGGGA
AGGCACTCGCACCGTGCTCTTGGGAAGGAAGGA
GACCTGGCCCTGCTCACCACGGACTGGGTGCCTC
GACCTCCTGAATCCCCAGAACACAACCCCCCTG GGCTGGGGTGGTCTGGGGAACCATCGTGCCCCC
GCCTCCCGCCTACTCCTTTTTAAGCTT 3UTR-015 Plod1;
TTGGCCAGGCCTGACCCTCTTGGACCTTTCTTCT 19 procollagen-
TTGCCGACAACCACTGCCCAGCAGCCTCTGGGA lysine, 2-
CCTCGGGGTCCCAGGGAACCCAGTCCAGCCTCC oxoglutarate
TGGCTGTTGACTTCCCATTGCTCTTGGAGCCACC 5-
AATCAAAGAGATTCAAAGAGATTCCTGCAGGCC dioxygenase 1
AGAGGCGGAACACACCTTTATGGCTGGGGCTCT CCGTGGTGTTCTGGACCCAGCCCCTGGAGACAC
CATTCACTTTTACTGCTTTGTAGTGACTCGTGCTC
TCCAACCTGTCTTCCTGAAAAACCAAGGCCCCCT
TCCCCCACCTCTTCCATGGGGTGAGACTTGAGCA
GAACAGGGGCTTCCCCAAGTTGCCCAGAAAGAC TGTCTGGGTGAGAAGCCATGGCCAGAGCTTCTC
CCAGGCACAGGTGTTGCACCAGGGACTTCTGCTT
CAAGTTTTGGGGTAAAGACACCTGGATCAGACT
CCAAGGGCTGCCCTGAGTCTGGGACTTCTGCCTC
CATGGCTGGTCATGAGAGCAAACCGTAGTCCCC TGGAGACAGCGACTCCAGAGAACCTCTTGGGAG
ACAGAAGAGGCATCTGTGCACAGCTCGATCTTC TACTTGCCTGTGGGGAGGGGAGTGACAGGTCCA
CACACCACACTGGGTCACCCTGTCCTGGATGCCT CTGAAGAGAGGGACAGACCGTCAGAAACTGGA
GAGTTTCTATTAAAGGTCATTTAAACCA 3UTR-016 Nucb1;
TCCTCCGGGACCCCAGCCCTCAGGATTCCTGATG 20 nucleobindin 1
CTCCAAGGCGACTGATGGGCGCTGGATGAAGTG
GCACAGTCAGCTTCCCTGGGGGCTGGTGTCATGT
TGGGCTCCTGGGGCGGGGGCACGGCCTGGCATT
TCACGCATTGCTGCCACCCCAGGTCCACCTGTCT
CCACTTTCACAGCCTCCAAGTCTGTGGCTCTTCC
CTTCTGTCCTCCGAGGGGCTTGCCTTCTCTCGTG
TCCAGTGAGGTGCTCAGTGATCGGCTTAACTTAG
AGAAGCCCGCCCCCTCCCCTTCTCCGTCTGTCCC
AAGAGGGTCTGCTCTGAGCCTGCGTTCCTAGGTG
GCTCGGCCTCAGCTGCCTGGGTTGTGGCCGCCCT
AGCATCCTGTATGCCCACAGCTACTGGAATCCCC
GCTGCTGCTCCGGGCCAAGCTTCTGGTTGATTAA
TGAGGGCATGGGGTGGTCCCTCAAGACCTTCCC
CTACCTTTTGTGGAACCAGTGATGCCTCAAAGAC
AGTGTCCCCTCCACAGCTGGGTGCCAGGGGCAG GGGATCCTCAGTATAGCCGGTGAACCCTGATAC
CAGGAGCCTGGGCCTCCCTGAACCCCTGGCTTCC
AGCCATCTCATCGCCAGCCTCCTCCTGGACCTCT
TGGCCCCCAGCCCCTTCCCCACACAGCCCCAGA AGGGTCCCAGAGCTGACCCCACTCCAGGACCTA
GGCCCAGCCCCTCAGCCTCATCTGGAGCCCCTGA
AGACCAGTCCCACCCACCTTTCTGGCCTCATCTG
ACACTGCTCCGCATCCTGCTGTGTGTCCTGTTCC
ATGTTCCGGTTCCATCCAAATACACTTTCTGGAA CAAA 3UTR-017 .alpha.-globin
GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTT 21
GGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCA
CCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAG TGGGCGGC
[0260] It should be understood that those listed in the previous
tables are examples and that any UTR from any gene may be
incorporated into the respective first or second flanking region of
the primary construct. Furthermore, multiple wild-type UTRs of any
known gene may be utilized. It is also within the scope of the
present invention to provide artificial UTRs which are not variants
of wild type genes. These UTRs or portions thereof may be placed in
the same orientation as in the transcript from which they were
selected or may be altered in orientation or location. Hence a 5'
or 3' UTR may be inverted, shortened, lengthened, made chimeric
with one or more other 5' UTRs or 3' UTRs. As used herein, the term
"altered" as it relates to a UTR sequence, means that the UTR has
been changed in some way in relation to a reference sequence. For
example, a 3' or 5' UTR may be altered relative to a wild type or
native UTR by the change in orientation or location as taught above
or may be altered by the inclusion of additional nucleotides,
deletion of nucleotides, swapping or transposition of nucleotides.
Any of these changes producing an "altered" UTR (whether 3' or 5')
comprise a variant UTR.
[0261] In one embodiment, a double, triple or quadruple UTR such as
a 5' or 3' UTR may be used. As used herein, a "double" UTR is one
in which two copies of the same UTR are encoded either in series or
substantially in series. For example, a double beta-globin 3' UTR
may be used as described in US Patent publication 20100129877, the
contents of which are incorporated herein by reference in its
entirety.
[0262] It is also within the scope of the present invention to have
patterned UTRs. As used herein "patterned UTRs" are those UTRs
which reflect a repeating or alternating pattern, such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice,
or more than 3 times. In these patterns, each letter, A, B, or C
represent a different UTR at the nucleotide level.
[0263] In one embodiment, flanking regions are selected from a
family of transcripts whose proteins share a common function,
structure, feature of property. For example, oncology-related
polypeptides of interest may belong to a family of proteins which
are expressed in a particular cell, tissue or at some time during
development. The UTRs from any of these genes may be swapped for
any other UTR of the same or different family of proteins to create
a new chimeric primary transcript. As used herein, a "family of
proteins" is used in the broadest sense to refer to a group of two
or more oncology-related polypeptides of interest which share at
least one function, structure, feature, localization, origin, or
expression pattern.
[0264] After optimization (if desired), the signal-sensor primary
construct components are reconstituted and transformed into a
vector such as, but not limited to, plasmids, viruses, cosmids, and
artificial chromosomes. For example, the optimized construct may be
reconstituted and transformed into chemically competent E. coli,
yeast, neurospora, maize, drosophila, etc. where high copy
plasmid-like or chromosome structures occur by methods described
herein.
Stop Codons
[0265] In one embodiment, the signal-sensor primary constructs of
the present invention may include at least two stop codons before
the 3' untranslated region (UTR). The stop codon may be selected
from TGA, TAA and TAG. In one embodiment, the signal-sensor primary
constructs of the present invention include the stop codon TGA and
one additional stop codon. In a further embodiment the addition
stop codon may be TAA.
Vector Amplification
[0266] The vector containing the signal-sensor primary construct is
then amplified and the plasmid isolated and purified using methods
known in the art such as, but not limited to, a maxi prep using the
Invitrogen PURELINK.TM. HiPure Maxiprep Kit (Carlsbad, Calif.).
Plasmid Linearization
[0267] The plasmid may then be linearized using methods known in
the art such as, but not limited to, the use of restriction enzymes
and buffers. The linearization reaction may be purified using
methods including, for example Invitrogen's PURELINK.TM. PCR Micro
Kit (Carlsbad, Calif.), and HPLC based purification methods such
as, but not limited to, strong anion exchange HPLC, weak anion
exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic
interaction HPLC (HIC-HPLC) and Invitrogen's standard PURELINK.TM.
PCR Kit (Carlsbad, Calif.). The purification method may be modified
depending on the size of the linearization reaction which was
conducted. The linearized plasmid is then used to generate cDNA for
in vitro transcription (IVT) reactions.
cDNA Template Synthesis
[0268] A cDNA template may be synthesized by having a linearized
plasmid undergo polymerase chain reaction (PCR). Table 4 is a
listing of primers and probes that may be useful in the PCR
reactions of the present invention. It should be understood that
the listing is not exhaustive and that primer-probe design for any
amplification is within the skill of those in the art. Probes may
also contain chemically modified bases to increase base-pairing
fidelity to the target molecule and base-pairing strength. Such
modifications may include 5-methyl-Cytidine, 2, 6-di-amino-purine,
2'-fluoro, phosphoro-thioate, or locked nucleic acids.
TABLE-US-00004 TABLE 4 Primers and Probes Primer/ SEQ Probe
Hybridization ID Identifier Sequence (5'-3') target NO. UFP
TTGGACCCTCGTACAGAAGCTAA cDNA Template 22 TACG URP
T.sub.x160CTTCCTACTCAGGCTTTATTC cDNA Template 23 AAAGACCA GBA1
CCTTGACCTTCTGGAACTTC Acid 24 glucocerebrosidase GBA2
CCAAGCACTGAAACGGATAT Acid 25 glucocerebrosidase LUC1
GATGAAAAGTGCTCCAAGGA Luciferase 26 LUC2 AACCGTGATGAAAAGGTACC
Luciferase 27 LUC3 TCATGCAGATTGGAAAGGTC Luciferase 28 GCSF1
CTTCTTGGACTGTCCAGAGG G-CSF 29 GCSF2 GCAGTCCCTGATACAAGAAC G-CSF 30
GCSF3 GATTGAAGGTGGCTCGCTAC G-CSF 31 *UFP is universal forward
primer; URP is universal reverse primer.
[0269] In one embodiment, the cDNA may be submitted for sequencing
analysis before undergoing transcription.
Signal-Sensor Polynucleotide Production (Signal-Sensor mRNA)
[0270] The process of signal-sensor polynucleotide production may
include, but is not limited to, in vitro transcription, cDNA
template removal and RNA clean-up, and capping and/or tailing
reactions.
In Vitro Transcription
[0271] The cDNA produced in the previous step may be transcribed
using an in vitro transcription (IVT) system. The system typically
comprises a transcription buffer, nucleotide triphosphates (NTPs),
an RNase inhibitor and a polymerase. The NTPs may be manufactured
in house, may be selected from a supplier, or may be synthesized as
described herein. The NTPs may be selected from, but are not
limited to, those described herein including natural and unnatural
(modified) NTPs. The polymerase may be selected from, but is not
limited to, T7 RNA polymerase, T3 RNA polymerase and mutant
polymerases such as, but not limited to, polymerases able to be
incorporated into modified nucleic acids.
RNA Polymerases
[0272] Any number of RNA polymerases or variants may be used in the
design of the signal-sensor primary constructs of the present
invention.
[0273] RNA polymerases may be modified by inserting or deleting
amino acids of the RNA polymerase sequence. As a non-limiting
example, the RNA polymerase may be modified to exhibit an increased
ability to incorporate a 2'-modified nucleotide triphosphate
compared to an unmodified RNA polymerase (see International
Publication WO2008078180 and U.S. Pat. No. 8,101,385; herein
incorporated by reference in their entireties).
[0274] Variants may be obtained by evolving an RNA polymerase,
optimizing the RNA polymerase amino acid and/or nucleic acid
sequence and/or by using other methods known in the art. As a
non-limiting example, T7 RNA polymerase variants may be evolved
using the continuous directed evolution system set out by Esvelt et
al. (Nature (2011) 472(7344):499-503; herein incorporated by
reference in its entirety) where clones of T7 RNA polymerase may
encode at least one mutation such as, but not limited to, lysine at
position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D,
A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H,
F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D,
M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C,
D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523L,
H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E,
N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limiting
example, T7 RNA polymerase variants may encode at least mutation as
described in U.S. Pub. Nos. 20100120024 and 20070117112; herein
incorporated by reference in their entireties. Variants of RNA
polymerase may also include, but are not limited to, substitutional
variants, conservative amino acid substitution, insertional
variants, deletional variants and/or covalent derivatives.
[0275] In one embodiment, the signal-sensor primary construct may
be designed to be recognized by the wild type or variant RNA
polymerases. In doing so, the signal-sensor primary construct may
be modified to contain sites or regions of sequence changes from
the wild type or parent primary construct.
[0276] In one embodiment, the signal-sensor primary construct may
be designed to include at least one substitution and/or insertion
upstream of an RNA polymerase binding or recognition site,
downstream of the RNA polymerase binding or recognition site,
upstream of the TATA box sequence, downstream of the TATA box
sequence of the signal-sensor primary construct but upstream of the
coding region of the primary construct, within the 5'UTR, before
the 5'UTR and/or after the 5'UTR.
[0277] In one embodiment, the 5'UTR of the signal-sensor primary
construct may be replaced by the insertion of at least one region
and/or string of nucleotides of the same base. The region and/or
string of nucleotides may include, but is not limited to, at least
3, at least 4, at least 5, at least 6, at least 7 or at least 8
nucleotides and the nucleotides may be natural and/or unnatural. As
a non-limiting example, the group of nucleotides may include 5-8
adenine, cytosine, thymine, a string of any of the other
nucleotides disclosed herein and/or combinations thereof
[0278] In one embodiment, the 5'UTR of the signal-sensor primary
construct may be replaced by the insertion of at least two regions
and/or strings of nucleotides of two different bases such as, but
not limited to, adenine, cytosine, thymine, any of the other
nucleotides disclosed herein and/or combinations thereof. For
example, the 5'UTR may be replaced by inserting 5-8 adenine bases
followed by the insertion of 5-8 cytosine bases. In another
example, the 5'UTR may be replaced by inserting 5-8 cytosine bases
followed by the insertion of 5-8 adenine bases.
[0279] In one embodiment, the signal-sensor primary construct may
include at least one substitution and/or insertion downstream of
the transcription start site which may be recognized by an RNA
polymerase. As a non-limiting example, at least one substitution
and/or insertion may occur downstream the transcription start site
by substituting at least one nucleic acid in the region just
downstream of the transcription start site (such as, but not
limited to, +1 to +6). Changes to region of nucleotides just
downstream of the transcription start site may affect initiation
rates, increase apparent nucleotide triphosphate (NTP) reaction
constant values, and increase the dissociation of short transcripts
from the transcription complex curing initial transcription (Brieba
et al, Biochemistry (2002) 41: 5144-5149; herein incorporated by
reference in its entirety). The modification, substitution and/or
insertion of at least one nucleic acid may cause a silent mutation
of the nucleic acid sequence or may cause a mutation in the amino
acid sequence.
[0280] In one embodiment, the signal-sensor primary construct may
include the substitution of at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 11, at least 12 or at least 13 guanine
bases downstream of the transcription start site.
[0281] In one embodiment, the signal-sensor primary construct may
include the substitution of at least 1, at least 2, at least 3, at
least 4, at least 5 or at least 6 guanine bases in the region just
downstream of the transcription start site. As a non-limiting
example, if the nucleotides in the region are GGGAGA the guanine
bases may be substituted by at least 1, at least 2, at least 3 or
at least 4 adenine nucleotides. In another non-limiting example, if
the nucleotides in the region are GGGAGA the guanine bases may be
substituted by at least 1, at least 2, at least 3 or at least 4
cytosine bases. In another non-limiting example, if the nucleotides
in the region are GGGAGA the guanine bases may be substituted by at
least 1, at least 2, at least 3 or at least 4 thymine, and/or any
of the nucleotides described herein.
[0282] In one embodiment, the signal-sensor primary construct may
include at least one substitution and/or insertion upstream of the
start codon. For the purpose of clarity, one of skill in the art
would appreciate that the start codon is the first codon of the
protein coding region whereas the transcription start site is the
site where transcription begins. The signal-sensor primary
construct may include, but is not limited to, at least 1, at least
2, at least 3, at least 4, at least 5, at least 6, at least 7 or at
least 8 substitutions and/or insertions of nucleotide bases. The
nucleotide bases may be inserted or substituted at 1, at least 1,
at least 2, at least 3, at least 4 or at least 5 locations upstream
of the start codon. The nucleotides inserted and/or substituted may
be the same base (e.g., all A or all C or all T or all G), two
different bases (e.g., A and C, A and T, or C and T), three
different bases (e.g., A, C and T or A, C and T) or at least four
different bases. As a non-limiting example, the guanine base
upstream of the coding region in the signal-sensor primary
construct may be substituted with adenine, cytosine, thymine, or
any of the nucleotides described herein. In another non-limiting
example the substitution of guanine bases in the signal-sensor
primary construct may be designed so as to leave one guanine base
in the region downstream of the transcription start site and before
the start codon (see Esvelt et al. Nature (2011) 472(7344):499-503;
herein incorporated by reference in its entirety). As a
non-limiting example, at least 5 nucleotides may be inserted at 1
location downstream of the transcription start site but upstream of
the start codon and the at least 5 nucleotides may be the same base
type.
cDNA Template Removal and Clean-Up
[0283] The cDNA template may be removed using methods known in the
art such as, but not limited to, treatment with Deoxyribonuclease I
(DNase I). RNA clean-up may also include a purification method such
as, but not limited to, AGENCOURT.RTM. CLEANSEQ.RTM. system from
Beckman Coulter (Danvers, Mass.), HPLC based purification methods
such as, but not limited to, strong anion exchange HPLC, weak anion
exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic
interaction HPLC (HIC-HPLC).
Capping and/or Tailing Reactions
[0284] The signal-sensor primary construct or mmRNA may also
undergo capping and/or tailing reactions. A capping reaction may be
performed by methods known in the art to add a 5' cap to the 5' end
of the signal-sensor primary construct. Methods for capping
include, but are not limited to, using a Vaccinia Capping enzyme
(New England Biolabs, Ipswich, Mass.).
[0285] A poly-A tailing reaction may be performed by methods known
in the art, such as, but not limited to, 2' O-methyltransferase and
by methods as described herein. If the signal-sensor primary
construct generated from cDNA does not include a poly-T, it may be
beneficial to perform the poly-A-tailing reaction before the
signal-sensor primary construct is cleaned.
Purification
[0286] Signal-sensor primary construct or mmRNA purification may
include, but is not limited to, mRNA or mmRNA clean-up, quality
assurance and quality control. mRNA or mmRNA clean-up may be
performed by methods known in the arts such as, but not limited to,
AGENCOURT.RTM. beads (Beckman Coulter Genomics, Danvers, Mass.),
poly-T beads, LNA.TM. oligo-T capture probes (EXIQON.RTM. Inc,
Vedbaek, Denmark) or HPLC based purification methods such as, but
not limited to, strong anion exchange HPLC, weak anion exchange
HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction
HPLC (HIC-HPLC). The term "purified" when used in relation to a
polynucleotide such as a "purified mRNA or signal-sensor mmRNA"
refers to one that is separated from at least one contaminant. As
used herein, a "contaminant" is any substance which makes another
unfit, impure or inferior. Thus, a purified signal-sensor
polynucleotide (e.g., DNA and RNA) is present in a form or setting
different from that in which it is found in nature, or a form or
setting different from that which existed prior to subjecting it to
a treatment or purification method.
[0287] A quality assurance and/or quality control check may be
conducted using methods such as, but not limited to, gel
electrophoresis, UV absorbance, or analytical HPLC.
[0288] In another embodiment, the signal-sensor mRNA or mmRNA may
be sequenced by methods including, but not limited to
reverse-transcriptase-PCR.
[0289] In one embodiment, the signal-sensor mRNA or mmRNA may be
quantified using methods such as, but not limited to, ultraviolet
visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis
spectrometer is a NANODROP.RTM. spectrometer (ThermoFisher,
Waltham, Mass.). The quantified signal-sensor mRNA or mmRNA may be
analyzed in order to determine if the signal-sensor mRNA or mmRNA
may be of proper size, check that no degradation of the
signal-sensor mRNA or mmRNA has occurred. Degradation of the
signal-sensor mRNA and/or mmRNA may be checked by methods such as,
but not limited to, agarose gel electrophoresis, HPLC based
purification methods such as, but not limited to, strong anion
exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid
chromatography-mass spectrometry (LCMS), capillary electrophoresis
(CE) and capillary gel electrophoresis (CGE).
Signal Peptides or Proteins
[0290] The signal-sensor primary constructs or mmRNA may also
encode additional features which facilitate trafficking of the
polypeptides to therapeutically relevant sites. One such feature
which aids in protein trafficking is the signal peptide sequence.
As used herein, a "signal sequence" or "signal peptide" is a
polynucleotide or polypeptide, respectively, which is from about 9
to 200 nucleotides (3-60 amino acids) in length which is
incorporated at the 5' (or N-terminus) of the coding region or
polypeptide encoded, respectively. Addition of these sequences
result in trafficking of the encoded oncology-related polypeptide
to the endoplasmic reticulum through one or more secretory
pathways. Some signal peptides are cleaved from the protein by
signal peptidase after the proteins are transported.
[0291] Table 5 is a representative listing of signal proteins or
peptides which may be incorporated for encoding by the
signal-sensor polynucleotides, primary constructs or mmRNA of the
invention.
TABLE-US-00005 TABLE 5 Signal Peptides SEQ SEQ NUCLEOTIDE SEQUENCE
ID ENCODED ID ID Description (5'-3') NO. PEPTIDE NO. SS-001
.alpha.-1- ATGATGCCATCCTCAGTCTCA 32 MMPSSVS 94 antitrypsin
TGGGGTATTTTGCTCTTGGCG WGILLAGL GGTCTGTGCTGTCTCGTGCCG CCLVPVSLA
GTGTCGCTCGCA SS-002 G-CSF ATGGCCGGACCGGCGACTCAG 33 MAGPATQ 95
TCGCCCATGAAACTCATGGCC SPMKLMA CTGCAGTTGTTGCTTTGGCAC LQLLLWH
TCAGCCCTCTGGACCGTCCAA SALWTVQ GAGGCG EA SS-003 Factor IX
ATGCAGAGAGTGAACATGATT 34 MQRVNMI 96 ATGGCCGAGTCCCCATCGCTC MAESPSLI
ATCACAATCTGCCTGCTTGGT TICLLGYL ACCTGCTTTCCGCCGAATGCA LSAECTVF
CTGTCTTTCTGGATCACGAGA LDHENAN ATGCGAATAAGATCTTGAACC KILNRPKR
GACCCAAACGG SS-004 Prolactin ATGAAAGGATCATTGCTGTTG 35 MKGSLLL 97
CTCCTCGTGTCGAACCTTCTG LLVSNLLL CTTTGCCAGTCCGTAGCCCCC CQSVAP SS-005
Albumin ATGAAATGGGTGACGTTCATC 36 MKWVTFI 98 TCACTGTTGTTTTTGTTCTCGT
SLLFLFSS CCGCCTACTCCAGGGGAGTAT AYSRG TCCGCCGA VFRR SS-006 HMMSP38
ATGTGGTGGCGGCTCTGGTGG 37 MWWRLW 99 CTGCTCCTGTTGCTCCTCTTGC WLLLLLLL
TGTGGCCCATGGTGTGGGCA LPMWA MLS- ornithine TGCTCTTTAACCTCCGCATCCT 38
MLFNLRIL 100 001 carbamoyltransferase GTTGAATAACGCTGCGTTCCG LNNAAFR
AAATGGGCATAACTTCATGGT NGHNFMV ACGCAACTTCAGATGCGGCCA RNFRCGQP
GCCACTCCAG LQ MLS- Cytochrome ATGTCCGTCTTGACACCCCTG 39 MSVLTPLL 101
002 C Oxidase CTCTTGAGAGGGCTGACGGGG LRGLTGSA subunit 8A
TCCGCTAGACGCCTGCCGGTA RRLPVPRA CCGCGAGCGAAGATCCACTCC KIHSL CTG MLS-
Cytochrome ATGAGCGTGCTCACTCCGTTG 40 MSVLTPLL 102 003 C Oxidase
CTTCTTCGAGGGCTTACGGGA LRGLTGSA subunit 8A TCGGCTCGGAGGTTGCCCGTC
RRLPVPRA CCGAGAGCGAAGATCCATTCG KIHSL TTG SS-007 Type III,
TGACAAAAATAACTTTATCTC 41 MVTKITLS 103 bacterial
CCCAGAATTTTAGAATCCAAA PQNFRIQK AACAGGAAACCACACTACTA QETTLLKE
AAAGAAAAATCAACCGAGAA KSTEKNSL AAATTCTTTAGCAAAAAGTAT AKSILAVK
TCTCGCAGTAAAAATCACTTC NHFIELRS ATCGAATTAAGGTCAAAATTA KLSERFIS
TCGGAACGTTTTATTTCGCAT HKNT AAGAACACT SS-008 Viral
ATGCTGAGCTTTGTGGATACC 42 MLSFVDT 104 CGCACCCTGCTGCTGCTGGCG RTLLLLAV
GTGACCAGCTGCCTGGCGACC TSCLATCQ TGCCAG SS-009 viral
ATGGGCAGCAGCCAGGCGCC 43 MGSSQAP 105 GCGCATGGGCAGCGTGGGCG RMGSVGG
GCCATGGCCTGATGGCGCTGC HGLMALL TGATGGCGGGCCTGATTCTGC MAGLILPG
CGGGCATTCTGGCG ILA SS-010 Viral ATGGCGGGCATTTTTTATTTTC 44 MAGIFYFL
106 TGTTTAGCTTTCTGTTTGGCAT FSFLFGICD TTGCGAT SS-011 Viral
ATGGAAAACCGCCTGCTGCGC 45 MENRLLR 107 GTGTTTCTGGTGTGGGCGGCG VFLVWAA
CTGACCATGGATGGCGCGAGC LTMDGASA GCG SS-012 Viral
ATGGCGCGCCAGGGCTGCTTT 46 MARQGCF 108 GGCAGCTATCAGGTGATTAGC GSYQVISL
CTGTTTACCTTTGCGATTGGC FTFAIGVN GTGAACCTGTGCCTGGGC LCLG SS-013
Bacillus ATGAGCCGCCTGCCGGTGCTG 47 MSRLPVLL 109
CTGCTGCTGCAGCTGCTGGTG LLQLLVRP CGCCCGGGCCTGCAG GLQ SS-014 Bacillus
ATGAAACAGCAGAAACGCCT 48 MKQQKRL 110 GTATGCGCGCCTGCTGACCCT YARLLTLL
GCTGTTTGCGCTGATTTTTCTG FALIFLLPH CTGCCGCATAGCAGCGCGAGC SSASA GCG
SS-015 Secretion ATGGCGACGCCGCTGCCTCCG 49 MATPLPPP 111 signal
CCCTCCCCGCGGCACCTGCGG SPRHLRLL CTGCTGCGGCTGCTGCTCTCC RLLLSG
GCCCTCGTCCTCGGC SS-016 Secretion ATGAAGGCTCCGGGTCGGCTC 50 MKAPGRL
112 signal GTGCTCATCATCCTGTGCTCC VLIILCSVV GTGGTCTTCTCT FS SS-017
Secretion ATGCTTCAGCTTTGGAAACTT 51 MLQLWKL 113 signal
GTTCTCCTGTGCGGCGTGCTC LCGVLT ACT SS-018 Secretion
ATGCTTTATCTCCAGGGTTGG 52 MLYLQGW 114 signal AGCATGCCTGCTGTGGCA
SMPAVA SS-019 Secretion ATGGATAACGTGCAGCCGAA 53 MDNVQPK 115 signal
AATAAAACATCGCCCCTTCTG IKHRPFCF CTTCAGTGTGAAAGGCCACGT SVKGHVK
GAAGATGCTGCGGCTGGATAT MLRLDIIN TATCAACTCACTGGTAACAAC SLVTTVFM
AGTATTCATGCTCATCGTATC LIVSVLALIP TGTGTTGGCACTGATACCA SS-020
Secretion ATGCCCTGCCTAGACCAACAG 54 MPCLDQQ 116 signal
CTCACTGTTCATGCCCTACCCT LTVHALPC GCCCTGCCCAGCCCTCCTCTC PAQPSSLA
TGGCCTTCTGCCAAGTGGGGT FCQVGFLTA TCTTAACAGCA SS-021 Secretion
ATGAAAACCTTGTTCAATCCA 55 MKTLFNP 117 signal GCCCCTGCCATTGCTGACCTG
APAIADLD GATCCCCAGTTCTACACCCTC PQFYTLSD TCAGATGTGTTCTGCTGCAAT
VFCCNESE GAAAGTGAGGCTGAGATTTTA AEILTGLT ACTGGCCTCACGGTGGGCAGC
VGSAADA GCTGCAGATGCT SS-022 Secretion ATGAAGCCTCTCCTTGTTGTG 56
MKPLLVV 118 signal TTTGTCTTTCTTTTCCTTTGGG FVFLFLWD ATCCAGTGCTGGCA
PVLA SS-023 Secretion ATGTCCTGTTCCCTAAAGTTT 57 MSCSLKFT 119 signal
ACTTTGATTGTAATTTTTTTTT LIVIFFTCT ACTGTTGGCTTTCATCCAGC LSSS SS-024
Secretion ATGGTTCTTACTAAACCTCTTC 58 MVLTKPL 120 signal
AAAGAAATGGCAGCATGATG QRNGSMM AGCTTTGAAAATGTGAAAGAA SFENVKEK
AAGAGCAGAGAAGGAGGGCC SREGGPHA CCATGCACACACACCCGAAGA HTPEEELC
AGAATTGTGTTTCGTGGTAAC FVVTHTPQ ACACTACCCTCAGGTTCAGAC VQTTLNLF
CACACTCAACCTGTTTTTCCAT FHIFKVLT ATATTCAAGGTTCTTACTCAA QPLSLLWG
CCACTTTCCCTTCTGTGGGGT SS-025 Secretion ATGGCCACCCCGCCATTCCGG 59
MATPPFRL 121 signal CTGATAAGGAAGATGTTTTCC IRKMFSFK
TTCAAGGTGAGCAGATGGATG VSRWMGL GGGCTTGCCTGCTTCCGGTCC ACFRSLAAS
CTGGCGGCATCC SS-026 Secretion ATGAGCTTTTTCCAACTCCTG 60 MSFFQLL 122
signal ATGAAAAGGAAGGAACTCAT MKRKELIP TCCCTTGGTGGTGTTCATGAC LVVFMTV
TGTGGCGGCGGGTGGAGCCTC AAGGASS ATCT SS-027 Secretion
ATGGTCTCAGCTCTGCGGGGA 61 MVSALRG 123 signal GCACCCCTGATCAGGGTGCAC
APLIRVHS TCAAGCCCTGTTTCTTCTCCTT SPVSSPSV CTGTGAGTGGACCACGGAGGC
SGPAALVS TGGTGAGCTGCCTGTCATCCC CLSSQSSA AAAGCTCAGCTCTGAGC LS SS-028
Secretion ATGATGGGGTCCCCAGTGAGT 62 MMGSPVS 124 signal
CATCTGCTGGCCGGCTTCTGT HLLAGFC GTGTGGGTCGTCTTGGGC VWVVLG SS-029
Secretion ATGGCAAGCATGGCTGCCGTG 63 MASMAAV 125 signal
CTCACCTGGGCTCTGGCTCTT LTWALAL CTTTCAGCGTTTTCGGCCACC LSAFSATQA
CAGGCA SS-030 Secretion ATGGTGCTCATGTGGACCAGT 64 MVLMWTS 126 signal
GGTGACGCCTTCAAGACGGCC GDAFKTA TACTTCCTGCTGAAGGGTGCC YFLLKGAP
CCTCTGCAGTTCTCCGTGTGC LQFSVCGL GGCCTGCTGCAGGTGCTGGTG LQVLVDL
GACCTGGCCATCCTGGGGCAG AILGQATA GCCTACGCC SS-031 Secretion
ATGGATTTTGTCGCTGGAGCC 65 MDFVAGA 127 signal ATCGGAGGCGTCTGCGGTGTT
IGGVCGV GCTGTGGGCTACCCCCTGGAC AVGYPLD ACGGTGAAGGTCAGGATCCA TVKVRIQT
GACGGAGCCAAAGTACACAG EPLYTGIW GCATCTGGCACTGCGTCCGGG HCVRDTY
ATACGTATCACCGAGAGCGCG HRERVWG TGTGGG FYRGLSLP GCTTCTACCGGGGCCTCTCGC
VCTVSLVSS TGCCCGTGTGCACGGTGTCCC TGGTATCTTCC SS-032 Secretion
ATGGAGAAGCCCCTCTTCCCA 66 MEKPLFPL 128 signal TTAGTGCCTTTGCATTGGTTTG
VPLHWFG GCTTTGGCTACACAGCACTGG FGYTALV TTGTTTCTGGTGGGATCGTTG
VSGGIVGY GCTATGTAAAAACAGGCAGC VKTGSVPS GTGCCGTCCCTGGCTGCAGGG
LAAGLLFG CTGCTCTTCGGCAGTCTAGCC SLA SS-033 Secretion
ATGGGTCTGCTCCTTCCCCTG 67 MGLLLPL 129 signal GCACTCTGCATCCTAGTCCTG
ALCILVLC TGC SS-034 Secretion ATGGGGATCCAGACGAGCCCC 68 MGIQTSPV 130
signal GTCCTGCTGGCCTCCCTGGGG LLASLGVG GTGGGGCTGGTCACTCTGCTC
LVTLLGLA GGCCTGGCTGTGGGC VG SS-035 Secretion ATGTCGGACCTGCTACTACTG
69 MSDLLLL 131 signal GGCCTGATTGGGGGCCTGACT GLIGGLTL
CTCTTACTGCTGCTGACGCTG LLLLTLLA CTAGCCTTTGCC FA SS-036 Secretion
ATGGAGACTGTGGTGATTGTT 70 METVVIV 132 signal GCCATAGGTGTGCTGGCCACC
AIGVLATI ATGTTTCTGGCTTCGTTTGCAG FLASFAAL CCTTGGTGCTGGTTTGCAGGC
VLVCRQ AG SS-037 Secretion ATGCGCGGCTCTGTGGAGTGC 71 MAGSVEC 133
signal ACCTGGGGTTGGGGGCACTGT TWGWGH GCCCCCAGCCCCCTGCTCCTT CAPSPLLL
TGGACTCTACTTCTGTTTGCA WTLLLFA GCCCCATTTGGCCTGCTGGGG APFGLLG SS-038
Secretion ATGATGCCGTCCCGTACCAAC 72 MMPSRTN 134 signal
CTGGCTACTGGAATCCCCAGT LATGIPSS AGTAAAGTGAAATATTCAAGG KVKYSRLS
CTCTCCAGCACAGACGATGGC STDDGYID TACATTGACCTTCAGTTTAAG LQFKKTPP
AAAACCCCTCCTAAGATCCCT KIPYKAIA TATAAGGCCATCGCACTTGCC LATVLFLI
ACTGTGCTGTTTTTGATTGGC GA GCC
SS-039 Secretion ATGGCCCTGCCCCAGATGTGT 73 MALPQMC 135 signal
GACGGGAGCCACTTGGCCTCC DGSHLAST ACCCTCCGCTATTGCATGACA LRYCMTV
GTCAGCGGCACAGTGGTTCTG SGTVVLV GTGGCCGGGACGCTCTGCTTC AGTLCFA GCT
SS-041 Vrg-6 TGAAAAAGTGGTTCGTTGCTG 74 MKKWFVA 136
CCGGCATCGGCGCTGCCGGAC AGIGAGLL TCATGCTCTCCAGCGCCGCCA MLSSAA SS-042
PhoA ATGAAACAGAGCACCATTGCG 75 MKQSTIAL 137 CTGGCGCTGCTGCCGCTGCTG
ALLPLLFT TTTACCCCGGTGACCAAAGCG PVTKA SS-043 OmpA
ATGAAAAAAACCGCGATTGC 76 MKKTAIAI 138 GATTGCGGTGGCGCTGGCGGG AVALAGF
CTTTGCGACCGTGGCGCAGGCG ATVAQA SS-044 STI ATGAAAAAACTGATGCTGGCG 77
MKKLMLA 139 ATTTTTTTTAGCGTGCTGAGCT IFFSVLSFP TTCCGAGCTTTAGCCAGAGC
SFSQS SS-045 STII ATGAAAAAAAACATTGCGTTT 78 MKKNIAFL 140
CTGCTGGCGAGCATGTTTGTG LASMFVFS TTTAGCATTGCGACCAACGCG IATNAYA TATGCG
SS-046 Amylase ATGTTTGCGAAACGCTTTAAA 79 MFAKRFK 141
ACCAGCCTGCTGCCGCTGTTT TSLLPLFA GCGGGCTTTCTGCTGCTGTTTC GFLLLFHL
ATCTGGTGCTGGCGGGCCCGG VLAGPAA CGGCGGCGAGC AS SS-047 Alpha
ATGCGCTTTCCGAGCATTTTT 80 MRFPSIFT 142 Factor ACCGCGGTGCTGTTTGCGGCG
AVLFAASS AGCAGCGCGCTGGCG ALA SS-048 Alpha ATGCGCTTTCCGAGCATTTTT 81
MRFPSIFT 143 Factor ACCACCGTGCTGTTTGCGGCG TVLFAASS AGCAGCGCGCTGGCG
ALA SS-049 Alpha ATGCGCTTTCCGAGCATTTTT 82 MRFPSIFTS 144 Factor
ACCAGCGTGCTGTTTGCGGCG VLFAASSA AGCAGCGCGCTGGCG LA SS-050 Alpha
ATGCGCTTTCCGAGCATTTTT 83 MRFPSIFT 145 Factor ACCCATGTGCTGTTTGCGGCG
HVLFAASS AGCAGCGCGCTGGCG ALA SS-051 Alpha ATGCGCTTTCCGAGCATTTTT 84
MRFPSIFTI 146 Factor ACCATTGTGCTGTTTGCGGCG VLFAASSA AGCAGCGCGCTGGCG
LA SS-052 Alpha ATGCGCTTTCCGAGCATTTTT 85 MRFPSIFTF 147 Factor
ACCTTTGTGCTGTTTGCGGCG VLFAASSA AGCAGCGCGCTGGCG LA SS-053 Alpha
ATGCGCTTTCCGAGCATTTTT 86 MRFPSIFT 148 Factor ACCGAAGTGCTGTTTGCGGCG
EVLFAASS AGCAGCGCGCTGGCG ALA SS-054 Alpha ATGCGCTTTCCGAGCATTTTT 87
MRFPSIFT 149 Factor ACCGGCGTGCTGTTTGCGGCG GVLFAASS AGCAGCGCGCTGGCG
ALA SS-055 Endoglucanase V ATGCGTTCCTCCCCCCTCCTCC 88 MRSSPLLR 150
GCTCCGCCGTTGTGGCCGCCC SAVVAAL TGCCGGTGTTGGCCCTTGCC PVLALA SS-056
Secretion ATGGGCGCGGCGGCCGTGCGC 89 MGAAAVR 151 signal
TGGCACTTGTGCGTGCTGCTG WHLCVLL GCCCTGGGCACACGCGGGCG ALGTRGRL GCTG
SS-057 Fungal ATGAGGAGCTCCCTTGTGCTG 90 MRSSLVLF 152
TTCTTTGTCTCTGCGTGGACG FVSAWTA GCCTTGGCCAG LA SS-058 Fibronectin
ATGCTCAGGGGTCCGGGACCC 91 MLRGPGP 153 GGGCGGCTGCTGCTGCTAGCA GRLLLLAV
GTCCTGTGCCTGGGGACATCG LCLGTSVR GTGCGCTGCACCGAAACCGGG CTETGKSKR
AAGAGCAAGAGG SS-059 Fibronectin ATGCTTAGGGGTCCGGGGCCC 92 MLRGPGP
154 GGGCTGCTGCTGCTGGCCGTC GLLLLAV CAGCTGGGGACAGCGGTGCCC QCLGTAV
TCCACG PSTGA SS-060 Fibronectin ATGCGCCGGGGGGCCCTGACC 93 MRRGALT
155 GGGCTGCTCCTGGTCCTGTGC GLLLVLCL CTGAGTGTTGTGCTACGTGCA SVVLRAAP
GCCCCCTCTGCAACAAGCAAG SATSKKRR AAGCGCAGG
[0292] In the table, SS is secretion signal and MLS is
mitochondrial leader signal. The signal-sensor primary constructs
or mmRNA of the present invention may be designed to encode any of
the signal peptide sequences of SEQ ID NOs 94-155, or fragments or
variants thereof. These sequences may be included at the beginning
of the oncology-related polypeptide coding region, in the middle or
at the terminus or alternatively into a flanking region. Further,
any of the signal-sensor polynucleotide primary constructs of the
present invention may also comprise one or more of the sequences
defined by SEQ ID NOs 32-93. These may be in the first region or
either flanking region.
[0293] Additional signal peptide sequences which may be utilized in
the present invention include those taught in, for example,
databases such as those found at http://www.signalpeptide.de/ or
http://proline.bic.nus.edu.sg/spdb/. Those described in U.S. Pat.
Nos. 8,124,379; 7,413,875 and 7,385,034 are also within the scope
of the invention and the contents of each are incorporated herein
by reference in their entirety.
[0294] In one embodiment, the signal-sensor polynucleotide, primary
constructs or mmRNA may include a nucleic acid sequence encoding a
nuclear localization signal (NLS) and/or a nuclear export signal
(NES). In one aspect, a signal-sensor polynucleotide, primary
constructs or mmRNA may include a nucleic acid sequence encoding a
nuclear localization signal (NLS). The signal-sensor
polynucleotide, primary construct or mmRNA encoding a NLS would be
able to traffic an oncology related polypeptide into the nucleus
and deliver a survival or death signal to the nuclear
microenvironment. In another aspect, the signal-sensor
polynucleotide, primary constructs or mmRNA may include a nucleic
acid sequence encoding a nuclear export signal such as NES 1 and/or
NES2. As a nonlimiting example, the signal-sensor polynucleotide,
primary constructs or mmRNA may encode a NES1, NES2 and a NLS
signal and an oncology related polypeptide or a scambled sequence
which is not translatable in order to interact with HIF1-alpha to
alter the transcritome of the cancer cells.
Target Selection
[0295] According to the present invention, the signal-sensor
primary constructs comprise at least a first region of linked
nucleosides encoding at least one oncology-related polypeptide of
interest. The oncology-related polypeptides of interest or
"targets" or oncology-related proteins and oncology-related
peptides of the present invention are listed in Table 6, Table 7
and Table 41. Oncology-related polypeptides may be divided into
classes based on their function and area of cancer intervention.
For example, the classes may include targets associated with (1)
apoptosis or Survival signal imbalance (AS targets). These may be
caspase dependent or caspase independent targets; (2) replicative
potential or anti-senescence (CC/S targets); (3) metabolic stress
including the involvement of acidosis or hypoxia (O.sub.0>1%) (M
targets); (4) immune response (I targets); and (5) DNA
damage/protection (DDR targets).
[0296] Shown in Table 6, in addition to the name and description of
the gene encoding the oncology-related polypeptide of interest are
the ENSEMBL Transcript ID (ENST), the ENSEMBL Protein ID (ENSP),
each present where applicable, and when available the optimized
sequence ID (OPT. SEQ ID). The targets are also categorized by
group where "AS" refers to targets involved in apoptotic signaling;
"M" refers to targets involved in metabolic processes and "CC/S"
refers to targets involved in cell cycle and senescense.
TABLE-US-00006 TABLE 6 Oncology Related Targets Prot. OPT. Trans.
SEQ SEQ ENST SEQ ENSP ID ID Cat. Target Target Description ID ID NO
ID NO NO AS 14-3-3 tyrosine 3- 238081 156 238081 1321
monooxygenase/tryptophan 5- monooxygenase activation protein, theta
polypeptide AS 14-3-3 tyrosine 3- 248975 157 248975 1322
monooxygenase/tryptophan 5- monooxygenase activation protein, eta
polypeptide AS 14-3-3 tyrosine 3- 264335 158 264335 1323
monooxygenase/tryptophan 5- monooxygenase activation protein,
epsilon polypeptide AS 14-3-3 tyrosine 3- 307630 159 306330 1324
monooxygenase/tryptophan 5- monooxygenase activation protein, gamma
polypeptide AS 14-3-3 tyrosine 3- 353245 160 309503 1325
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 353703 161 300161 1326
monooxygenase/tryptophan 5- monooxygenase activation protein, beta
polypeptide AS 14-3-3 tyrosine 3- 372839 162 361930 1327
monooxygenase/tryptophan 5- monooxygenase activation protein, beta
polypeptide AS 14-3-3 tyrosine 3- 381844 163 371267 1328
monooxygenase/tryptophan 5- monooxygenase activation protein, theta
polypeptide AS 14-3-3 tyrosine 3- 395948 164 379278 1329
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 395951 165 379281 1330
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 395953 166 379283 1331
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 395956 167 379286 1332
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 395957 168 379287 1333
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 395958 169 379288 1334
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 414131 170 406058 1335
monooxygenase/tryptophan 5- monooxygenase activation protein,
epsilon polypeptide AS 14-3-3 tyrosine 3- 418997 171 416551 1336
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 419477 172 395114 1337
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 428262 173 394729 1338
monooxygenase/tryptophan 5- monooxygenase activation protein, beta
polypeptide AS 14-3-3 tyrosine 3- 437293 174 394880 1339
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 445830 175 394558 1340
monooxygenase/tryptophan 5- monooxygenase activation protein, beta
polypeptide AS 14-3-3 tyrosine 3- 446619 176 398990 1341
monooxygenase/tryptophan 5- monooxygenase activation protein, theta
polypeptide AS 14-3-3 tyrosine 3- 453207 177 390645 1342
monooxygenase/tryptophan 5- monooxygenase activation protein, gamma
polypeptide AS 14-3-3 tyrosine 3- 457309 178 398599 1343
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 517797 179 427801 1344
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 521309 180 429623 1345
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 521328 181 429041 1346
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 521607 182 430058 1347
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 522542 183 430072 1348
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 522819 184 428775 1349
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 523131 185 428381 1350
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 523848 186 428860 1351
monooxygenase/tryptophan 5- monooxygenase activation protein, zeta
polypeptide AS 14-3-3 tyrosine 3- 536755 187 443803 1352
monooxygenase/tryptophan 5- monooxygenase activation protein, gamma
polypeptide AS 14-3-3 tyrosine 3- 539979 188 443226 1353
monooxygenase/tryptophan 5- monooxygenase activation protein, theta
polypeptide AS AIF apoptosis-inducing factor, 287295 189 287295
1354 mitochondrion-associated, 1 AS AIF apoptosis-inducing factor,
307864 190 312370 1355 mitochondrion-associated, 2 AS AIF
apoptosis-inducing factor, 319908 191 315122 1356
mitochondrion-associated, 1 AS AIF apoptosis-inducing factor,
333607 192 327671 1357 mitochondrion-associated, 3 AS AIF
apoptosis-inducing factor, 335375 193 335369 1358
mitochondrion-associated, 3 AS AIF apoptosis-inducing factor,
346424 194 316320 1359 mitochondrion-associated, 1 AS AIF
apoptosis-inducing factor, 373248 195 362345 1360
mitochondrion-associated, 2 AS AIF apoptosis-inducing factor,
395039 196 378480 1361 mitochondrion-associated, 2 AS AIF
apoptosis-inducing factor, 399163 197 382116 1362
mitochondrion-associated, 3 AS AIF apoptosis-inducing factor,
399167 198 382120 1363 mitochondrion-associated, 3 AS AIF
apoptosis-inducing factor, 405089 199 385800 1364
mitochondrion-associated, 3 AS AIF apoptosis-inducing factor,
434714 200 399657 1365 mitochondrion-associated, 3 AS AIF
apoptosis-inducing factor, 440238 201 390798 1366
mitochondrion-associated, 3 AS AIF apoptosis-inducing factor,
440263 202 405879 1367 mitochondrion-associated, 1 AS AIF
apoptosis-inducing factor, 441376 203 402067 1368
mitochondrion-associated, 3 AS AIF apoptosis-inducing factor,
460436 204 431222 1369 mitochondrion-associated, 1 AS AIF
apoptosis-inducing factor, 535724 205 446113 1370
mitochondrion-associated, 1 AS AKT v-akt murine thymoma viral
263826 206 263826 1371 (PKB) oncogene homolog 3 (protein kinase B,
gamma) AS AKT v-akt murine thymoma viral 311278 207 309428 1372
(PKB) oncogene homolog 2 AS AKT v-akt murine thymoma viral 336199
208 336943 1373 (PKB) oncogene homolog 3 (protein kinase B, gamma)
AS AKT v-akt murine thymoma viral 349310 209 270202 1374 (PKB)
oncogene homolog 1 AS AKT v-akt murine thymoma viral 358335 210
351095 1375 (PKB) oncogene homolog 2 AS AKT v-akt murine thymoma
viral 366539 211 355497 1376 (PKB) oncogene homolog 3 (protein
kinase B, gamma) AS AKT v-akt murine thymoma viral 366540 212
355498 1377 (PKB) oncogene homolog 3 (protein kinase B, gamma) AS
AKT v-akt murine thymoma viral 391844 213 375719 1378 (PKB)
oncogene homolog 2 AS AKT v-akt murine thymoma viral 392037 214
375891 1379 (PKB) oncogene homolog 2 AS AKT v-akt murine thymoma
viral 392038 215 375892 1380 (PKB) oncogene homolog 2 AS AKT v-akt
murine thymoma viral 402615 216 385326 1381 (PKB) oncogene homolog
1 AS AKT v-akt murine thymoma viral 407796 217 384293 1382 (PKB)
oncogene homolog 1 AS AKT v-akt murine thymoma viral 416362 218
407999 1383 (PKB) oncogene homolog 2 AS AKT v-akt murine thymoma
viral 416994 219 392458 1384 (PKB) oncogene homolog 2 AS AKT v-akt
murine thymoma viral 423127 220 403842 1385 (PKB) oncogene homolog
2 AS AKT v-akt murine thymoma viral 424901 221 399532 1386 (PKB)
oncogene homolog 2 AS AKT v-akt murine thymoma viral 427375 222
403890 1387 (PKB) oncogene homolog 2 AS AKT v-akt murine thymoma
viral 452077 223 404083 1388 (PKB) oncogene homolog 2 AS AKT v-akt
murine thymoma viral 456441 224 396532 1389 (PKB) oncogene homolog
2 AS AKT v-akt murine thymoma viral 537834 225 441591 1390 (PKB)
oncogene homolog 2 AS AKT v-akt murine thymoma viral 544168 226
443897 1391 (PKB) oncogene homolog 1 AS AKT v-akt murine thymoma
viral 552631 227 447820 1392 (PKB) oncogene homolog 3 (protein
kinase B, gamma) AS AKT v-akt murine thymoma viral 554581 228
451828 1393 (PKB) oncogene homolog 1 AS AKT v-akt murine thymoma
viral 554848 229 451166 1394 (PKB) oncogene homolog 1 AS AKT v-akt
murine thymoma viral 555528 230 450688 1395 (PKB) oncogene homolog
1 AS AKT v-akt murine thymoma viral 555926 231 451824 1396 (PKB)
oncogene homolog 1 AS ANT solute carrier family 25 281456 232
281456 1397 (mitochondrial carrier; adenine nucleotide
translocator), member 4 AS Apaf-1 apoptotic peptidase activating
333991 233 334558 1398 factor 1 AS Apaf-1 apoptotic peptidase
activating 339433 234 341830 1399 factor 1 AS Apaf-1 apoptotic
peptidase activating 357310 235 349862 1400 factor 1 AS Apaf-1
apoptotic peptidase activating 359972 236 353059 1401 factor 1 AS
Apaf-1 apoptotic peptidase activating 547045 237 449791 1402 factor
1 AS Apaf-1 apoptotic peptidase activating 549007 238 448161 1403
factor 1 AS Apaf-1 apoptotic peptidase activating 550527 239 448449
1404 factor 1 AS Apaf-1 apoptotic peptidase activating 551964 240
448165 1405
factor 1 AS Apaf-1 apoptotic peptidase activating 552268 241 448826
1406 factor 1 AS APRIL tumor necrosis factor (ligand) 338784 242
343505 1407 (TNFSF13) superfamily, member 13 AS APRIL tumor
necrosis factor (ligand) 349228 243 314455 1408 (TNFSF13)
superfamily, member 13 AS APRIL tumor necrosis factor (ligand)
380535 244 369908 1409 (TNFSF13) superfamily, member 13 AS APRIL
tumor necrosis factor (ligand) 396545 245 379794 1410 (TNFSF13)
superfamily, member 13 AS ARTS phosphoribosyl pyrophosphate 372418
246 361495 1411 synthetase 1 AS ARTS phosphoribosyl pyrophosphate
372419 247 361496 1412 synthetase 1 AS ARTS phosphoribosyl
pyrophosphate 372428 248 361505 1413 synthetase 1 AS ARTS
phosphoribosyl pyrophosphate 372435 249 361512 1414 synthetase 1 AS
ARTS phosphoribosyl pyrophosphate 543248 250 443185 1415 synthetase
1 AS ASK1 mitogen-activated protein 355845 251 348104 1416 (MAP3K5)
kinase kinase kinase 5 AS ASK1 mitogen-activated protein 359015 252
351908 1417 (MAP3K5) kinase kinase kinase 5 AS ASK1
mitogen-activated protein 367768 253 356742 1418 (MAP3K5) kinase
kinase kinase 5 AS BAD BCL2-associated agonist of 309032 254 309103
1419 cell death AS BAD BCL2-associated agonist of 394532 255 378040
1420 cell death AS BAD BCL2-associated agonist of 540152 256 440807
1421 cell death AS BAFF(TNFSF13B) tumor necrosis factor (ligand)
375887 257 365048 1422 superfamily, member 13b AS BAFF(TNFSF13B)
tumor necrosis factor (ligand) 430559 258 389540 1423 superfamily,
member 13b AS BAFF(TNFSF13B) tumor necrosis factor (ligand) 542136
259 445334 1424 superfamily, member 13b AS Bak
BCL2-antagonist/killer 1 360661 260 353878 1425 AS Bak
BCL2-antagonist/killer 1 374460 261 363584 1426 AS Bak
BCL2-antagonist/killer 1 374467 262 363591 1427 AS Bak
BCL2-antagonist/killer 1 442998 263 391258 1428 AS BAX
BCL2-associated X protein 293288 264 293288 1429 AS BAX
BCL2-associated X protein 345358 265 263262 1430 AS BAX
BCL2-associated X protein 354470 266 346461 1431 AS BAX
BCL2-associated X protein 391871 267 375744 1432 AS BAX
BCL2-associated X protein 415969 268 389971 1433 AS BAX
BCL2-associated X protein 539787 269 441413 1434 AS Bcl-2 B-cell
CLL/lymphoma 2 333681 270 329623 1435 AS Bcl-2 B-cell CLL/lymphoma
2 398117 271 381185 1436 AS Bcl-2 B-cell CLL/lymphoma 2 444484 272
404214 1437 AS Bcl-B BCL2-like 10 (apoptosis 260442 273 260442 1438
facilitator) AS Bcl-W BCL2-like 2 250405 274 250405 1439 AS Bcl-W
BCL2-like 2 554635 275 451234 1440 AS Bcl-W BCL2-like 2 557236 276
451701 1441 AS Bcl-W BCL2-like 2 557579 277 452265 1442 AS Bcl-XL
BCL2-like 1 307677 278 302564 1443 AS Bcl-XL BCL2-like 1 376055 279
365223 1444 AS Bcl-XL BCL2-like 1 376062 280 365230 1445 AS Bcl-XL
BCL2-like 1 420488 281 390760 1446 AS Bcl-XL BCL2-like 1 420653 282
405563 1447 AS Bcl-XL BCL2-like 1 422920 283 411252 1448 AS Bcl-XL
BCL2-like 1 439267 284 389688 1449 AS Bcl-XL BCL2-like 1 450273 285
406203 1450 AS Bcl-XL BCL2-like 1 456404 286 395545 1451 AS BCMA
tumor necrosis factor receptor 53243 287 53243 1452 superfamily,
member 17 AS BCMA tumor necrosis factor receptor 396495 288 379753
1453 superfamily, member 17 AS BCMA tumor necrosis factor receptor
435355 289 401782 1454 superfamily, member 17 AS BFL1 BCL2-related
protein A1 267953 290 267953 1455 AS BFL1 BCL2-related protein A1
335661 291 335250 1456 AS Bid BH3 interacting domain death 317361
292 318822 1457 agonist AS Bid BH3 interacting domain death 342111
293 344594 1458 agonist AS Bid BH3 interacting domain death 399765
294 382667 1459 agonist AS Bid BH3 interacting domain death 399767
295 382669 1460 agonist AS Bid BH3 interacting domain death 399774
296 382674 1461 agonist AS Bid BH3 interacting domain death 551952
297 449236 1462 agonist AS Bik BCL2-interacting killer 216115 298
216115 1463 (apoptosis-inducing) AS Bim BCL2-like 11 (apoptosis
308659 299 309226 1464 facilitator) AS Bim BCL2-like 11 (apoptosis
337565 300 338374 1465 facilitator) AS Bim BCL2-like 11 (apoptosis
357757 301 350398 1466 facilitator) AS Bim BCL2-like 11 (apoptosis
393252 302 376941 1467 facilitator) AS Bim BCL2-like 11 (apoptosis
393253 303 376942 1468 facilitator) AS Bim BCL2-like 11 (apoptosis
393256 304 376943 1469 facilitator) AS Bim BCL2-like 11 (apoptosis
432179 305 411870 1470 facilitator) AS Bim BCL2-like 11 (apoptosis
452033 306 403666 1471 facilitator) AS BMF Bcl2 modifying factor
220446 307 220446 1472 AS BMF Bcl2 modifying factor 354670 308
346697 1473 AS BMF Bcl2 modifying factor 397573 309 380703 1474 AS
BMF Bcl2 modifying factor 431415 310 396511 1475 AS BMF Bcl2
modifying factor 559701 311 453919 1476 AS BMF Bcl2 modifying
factor 561282 312 453522 1477 AS BMF Bcl2 modifying factor 561360
313 453892 1478 AS BRE brain and reproductive organ- 342045 314
339371 1479 expressed (TNFRSF1A modulator) AS BRE brain and
reproductive organ- 344773 315 343412 1480 expressed (TNFRSF1A
modulator) AS BRE brain and reproductive organ- 361704 316 354699
1481 expressed (TNFRSF1A modulator) AS BRE brain and reproductive
organ- 379623 317 368944 1482 expressed (TNFRSF1A modulator) AS BRE
brain and reproductive organ- 379624 318 368945 1483 expressed
(TNFRSF1A modulator) AS BRE brain and reproductive organ- 379632
319 368953 1484 expressed (TNFRSF1A modulator) AS BRE brain and
reproductive organ- 436924 320 392345 1485 expressed (TNFRSF1A
modulator) AS Calcineurin A protein phosphatase 3, catalytic 323055
321 320580 1486 subunit, alpha isozyme AS Calcineurin A protein
phosphatase 3, catalytic 394853 322 378322 1487 subunit, alpha
isozyme AS Calcineurin A protein phosphatase 3, catalytic 394854
323 378323 1488 subunit, alpha isozyme AS Calcineurin A protein
phosphatase 3, catalytic 507176 324 422990 1489 subunit, alpha
isozyme AS Calcineurin A protein phosphatase 3, catalytic 512215
325 422781 1490 subunit, alpha isozyme AS Calcineurin A protein
phosphatase 3, catalytic 523694 326 429350 1491 subunit, alpha
isozyme AS Calcineurin A protein phosphatase 3, catalytic 525819
327 434599 1492 subunit, alpha isozyme AS Calcineurin A protein
phosphatase 3, catalytic 529324 328 431619 1493 subunit, alpha
isozyme AS Caspase-1 caspase 1, apoptosis-related 353247 329 344132
1494 cysteine peptidase (interleukin 1, beta, convertase) AS
Caspase-1 caspase 1, apoptosis-related 393136 330 376844 1495
cysteine peptidase (interleukin 1, beta, convertase) AS Caspase-1
caspase 1, apoptosis-related 415981 331 408446 1496 cysteine
peptidase (interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 436863 332 410076 1497 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 446369 333 403260 1498 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 525825 334 434779 1499 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 526568 335 434250 1500 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 528974 336 434259 1501 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 529871 337 431947 1502 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 531166 338 434303 1503 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 533400 339 433138 1504 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase-1 caspase 1,
apoptosis-related 534497 340 436875 1505 cysteine peptidase
(interleukin 1, beta, convertase) AS Caspase- caspase 10,
apoptosis-related 272879 341 272879 1506 10 cysteine peptidase AS
Caspase- caspase 10, apoptosis-related 286186 342 286186 1507 10
cysteine peptidase AS Caspase- caspase 10, apoptosis-related 346817
343 237865 1508 10 cysteine peptidase AS Caspase- caspase 10,
apoptosis-related 360132 344 353250 1509 10 cysteine peptidase AS
Caspase-2 caspase 2, apoptosis-related 310447 345 312664 1510
cysteine peptidase AS Caspase-2 caspase 2, apoptosis-related 350623
346 340030 1511 cysteine peptidase AS Caspase-2 caspase 2,
apoptosis-related 392923 347 376654 1512 cysteine peptidase AS
Caspase-3 caspase 3, apoptosis-related 308394 348 311032 1513
cysteine peptidase AS Caspase-3 caspase 3, apoptosis-related 438467
349 390792 1514 cysteine peptidase AS Caspase-3 caspase 3,
apoptosis-related 447121 350 407142 1515 cysteine peptidase AS
Caspase-3 caspase 3, apoptosis-related 523916 351 428929 1516
cysteine peptidase AS Caspase-4 caspase 4, apoptosis-related 355546
352 347741 1517 cysteine peptidase AS Caspase-4 caspase 4,
apoptosis-related 417440 353 401673 1518 cysteine peptidase AS
Caspase-4 caspase 4, apoptosis-related 444739 354 388566 1519
cysteine peptidase AS Caspase-5 caspase 5, apoptosis-related 260315
355 260315 1520 cysteine peptidase AS Caspase-5 caspase 5,
apoptosis-related 393139 356 376847 1521 cysteine peptidase AS
Caspase-5 caspase 5, apoptosis-related 393141 357 376849 1522
cysteine peptidase AS Caspase-5 caspase 5, apoptosis-related 418434
358 398130 1523 cysteine peptidase AS Caspase-5 caspase 5,
apoptosis-related 444749 359 388365 1524 cysteine peptidase AS
Caspase-5 caspase 5, apoptosis-related 526056 360 436877 1525
cysteine peptidase AS Caspase-5 caspase 5, apoptosis-related 531367
361 434471 1526 cysteine peptidase AS Caspase-6 caspase 6,
apoptosis-related 265164 362 265164 1527 cysteine peptidase AS
Caspase-6 caspase 6, apoptosis-related 352981 363 285333 1528
cysteine peptidase AS Caspase-7 caspase 7, apoptosis-related 345633
364 298701 1529 cysteine peptidase AS Caspase-7 caspase 7,
apoptosis-related 369315 365 358321 1530 cysteine peptidase AS
Caspase-7 caspase 7, apoptosis-related 369316 366 358322 1531
cysteine peptidase AS Caspase-7 caspase 7, apoptosis-related 369318
367 358324 1532 cysteine peptidase AS Caspase-7 caspase 7,
apoptosis-related 369319 368 358325 1533 cysteine peptidase AS
Caspase-7 caspase 7, apoptosis-related 369321 369 358327 1534
cysteine peptidase AS Caspase-7 caspase 7, apoptosis-related 369331
370 358337 1535 cysteine peptidase AS Caspase-7 caspase 7,
apoptosis-related 429617 371 400094 1536 cysteine peptidase AS
Caspase-7 caspase 7, apoptosis-related 442393 372 394482 1537
cysteine peptidase AS Caspase-7 caspase 7, apoptosis-related 452490
373 398107 1538 cysteine peptidase
AS Caspase-8 caspase 8, apoptosis-related 264274 374 264274 1539
cysteine peptidase AS Caspase-8 caspase 8, apoptosis-related 264275
375 264275 1540 cysteine peptidase AS Caspase-8 caspase 8,
apoptosis-related 323492 376 325722 1541 cysteine peptidase AS
Caspase-8 caspase 8, apoptosis-related 358485 377 351273 1542
cysteine peptidase AS Caspase-8 caspase 8, apoptosis-related 392258
378 376087 1543 cysteine peptidase AS Caspase-8 caspase 8,
apoptosis-related 392259 379 376088 1544 cysteine peptidase AS
Caspase-8 caspase 8, apoptosis-related 392261 380 376089 1545
cysteine peptidase AS Caspase-8 caspase 8, apoptosis-related 392263
381 376091 1546 cysteine peptidase AS Caspase-8 caspase 8,
apoptosis-related 392266 382 376094 1547 cysteine peptidase AS
Caspase-8 caspase 8, apoptosis-related 413726 383 397528 1548
cysteine peptidase AS Caspase-8 caspase 8, apoptosis-related 429881
384 390641 1549 cysteine peptidase AS Caspase-8 caspase 8,
apoptosis-related 432109 385 412523 1550 cysteine peptidase AS
Caspase-8 caspase 8, apoptosis-related 440732 386 396869 1551
cysteine peptidase AS Caspase-8 caspase 8, apoptosis-related 447616
387 388306 1552 cysteine peptidase AS Caspase-9 caspase 9,
apoptosis-related 333868 388 330237 1553 cysteine peptidase AS
Caspase-9 caspase 9, apoptosis-related 348549 389 255256 1554
cysteine peptidase AS Caspase-9 caspase 9, apoptosis-related 375874
390 365034 1555 cysteine peptidase AS Caspase-9 caspase 9,
apoptosis-related 375890 391 365051 1556 cysteine peptidase AS
Caspase-9 caspase 9, apoptosis-related 440484 392 411304 1557
cysteine peptidase AS Caspase-9 caspase 9, apoptosis-related 447522
393 396540 1558 cysteine peptidase AS Caspase-9 caspase 9,
apoptosis-related 546424 394 449584 1559 cysteine peptidase AS CD27
CD27 molecule 266557 395 266557 1560 AS CD30 tumor necrosis factor
receptor 263932 396 263932 1561 superfamily, member 8 AS CD30 tumor
necrosis factor receptor 413146 397 398337 1562 superfamily, member
8 AS CD30 tumor necrosis factor receptor 417814 398 390650 1563
superfamily, member 8 AS CD30L tumor necrosis factor (ligand)
223795 399 223795 1564 superfamily, member 8 AS CD40 CD40 molecule,
TNF receptor 372278 400 361352 1565 superfamily member 5 AS CD40L
CD40 ligand 370628 401 359662 1566 (TNFSF5) AS CD40L CD40 ligand
370629 402 359663 1567 (TNFSF5) AS CD41 CD40 molecule, TNF receptor
372276 403 361350 1568 superfamily member 5 AS CD42 CD40 molecule,
TNF receptor 372285 404 361359 1569 superfamily member 5 AS
CD70(TNFSF7) CD70 molecule 245903 405 245903 1570 AS CD70(TNFSF7)
CD70 molecule 423145 406 395294 1571 AS CDK1 cyclin-dependent
kinase 1 316629 407 325970 1572 (p34) AS CDK1 cyclin-dependent
kinase 1 373809 408 362915 1573 (p34) AS CDK1 cyclin-dependent
kinase 1 395284 409 378699 1574 (p34) AS CDK1 cyclin-dependent
kinase 1 448257 410 397973 1575 (p34) AS CDK1 cyclin-dependent
kinase 1 519078 411 430665 1576 (p34) AS CDK5 cyclin-dependent
kinase 5 485972 412 419782 1577 AS CDK5R1 cyclin-dependent kinase
5, 313401 413 318486 1578 (p35) regulatory subunit 1 (p35) AS c-
CASP8 and FADD-like 309955 414 312455 1579 FLIP(S) apoptosis
regulator AS c- CASP8 and FADD-like 340870 415 339326 1580 FLIP(S)
apoptosis regulator AS c- CASP8 and FADD-like 343375 416 339391
1581 FLIP(S) apoptosis regulator AS c- CASP8 and FADD-like 355558
417 347757 1582 FLIP(S) apoptosis regulator AS c- CASP8 and
FADD-like 395148 418 378580 1583 FLIP(S) apoptosis regulator AS c-
CASP8 and FADD-like 417748 419 412882 1584 FLIP(S) apoptosis
regulator AS c- CASP8 and FADD-like 423241 420 399420 1585 FLIP(S)
apoptosis regulator AS c- CASP8 and FADD-like 433445 421 391029
1586 FLIP(S) apoptosis regulator AS c- CASP8 and FADD-like 441224
422 411897 1587 FLIP(S) apoptosis regulator AS c- CASP8 and
FADD-like 443227 423 413270 1588 FLIP(S) apoptosis regulator AS
cIAP1 baculoviral IAP repeat NA 424 NA 1589 2488 containing 3 AS
c-IAP1 baculoviral IAP repeat 263464 425 263464 1590 containing 3
AS c-IAP1 baculoviral IAP repeat 532808 426 432907 1591 containing
3 AS cIAP2 baculoviral IAP repeat NA 427 NA 1592 containing 2 AS
C-IAP2 baculoviral IAP repeat 227758 428 227758 1593 containing 2
AS C-IAP2 baculoviral IAP repeat 530675 429 431723 1594 containing
2 AS C-IAP2 baculoviral IAP repeat 532672 430 434979 1595
containing 2 AS C-IAP2 baculoviral IAP repeat 541741 431 440771
1596 containing 2 AS c-Jun jun proto-oncogene 371222 432 360266
1597 AS c-Raf-1 v-raf-1 murine leukemia viral 251849 433 251849
1598 oncogene homolog 1 AS c-Raf-1 v-raf-1 murine leukemia viral
442415 434 401888 1599 oncogene homolog 1 AS c-Raf-1 v-raf-1 murine
leukemia viral 534997 435 441186 1600 oncogene homolog 1 AS c-Raf-1
v-raf-1 murine leukemia viral 542177 436 443567 1601 oncogene
homolog 1 AS Cytochrome c cytochrome c, somatic 305786 437 307786
1602 AS Cytochrome c cytochrome c, somatic 409409 438 386270 1603
AS Cytochrome c cytochrome c, somatic 409764 439 387279 1604 AS
Cytochrome c cytochrome c, somatic 413447 440 416479 1605 AS DAXX
death-domain associated 266000 441 266000 1606 protein AS DAXX
death-domain associated 374542 442 363668 1607 protein AS DAXX
death-domain associated 383062 443 372539 1608 protein AS DAXX
death-domain associated 383194 444 372681 1609 protein AS DAXX
death-domain associated 399060 445 382014 1610 protein AS DAXX
death-domain associated 399344 446 382281 1611 protein AS DAXX
death-domain associated 414083 447 396876 1612 protein AS DAXX
death-domain associated 414272 448 409756 1613 protein AS DAXX
death-domain associated 419855 449 397612 1614 protein AS DAXX
death-domain associated 428268 450 408215 1615 protein AS DAXX
death-domain associated 429531 451 415898 1616 protein AS DAXX
death-domain associated 433482 452 404623 1617 protein AS DAXX
death-domain associated 436311 453 404376 1618 protein AS DAXX
death-domain associated 438332 454 411700 1619 protein AS DAXX
death-domain associated 440500 455 403986 1620 protein AS DAXX
death-domain associated 445009 456 394108 1621 protein AS DAXX
death-domain associated 446403 457 406008 1622 protein AS DAXX
death-domain associated 453407 458 408499 1623 protein AS DAXX
death-domain associated 453931 459 412433 1624 protein AS DAXX
death-domain associated 454197 460 412177 1625 protein AS DAXX
death-domain associated 455860 461 410772 1626 protein AS DAXX
death-domain associated 547663 462 447115 1627 protein AS DAXX
death-domain associated 548604 463 448337 1628 protein AS DAXX
death-domain associated 550822 464 447861 1629 protein AS DAXX
death-domain associated 552944 465 447833 1630 protein AS DcR3
tumor necrosis factor receptor 342852 466 342328 1631 superfamily,
member 6b, decoy AS DcR3 tumor necrosis factor receptor 369996 467
359013 1632 superfamily, member 6b, decoy AS DcR3 tumor necrosis
factor receptor 370006 468 359023 1633 superfamily, member 6b,
decoy AS DFF40 DNA fragmentation factor, 338895 469 339524 1634
(CAD) 40 kDa, beta polypeptide (caspase-activated DNase) AS DFF40
DNA fragmentation factor, 339350 470 343218 1635 (CAD) 40 kDa, beta
polypeptide (caspase-activated DNase) AS DFF40 DNA fragmentation
factor, 341385 471 345906 1636 (CAD) 40 kDa, beta polypeptide
(caspase-activated DNase) AS DFF40 DNA fragmentation factor, 378206
472 367448 1637 (CAD) 40 kDa, beta polypeptide (caspase-activated
DNase) AS DFF40 DNA fragmentation factor, 378209 473 367454 1638
(CAD) 40 kDa, beta polypeptide (caspase-activated DNase) AS DFF40
DNA fragmentation factor, 378212 474 367457 1639 (CAD) 40 kDa, beta
polypeptide (caspase-activated DNase) AS DFF40 DNA fragmentation
factor, 430539 475 389502 1640 (CAD) 40 kDa, beta polypeptide
(caspase-activated DNase) AS DFF40 DNA fragmentation factor, 448632
476 411635 1641 (CAD) 40 kDa, beta polypeptide (caspase-activated
DNase) AS DFF40 DNA fragmentation factor, 491998 477 436775 1642
(CAD) 40 kDa, beta polypeptide (caspase-activated DNase) AS DR3
tumor necrosis factor receptor 348333 478 314451 1643 superfamily,
member 25 AS DR3 tumor necrosis factor receptor 351748 479 326762
1644 superfamily, member 25 AS DR3 tumor necrosis factor receptor
351959 480 337713 1645 superfamily, member 25 AS DR3 tumor necrosis
factor receptor 356876 481 349341 1646 superfamily, member 25 AS
DR3 tumor necrosis factor receptor 377782 482 367013 1647
superfamily, member 25 AS DR4 tumor necrosis factor receptor 221132
483 221132 1648 superfamily, member 10a AS DR5 tumor necrosis
factor receptor 276431 484 276431 1649 superfamily, member 10b AS
DR5 tumor necrosis factor receptor 347739 485 317859 1650
superfamily, member 10b AS DR5 tumor necrosis factor receptor
542226 486 443386 1651 superfamily, member 10b AS DR6 tumor
necrosis factor receptor 296861 487 296861 1652 superfamily, member
21 AS DR6 tumor necrosis factor receptor 419206 488 390032 1653
superfamily, member 21 AS EGFR epidermal growth factor 275493 489
275493 1654 receptor AS EGFR epidermal growth factor 342916 490
342376 1655 receptor AS EGFR epidermal growth factor 344576 491
345973 1656 receptor AS EGFR epidermal growth factor 395504 492
378880 1657 receptor AS EGFR epidermal growth factor 420316 493
413843 1658 receptor AS EGFR epidermal growth factor 442591 494
410031 1659 receptor AS EGFR epidermal growth factor 454757 495
395243 1660 receptor AS EGFR epidermal growth factor 455089 496
415559 1661 receptor AS EGFR epidermal growth factor 533450 497
435262 1662 receptor
AS ErbB2 v-erb-b2 erythroblastic 269571 498 269571 1663 leukemia
viral oncogene homolog 2, neuro/glioblastoma derived oncogene
homolog (avian) AS ErbB2 v-erb-b2 erythroblastic 406381 499 385185
1664 leukemia viral oncogene homolog 2, neuro/glioblastoma derived
oncogene homolog (avian) AS ErbB2 v-erb-b2 erythroblastic 445658
500 404047 1665 leukemia viral oncogene homolog 2,
neuro/glioblastoma derived oncogene homolog (avian) AS ErbB2
v-erb-b2 erythroblastic 540042 501 446382 1666 leukemia viral
oncogene homolog 2, neuro/glioblastoma derived oncogene homolog
(avian) AS ErbB2 v-erb-b2 erythroblastic 540147 502 443562 1667
leukemia viral oncogene homolog 2, neuro/glioblastoma derived
oncogene homolog (avian) AS ErbB2 v-erb-b2 erythroblastic 541774
503 446466 1668 leukemia viral oncogene homolog 2,
neuro/glioblastoma derived oncogene homolog (avian) AS ErbB3
v-erb-b2 erythroblastic 267101 504 267101 1669 leukemia viral
oncogene homolog 3 (avian) AS ErbB3 v-erb-b2 erythroblastic 394099
505 377659 1670 leukemia viral oncogene homolog 3 (avian) AS ErbB3
v-erb-b2 erythroblastic 411731 506 415753 1671 leukemia viral
oncogene homolog 3 (avian) AS ErbB3 v-erb-b2 erythroblastic 415288
507 408340 1672 leukemia viral oncogene homolog 3 (avian) AS ErbB3
v-erb-b2 erythroblastic 450146 508 399178 1673 leukemia viral
oncogene homolog 3 (avian) AS ErbB3 v-erb-b2 erythroblastic 549282
509 448636 1674 leukemia viral oncogene homolog 3 (avian) AS ErbB3
v-erb-b2 erythroblastic 551085 510 448483 1675 leukemia viral
oncogene homolog 3 (avian) AS Erk(MAPK1/ mitogen-activated protein
215832 511 215832 1676 3) kinase 1 AS Erk(MAPK1/ mitogen-activated
protein 263025 512 263025 1677 3) kinase 3 AS Erk(MAPK1/
mitogen-activated protein 322266 513 327293 1678 3) kinase 3 AS
Erk(MAPK1/ mitogen-activated protein 395200 514 378626 1679 3)
kinase 3 AS Erk(MAPK1/ mitogen-activated protein 395202 515 378628
1680 3) kinase 3 AS Erk(MAPK1/ mitogen-activated protein 398822 516
381803 1681 3) kinase 1 AS Erk(MAPK1/ mitogen-activated protein
403394 517 384895 1682 3) kinase 3 AS Erk(MAPK1/ mitogen-activated
protein 415911 518 409149 1683 3) kinase 1 AS Erk(MAPK1/
mitogen-activated protein 484663 519 432742 1684 3) kinase 3 AS
Erk(MAPK1/ mitogen-activated protein 544786 520 440842 1685 3)
kinase 1 AS FADD Fas (TNFRSF6)-associated via 301838 521 301838
1686 death domain AS FLASH caspase 8 associated protein 2 237177
522 NA 1687 AS FLASH caspase 8 associated protein 2 419040 523 NA
AS FLASH caspase 8 associated protein 2 444163 524 NA AS FLASH
caspase 8 associated protein 2 547893 525 NA AS FLASH caspase 8
associated protein 2 548224 526 NA AS FLASH caspase 8 associated
protein 2 551025 527 NA AS FLASH caspase 8 associated protein 2
552401 528 NA AS FN14 tumor necrosis factor receptor 326577 529
326737 1688 superfamily, member 12A AS FN14 tumor necrosis factor
receptor 341627 530 343894 1689 superfamily, member 12A AS GCK
mitogen-activated protein 294066 531 294066 1690 (MAP4K2) kinase
kinase kinase kinase 2 AS GRB2 growth factor receptor-bound 316615
532 317360 1691 protein 2 AS GRB2 growth factor receptor-bound
316804 533 339007 1692 protein 2 AS GRB2 growth factor
receptor-bound 392562 534 376345 1693 protein 2 AS GRB2 growth
factor receptor-bound 392564 535 376347 1694 protein 2 AS H-Ras
v-Ha-ras Harvey rat sarcoma 311189 536 309845 1695 viral oncogene
homolog AS H-Ras v-Ha-ras Harvey rat sarcoma 397594 537 380722 1696
viral oncogene homolog AS H-Ras v-Ha-ras Harvey rat sarcoma 397596
538 380723 1697 viral oncogene homolog AS H-Ras v-Ha-ras Harvey rat
sarcoma 417302 539 388246 1698 viral oncogene homolog AS H-Ras
v-Ha-ras Harvey rat sarcoma 451590 540 407586 1699 viral oncogene
homolog AS H-Ras v-Ha-ras Harvey rat sarcoma 493230 541 434023 1700
viral oncogene homolog AS HRK harakiri, BCL2 interacting 257572 542
257572 1701 protein (contains only BH3 domain) AS HSP27 heat shock
27 kDa protein 1 248553 543 248553 1702 AS HSP27 heat shock 27 kDa
protein 3 302005 544 303394 1703 AS HSP27 Heat shock protein beta-2
304298 545 302476 1704 AS HSP27 heat shock 27 kDa protein 1 432276
546 406545 1705 AS HSP27 Heat shock protein beta-2 537382 547
445585 1706 AS HtrA2/Omi HtrA serine peptidase 2 258080 548 258080
1707 AS HtrA2/Omi HtrA serine peptidase 2 352222 549 312893 1708 AS
Humanin MT-RNR2-like 4 399974 550 382856 1709 AS Humanin
MT-RNR2-like 5 512524 551 437910 1710 AS Humanin MT-RNR2-like 8
536684 552 439666 1711 AS Humanin MT-RNR2-like 1 540040 553 439228
1712 AS Humanin MT-RNR2-like 3 543500 554 443339 1713 AS Humanin
MT-RNR2-like 7 544824 555 439985 1714 AS Humanin MT-RNR2-like 10
545075 556 442159 1715 AS Humanin MT-RNR2-like 6 570419 557 461075
1716 AS ICAD DNA fragmentation factor, 377036 558 366235 1717 45
kDa, alpha polypeptide AS ICAD DNA fragmentation factor, 377038 559
366237 1718 45 kDa, alpha polypeptide AS IGF-1R insulin-like growth
factor 1 268035 560 268035 1719 receptor AS IKK conserved
helix-loop-helix 370397 561 359424 1720 (alpha) ubiquitous kinase
AS IKK inhibitor of kappa light 379708 562 369030 1721 (beta)
polypeptide gene enhancer in B-cells, kinase beta AS IKK inhibitor
of kappa light 416505 563 404920 1722 (beta) polypeptide gene
enhancer in B-cells, kinase beta AS IKK inhibitor of kappa light
520810 564 430684 1723 (beta) polypeptide gene enhancer in B-cells,
kinase beta AS IKK- inhibitor of kappa light 263518 565 263518 1724
gamma polypeptide gene enhancer in B-cells, kinase gamma AS IKK-
inhibitor of kappa light 369601 566 358614 1725 gamma polypeptide
gene enhancer in B-cells, kinase gamma AS IKK- inhibitor of kappa
light 369606 567 358619 1726 gamma polypeptide gene enhancer in
B-cells, kinase gamma AS IKK- inhibitor of kappa light 369607 568
358620 1727 gamma polypeptide gene enhancer in B-cells, kinase
gamma AS IKK- inhibitor of kappa light 369609 569 358622 1728 gamma
polypeptide gene enhancer in B-cells, kinase gamma AS IKK-
inhibitor of kappa light 422680 570 390368 1729 gamma polypeptide
gene enhancer in B-cells, kinase gamma AS IKK- inhibitor of kappa
light 440286 571 394934 1730 gamma polypeptide gene enhancer in
B-cells, kinase gamma AS IKK- inhibitor of kappa light 445622 572
395205 1731 gamma polypeptide gene enhancer in B-cells, kinase
gamma AS IKK- inhibitor of kappa light 455588 573 400769 1732 gamma
polypeptide gene enhancer in B-cells, kinase gamma AS IRAK1
interleukin-1 receptor- 369980 574 358997 1733 associated kinase 1
AS IRAK1 interleukin-1 receptor- 393682 575 377287 1734 associated
kinase 1 AS IRAK1 interleukin-1 receptor- 393687 576 377291 1735
associated kinase 1 AS IRAK1 interleukin-1 receptor- 429936 577
392662 1736 associated kinase 1 AS IRAK2 interleukin-1 receptor-
256458 578 256458 1737 associated kinase 2 AS IRS-1 insulin
receptor substrate 1 305123 579 304895 1738 AS jBid; jBID NA NA
1739 formed after cleaving BID at position 25 AS JNK1(MAPK8)
mitogen-activated protein 360332 580 353483 1740 kinase 8 AS
JNK1(MAPK8) mitogen-activated protein 374174 581 363289 1741 kinase
8 AS JNK1(MAPK8) mitogen-activated protein 374176 582 363291 1742
kinase 8 AS JNK1(MAPK8) mitogen-activated protein 374179 583 363294
1743 kinase 8 AS JNK1(MAPK8) mitogen-activated protein 374182 584
363297 1744 kinase 8 AS JNK1(MAPK8) mitogen-activated protein
374189 585 363304 1745 kinase 8 AS JNK1(MAPK8) mitogen-activated
protein 395611 586 378974 1746 kinase 8 AS JNK1(MAPK8)
mitogen-activated protein 426557 587 397729 1747 kinase 8 AS
JNK1(MAPK8) mitogen-activated protein 429041 588 393223 1748 kinase
8 AS JNK1(MAPK8) mitogen-activated protein 432379 589 387936 1749
kinase 8 AS JNK3(MAPK10) mitogen-activated protein 359221 590
352157 1750 kinase 10 AS JNK3(MAPK10) mitogen-activated protein
361569 591 355297 1751 kinase 10 AS JNK3(MAPK10) mitogen-activated
protein 395157 592 378586 1752 kinase 10 AS JNK3(MAPK10)
mitogen-activated protein 395160 593 378589 1753 kinase 10 AS
JNK3(MAPK10) mitogen-activated protein 395161 594 378590 1754
kinase 10 AS JNK3(MAPK10) mitogen-activated protein 395166 595
378595 1755 kinase 10 AS JNK3(MAPK10) mitogen-activated protein
395169 596 378598 1756 kinase 10 AS JNK3(MAPK10) mitogen-activated
protein 449047 597 414469 1757 kinase 10 AS JNK3(MAPK10)
mitogen-activated protein 502302 598 423918 1758 kinase 10 AS
JNK3(MAPK10) mitogen-activated protein 503911 599 421409 1759
kinase 10 AS JNK3(MAPK10) mitogen-activated protein 506773 600
421359 1760 kinase 10 AS JNK3(MAPK10) mitogen-activated protein
509464 601 424128 1761 kinase 10 AS JNK3(MAPK10) mitogen-activated
protein 511167 602 422277 1762 kinase 10 AS JNK3(MAPK10)
mitogen-activated protein 511328 603 421762 1763 kinase 10 AS
JNK3(MAPK10) mitogen-activated protein 512017 604 424755 1764
kinase 10 AS JNK3(MAPK10) mitogen-activated protein 512564 605
422985 1765 kinase 10 AS JNK3(MAPK10) mitogen-activated protein
515400 606 424154 1766 kinase 10 AS MAP1 mannan-binding lectin
serine 169293 607 169293 1767 peptidase 1 (C4/C2 activating
component of Ra-reactive factor) AS MAP1 mannan-binding lectin
serine 296280 608 296280 1768 peptidase 1 (C4/C2 activating
component of Ra-reactive factor) AS MAP1 mannan-binding lectin
serine 337774 609 336792 1769 peptidase 1 (C4/C2 activating
component of Ra-reactive
factor) AS MAP1 mannan-binding lectin serine 392472 610 376264 1770
peptidase 1 (C4/C2 activating component of Ra-reactive factor) AS
MAP1 mannan-binding lectin serine 541811 611 440446 1771 peptidase
1 (C4/C2 activating component of Ra-reactive factor) AS MAP1
mannan-binding lectin serine 541896 612 446240 1772 peptidase 1
(C4/C2 activating component of Ra-reactive factor) AS Mcl-1 myeloid
cell leukemia 307940 613 309973 1773 sequence 1 (BCL2-related) AS
Mcl-1 myeloid cell leukemia 369026 614 358022 1774 sequence 1
(BCL2-related) AS Mcl-1 myeloid cell leukemia 439749 615 411395
1775 sequence 1 (BCL2-related) AS MEK1 mitogen-activated protein
215832 616 215832 1776 (MAP2K1) kinase 1 AS MEK1 mitogen-activated
protein 307102 617 302486 1777 (MAP2K1) kinase kinase 1 AS MEK1
mitogen-activated protein 415911 618 409149 1778 (MAP2K1) kinase 1
AS MEK1 mitogen-activated protein 544786 619 440842 1779 (MAP2K1)
kinase 1 AS MEK2 mitogen-activated protein 262948 620 262948 1780
(MAP2K2) kinase kinase 2 AS MEK4 mitogen-activated protein 353533
621 262445 1781 (MAP2K4) kinase kinase 4 AS MEK4 mitogen-activated
protein 415385 622 410402 1782 (MAP2K4) kinase kinase 4 AS MEK4
mitogen-activated protein 536413 623 441610 1783 (MAP2K4) kinase
kinase 4 AS MEK4 mitogen-activated protein 538465 624 444874 1784
(MAP2K4) kinase kinase 4 AS MEKK1 mitogen-activated protein 399503
625 382423 1785 (MAP3K1) kinase kinase kinase 1 AS NADE nerve
growth factor receptor 299872 626 299872 1786 (NGFRAP1) (TNFRSF16)
associated protein 1 AS NADE nerve growth factor receptor 361298
627 354843 1787 (NGFRAP1) (TNFRSF16) associated protein 1 AS NADE
nerve growth factor receptor 372634 628 361717 1788 (NGFRAP1)
(TNFRSF16) associated protein 1 AS NADE nerve growth factor
receptor 372635 629 361718 1789 (NGFRAP1) (TNFRSF16) associated
protein 1 AS NADE nerve growth factor receptor 372645 630 361728
1790 (NGFRAP1) (TNFRSF16) associated protein 1 AS NGF nerve growth
factor (beta 369512 631 358525 1791 polypeptide) AS NGFR nerve
growth factor receptor 172229 632 172229 1792 AS NGFR nerve growth
factor receptor 504201 633 421731 1793 AS NIK mitogen-activated
protein 344686 634 342059 1794 (MAP3K14) kinase kinase kinase 14 AS
NIK mitogen-activated protein 376926 635 366125 1795 (MAP3K14)
kinase kinase kinase 14 AS NOXA phorbol-12-myristate-13- 269518 636
269518 1796 acetate-induced protein 1 AS NOXA
phorbol-12-myristate-13- 316660 637 326119 1797 acetate-induced
protein 1 AS OX40 tumor necrosis factor receptor 379236 638 368538
1798 superfamily, member 4 AS OX40 tumor necrosis factor receptor
453580 639 390907 1799 superfamily, member 4 AS OX40L tumor
necrosis factor (ligand) 281834 640 281834 1800 (TNFSF4)
superfamily, member 4 AS OX40L tumor necrosis factor (ligand)
367718 641 356691 1801 (TNFSF4) superfamily, member 4 AS OX40L
tumor necrosis factor (ligand) 545292 642 439704 1802 (TNFSF4)
superfamily, member 4 AS p53 tumor protein p53 269305 643 269305
1803 AS p53 tumor protein p53 269305 644 269305 1804 2489 AS p53
tumor protein p53 359597 645 352610 1805 AS p53 tumor protein p53
396473 646 379735 1806 AS p53 tumor protein p53 413465 647 410739
1807 AS p53 tumor protein p53 414315 648 394195 1808 AS p53 tumor
protein p53 419024 649 402130 1809 AS p53 tumor protein p53 420246
650 391127 1810 AS p53 tumor protein p53 445888 651 391478 1811
2490 AS p53 tumor protein p53 455263 652 398846 1812 AS p53 tumor
protein p53 503591 653 426252 1813 AS p53 tumor protein p53 508793
654 424104 1814 AS p53 tumor protein p53 509690 655 425104 1815 AS
p53 tumor protein p53 514944 656 423862 1816 AS p53 tumor protein
p53 545858 657 437792 1817 AS p70 S6 ribosomal protein S6 kinase,
225577 658 225577 1818 kinase 1 70 kDa, polypeptide 1 AS p70 S6
ribosomal protein S6 kinase, 393021 659 376744 1819 kinase 1 70
kDa, polypeptide 1 AS p70 S6 ribosomal protein S6 kinase, 406116
660 384335 1820 kinase 1 70 kDa, polypeptide 1 AS p70 S6 ribosomal
protein S6 kinase, 443572 661 441993 1821 kinase 1 70 kDa,
polypeptide 1 AS p70 S6 ribosomal protein S6 kinase, 312629 662
308413 1822 kinase 2 70 kDa, polypeptide 2 AS p70 S6 ribosomal
protein S6 kinase, 528964 663 432847 1823 kinase 2 70 kDa,
polypeptide 2 AS p70 S6 ribosomal protein S6 kinase, 539188 664
442949 1824 kinase 2 70 kDa, polypeptide 2 AS p90Rsk ribosomal
protein S6 kinase, 374162 665 363277 1825 90 kDa, polypeptide 1 AS
p90Rsk ribosomal protein S6 kinase, 374164 666 363279 1826 90 kDa,
polypeptide 1 AS p90Rsk ribosomal protein S6 kinase, 374168 667
363283 1827 90 kDa, polypeptide 1 AS p90Rsk ribosomal protein S6
kinase, 403732 668 383967 1828 90 kDa, polypeptide 1 AS p90Rsk
ribosomal protein S6 kinase, 530003 669 432281 1829 90 kDa,
polypeptide 1 AS p90Rsk ribosomal protein S6 kinase, 531382 670
435412 1830 90 kDa, polypeptide 1 AS PAK2 p21 protein (Cdc42/Rac)-
327134 671 314067 1831 activated kinase 2 AS PARP-1 poly
(ADP-ribose) polymerase 1 366790 672 355755 1832 AS PARP-1 poly
(ADP-ribose) polymerase 1 366791 673 355756 1833 AS PARP-1 poly
(ADP-ribose) polymerase 1 366792 674 355757 1834 AS PARP-1 poly
(ADP-ribose) polymerase 1 366794 675 355759 1835 AS PARP-1 poly
(ADP-ribose) polymerase 1 432338 676 412774 1836 AS PDPK1
3-phosphoinositide dependent 342085 677 344220 1837 protein
kinase-1 AS PDPK1 3-phosphoinositide dependent 354836 678 346895
1838 protein kinase-1 AS PDPK1 3-phosphoinositide dependent 441549
679 395357 1839 protein kinase-1 AS PI3K phosphoinositide-3-kinase,
263967 680 263967 1840 catalytic, alpha polypeptide AS PI3K
phosphoinositide-3-kinase, 289153 681 289153 1841 catalytic, beta
polypeptide AS PI3K phosphoinositide-3-kinase, 359195 682 352121
1842 catalytic, gamma polypeptide AS PI3K
phosphoinositide-3-kinase, 360563 683 353766 1843 catalytic, delta
polypeptide AS PI3K phosphoinositide-3-kinase, 361110 684 354410
1844 catalytic, delta polypeptide AS PI3K
phosphoinositide-3-kinase, 377346 685 366563 1845 catalytic, delta
polypeptide AS PI3K phosphoinositide-3-kinase, 440650 686 392258
1846 catalytic, gamma polypeptide AS PI3K
phosphoinositide-3-kinase, 461451 687 420399 1847 catalytic, beta
polypeptide AS PI3K phosphoinositide-3-kinase, 468036 688 417479
1848 catalytic, alpha polypeptide AS PI3K
phosphoinositide-3-kinase, 477593 689 418143 1849 catalytic, beta
polypeptide AS PI3K phosphoinositide-3-kinase, 483968 690 419857
1850 catalytic, beta polypeptide AS PI3K phosphoinositide-3-kinase,
493568 691 417869 1851 catalytic, beta polypeptide AS PI3K
phosphoinositide-3-kinase, 496166 692 419260 1852 catalytic, gamma
polypeptide AS PI3K phosphoinositide-3-kinase, 536656 693 446444
1853 catalytic, delta polypeptide AS PI3K
phosphoinositide-3-kinase, 543390 694 443811 1854 catalytic, delta
polypeptide AS PI3K phosphoinositide-3-kinase, 544716 695 438259
1855 catalytic, beta polypeptide AS PKA-cat protein kinase, cAMP-
308677 696 309591 1856 dependent, catalytic, alpha AS PKA-cat
protein kinase, cAMP- 350356 697 340940 1857 dependent, catalytic,
alpha AS PKA-cat protein kinase, cAMP- 370679 698 359713 1858
dependent, catalytic, beta AS PKA-cat protein kinase, cAMP- 370680
699 359714 1859 dependent, catalytic, beta AS PKA-cat protein
kinase, cAMP- 370681 700 359715 1860 dependent, catalytic, beta AS
PKA-cat protein kinase, cAMP- 370682 701 359716 1861 dependent,
catalytic, beta AS PKA-cat protein kinase, cAMP- 370684 702 359718
1862 dependent, catalytic, beta AS PKA-cat protein kinase, cAMP-
370685 703 359719 1863 dependent, catalytic, beta AS PKA-cat
protein kinase, cAMP- 370688 704 359722 1864 dependent, catalytic,
beta AS PKA-cat protein kinase, cAMP- 370689 705 359723 1865
dependent, catalytic, beta AS PKA-cat protein kinase, cAMP- 377276
706 366488 1866 dependent, catalytic, gamma AS PKA-cat protein
kinase, cAMP- 394838 707 378314 1867 dependent, catalytic, beta AS
PKA-cat protein kinase, cAMP- 394839 708 378315 1868 dependent,
catalytic, beta AS PKA-cat protein kinase, cAMP- 413538 709 397175
1869 dependent, catalytic, beta AS PKA-cat protein kinase, cAMP-
417530 710 399326 1870 dependent, catalytic, beta AS PKA-cat
protein kinase, cAMP- 432111 711 392275 1871 dependent, catalytic,
beta AS PKA-cat protein kinase, cAMP- 436133 712 390906 1872
dependent, catalytic, beta AS PKA-cat protein kinase, cAMP- 446538
713 401252 1873 dependent, catalytic, beta AS PKA-cat protein
kinase, cAMP- 450730 714 393654 1874 dependent, catalytic, beta AS
PKA-cat protein kinase, cAMP- 535695 715 441654 1875 dependent,
catalytic, alpha AS PKA-cat protein kinase, cAMP- 536649 716 440418
1876 dependent, catalytic, alpha AS PKC- protein kinase C, delta
330452 717 331602 1877 delta AS PKC- protein kinase C, delta 394729
718 378217 1878 delta AS PKC- protein kinase C, delta 478843 719
419726 1879 delta AS PKC- protein kinase C, delta 487897 720 418106
1880 delta AS PKC- protein kinase C, zeta 378567 721 367830 1881
Zeta AS PKC- protein kinase C, zeta 400920 722 383711 1882 Zeta AS
PKC- protein kinase C, zeta 400921 723 383712 1883 Zeta AS PKC-
protein kinase C, zeta 461106 724 426412 1884 Zeta AS PKC- protein
kinase C, zeta 470511 725 421350 1885 Zeta AS PKC- protein kinase
C, zeta 470596 726 424228 1886 Zeta AS PKC- protein kinase C, zeta
470986 727 421219 1887 Zeta AS PKC- protein kinase C, zeta 482686
728 425317 1888 Zeta AS PKC- protein kinase C, zeta 496325 729
421869 1889 Zeta AS PP1-cat protein phosphatase 1, catalytic 312989
730 326031 1890 alpha subunit, alpha isozyme AS PP1-cat protein
phosphatase 1, catalytic 376745 731 365936 1891 alpha subunit,
alpha isozyme AS PP1-cat protein phosphatase 1, catalytic 451458
732 405603 1892 alpha subunit, alpha isozyme AS PP2a protein
phosphatase 2, catalytic 481195 733 418447 1893 catalytic subunit,
alpha isozyme AS PP2C protein phosphatase, 228705 734 228705 1894
Mg2+/Mn2+ dependent, 1H AS PP2C protein phosphatase, 263212 735
263212 1895 Mg2+/Mn2+ dependent, 1F AS PP2C protein phosphatase,
282412 736 282412 1896 Mg2+/Mn2+ dependent, 1B AS PP2C protein
phosphatase, 295908 737 295908 1897 Mg2+/Mn2+ dependent, 1K AS PP2C
protein phosphatase, 296487 738 296487 1898 Mg2+/Mn2+ dependent, 1M
AS PP2C protein phosphatase, 305921 739 306682 1899 Mg2+/Mn2+
dependent, 1D AS PP2C protein phosphatase, 308249 740 312411
1900
Mg2+/Mn2+ dependent, 1E AS PP2C protein phosphatase, 309276 741
308926 1901 Mg2+/Mn2+ dependent, 1J AS PP2C protein phosphatase,
315194 742 324761 1902 Mg2+/Mn2+ dependent, 1K AS PP2C protein
phosphatase, 323588 743 319894 1903 Mg2+/Mn2+ dependent, 1M AS PP2C
protein phosphatase, 324688 744 321761 1904 Mg2+/Mn2+ dependent, 1N
(putative) AS PP2C protein phosphatase, 325642 745 327255 1905
Mg2+/Mn2+ dependent, 1A AS PP2C protein phosphatase, 325658 746
314850 1906 Mg2+/Mn2+ dependent, 1A AS PP2C protein phosphatase,
344034 747 342778 1907 Mg2+/Mn2+ dependent, 1G AS PP2C protein
phosphatase, 345249 748 326089 1908 Mg2+/Mn2+ dependent, 1B AS PP2C
protein phosphatase, 350803 749 264714 1909 Mg2+/Mn2+ dependent, 1G
AS PP2C protein phosphatase, 359994 750 353088 1910 Mg2+/Mn2+
dependent, 1J AS PP2C protein phosphatase, 378551 751 367813 1911
Mg2+/Mn2+ dependent, 1B AS PP2C protein phosphatase, 392995 752
376720 1912 Mg2+/Mn2+ dependent, 1D AS PP2C protein phosphatase,
395076 753 378514 1913 Mg2+/Mn2+ dependent, 1A AS PP2C protein
phosphatase, 395543 754 378913 1914 Mg2+/Mn2+ dependent, 1G AS PP2C
protein phosphatase, 396734 755 379960 1915 Mg2+/Mn2+ dependent, 1N
(putative) AS PP2C protein phosphatase, 397495 756 380632 1916
Mg2+/Mn2+ dependent, 1F AS PP2C protein phosphatase, 406981 757
384715 1917 Mg2+/Mn2+ dependent, 1F AS PP2C protein phosphatase,
407142 758 384930 1918 Mg2+/Mn2+ dependent, 1F AS PP2C protein
phosphatase, 409432 759 387287 1919 Mg2+/Mn2+ dependent, 1B AS PP2C
protein phosphatase, 409502 760 387046 1920 Mg2+/Mn2+ dependent, 1M
AS PP2C protein phosphatase, 409895 761 387341 1921 Mg2+/Mn2+
dependent, 1B AS PP2C protein phosphatase, 419807 762 390087 1922
Mg2+/Mn2+ dependent, 1B AS PP2C protein phosphatase, 443121 763
390257 1923 Mg2+/Mn2+ dependent, 1E AS PP2C protein phosphatase,
457351 764 393747 1924 Mg2+/Mn2+ dependent, 1M AS PP2C protein
phosphatase, 497343 765 420354 1925 Mg2+/Mn2+ dependent, 1L AS PP2C
protein phosphatase, 498165 766 417659 1926 Mg2+/Mn2+ dependent, 1L
AS PP2C protein phosphatase, 506423 767 424155 1927 Mg2+/Mn2+
dependent, 1K AS PP2C protein phosphatase, 525399 768 435398 1928
Mg2+/Mn2+ dependent, 1A AS PP2C protein phosphatase, 528241 769
431453 1929 Mg2+/Mn2+ dependent, 1A AS PP2C protein phosphatase,
529574 770 432966 1930 Mg2+/Mn2+ dependent, 1A AS PP2C protein
phosphatase, 531937 771 435575 1931 Mg2+/Mn2+ dependent, 1A AS PP2C
protein phosphatase, 538191 772 439915 1932 Mg2+/Mn2+ dependent, 1F
AS PP2C protein phosphatase, 544412 773 442536 1933 Mg2+/Mn2+
dependent, 1G AS PP2C protein phosphatase, 544712 774 438518 1934
Mg2+/Mn2+ dependent, 1D AS Puma BCL2 binding component 3 300880 775
300880 1935 AS Puma BCL2 binding component 3 341983 776 341155 1936
AS Puma BCL2 binding component 3 439096 777 395862 1937 AS Puma
BCL2 binding component 3 449228 778 404503 1938 AS RAIDD CASP2 and
RIPK1 domain 332896 779 327647 1939 containing adaptor with death
domain AS RAIDD CASP2 and RIPK1 domain 541813 780 442624 1940
containing adaptor with death domain AS RAIDD CASP2 and RIPK1
domain 542893 781 439068 1941 containing adaptor with death domain
AS RAIDD CASP2 and RIPK1 domain 551065 782 448425 1942 containing
adaptor with death domain AS RANK tumor necrosis factor receptor
269485 783 269485 1943 superfamily, member 11a, NFKB activator AS
RANK tumor necrosis factor receptor 382790 784 372240 1944
superfamily, member 11a, NFKB activator AS RANKL tumor necrosis
factor (ligand) 239849 785 239849 1945 superfamily, member 11 AS
RANKL tumor necrosis factor (ligand) 358545 786 351347 1946
superfamily, member 11 AS RANKL tumor necrosis factor (ligand)
398795 787 381775 1947 superfamily, member 11 AS RANKL tumor
necrosis factor (ligand) 405262 788 384042 1948 superfamily, member
11 AS RANKL tumor necrosis factor (ligand) 544862 789 444913 1949
superfamily, member 11 AS ReIA v-rel reticuloendotheliosis viral
308639 790 311508 1950 (p65 oncogene homolog A (avian) NF- kappaB
subunit) AS ReIA v-rel reticuloendotheliosis viral 406246 791
384273 1951 (p65 oncogene homolog A (avian) NF- kappaB subunit) AS
ReIA v-rel reticuloendotheliosis viral 426617 792 437980 1952 (p65
oncogene homolog A (avian) NF- kappaB subunit) AS ReIA v-rel
reticuloendotheliosis viral 525693 793 432537 1953 (p65 oncogene
homolog A (avian) NF- kappaB subunit) AS ReIA v-rel
reticuloendotheliosis viral 526283 794 435290 1954 (p65 oncogene
homolog A (avian) NF- kappaB subunit) AS ReIA v-rel
reticuloendotheliosis viral 545816 795 443700 1955 (p65 oncogene
homolog A (avian) NF- kappaB subunit) AS RIPK1 receptor
(TNFRSF)-interacting 259808 796 259808 1956 serine-threonine kinase
1 AS RIPK1 receptor (TNFRSF)-interacting 380409 797 369773 1957
serine-threonine kinase 1 AS RIPK1 receptor (TNFRSF)-interacting
453483 798 415981 1958 serine-threonine kinase 1 AS RIPK1 receptor
(TNFRSF)-interacting 541791 799 442294 1959 serine-threonine kinase
1 AS Sequestosome 1 sequestosome 1 360718 800 353944 1960 (p62) AS
Sequestosome 1 sequestosome 1 376929 801 366128 1961 (p62) AS
Sequestosome 1 sequestosome 1 389805 802 374455 1962 (p62) AS
Sequestosome 1 sequestosome 1 402874 803 385553 1963 (p62) AS
Sequestosome 1 sequestosome 1 422245 804 394534 1964 (p62) AS
Sequestosome 1 sequestosome 1 454378 805 408107 1965 (p62) AS
Sequestosome 1 sequestosome 1 514093 806 427308 1966 (p62) AS Shc
SHC (Src homology 2 domain 264554 807 264554 1967 containing)
transforming protein 2 AS Shc SHC (Src homology 2 domain 366442 808
396162 1968 containing) transforming protein 1 AS Shc SHC (Src
homology 2 domain 368441 809 357426 1969 containing) transforming
protein 1 AS Shc SHC (Src homology 2 domain 368443 810 357428 1970
containing) transforming protein 1 AS Shc SHC (Src homology 2
domain 368445 811 357430 1971 containing) transforming protein 1 AS
Shc SHC (Src homology 2 domain 368449 812 357434 1972 containing)
transforming protein 1 AS Shc SHC (Src homology 2 domain 368450 813
357435 1973 containing) transforming protein 1 AS Shc SHC (Src
homology 2 domain 368453 814 357438 1974 containing) transforming
protein 1 AS Shc SHC (Src homology 2 domain 375830 815 364990 1975
containing) transforming protein 3 AS Shc SHC (Src homology 2
domain 375831 816 364991 1976 containing) transforming protein 3 AS
Shc SHC (Src homology 2 domain 375835 817 364995 1977 containing)
transforming protein 3 AS Shc SHC (Src homology 2 domain 412170 818
398441 1978 containing) transforming protein 1 AS Shc SHC (Src
homology 2 domain 414115 819 404908 1979 containing) transforming
protein 1 AS Shc SHC (Src homology 2 domain 444179 820 398864 1980
containing) transforming protein 1 AS Shc SHC (Src homology 2
domain 444664 821 396333 1981 containing) transforming protein 1 AS
Shc SHC (Src homology 2 domain 448116 822 401303 1982 containing)
transforming protein 1 AS Siah-1 seven in absentia homolog 1 356721
823 349156 1983 (Drosophila) AS Siah-1 seven in absentia homolog 1
380006 824 369343 1984 (Drosophila) AS Siah-1 seven in absentia
homolog 1 394725 825 378214 1985 (Drosophila) AS SMAC diablo,
IAP-binding NA 826 NA 1986 mitochondrial protein AS Smac/Diablo
diablo, IAP-binding 267169 827 267169 1987 mitochondrial protein AS
Smac/Diablo diablo, IAP-binding 353548 828 320343 1988
mitochondrial protein AS Smac/Diablo diablo, IAP-binding 413918 829
411638 1989 mitochondrial protein AS Smac/Diablo diablo,
IAP-binding 443649 830 398495 1990 mitochondrial protein AS
Smac/Diablo diablo, IAP-binding 464942 831 442360 1991
mitochondrial protein AS SODD BCL2-associated athanogene 4 287322
832 287322 1992 AS SODD BCL2-associated athanogene 4 432471 833
393298 1993 AS SOS son of sevenless homolog 2 216373 834 216373
1994 (Drosophila) AS SOS son of sevenless homolog 1 263879 835
263879 1995 (Drosophila) AS SOS son of sevenless homolog 1 395038
836 378479 1996 (Drosophila) AS SOS son of sevenless homolog 1
402219 837 384675 1997 (Drosophila) AS SOS son of sevenless homolog
1 426016 838 387784 1998 (Drosophila) AS SOS son of sevenless
homolog 1 428721 839 399992 1999 (Drosophila) AS SOS son of
sevenless homolog 2 543680 840 445328 2000 (Drosophila) AS SOS son
of sevenless homolog 1 543698 841 441172 2001 (Drosophila) AS
SUMO-1 SMT3 suppressor of mif two 3 392244 842 376075 2002 homolog
1 (S. cerevisiae) AS SUMO-1 SMT3 suppressor of mif two 3 392245 843
376076 2003 homolog 1 (S. cerevisiae) AS SUMO-1 SMT3 suppressor of
mif two 3 392246 844 376077 2004 homolog 1 (S. cerevisiae) AS
SUMO-1 SMT3 suppressor of mif two 3 409205 845 386267 2005 homolog
1 (S. cerevisiae) AS SUMO-1 SMT3 suppressor of mif two 3 409498 846
386472 2006 homolog 1 (S. cerevisiae) AS Survivin baculoviral IAP
repeat 301633 847 301633 2007 containing 5
AS Survivin baculoviral IAP repeat 350051 848 324180 2008
containing 5 AS Survivin baculoviral IAP repeat 374948 849 364086
2009 containing 5 AS Survivin baculoviral IAP repeat 432014 850
389088 2010 containing 5 AS TACI tumor necrosis factor receptor
261652 851 261652 2011 superfamily, member 13B AS TACI tumor
necrosis factor receptor 437538 852 413453 2012 superfamily, member
13B AS tBid tBID NA NA 2013 AS TL1A tumor necrosis factor (ligand)
374044 853 363156 2014 superfamily, member 15 AS TL1A tumor
necrosis factor (ligand) 374045 854 363157 2015 superfamily, member
15 AS TNF- tumor necrosis factor 376122 855 365290 2016 alpha AS
TNF- tumor necrosis factor 383496 856 372988 2017 alpha AS TNF-
tumor necrosis factor 412275 857 392858 2018 alpha AS TNF- tumor
necrosis factor 420425 858 410668 2019 alpha AS TNF- tumor necrosis
factor 443707 859 389492 2020 alpha AS TNF- tumor necrosis factor
445232 860 389265 2021 alpha AS TNF- tumor necrosis factor 448781
861 389490 2022 alpha AS TNF- tumor necrosis factor 449264 862
398698 2023 alpha AS TNF-R1 tumor necrosis factor receptor 162749
863 162749 2024 superfamily, member 1A AS TNF-R1 tumor necrosis
factor receptor 366159 864 380389 2025 superfamily, member 1A AS
TNF-R2 tumor necrosis factor receptor 376259 865 365435 2026
superfamily, member 1B AS TNF-R2 tumor necrosis factor receptor
376259 866 365435 2027 2491 superfamily, member 1B AS TNF-R2 tumor
necrosis factor receptor 400863 867 383660 2028 superfamily, member
1B AS TNF-R2 tumor necrosis factor receptor 536782 868 440425 2029
superfamily, member 1B AS TRADD TNFRSF1A-associated via 345057 869
341268 2030 death domain AS TRAF2 TNF receptor-associated factor 2
247668 870 247668 2031 AS TRAF2 TNF receptor-associated factor 2
359662 871 352685 2032 AS TRAF2 TNF receptor-associated factor 2
371645 872 360708 2033 AS TRAF2 TNF receptor-associated factor 2
414589 873 397653 2034 AS TRAF2 TNF receptor-associated factor 2
419057 874 405860 2035 AS TRAF2 TNF receptor-associated factor 2
429509 875 406524 2036 AS TRAF2 TNF receptor-associated factor 2
432785 876 400061 2037 AS TRAF2 TNF receptor-associated factor 2
536468 877 446414 2038 AS TRAF3 TNF receptor-associated factor 2
347662 878 328003 2039 AS TRAF3 TNF receptor-associated factor 3
351691 879 332468 2040 AS TRAF3 TNF receptor-associated factor 3
392745 880 376500 2041 AS TRAF3 TNF receptor-associated factor 3
539721 881 445998 2042 AS TRAF3 TNF receptor-associated factor 3
560371 882 454207 2043 AS TRAF3 TNF receptor-associated factor 3
560463 883 453623 2044 AS TRAF5 TNF receptor-associated factor 5
261464 884 261464 2045 AS TRAF5 TNF receptor-associated factor 5
336184 885 336825 2046 AS TRAF5 TNF receptor-associated factor 5
367004 886 355971 2047 AS TRAF5 TNF receptor-associated factor 5
427925 887 389891 2048 AS TRAF6 TNF receptor-associated factor 6
348124 888 337853 2049 AS TRAF6 TNF receptor-associated factor 6
526995 889 433623 2050 AS TrkA neurotrophic tyrosine kinase, 368196
890 357179 2051 receptor, type 1 AS TrkA neurotrophic tyrosine
kinase, 392302 891 376120 2052 receptor, type 1 AS TrkA
neurotrophic tyrosine kinase, 524377 892 431418 2053 receptor, type
1 AS TWEAK tumor necrosis factor (ligand) 293825 893 293825 2054
(TNFSF12) superfamily, member 12 AS TWEAK tumor necrosis factor
(ligand) 557233 894 451451 2055 (TNFSF12) superfamily, member 12 AS
VDAC 1 voltage-dependent anion 265333 895 265333 2056 channel 1 AS
VDAC 1 voltage-dependent anion 395044 896 378484 2057 channel 1 AS
VDAC 1 voltage-dependent anion 395047 897 378487 2058 channel 1 AS
VDAC 2 voltage-dependent anion 298468 898 298468 2059 channel 2 AS
VDAC 2 voltage-dependent anion 313132 899 361635 2060 channel 2 AS
VDAC 2 voltage-dependent anion 332211 900 361686 2061 channel 2 AS
VDAC 2 voltage-dependent anion 344036 901 344876 2062 channel 2 AS
VDAC 2 voltage-dependent anion 413289 902 389551 2063 channel 2 AS
VDAC 2 voltage-dependent anion 447677 903 401492 2064 channel 2 AS
VDAC 2 voltage-dependent anion 535553 904 445901 2065 channel 2 AS
VDAC 2 voltage-dependent anion 543351 905 443092 2066 channel 2 AS
XIAP X-linked inhibitor of apoptosis 355640 906 347858 2067 AS XIAP
X-linked inhibitor of apoptosis 371199 907 360242 2068 AS XIAP
X-linked inhibitor of apoptosis 430625 908 400637 2069 AS XIAP
X-linked inhibitor of apoptosis 434753 909 395230 2070 AS XIAP
X-linked inhibitor of apoptosis NA 910 NA 2071 CC/S ATM ataxia
telangiectasia mutated 278616 911 278616 2072 CC/S ATM ataxia
telangiectasia mutated 389511 912 374162 2073 CC/S ATM ataxia
telangiectasia mutated 452508 913 388058 2074 CC/S ATM ataxia
telangiectasia mutated 532931 914 432318 2075 CC/S ATR ataxia
telangiectasia and Rad3 350721 915 343741 2076 related CC/S ATR
ataxia telangiectasia and Rad3 383101 916 372581 2077 related CC/S
ATRIP ATR interacting protein 320211 917 323099 2078 CC/S ATRIP ATR
interacting protein 346691 918 302338 2079 CC/S ATRIP ATR
interacting protein 357105 919 349620 2080 CC/S ATRIP ATR
interacting protein 412052 920 400930 2081 CC/S ATRIP ATR
interacting protein 421175 921 406664 2082 CC/S Bard1 BRCA1
associated RING 260947 922 260947 2083 domain 1 CC/S Bard1 BRCA1
associated RING 449967 923 406752 2084 domain 1 CC/S BLM Bloom
syndrome, RecQ 355112 924 347232 2085 helicase-like CC/S BLM Bloom
syndrome, RecQ 536925 925 442330 2086 helicase-like CC/S BLM Bloom
syndrome, RecQ 543977 926 439075 2087 helicase-like CC/S Brca1
breast cancer 1, early onset 309486 927 310938 2088 CC/S Brca1
breast cancer 1, early onset 346315 928 246907 2089 CC/S Brca1
breast cancer 1, early onset 351666 929 338007 2090 CC/S Brca1
breast cancer 1, early onset 352993 930 312236 2091 CC/S Brca1
breast cancer 1, early onset 354071 931 326002 2092 CC/S Brca1
breast cancer 1, early onset 357654 932 350283 2093 CC/S Brca1
breast cancer 1, early onset 393691 933 377294 2094 CC/S Brca1
breast cancer 1, early onset 412061 934 397145 2095 CC/S Brca1
breast cancer 1, early onset 461221 935 418548 2096 CC/S Brca1
breast cancer 1, early onset 461798 936 417988 2097 CC/S Brca1
breast cancer 1, early onset 468300 937 417148 2098 CC/S Brca1
breast cancer 1, early onset 470026 938 419274 2099 CC/S Brca1
breast cancer 1, early onset 471181 939 418960 2100 CC/S Brca1
breast cancer 1, early onset 476777 940 417554 2101 CC/S Brca1
breast cancer 1, early onset 477152 941 419988 2102 CC/S Brca1
breast cancer 1, early onset 478531 942 420412 2103 CC/S Brca1
breast cancer 1, early onset 484087 943 419481 2104 CC/S Brca1
breast cancer 1, early onset 489037 944 420781 2105 CC/S Brca1
breast cancer 1, early onset 491747 945 420705 2106 CC/S Brca1
breast cancer 1, early onset 492859 946 420253 2107 CC/S Brca1
breast cancer 1, early onset 493795 947 418775 2108 CC/S Brca1
breast cancer 1, early onset 493919 948 418819 2109 CC/S Brca1
breast cancer 1, early onset 494123 949 419103 2110 CC/S Brca1
breast cancer 1, early onset 497488 950 418986 2111 CC/S c-Abl
c-abl oncogene 1, non-receptor 318560 951 323315 2112 tyrosine
kinase CC/S c-Abl c-abl oncogene 1, non-receptor 372348 952 361423
2113 tyrosine kinase CC/S c-Abl c-abl oncogene 1, non-receptor
393293 953 376971 2114 tyrosine kinase CC/S c-Abl c-abl oncogene 1,
non-receptor 438426 954 407756 2115 tyrosine kinase CC/S c-Abl
c-abl oncogene 1, non-receptor 444970 955 400412 2116 tyrosine
kinase CC/S CDC25A cell division cycle 25 homolog 302506 956 303706
2117 A (S. pombe) CC/S CDC25A cell division cycle 25 homolog 351231
957 343166 2118 A (S. pombe) CC/S CDC25A cell division cycle 25
homolog 437972 958 404285 2119 A (S. pombe) CC/S CDC25B cell
division cycle 25 homolog 245960 959 245960 2120 B (S. pombe) CC/S
CDC25B cell division cycle 25 homolog 340833 960 339170 2121 B (S.
pombe) CC/S CDC25B cell division cycle 25 homolog 344256 961 339125
2122 B (S. pombe) CC/S CDC25B cell division cycle 25 homolog 379598
962 368918 2123 B (S. pombe) CC/S CDC25B cell division cycle 25
homolog 439880 963 405972 2124 B (S. pombe) CC/S CDC25C cell
division cycle 25 homolog 323760 964 321656 2125 C (S. pombe) CC/S
CDC25C cell division cycle 25 homolog 348983 965 345205 2126 C (S.
pombe) CC/S CDC25C cell division cycle 25 homolog 356505 966 348898
2127 C (S. pombe) CC/S CDC25C cell division cycle 25 homolog 357274
967 349821 2128 C (S. pombe) CC/S CDC25C cell division cycle 25
homolog 415130 968 392631 2129 C (S. pombe) CC/S CDC25C cell
division cycle 25 homolog 503022 969 427251 2130 C (S. pombe) CC/S
CDC25C cell division cycle 25 homolog 513970 970 424795 2131 C (S.
pombe) CC/S CDC25C cell division cycle 25 homolog 534892 971 443196
2132 C (S. pombe) CC/S CDK2 cyclin-dependent kinase 2 266970 972
266970 2133 CC/S CDK2 cyclin-dependent kinase 2 354056 973 243067
2134 CC/S CDK4 cyclin-dependent kinase 4 257904 974 257904 2135
CC/S CDK4 cyclin-dependent kinase 4 312990 975 316889 2136 CC/S
CDK4 cyclin-dependent kinase 4 540325 976 439076 2137 CC/S CDK4
cyclin-dependent kinase 4 552254 977 449179 2138 CC/S CDK4
cyclin-dependent kinase 4 552388 978 448963 2139 CC/S CDK4
cyclin-dependent kinase 4 552862 979 446763 2140 CC/S CDK6
cyclin-dependent kinase 6 265734 980 265734 2141 CC/S CDK6
cyclin-dependent kinase 6 424848 981 397087 2142 CC/S Chk1
checkpoint kinase 1 278916 982 278916 2143 CC/S Chk1 checkpoint
kinase 1 428830 983 412504 2144 CC/S Chk1 checkpoint kinase 1
438015 984 388648 2145 CC/S Chk1 checkpoint kinase 1 524737 985
432890 2146 CC/S Chk1 checkpoint kinase 1 525396 986 434141 2147
CC/S Chk1 checkpoint kinase 1 526937 987 431815 2148 CC/S Chk1
checkpoint kinase 1 527013 988 431525 2149 CC/S Chk1 checkpoint
kinase 1 534070 989 435371 2150 CC/S Chk1 checkpoint kinase 1
534685 990 432470 2151 CC/S Chk1 checkpoint kinase 1 544373 991
442317 2152 CC/S Chk2 checkpoint kinase 2 328354 992 329178 2153
CC/S Chk2 checkpoint kinase 2 348295 993 329012 2154 CC/S Chk2
checkpoint kinase 2 382563 994 372003 2155 CC/S Chk2 checkpoint
kinase 2 382565 995 372006 2156 CC/S Chk2 checkpoint kinase 2
382566 996 372007 2157 CC/S Chk2 checkpoint kinase 2 382578 997
372021 2158 CC/S Chk2 checkpoint kinase 2 382580 998 372023 2159
CC/S Chk2 checkpoint kinase 2 402731 999 384835 2160 CC/S Chk2
checkpoint kinase 2 403642 1000 384919 2161 CC/S Chk2 checkpoint
kinase 2 404276 1001 385747 2162 CC/S Chk2 checkpoint kinase 2
405598 1002 386087 2163 CC/S Chk2 checkpoint kinase 2 544772 1003
442458 2164 CC/S Claspin claspin 251195 1004 251195 2165 CC/S
Claspin claspin 318121 1005 312995 2166 CC/S Claspin claspin 373220
1006 362317 2167 CC/S Claspin claspin 544356 1007 442335 2168 CC/S
Cyclin A cyclin A2 274026 1008 274026 2169 CC/S Cyclin B cyclin B1
256442 1009 256442 2170 CC/S Cyclin B cyclin B3 276014 1010 276014
2171 CC/S Cyclin B cyclin B2 288207 1011 288207 2172 CC/S Cyclin B
cyclin B3 348603 1012 338682 2173 CC/S Cyclin B cyclin B3 376038
1013 365206 2174 CC/S Cyclin B cyclin B3 376042 1014 365210 2175
CC/S Cyclin B cyclin B3 396540 1015 379790 2176 CC/S Cyclin B
cyclin B1 505500 1016 424588 2177 CC/S Cyclin B cyclin B1 506572
1017 423387 2178 CC/S Cyclin D cyclin D1 227507 1018 227507 2179
CC/S Cyclin D cyclin D2 261254 1019 261254 2180 CC/S Cyclin D
cyclin D3 372987 1020 362078 2181 CC/S Cyclin D cyclin D3 372988
1021 362079 2182 CC/S Cyclin D cyclin D3 372991 1022 362082 2183
CC/S Cyclin D cyclin D3 414200 1023 397545 2184 CC/S Cyclin D
cyclin D3 415497 1024 401595 2185 CC/S Cyclin D cyclin D3 505064
1025 425830 2186 CC/S Cyclin D cyclin D3 511642 1026 426212 2187
CC/S Cyclin D cyclin D1 542897 1027 441863 2188 CC/S Cyclin E
cyclin E1 262643 1028 262643 2189 CC/S Cyclin E cyclin E2 308108
1029 309181 2190 CC/S Cyclin E cyclin E1 357943 1030 350625 2191
CC/S Cyclin E cyclin E2 396133 1031 379437 2192
CC/S Cyclin E cyclin E1 444983 1032 410179 2193 CC/S Cyclin E
cyclin E2 520509 1033 429089 2194 CC/S Cyclin E cyclin E2 542725
1034 445726 2195 CC/S DNA- protein kinase, DNA-activated, 314191
1035 313420 2196 PK catalytic polypeptide CC/S DNA- protein kinase,
DNA-activated, 338368 1036 345182 2197 PK catalytic polypeptide
CC/S E2F1/2/ E2F transcription factor 5, 256117 1037 256117 2198
3/4/5/6 p130-binding CC/S E2F1/2/ E2F transcription factor 6 307236
1038 302159 2199 3/4/5/6 CC/S E2F1/2/ E2F transcription factor 1
343380 1039 345571 2200 3/4/5/6 CC/S E2F1/2/ E2F transcription
factor 3 346618 1040 262904 2201 3/4/5/6 CC/S E2F1/2/ E2F
transcription factor 2 361729 1041 355249 2202 3/4/5/6 CC/S E2F1/2/
E2F transcription factor 6 362009 1042 355036 2203 3/4/5/6 CC/S
E2F1/2/ E2F transcription factor 3 378646 1043 367914 2204 3/4/5/6
CC/S E2F1/2/ E2F transcription factor 4, 379378 1044 368686 2205
3/4/5/6 p107/p130-binding CC/S E2F1/2/ E2F transcription factor 6
381525 1045 370936 2206 3/4/5/6 CC/S E2F1/2/ E2F transcription
factor 5, 416274 1046 398124 2207 3/4/5/6 p130-binding CC/S E2F1/2/
E2F transcription factor 5, 418930 1047 414312 2208 3/4/5/6
p130-binding CC/S E2F1/2/ E2F transcription factor 5, 517476 1048
429120 2209 3/4/5/6 p130-binding CC/S E2F1/2/ E2F transcription
factor 5, 518234 1049 429669 2210 3/4/5/6 p130-binding CC/S E2F1/2/
E2F transcription factor 3 535432 1050 443418 2211 3/4/5/6 CC/S
E2F1/2/ E2F transcription factor 6 542100 1051 446315 2212 3/4/5/6
CC/S E2F1/2/ E2F transcription factor 6 546212 1052 438864 2213
3/4/5/6 CC/S FANCD2 Fanconi anemia, 287647 1053 287647 2214
complementation group D2 CC/S FANCD2 Fanconi anemia, 383806 1054
373317 2215 complementation group D2 CC/S FANCD2 Fanconi anemia,
383807 1055 373318 2216 complementation group D2 CC/S FANCD2
Fanconi anemia, 419585 1056 398754 2217 complementation group D2
CC/S FANCL Fanconi anemia, 233741 1057 233741 2218 complementation
group L CC/S FANCL Fanconi anemia, 540646 1058 441431 2219
complementation group L CC/S GADD4 growth arrest and DNA- 370986
1059 360025 2220 5 alpha damage-inducible, alpha CC/S GADD4 growth
arrest and DNA- 215631 1060 215631 2221 5 beta damage-inducible,
beta CC/S GADD4 growth arrest and DNA- 370985 1061 360024 2222 5
beta damage-inducible, alpha CC/S MDM2 Mdm2 p53 binding protein
258148 1062 258148 2223 homolog (mouse) CC/S MDM2 Mdm2 p53 binding
protein 258149 1063 258149 2224 homolog (mouse) CC/S MDM2 Mdm2 p53
binding protein 299252 1064 299252 2225 homolog (mouse) CC/S MDM2
Mdm2 p53 binding protein 311420 1065 310742 2226 homolog (mouse)
CC/S MDM2 Mdm2 p53 binding protein 311440 1066 311302 2227 homolog
(mouse) CC/S MDM2 Mdm2 p53 binding protein 348801 1067 335096 2228
homolog (mouse) CC/S MDM2 Mdm2 p53 binding protein 350057 1068
266624 2229 homolog (mouse) CC/S MDM2 Mdm2 p53 binding protein
356290 1069 348637 2230 homolog (mouse) CC/S MDM2 Mdm2 p53 binding
protein 358483 1070 351270 2231 homolog (mouse) CC/S MDM2 Mdm2 p53
binding protein 360430 1071 353611 2232 homolog (mouse) CC/S MDM2
Mdm2 p53 binding protein 393410 1072 377062 2233 homolog (mouse)
CC/S MDM2 Mdm2 p53 binding protein 393412 1073 377064 2234 homolog
(mouse) CC/S MDM2 Mdm2 p53 binding protein 393413 1074 377065 2235
homolog (mouse) CC/S MDM2 Mdm2 p53 binding protein 393415 1075
377067 2236 homolog (mouse) CC/S MDM2 Mdm2 p53 binding protein
428863 1076 410694 2237 homolog (mouse) CC/S MDM2 Mdm2 p53 binding
protein 462284 1077 417281 2238 homolog (mouse) CC/S MDM2 Mdm2 p53
binding protein 517852 1078 430257 2239 homolog (mouse) CC/S MDM2
Mdm2 p53 binding protein 539479 1079 444430 2240 homolog (mouse)
CC/S MDM2 Mdm2 p53 binding protein 540827 1080 440932 2241 homolog
(mouse) CC/S MDM2 Mdm2 p53 binding protein 544648 1081 443274 2242
homolog (mouse) CC/S NFBD1 mediator of DNA-damage 376405 1082
365587 2243 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 376406
1083 365588 2244 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
383566 1084 373060 2245 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 412395 1085 392833 2246 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 413973 1086 408831 2247 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 416368 1087 410383 2248 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 416571 1088 400979 2249 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 417033 1089 408962 2250
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 417228 1090 400305
2251 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 419172 1091
398474 2252 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 419675
1092 397642 2253 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
420019 1093 396484 2254 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 420320 1094 416511 2255 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 422104 1095 390375 2256 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 422195 1096 407703 2257 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 422266 1097 411310 2258 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 423726 1098 391230 2259
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 424437 1099 398151
2260 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 424507 1100
388355 2261 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 424638
1101 394074 2262 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
425029 1102 397126 2263 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 425072 1103 396989 2264 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 425790 1104 397021 2265 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 427406 1105 387429 2266 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 429610 1106 406850 2267 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 430358 1107 414163 2268
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 431441 1108 392784
2269 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 432998 1109
405991 2270 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 435664
1110 404318 2271 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
435797 1111 400677 2272 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 437759 1112 387743 2273 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 438165 1113 387706 2274 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 440369 1114 415212 2275 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 441397 1115 390489 2276 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 444412 1116 413610 2277
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 445130 1117 396124
2278 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 445764 1118
393886 2279 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 447192
1119 405806 2280 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
447640 1120 396389 2281 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 448895 1121 396121 2282 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 449153 1122 409167 2283 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 450033 1123 390040 2284 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 452213 1124 404936 2285 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 455729 1125 404954 2286
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 456589 1126 405350
2287 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 546487 1127
448679 2288 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 546539
1128 448232 2289 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
547047 1129 449059 2290 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 547353 1130 447883 2291 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 547681 1131 447851 2292 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 547700 1132 449083 2293 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 547874 1133 447682 2294 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 548103 1134 449499 2295
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 548112 1135 448434
2296 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 548248 1136
448080 2297 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 548433
1137 449971 2298 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
548542 1138 446597 2299 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 548805 1139 446924 2300 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 548827 1140 449201 2301 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 548893 1141 447943 2302 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 548947 1142 447711 2303 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 549228 1143 447517 2304
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 549382 1144 449177
2305 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 549428 1145
447038 2306 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 549771
1146 448812 2307 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
550004 1147 447084 2308 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 550110 1148 446980 2309 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 550210 1149 447697 2310 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 550408 1150 447136 2311 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 550500 1151 450002 2312 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 550688 1152 448066 2313
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 551204 1153 447799
2314 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 551267 1154
450198 2315 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 551460
1155 449274 2316 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
551554 1156 448538 2317 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 551621 1157 448285 2318 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 551740 1158 450037 2319 checkpoint 1
CC/S NFBD1 mediator of DNA-damage 552263 1159 447069 2320
checkpoint 1 CC/S NFBD1 mediator of DNA-damage 552349 1160 449892
2321 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 552474 1161
447771 2322 checkpoint 1 CC/S NFBD1 mediator of DNA-damage 552522
1162 449936 2323 checkpoint 1 CC/S NFBD1 mediator of DNA-damage
552776 1163 447825 2324 checkpoint 1 CC/S NFBD1 mediator of
DNA-damage 553047 1164 447247 2325 checkpoint 1 CC/S NFBD1 mediator
of DNA-damage 553048 1165 447787 2326 checkpoint 1 CC/S NFBD1
mediator of DNA-damage 553130 1166 446809 2327 checkpoint 1 CC/S
NFBD1 mediator of DNA-damage 553196 1167 449586 2328 checkpoint 1
CC/S Nibrin nibrin 265433 1168 265433 2329 CC/S Nibrin nibrin
452387 1169 445213 2330 CC/S p107 retinoblastoma-like 1 (p107)
344359 1170 343646 2331 CC/S p107 retinoblastoma-like 1 (p107)
373664 1171 362768 2332 CC/S p130 retinoblastoma-like 2 (p130)
262133 1172 262133 2333 CC/S p130 retinoblastoma-like 2 (p130)
379935 1173 369267 2334 CC/S p130 retinoblastoma-like 2 (p130)
544405 1174 443744 2335 CC/S p130 retinoblastoma-like 2 (p130)
544545 1175 444685 2336 CC/S p21 P21 NA 1176 NA 2337 CC/S PCNA
proliferating cell nuclear 379143 1177 368438 2338 antigen CC/S
PCNA proliferating cell nuclear 379160 1178 368458 2339 antigen
CC/S RAD9 RAD9 homolog A (S. pombe) 307980 1179 311360 2340 CC/S Rb
retinoblastoma 1 267163 1180 267163 2341 protein CC/S Rb
retinoblastoma 1 467505 1181 434702 2342 protein CC/S Rb
retinoblastoma 1 542917 1182 437642 2343 protein CC/S SMC1
structural maintenance of 322213 1183 323421 2344 chromosomes 1A
CC/S SMC1 structural maintenance of 340213 1184 344906 2345
chromosomes 1A CC/S SMC1 structural maintenance of 375340 1185
364489 2346 chromosomes 1A CC/S SMC1 structural maintenance of
428014 1186 413509 2347 chromosomes 1A CC/S USP1 ubiquitin specific
peptidase 1 339950 1187 343526 2348 CC/S USP1 ubiquitin specific
peptidase 1 371146 1188 360188 2349 CC/S USP1 ubiquitin specific
peptidase 1 452143 1189 403662 2350 M 4EBP-1 eukaryotic translation
initiation 338825 1190 340691 2351 factor 4E binding protein 1 M
ARNT aryl hydrocarbon receptor 354396 1191 346372 2352 nuclear
translocator M ARNT aryl hydrocarbon receptor 358595 1192 351407
2353 nuclear translocator M ARNT aryl hydrocarbon receptor 368975
1193 357971 2354 nuclear translocator M ARNT aryl hydrocarbon
receptor 394700 1194 378190 2355 nuclear translocator M ARNT aryl
hydrocarbon receptor 471844 1195 425899 2356 nuclear translocator M
ARNT aryl hydrocarbon receptor 505755 1196 427571 2357 nuclear
translocator M ARNT aryl hydrocarbon receptor 515192 1197 423851
2358 nuclear translocator M CAIX carbonic anhydrase IX 378357 1198
367608 2359 M CAIX carbonic anhydrase IX 544074 1199 438541 2360 M
CBP CREB binding protein 262367 1200 262367 2361 M CBP CREB binding
protein 323508 1201 323550 2362 M CBP CREB binding protein 382070
1202 371502 2363 M CBP CREB binding protein 543883 1203 441978 2364
M CITED1 Cbp/p300-interacting 246139 1204 246139 2365
transactivator, with Glu/Asp- rich carboxy-terminal domain, 1 M
CITED1 Cbp/p300-interacting 373619 1205 362721 2366 transactivator,
with Glu/Asp- rich carboxy-terminal domain, 1 M CITED1
Cbp/p300-interacting 417400 1206 414781 2367 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 1 M CITED1
Cbp/p300-interacting 427412 1207 391407 2368 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 1 M CITED1
Cbp/p300-interacting 429794 1208 407496 2369 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 1 M CITED1
Cbp/p300-interacting 431381 1209 388548 2370 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 1 M CITED1
Cbp/p300-interacting 445983 1210 403274 2371 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 1 M CITED1
Cbp/p300-interacting 450875 1211 405765 2372 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 1 M CITED1
Cbp/p300-interacting 453707 1212 401764 2373 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 1 M CITED2
Cbp/p300-interacting 367651 1213 356623 2374 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 2 M CITED2
Cbp/p300-interacting 392312 1214 376126 2375 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 2 M CITED2
Cbp/p300-interacting 536159 1215 442831 2376 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 2 M CITED2
Cbp/p300-interacting 537332 1216 444198 2377 transactivator, with
Glu/Asp- rich carboxy-terminal domain, 2 M CITED4
Cbp/p300-interacting NA 1217 NA 2378 transactivator, with Glu/Asp-
rich carboxy-terminal domain, 4 M CITED4 Cbp/p300-interacting
372638 1218 361721 2379 transactivator, with Glu/Asp- rich
carboxy-terminal domain, 4 (CBP/p300 interacting transactivator
with ED-rich tail) M COMMD1 copper metabolism (Murr1) 311832 1219
308236 2380 domain containing 1 M COMMD1 copper metabolism (Murr1)
427417 1220 413207 2381 domain containing 1 M COMMD1 copper
metabolism (Murr1) 444166 1221 410050 2382 domain containing 1 M
COMMD1 copper metabolism (Murr1) 458337 1222 401236 2383 domain
containing 1 M COMMD1 copper metabolism (Murr1) 538736 1223 438961
2384 domain containing 1 M CREB cAMP responsive element 236996 1224
236996 2385 binding protein 1 M CREB cAMP responsive element 353267
1225 236995 2386 binding protein 1 M CREB cAMP responsive element
353704 1226 342136 2387 binding protein 3 M CREB cAMP responsive
element 374397 1227 363518 2388 binding protein 1 M CREB cAMP
responsive element 430624 1228 405539 2389 binding protein 1 M CREB
cAMP responsive element 432329 1229 387699 2390 binding protein 1 M
CREB cAMP responsive element 445803 1230 407227 2391 binding
protein 1 M CREB cAMP responsive element 452474 1231 392428 2392
binding protein 1 M CREB cAMP responsive element 536726 1232 445892
2393 binding protein 1 M CREB cAMP responsive element 539789 1233
440809 2394 binding protein 1 M eIF4E eukaryotic translation
initiation 280892 1234 280892 2395 factor 4E M eIF4E eukaryotic
translation initiation 450253 1235 389624 2396 factor 4E M HIF3-
hypoxia inducible factor 3, 244302 1236 244302 2397 alpha alpha
subunit M HIF3- hypoxia inducible factor 3, 291300 1237 291300 2398
alpha alpha subunit M FIH hypoxia inducible factor 1, 299163 1238
299163 2399 alpha subunit inhibitor (factor inhibiting HIF) M HIF3-
hypoxia inducible factor 3, 300862 1239 300862 2400 alpha) alpha
subunit M HIF3- hypoxia inducible factor 3, 339613 1240 341877 2401
alpha alpha subunit M HIF3- hypoxia inducible factor 3, 377670 1241
366898 2402 alpha alpha subunit M HIF3- hypoxia inducible factor 3,
414707 1242 412808 2403 alpha alpha subunit M HIF3- hypoxia
inducible factor 3, 420102 1243 407771 2404 alpha alpha subunit M
FIH hypoxia inducible factor 1, 442724 1244 399734 2405 (factor
alpha subunit inhibitor inhibiting HIF) M HIF3- hypoxia inducible
factor 3, 457771 1245 408008 2406 alpha alpha subunit M HIF3-
hypoxia inducible factor 3, 457865 1246 394052 2407 alpha alpha
subunit M HIF3- hypoxia inducible factor 3, 475432 1247 432578 2408
alpha alpha subunit M FIH hypoxia inducible factor 1, 533589 1248
433360 2409 (factor alpha subunit inhibitor inhibiting HIF) M Grb2
growth factor receptor-bound 316615 1249 317360 2410 protein 2 M
Grb2 growth factor receptor-bound 316804 1250 339007 2411 protein 2
M Grb2 growth factor receptor-bound 392562 1251 376345 2412 protein
2 M Grb2 growth factor receptor-bound 392564 1252 376347 2413
protein 2 M HNF4alpha hepatocyte nuclear factor 4, 316099 1253
312987 2414 alpha M HNF4alpha hepatocyte nuclear factor 4, 316673
1254 315180 2415 alpha M HNF4alpha hepatocyte nuclear factor 4,
338692 1255 343807 2416 alpha M HNF4alpha hepatocyte nuclear factor
4, 415691 1256 412111 2417 alpha M HNF4alpha hepatocyte nuclear
factor 4, 443598 1257 410911 2418 alpha M HNF4alpha hepatocyte
nuclear factor 4, 457232 1258 396216 2419 alpha M HNF4alpha2 Homo
sapiens hepatocyte NA 1259 NA 2420 nuclear factor 4, alpha (HNF4A),
transcript variant 2, mRNA M IBP3 insulin-like growth factor 275521
1260 275521 2421 binding protein 3 M IBP3 insulin-like growth
factor 381083 1261 370473 2422 binding protein 3 M IBP3
insulin-like growth factor 381086 1262 370476 2423 binding protein
3 M IBP3 insulin-like growth factor 417621 1263 399116 2424 binding
protein 3 M IBP3 insulin-like growth factor 428530 1264 390298 2425
binding protein 3 M IBP3 insulin-like growth factor 433047 1265
404461 2426 binding protein 3 M IBP3 insulin-like growth factor
438491 1266 393740 2427 binding protein 3 M IBP3 insulin-like
growth factor 442142 1267 392472 2428 binding protein 3 M IBP3
insulin-like growth factor 545032 1268 439999 2429 binding protein
3 M JAB1 COP9 constitutive 357849 1269 350512 2430 photomorphogenic
homolog subunit 5 (Arabidopsis) M MNK1 MAP kinase interacting
341183 1270 339573 2431 serine/threonine kinase 1 M MNK1 MAP kinase
interacting 371944 1271 361012 2432 serine/threonine kinase 1 M
MNK1 MAP kinase interacting 371945 1272 361013 2433
serine/threonine kinase 1 M MNK1 MAP kinase interacting 371946 1273
361014 2434 serine/threonine kinase 1 M MNK1 MAP kinase interacting
428112 1274 411135 2435 serine/threonine kinase 1 M MNK1 MAP kinase
interacting 496619 1275 436709 2436 serine/threonine kinase 1 M
MNK1 MAP kinase interacting 545730 1276 440974 2437
serine/threonine kinase 1 M MNK2 MAP kinase interacting 250896 1277
250896 2438 serine/threonine kinase 2 M MNK2 MAP kinase interacting
309340 1278 309485 2439 serine/threonine kinase 2 M MNK2 MAP kinase
interacting 541165 1279 438904 2440 serine/threonine kinase 2 M
MNK2 MAP kinase interacting 545627 1280 441245 2441
serine/threonine kinase 2
M p15(INK4A) cyclin-dependent kinase 276925 1281 276925 2442
inhibitor 2B (p15, inhibits CDK4) M p15(INK4A) cyclin-dependent
kinase 380142 1282 369487 2443 inhibitor 2B (p15, inhibits CDK4) M
p300 E1A binding protein p300 263253 1283 263253 2444 M Per1 period
homolog 1 (Drosophila) 317276 1284 314420 2445 M Per1 period
homolog 1 (Drosophila) 354903 1285 346979 2446 M RPS6 ribosomal
protein S6 315377 1286 369743 2447 M RPS6 ribosomal protein S6
380381 1287 369741 2448 M RPS6 ribosomal protein S6 380384 1288
369745 2449 M RPS6 ribosomal protein S6 380394 1289 369757 2450 M
SHARP1 basic helix-loop-helix family, NA 1290 NA 2451 member e41 M
SHARP1 basic helix-loop-helix family, 242728 1291 242728 2452
(BHLHE41) member e41 M SHARP1 basic helix-loop-helix family, 540731
1292 437369 2453 (BHLHE41) member e41 M SRC1 nuclear receptor
coactivator 1 288599 1293 288599 2454 M SRC1 nuclear receptor
coactivator 1 348332 1294 320940 2455 M SRC1 nuclear receptor
coactivator 1 395856 1295 379197 2456 M SRC1 nuclear receptor
coactivator 1 405141 1296 385097 2457 M SRC1 nuclear receptor
coactivator 1 406961 1297 385216 2458 M SRC1 nuclear receptor
coactivator 1 538539 1298 444039 2459 M tuberin tuberous sclerosis
2 219476 1299 219476 2460 M tuberin tuberous sclerosis 2 350773
1300 344383 2461 M tuberin tuberous sclerosis 2 353929 1301 248099
2462 M tuberin tuberous sclerosis 2 382538 1302 371978 2463 M
tuberin tuberous sclerosis 2 401874 1303 384468 2464 M tuberin
tuberous sclerosis 2 439673 1304 399232 2465 AIFSH
apoptosis-inducing factor, NA 1305 NA 2466 short Angiopoietin1
Angiopoietin 1 NA 1306 NA 2467 2492 BMP2 BMP2 CO NA 1307 NA 2468
2493 CO c-MYC v-myc myelocytomatosis viral NA 1308 NA 2469 oncogene
homolog (avian) COMMD1 COMMD1 NA 1309 NA COMMD1 COMMD1 with nuclear
export NA NA 2470 NES seqences deleted deleted COMMD1 COMMD1 with
nuclear export NA NA 2471 NES1 sequences deleted and nuclear
deleted localization signals added and NLS added COMMD1 COMMD1 with
SV40 and NA NA 2472 SV40 nuclear localization signals NLS COMMD1wt
COMMD1 wild-type NA NA 2473 GLUT1 solute carrier family 2 NA 1310
NA 2474 (facilitated glucose transporter), member 1 Granulysin
Granulysin FL15 NA 1311 NA 2475 FL15 Granulysin Granulysin NS9 NA
NA 2476 2494 NS9 Granulysin Granulysin S9 NA NA 2477 2495 S9 HIF1 a
hypoxia inducible factor 1, NA 1312 NA 2478 alpha subunit (basic
helix- loop-helix transcription factor) IL15 interleukin 15 NA 1313
NA 2479 KGF fibroblast growth factor 7, NA 1314 NA 2480 precursor;
mature is 32-194 MCT4 solute carrier family 16, NA 1315 NA 2481
2496 member 4 (monocarboxylic acid transporter 5) MYC MYC inhibitor
D (OMOMyc) NA 1316 NA 2482 inhibitor D MYC MYC inhibitor D_90 NA NA
2483 inhibitor (OmoMyc_90) D_90 C.A. Constitutively active (C.A.)
NA 1317 NA 2484 caspase caspase 3 cleavable 3_cleavable
(RevCasp3_Cleavable) C.A. Constitutively active (C.A.) NA 1318 NA
2485 caspase caspase 3 uncleavable 3_uncleavable
(RevCasp3_UnCleavable) C.A. Constitutively active (C.A.) NA 1319 NA
2486 caspase 6 caspase 6 (RevCasp6) SIAh1 siah E3 ubiquitin protein
ligase 1 NA 1320 NA 2487 HSV1-tk Herpes simplex virus 1- thymidine
kinase
[0297] Shown in Table 7, are familiar cancer syndromes, tumor
suppressor genes, function of the tumor suppressor gene,
chromosomal location, and tumor type observed. Signal-sensor
polynucleotides of the present invention can be designed as a
therapeutic for any of those listed in the table.
TABLE-US-00007 TABLE 7 Familial Cancer Syndrome Targets Familial
Tumor Cancer Suppressor Chromosomal Tumor Types Syndrome Gene
Function Location Observed Li-Fraumeni P53 cell cycle 17p13.1 brain
tumors, Syndrome regulation, sarcomas, apoptosis leukemia, breast
cancer Familial RB1 cell cycle 13q14.1-q14.2 retinoblastoma,
Retinoblastoma regulation osteogenic sarcoma Wilms WT1
transcriptional 11p13 pediatric kidney Tumor regulation cancer,
most common form of childhood solid tumor Neurofibro- NF1 catalysis
of 17q11.2 neurofibromas, matosis Type RAS sarcomas, 1 inactivation
gliomas Neurofibro- NF2 linkage of cell 22q12.2 Schwann cell
matosis Type membrane to tumors, 2 actin astrocytomas, cytoskeleton
meningiomas, ependymonas Familial APC signaling 5q21-q22 colon
cancer Adenomatous through Polyposis adhesion molecules to nucleus
Tuberous TSC1 forms 9q34 seizures, mental sclerosis 1 complex with
retardation, TSC2 protein, facial inhibits angiofibromas signaling
to downstream effectors of mTOR Tuberous TSC2 forms 16p13.3 benign
growths sclerosis 2 complex with (hamartomas) TSC1 protein, in many
tissues, inhibits astrocytomas, signaling to rhabdomyosarcomas
downstream effectors of mTOR Deleted in DPC4, also regulation of
18q21.1 pancreatic Pancreatic known as TGF-.beta./BMP carcinoma,
Carcinoma SMAD4 signal colon cancer 4, Familial transduction
juvenile polyposis syndrome Deleted in DCC transmembrane 18q21.3
colorectal Colorectal receptor cancer Carcinoma involved in axonal
guidance via netrins Familial BRCA1 functions in 17q21 breast and
Breast transcription, ovarian cancer Cancer DNA binding,
transcription coupled DNA repair, homologous recombination,
chromosomal stability, ubiquitination of proteins, and centrosome
replication Familial BRCA2 transcriptional 13q12.3 breast and
Breast (FANCD1) regulation of ovarian cancer Cancer genes involved
in DNA repair and homologous recombination Cowden PTEN
phosphoinositide 10q23.3 gliomas, breast syndrome 3-phosphatase,
cancer, thyroid protein cancer, head & tyrosine neck squamous
phosphatase carcinoma Peutz-Jeghers STK11 phosphorylates 19p13.3
hyperpigmentation, Syndrome (serine- and activates multiple (PJS)
threonine AMP-activated hamartomatous kinase 11) kinase (AMPK),
polyps, AMPK involved colorectal, in stress breast and responses,
lipid ovarian cancers and glucose meatabolism Hereditary MSH2 DNA
2p22-p21 colon cancer Nonpolyposis mismatch Colon repair Cancer
type 1, HNPCC1 Hereditary MLH1 DNA 3p21.3 colon cancer Nonpolyposis
mismatch Colon repair Cancer type 2, HNPCC2 Familial CDH1 cell-cell
16q22.1 gastric cancer, diffuse-type adhesion lobular breast
gastric protein cancer cancer von Hippel- VHL regulation of
3p26-p25 renal cancers, Lindau transcription hemangioblastomas,
Syndrome elongation pheochromocytoma, through retinal angioma
activation of a ubiquitin ligase complex Familial CDKN2A p16INK4
9p21 melanoma, Melanoma inhibits cell- pancreatic cycle kinases
cancer, others CDK4 and CDK6; p14ARF binds the p53 stabilizing
protein MDM2 Gorlin PTCH (e.g., transmembrane 9q22.3 basal cell
skin Syndrome: PTCH1, receptor for carcinoma Nevoid basal PTCH2)
sonic hedgehog cell carcinoma (shh), involved syndrome in early
(NBCCS) development through repression of action of smoothened
Multiple MEN1 intrastrand 11q13 parathyroid and Endocrine DNA
crosslink pituitary adenomas, Neoplasia repair islet cell tumors,
Type 1 carcinoid
[0298] In additional to the above mentioned targets, the the
oncology-related polypeptides may include any "death signal"
protein that can be recognized by active T cells of immune system.
Such suicide signal proteins encoded by the sensor-signal
polynucleotides can be selectively expressed in particular tissues
or cells (e.g. cancer cells) through engineered microRNA binding
sites and/or other regulatory elements as described herein. The
group of proteins, when they are expressed on the surface of a
cancer cell, can prime T cell to induce T cell mediated immune
response, thus killing the cancer cell. As a non-limiting example,
a group of proteins that are known to present a "death signal",
include, CD80, CD86, B7 and MHC II, etc.
Protein Cleavage Signals and Sites
[0299] In one embodiment, the oncology-related polypeptides of the
present invention may include at least one protein cleavage signal
containing at least one protein cleavage site. The protein cleavage
site may be located at the N-terminus, the C-terminus, at any space
between the N- and the C-termini such as, but not limited to,
half-way between the N- and C-termini, between the N-terminus and
the half way point, between the half way point and the C-terminus,
and combinations thereof.
[0300] The oncology-related polypeptides of the present invention
may include, but is not limited to, a proprotein convertase (or
prohormone convertase), thrombin or Factor Xa protein cleavage
signal. Proprotein convertases are a family of nine proteinases,
comprising seven basic amino acid-specific subtilisin-like serine
proteinases related to yeast kexin, known as prohormone convertase
1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basic amino-acid
cleaving enzyme 4 (PACE4) and PC7, and two other subtilases that
cleave at non-basic residues, called subtilisin kexin isozyme 1
(SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9).
Non-limiting examples of protein cleavage signal amino acid
sequences are listing in Table 8. In Table 8, "X" refers to any
amino acid, "n" may be 0, 2, 4 or 6 amino acids and "*" refers to
the protein cleavage site. In Table 8, SEQ ID NO: 2499 refers to
when n=4 and SEQ ID NO: 2500 refers to when n=6.
TABLE-US-00008 TABLE 8 Protein Cleavage Site Sequences Protein
Cleavage Amino Acid Signal Cleavage Sequence SEQ ID NO Proprotein
R-X-X-R* 2497 convertase R-X-K/R-R* 2498 K/R-Xn-K/R* 2499 or 2500
Thrombin L-V-P-R*-G-S 2501 L-V-P-R* 2502 A/F/G/I/L/T/V/M-A/F/G/I/
2503 L/T/V/W/A-P-R* Factor Xa I-E-G-R* 2504 I-D-G-R* 2505 A-E-G-R*
2506 A/F/G/I/L/T/V/M-D/E-G-R* 2507
[0301] In one embodiment, the signal-sensor primary constructs and
the mmRNA of the present invention may be engineered such that the
primary construct or mmRNA contains at least one encoded protein
cleavage signal. The encoded protein cleavage signal may be located
before the start codon, after the start codon, before the coding
region, within the coding region such as, but not limited to, half
way in the coding region, between the start codon and the half way
point, between the half way point and the stop codon, after the
coding region, before the stop codon, between two stop codons,
after the stop codon and combinations thereof
[0302] In one embodiment, the signal-sensor primary constructs or
mmRNA of the present invention may include at least one encoded
protein cleavage signal containing at least one protein cleavage
site. The encoded protein cleavage signal may include, but is not
limited to, a proprotein convertase (or prohormone convertase),
thrombin and/or Factor Xa protein cleavage signal. One of skill in
the art may use Table 1 above or other known methods to determine
the appropriate encoded protein cleavage signal to include in the
signal-sensor primary constructs or mmRNA of the present invention.
For example, starting with the signal of Table 8 and considering
the codons of Table 1 one can design a signal for the signal-sensor
primary construct which can produce a protein signal in the
resulting oncology-related polypeptide.
[0303] In one embodiment, the oncology-related polypeptides of the
present invention include at least one protein cleavage signal
and/or site.
[0304] As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S.
Pub. No. 20090227660, herein incorporated by reference in their
entireties, use a furin cleavage site to cleave the N-terminal
methionine of GLP-1 in the expression product from the Golgi
apparatus of the cells. In one embodiment, the polypeptides of the
present invention include at least one protein cleavage signal
and/or site with the proviso that the polypeptide is not GLP-1.
[0305] In one embodiment, the signal-sensor primary constructs or
mmRNA of the present invention includes at least one encoded
protein cleavage signal and/or site.
[0306] In one embodiment, the signal-sensor primary constructs or
mmRNA of the present invention includes at least one encoded
protein cleavage signal and/or site with the proviso that the
signal-sensor primary construct or mmRNA does not encode GLP-1.
[0307] In one embodiment, the signal-sensor primary constructs or
mmRNA of the present invention may include more than one coding
region. Where multiple coding regions are present in the
signal-sensor primary construct or mmRNA of the present invention,
the multiple coding regions may be separated by encoded protein
cleavage sites. As a non-limiting example, the signal-sensor
primary construct or mmRNA may be signed in an ordered pattern. On
such pattern follows AXBY form where A and B are coding regions
which may be the same or different coding regions and/or may encode
the same or different oncology-related polypeptides, and X and Y
are encoded protein cleavage signals which may encode the same or
different protein cleavage signals. A second such pattern follows
the form AXYBZ where A and B are coding regions which may be the
same or different coding regions and/or may encode the same or
different oncology-related polypeptides, and X, Y and Z are encoded
protein cleavage signals which may encode the same or different
protein cleavage signals. A third pattern follows the form ABXCY
where A, B and C are coding regions which may be the same or
different coding regions and/or may encode the same or different
oncology-related polypeptides, and X and Y are encoded protein
cleavage signals which may encode the same or different protein
cleavage signals.
[0308] In one embodiment, the oncology-related polypeptides,
signal-sensor primary constructs and mmRNA can also contain
sequences that encode protein cleavage sites so that the
polypeptides, signal-sensor primary constructs and mmRNA can be
released from a carrier region or a fusion partner by treatment
with a specific protease for said protein cleavage site.
microRNA
[0309] microRNAs (or miRNA) are 19-25 nucleotide long noncoding
RNAs that bind to the 3'UTR of nucleic acid molecules and
down-regulate gene expression either by reducing nucleic acid
molecule stability or by inhibiting translation. The modified
nucleic acids (mRNA), enhanced modified RNA or ribonucleic acids of
the invention may comprise one or more microRNA target sequences,
microRNA sequences, or microRNA seeds. Such sequences may
correspond to any known microRNA such as those taught in US
Publication US2005/0261218 and US Publication US2005/0059005, the
contents of which are incorporated herein by reference in their
entirety. As a non-limiting embodiment, known microRNAs, their
sequences and their binding site sequences in the human genome are
listed below in Table 9.
[0310] A microRNA sequence comprises a "seed" region, i.e., a
sequence in the region of positions 2-8 of the mature microRNA,
which sequence has perfect Watson-Crick complementarity to the
miRNA target sequence. A microRNA seed may comprise positions 2-8
or 2-7 of the mature microRNA. In some embodiments, a microRNA seed
may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature
microRNA), wherein the seed-complementary site in the corresponding
miRNA target is flanked by an adenine (A) opposed to microRNA
position 1. In some embodiments, a microRNA seed may comprise 6
nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein
the seed-complementary site in the corresponding miRNA target is
flanked by an adenine (A) opposed to microRNA position 1. See for
example, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L
P, Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of
the microRNA seed have complete complementarity with the target
sequence. By engineering microRNA target sequences into the 3'UTR
of nucleic acids or mRNA of the invention one can target the
molecule for degradation or reduced translation, provided the
microRNA in question is available. This process will reduce the
hazard of off target effects upon nucleic acid molecule delivery.
Identification of microRNA, microRNA target regions, and their
expression patterns and role in biology have been reported (Bonauer
et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr
Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012
26:404-413 (2011 Dec. 20. doi: 10.1038/1eu.2011.356); Bartel Cell
2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner
and Naldini, Tissue Antigens. 2012 80:393-403 and all references
therein; each of which is herein incorporated by reference in its
entirety).
[0311] For example, if the signal-sensor polynucleotide is not
intended to be delivered to the liver but ends up there, then
miR-122, a microRNA abundant in liver, can inhibit the expression
of the gene of interest if one or multiple target sites of miR-122
are engineered into the 3'UTR of the signal-sensor polynucleotide.
Introduction of one or multiple binding sites for different
microRNA can be engineered to further decrease the longevity,
stability, and protein translation of a signal-sensor
polynucleotide. As used herein, the term "microRNA site" refers to
a microRNA target site or a microRNA recognition site, or any
nucleotide sequence to which a microRNA binds or associates. It
should be understood that "binding" may follow traditional
Watson-Crick hybridization rules or may reflect any stable
association of the microRNA with the target sequence at or adjacent
to the microRNA site.
[0312] Conversely, for the purposes of the signal-sensor
polynucleotides of the present invention, microRNA binding sites
can be engineered out of (i.e. removed from) sequences in which
they naturally occur in order to increase protein expression in
specific tissues. For example, miR-122 binding sites may be removed
to improve protein expression in the liver.
[0313] In one embodiment, signal-sensor polynucleotides may include
at least one miRNA-binding site in the 3'UTR in order to direct
cytotoxic or cytoprotective mRNA therapeutics to specific cells
such as, but not limited to, normal and/or cancerous cells (e.g.,
HEP3B or SNU449). As a non-limiting example, a strong apoptotic
signal and at least one miR-122a binding site is encoded by the
signal-sensor polynucleotide where the at least one miR-122a
binding site is located in the 3'UTR. As another non-limiting
example, apoptosis inducing factor short isoform (AIFsh) and at
least one miR-122a binding site is encoded by the signal-sensor
polynucleotide where the at least one miR-122a binding site is
located in the 3'UTR. As yet another non-limiting example,
constitutively active (C.A.) caspase 6 and at least one miR-122a
binding site is encoded by the signal-sensor polynucleotide where
the at least one miR-122a binding site is located in the 3'UTR. As
another non-limiting example, HSV1-tk and at least one miR-122a
binding site is encoded by the signal-sensor polynucleotide where
the at least one miR-122a binding site is located in the 3'UTR.
[0314] In another embodiment, signal-sensor polynucleotides may
include three miRNA-binding sites in the 3'UTR in order to direct
cytotoxic or cytoprotective mRNA therapeutics to specific cells
such as, but not limited to, normal and/or cancerous cells (e.g.,
HEP3B or SNU449). As a non-limiting example, a strong apoptotic
signal and three miR-122a binding sites are encoded by the
signal-sensor polynucleotide where the three miR-122a binding sites
are located in the 3'UTR. As another non-limiting example,
apoptosis inducing factor short isoform (AIFsh) and three miR-122a
binding sites are encoded by the signal-sensor polynucleotide where
the three miR-122a binding sites are located in the 3'UTR. As yet
another non-limiting example, constitutively active (C.A.) caspase
6 and three miR-122a binding sites are encoded by the signal-sensor
polynucleotide where the three miR-122a binding sites are located
in the 3'UTR. As another non-limiting example, HSV1-tk and and
three miR-122a binding sites are encoded by the signal-sensor
polynucleotide where the three miR-122a binding sites are located
in the 3'UTR.
[0315] Regulation of expression in multiple tissues can be
accomplished through introduction or removal or one or several
microRNA binding sites. Shown below in Table 10 are microRNAs which
are differentially expressed in different tissues and cells, and
often associated with different types of dieases (e.g.cancer
cells). The decision of removal or insertion of microRNA binding
sites, or any combination, is dependent on microRNA expression
patterns and their profilings in cancer cells.
[0316] Examples of tissues where microRNA are known to regulate
mRNA, and thereby protein expression, include, but are not limited
to, liver (miR-122), muscle (miR-133, miR-206, miR-208),
endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p,
miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), nervous
system (mir-124a, miR-9), pluripotent cells (miR-302, miR-367,
miR-290, miR-371, miR-373), pancreatic islet cells (miR-375),
adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney
(miR-192, miR-194, miR-204), and lung epithelial cells (let-7,
miR-133, miR-126).
[0317] Specifically, microRNAs are known to be differentially
expressed in immune cells (also called hematopoietic cells), such
as antigen presenting cells (APCs) (e.g. dendritic cells and
macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes,
granuocytes, natural killer cells, etc. Immune cell specific
microRNAs are involved in immunogenicity, autoimmunity, the
immune-response to infection, inflammation, as well as unwanted
immune response after gene therapy and tissue/organ
transplantation. Immune cells specific microRNAs also regulate many
aspects of development, proliferation, differentiation and
apoptosis of hematopoietic cells (immune cells). For example,
miR-142 and miR-146 are exclusively expressed in the immune cells,
particularly abundant in myeloid dendritic cells. Introducing the
miR-142 binding site into the 3'-UTR of a signal-sensor polypeptide
of the present invention can selectively suppress the gene
expression in the antigen presenting cells through miR-142 mediated
mRNA degradation, limiting antigen presentation in professional
APCs (e.g. dendritic cells) and thereby preventing antigen-mediated
immune response after gene delivery (see, Annoni A et al., blood,
2009, 114, 5152-5161, the content of which is herein incorporated
by reference in its entirety.)
[0318] In one embodiment, microRNAs binding sites that are known to
be expressed in immune cells, in particular, the antigen presenting
cells, can be engineered into the signal-sensor polynucleotides to
suppress the expression of the sensor-signal polynucleotide in APCs
through microRNA mediated RNA degradation, subduing the
antigen-mediated immune response, while the expression of the
sensor-signal polynucleotide is maintained in non-immune cells
where the immune cell specific microRNAs are not expressed. For
example, to prevent the immunogenic reaction caused by a liver
specific protein expression, the miR-122 binding site can be
removed and the miR-142 (and/or mirR-146) binding sites can be
engineered into the 3-UTR of the signal-sensor polynucleotide
(e.g., see the constructs described in Example 38 and the
experiment outlined in Examples 39 and 40).
[0319] To further drive the selective degradation and suppression
of mRNA in APCs and macrophage, the signal-sensor polynucleotide
may include another negative regulatory element in the 3-UTR,
either alone or in combination with mir-142 and/or mir-146 binding
sites. As a non-limiting example, one regulatory element is the
Constitutive Decay Elements (CDEs).
[0320] Immune cells specific microRNAs include, but are not limited
to, hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c,
hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p,
hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184,
hsa-let-7f-1-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p,
miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p,
miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-143-3p,
miR-143-5p, miR-146a-3p, miR-146a-5p, miR-146b-3p, miR-146b-5p,
miR-147a, miR-147b, miR-148a-5p, miR-148a-3p, miR-150-3p,
miR-150-5p, miR-151b, miR-155-3p, miR-155-5p, miR-15a-3p,
miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p, miR-16-2-3p,
miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p, miR-181a-2-3p,
miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p, miR-21-5p,
miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,
miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,
miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,
miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p,
miR-27b-5p, miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p,
miR-29b-1-5p, miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p,
miR-30e-5p, miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p,
miR-345-5p, miR-346, miR-34a-3p, miR-34a-5p, miR-363-3p,
miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR-493-3p,
miR-493-5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j,
miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p,
miR-99a-5p, miR-99b-3p and miR-99b-5p. Shown below in Table 11 are
microRNAs that are enriched in specific types of immune cells.
Furthermore, novel miroRNAs are discovered in the immune cells in
the art through micro-array hybridization and microtome analysis
(Jima D D et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC
Genomics, 2010, 11,288, the content of each of which is
incorporated herein by reference in its entirety).
[0321] MicroRNAs that are known to be expressed in the liver
include, but are not limited to, miR-107, miR-122-3p, miR-122-5p,
miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303,
miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p,
miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p,
miR-557, miR-581, miR-939-3p, miR-939-5p. microRNA binding sites
from any liver specific microRNA can be introduced to or removed
from the signal-sensor polynucleotides to regulate the expression
of the signal-sensor polynucleotides in the liver. Liver specific
microRNAs binding sites can be engineered alone or further in
combination with immune cells (e.g. APCs) microRNA binding sites in
order to prevent an immune reaction against protein expression in
the liver.
[0322] MicroRNAs that are known to be expressed in the lung
include, but are not limited to, let-7a-2-3p, let-7a-3p, let-7a-5p,
miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p,
miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134,
miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p,
miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p,
miR-337-3p, miR-337-5p, miR-381-3p, miR-381-5p. MicroRNA binding
sites from any lung specific microRNA can be introduced to or
removed from the signal-sensor polynucleotide to regulate the
expression of the signal-sensor polynucleotide in the lung. Lung
specific microRNAs binding sites can be engineered alone or further
in combination with immune cells (e.g. APCs) microRNA binding sites
in order to prevent an immune reaction against protein expression
in the lung.
[0323] MicroRNAs that are known to be expressed in the heart
include, but are not limited to, miR-1, miR-133a, miR-133b,
miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b,
miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p,
miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p,
miR-92b-3p and miR-92b-5p. microRNA binding sites from any heart
specific microRNA can be introduced to or removed from the
signal-sensor polynucleotides to regulate the expression of the
signal-sensor polynucleotides in the heart. Heart specific
microRNAs binding sites can be engineered alone or further in
combination with immune cells (e.g. APCs) microRNA binding sites in
order to prevent an immune reaction against protein expression in
the heart.
[0324] MicroRNAs that are known to be expressed in the nervous
system include, but are not limited to, miR-124-5p, miR-125a-3p,
miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p,
miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p,
miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p,
miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,
miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b,
miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p,
miR-23a-5p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p,
miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329,
miR-342-3p, miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383,
miR-410, miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483,
miR-510, miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571,
miR-7-1-3p, miR-7-2-3p, miR-7-5p, miR-802, miR-922, miR-9-3p and
miR-9-5p. microRNAs enriched in the nervous system further include
those specifically expressed in neurons, including, but not limited
to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p,
miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e,
miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328,
miR-922 and those specifically expressed in glial cells, including,
but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,
miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,
miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, miR-657. microRNA
binding sites from any CNS specific microRNA can be introduced to
or removed from the signal-sensor polynucleotides to regulate the
expression of the signal-sensor polynucleotide in the nervous
system. Nervous system specific microRNAs binding sites can be
engineered alone or further in combination with immune cells (e.g.
APCs) microRNA binding sites in order to prevent an immune reaction
against protein expression in the nervous system.
[0325] MicroRNAs that are known to be expressed in the pancreas
include, but are not limited to, miR-105-3p, miR-105-5p, miR-184,
miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p,
miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p,
miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p
and miR-944. MicroRNA binding sites from any pancreas specific
microRNA can be introduced to or removed from the signal-sensor
polynucleotide to regulate the expression of the signal-sensor
polynucleotide in the pancreas. Pancreas specific microRNAs binding
sites can be engineered alone or further in combination with immune
cells (e.g. APCs) microRNA binding sites in order to prevent immune
reaction against protein expression in the pancreas.
[0326] MicroRNAs that are known to be expressed in the kidney
further include, but are not limited to, miR-122-3p, miR-145-5p,
miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p,
miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210,
miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p,
miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p,
miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p and
miR-562. MicroRNA binding sites from any kidney specific microRNA
can be introduced to or removed from the signal-sensor
polynucleotide to regulate the expression of the signal-sensor
polynucleotide in the kidney. Kidney specific microRNAs binding
sites can be engineered alone or further in combination with immune
cells (e.g. APCs) microRNA binding sites in order to prevent immune
reaction against protein expression in the kidney.
[0327] MicroRNAs that are known to be expressed in the muscle
further include, but are not limited to, let-7g-3p, let-7g-5p,
miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p,
miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p,
miR-206, miR-208a, miR-208b, miR-25-3p and miR-25-5p. MicroRNA
binding sites from any muscle specific microRNA can be introduced
to or removed from the signal-sensor polynucleotide to regulate the
expression of the signal-sensor polynucleotide in the muscle.
Muscle specific microRNAs binding sites can be engineered alone or
further in combination with immune cells (e.g. APCs) microRNA
binding sites in order to prevent an immune reaction against
protein expression in the muscle.
[0328] MicroRNAs are differentially expressed in different types of
cells, such as endothelial cells, epithelial cells and adipocytes.
For example, microRNAs that are expressed in endothelial cells
include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p,
miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p,
miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p,
miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p,
miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p,
miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p,
miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p,
miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p,
miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p and
miR-92b-5p. Many novel microRNAs were discovered in endothelial
cells from deep-sequencing analysis (Voellenkle C e tal., RNA,
2012, 18, 472-484, herein incorporated by reference in its
entirety). MicroRNA binding sites from any endothelial cell
specific microRNA can be introduced to or removed from the
signal-sensor polynucleotide in order to modulate the expression of
the signal-sensor polynucleotide in the endothelial cells in
various conditions.
[0329] For further example, microRNAs that are expressed in
epithelial cells include, but are not limited to, let-7b-3p,
let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p,
miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429,
miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p,
miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in
respiratory ciliated epithelial cells; let-7 family, miR-133a,
miR-133b, miR-126 specific in lung epithelial cells; miR-382-3p,
miR-382-5p specific in renal epithelial cells and miR-762 specific
in corneal epithelial cells. MicroRNA binding sites from any
epithelial cell specific microRNA can be introduced to or removed
from the signal-sensor polynucleotide in order to modulate the
expression of the signal-sensor polynucleotide in the epithelial
cells in various conditions.
[0330] In addition, a large group of microRNAs are enriched in
embryonic stem cells, controlling stem cell self-renewal as well as
the development and/or differentiation of various cell lineages,
such as neural cells, cardiac, hematopoietic cells, skin cells,
osteogenic cells and muscle cells (Kuppusamy K T et al., Curr. Mol
Med, 2013, 13(5), 757-764; Vidigal J A and Ventura A, Semin Cancer
Biol. 2012, 22(5-6), 428-436; Goff L A et al., PLoS One, 2009,
4:e7192; Morin R D et al., Genome Res, 2008,18, 610-621; Yoo J K et
al., Stem Cells Dev. 2012, 21(11), 2049-2057, each of which is
herein incorporated by reference in its entirety). MicroRNAs
abundant in embryonic stem cells include, but are not limited to,
let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,
miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,
miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,
miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p,
miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p,
miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e,
miR-367-3p, miR-367-5p, miR-369-3p, miR-369-5p, miR-370, miR-371,
miR-373, miR-380-5p, miR-423-3p, miR-423-5p, miR-486-5p,
miR-520c-3p, miR-548e, miR-548f, miR-548g-3p, miR-548g-5p,
miR-548i, miR-548k, miR-5481, miR-548m, miR-548n, miR-548o-3p,
miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p, miR-664b-3p,
miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p, miR-885-5p,
miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p, miR-99b-3p and
miR-99b-5p. Many predicted novel microRNAs are discovered by deep
sequencing in human embryonic stem cells (Morin R D et al., Genome
Res, 2008,18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;
Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each
of which is incorporated herein by references in its entirety).
[0331] In one embodiment, the binding sites of embryonic stem cell
specific microRNAs can be included in or removed from the 3-UTR of
the signal-sensor polynucleotide to modulate the development and/or
differentiation of embryonic stem cells, to inhibit the senescence
of stem cells in a degenerative condition (e.g. degenerative
diseases), or to stimulate the senescence and apoptosis of stem
cells in a disease condition (e.g. cancer stem cell).
[0332] Many microRNA expression studies have been conducted, and
are described in the art, to profile the differential expression of
microRNAs in various cancer cells/tissues and other diseases. Some
microRNAs are abnormally over-expressed in certain cancer cells and
others are under-expressed. For example, microRNAs are
differentially expressed in cancer cells (WO2008/154098,
US2013/0059015, US2013/0042333, WO2011/157294); cancer stem cells
(US2012/0053224); pancreatic cancers and diseases (US2009/0131348,
US2011/0171646, US2010/0286232, U.S. Pat. No. 8,389,210); asthma
and inflammation (U.S. Pat. No. 8,415,096); prostate cancer
(US2013/0053264); hepatocellular carcinoma (WO2012/151212,
US2012/0329672, WO2008/054828, U.S. Pat. No. 8,252,538); lung
cancer cells (WO2011/076143, WO2013/033640, WO2009/070653,
US2010/0323357); cutaneous T cell lymphoma (WO2013/011378);
colorectal cancer cells (WO2011/0281756, WO2011/076142); cancer
positive lympho nodes (WO2009/100430, US2009/0263803);
nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary
disease (U52012/0264626, US2013/0053263); thyroid cancer
(WO2013/066678); ovarian cancer cells (US2012/0309645,
WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,
US2012/0214699), leukemia and lymphoma (WO2008/073915,
US2009/0092974, US2012/0316081, US2012/0283310, WO2010/018563, the
content of each of which is incorporated herein by reference in
their entirety).
[0333] Specifically, microRNA sites that are over-expressed in
certain cancer and/or tumor cells can be removed from the 3-UTR of
the signal-sensor polynucleotide encoding the oncology-related
polypeptide, restoring the expression suppressed by the
over-expressed microRNAs in cancer cells, thus ameliorating the
corresponsive biological function, for instance, transcription
stimulation and/or repression, cell cycle arrest, apoptosis and
cell death. Normal cells and tissues, wherein microRNA expression
is not up-regulated, will remain unaffected.
[0334] MicroRNA can also regulate complex biological processes such
as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011
18:171-176). In the signal-sensor polynucleotides of the invention,
binding sites for microRNAs that are involved in such processes may
be removed or introduced, in order to tailor the expression of the
signal-sensor polynucleotides expression to biologically relevant
cell types or to the context of relevant biological processes. In
this context, the signal-sensor polynucleotideare defined as
auxotrophic signal-sensor polynucleotides.
[0335] Table 9 is a non-exhaustive listing of miRs and miR binding
sites (miR BS) and their sequences which may be used with the
present invention.
TABLE-US-00009 TABLE 9 Mirs and mir binding sites mir SEQ BS SEQ
microRNA ID ID hsa-let-7a-2-3p 2508 3529 hsa-let-7a-3p 2509 3530
hsa-let-7a-5p 2510 3531 hsa-let-7b-3p 2511 3532 hsa-let-7b-5p 2512
3533 hsa-let-7c 2513 3534 hsa-let-7d-3p 2514 3535 hsa-let-7d-5p
2515 3536 hsa-let-7e-3p 2516 3537 hsa-let-7e-5p 2517 3538
hsa-let-7f-1-3p 2518 3539 hsa-let-7f-2-3p 2519 3540 hsa-let-7f-5p
2520 3541 hsa-let-7g-3p 2521 3542 hsa-let-7g-5p 2522 3543
hsa-let-7i-3p 2523 3544 hsa-let-7i-5p 2524 3545 hsa-miR-1 2525 3546
hsa-miR-100-3p 2526 3547 hsa-miR-100-5p 2527 3548 hsa-miR-101-3p
2528 3549 hsa-miR-101-5p 2529 3550 hsa-miR-103a-2-5p 2530 3551
hsa-miR-103a-3p 2531 3552 hsa-miR-103b 2532 3553 hsa-miR-105-3p
2533 3554 hsa-miR-105-5p 2534 3555 hsa-miR-106a-3p 2535 3556
hsa-miR-106a-5p 2536 3557 hsa-miR-106b-3p 2537 3558 hsa-miR-106b-5p
2538 3559 hsa-miR-107 2539 3560 hsa-miR-10a-3p 2540 3561
hsa-miR-10a-5p 2541 3562 hsa-miR-10b-3p 2542 3563 hsa-miR-10b-5p
2543 3564 hsa-miR-1178-3p 2544 3565 hsa-miR-1178-5p 2545 3566
hsa-miR-1179 2546 3567 hsa-miR-1180 2547 3568 hsa-miR-1181 2548
3569 hsa-miR-1182 2549 3570 hsa-miR-1183 2550 3571 hsa-miR-1184
2551 3572 hsa-miR-1185-1-3p 2552 3573 hsa-miR-1185-2-3p 2553 3574
hsa-miR-1185-5p 2554 3575 hsa-miR-1193 2555 3576 hsa-miR-1197 2556
3577 hsa-miR-1200 2557 3578 hsa-miR-1202 2558 3579 hsa-miR-1203
2559 3580 hsa-miR-1204 2560 3581 hsa-miR-1205 2561 3582
hsa-miR-1206 2562 3583 hsa-miR-1207-3p 2563 3584 hsa-miR-1207-5p
2564 3585 hsa-miR-1208 2565 3586 hsa-miR-122-3p 2566 3587
hsa-miR-1224-3p 2567 3588 hsa-miR-1224-5p 2568 3589 hsa-miR-1225-3p
2569 3590 hsa-miR-1225-5p 2570 3591 hsa-miR-122-5p 2571 3592
hsa-miR-1226-3p 2572 3593 hsa-miR-1226-5p 2573 3594 hsa-miR-1227-3p
2574 3595 hsa-miR-1227-5p 2575 3596 hsa-miR-1228-3p 2576 3597
hsa-miR-1228-5p 2577 3598 hsa-miR-1229-3p 2578 3599 hsa-miR-1229-5p
2579 3600 hsa-miR-1231 2580 3601 hsa-miR-1233-1-5p 2581 3602
hsa-miR-1233-3p 2582 3603 hsa-miR-1234-3p 2583 3604 hsa-miR-1234-5p
2584 3605 hsa-miR-1236-3p 2585 3606 hsa-miR-1236-5p 2586 3607
hsa-miR-1237-3p 2587 3608 hsa-miR-1237-5p 2588 3609 hsa-miR-1238-3p
2589 3610 hsa-miR-1238-5p 2590 3611 hsa-miR-1243 2591 3612
hsa-miR-124-3p 2592 3613 hsa-miR-1244 2593 3614 hsa-miR-1245a 2594
3615 hsa-miR-1245b-3p 2595 3616 hsa-miR-1245b-5p 2596 3617
hsa-miR-124-5p 2597 3618 hsa-miR-1246 2598 3619 hsa-miR-1247-3p
2599 3620 hsa-miR-1247-5p 2600 3621 hsa-miR-1248 2601 3622
hsa-miR-1249 2602 3623 hsa-miR-1250 2603 3624 hsa-miR-1251 2604
3625 hsa-miR-1252 2605 3626 hsa-miR-1253 2606 3627 hsa-miR-1254
2607 3628 hsa-miR-1255a 2608 3629 hsa-miR-1255b-2-3p 2609 3630
hsa-miR-1255b-5p 2610 3631 hsa-miR-1256 2611 3632 hsa-miR-1257 2612
3633 hsa-miR-1258 2613 3634 hsa-miR-125a-3p 2614 3635
hsa-miR-125a-5p 2615 3636 hsa-miR-125b-1-3p 2616 3637
hsa-miR-125b-2-3p 2617 3638 hsa-miR-125b-5p 2618 3639 hsa-miR-1260a
2619 3640 hsa-miR-1260b 2620 3641 hsa-miR-1261 2621 3642
hsa-miR-1262 2622 3643 hsa-miR-1263 2623 3644 hsa-miR-126-3p 2624
3645 hsa-miR-1264 2625 3646 hsa-miR-1265 2626 3647 hsa-miR-126-5p
2627 3648 hsa-miR-1266 2628 3649 hsa-miR-1267 2629 3650
hsa-miR-1268a 2630 3651 hsa-miR-1268b 2631 3652 hsa-miR-1269a 2632
3653 hsa-miR-1269b 2633 3654 hsa-miR-1270 2634 3655 hsa-miR-1271-3p
2635 3656 hsa-miR-1271-5p 2636 3657 hsa-miR-1272 2637 3658
hsa-miR-1273a 2638 3659 hsa-miR-1273c 2639 3660 hsa-miR-1273d 2640
3661 hsa-miR-1273e 2641 3662 hsa-miR-1273f 2642 3663
hsa-miR-1273g-3p 2643 3664 hsa-miR-1273g-5p 2644 3665
hsa-miR-127-3p 2645 3666 hsa-miR-1275 2646 3667 hsa-miR-127-5p 2647
3668 hsa-miR-1276 2648 3669 hsa-miR-1277-3p 2649 3670
hsa-miR-1277-5p 2650 3671 hsa-miR-1278 2651 3672 hsa-miR-1279 2652
3673 hsa-miR-128 2653 3674 hsa-miR-1281 2654 3675 hsa-miR-1282 2655
3676 hsa-miR-1283 2656 3677 hsa-miR-1284 2657 3678 hsa-miR-1285-3p
2658 3679 hsa-miR-1285-5p 2659 3680 hsa-miR-1286 2660 3681
hsa-miR-1287 2661 3682 hsa-miR-1288 2662 3683 hsa-miR-1289 2663
3684 hsa-miR-1290 2664 3685 hsa-miR-1291 2665 3686 hsa-miR-129-1-3p
2666 3687 hsa-miR-1292-3p 2667 3688 hsa-miR-129-2-3p 2668 3689
hsa-miR-1292-5p 2669 3690 hsa-miR-1293 2670 3691 hsa-miR-1294 2671
3692 hsa-miR-1295a 2672 3693 hsa-miR-1295b-3p 2673 3694
hsa-miR-1295b-5p 2674 3695 hsa-miR-129-5p 2675 3696 hsa-miR-1296
2676 3697 hsa-miR-1297 2677 3698 hsa-miR-1298 2678 3699
hsa-miR-1299 2679 3700 hsa-miR-1301 2680 3701 hsa-miR-1302 2681
3702 hsa-miR-1303 2682 3703 hsa-miR-1304-3p 2683 3704
hsa-miR-1304-5p 2684 3705 hsa-miR-1305 2685 3706 hsa-miR-1306-3p
2686 3707 hsa-miR-1306-5p 2687 3708 hsa-miR-1307-3p 2688 3709
hsa-miR-1307-5p 2689 3710 hsa-miR-130a-3p 2690 3711 hsa-miR-130a-5p
2691 3712 hsa-miR-130b-3p 2692 3713 hsa-miR-130b-5p 2693 3714
hsa-miR-1321 2694 3715 hsa-miR-1322 2695 3716 hsa-miR-1323 2696
3717 hsa-miR-132-3p 2697 3718 hsa-miR-1324 2698 3719 hsa-miR-132-5p
2699 3720 hsa-miR-133a 2700 3721 hsa-miR-133b 2701 3722 hsa-miR-134
2702 3723 hsa-miR-1343 2703 3724 hsa-miR-135a-3p 2704 3725
hsa-miR-135a-5p 2705 3726 hsa-miR-135b-3p 2706 3727 hsa-miR-135b-5p
2707 3728 hsa-miR-136-3p 2708 3729 hsa-miR-136-5p 2709 3730
hsa-miR-137 2710 3731 hsa-miR-138-1-3p 2711 3732 hsa-miR-138-2-3p
2712 3733 hsa-miR-138-5p 2713 3734 hsa-miR-139-3p 2714 3735
hsa-miR-139-5p 2715 3736 hsa-miR-140-3p 2716 3737 hsa-miR-140-5p
2717 3738 hsa-miR-141-3p 2718 3739 hsa-miR-141-5p 2719 3740
hsa-miR-142-3p 2720 3741 hsa-miR-142-5p 2721 3742 hsa-miR-143-3p
2722 3743 hsa-miR-143-5p 2723 3744 hsa-miR-144-3p 2724 3745
hsa-miR-144-5p 2725 3746 hsa-miR-145-3p 2726 3747 hsa-miR-145-5p
2727 3748 hsa-miR-1468 2728 3749 hsa-miR-1469 2729 3750
hsa-miR-146a-3p 2730 3751 hsa-miR-146a-5p 2731 3752 hsa-miR-146b-3p
2732 3753 hsa-miR-146b-5p 2733 3754 hsa-miR-1470 2734 3755
hsa-miR-1471 2735 3756 hsa-miR-147a 2736 3757 hsa-miR-147b 2737
3758 hsa-miR-148a-3p 2738 3759 hsa-miR-148a-5p 2739 3760
hsa-miR-148b-3p 2740 3761 hsa-miR-148b-5p 2741 3762 hsa-miR-149-3p
2742 3763 hsa-miR-149-5p 2743 3764 hsa-miR-150-3p 2744 3765
hsa-miR-150-5p 2745 3766 hsa-miR-151a-3p 2746 3767 hsa-miR-151a-5p
2747 3768 hsa-miR-151b 2748 3769 hsa-miR-152 2749 3770 hsa-miR-153
2750 3771 hsa-miR-1537 2751 3772
hsa-miR-1538 2752 3773 hsa-miR-1539 2753 3774 hsa-miR-154-3p 2754
3775 hsa-miR-154-5p 2755 3776 hsa-miR-155-3p 2756 3777
hsa-miR-155-5p 2757 3778 hsa-miR-1587 2758 3779 hsa-miR-15a-3p 2759
3780 hsa-miR-15a-5p 2760 3781 hsa-miR-15b-3p 2761 3782
hsa-miR-15b-5p 2762 3783 hsa-miR-16-1-3p 2763 3784 hsa-miR-16-2-3p
2764 3785 hsa-miR-16-5p 2765 3786 hsa-miR-17-3p 2766 3787
hsa-miR-17-5p 2767 3788 hsa-miR-181a-2-3p 2768 3789 hsa-miR-181a-3p
2769 3790 hsa-miR-181a-5p 2770 3791 hsa-miR-181b-3p 2771 3792
hsa-miR-181b-5p 2772 3793 hsa-miR-181c-3p 2773 3794 hsa-miR-181c-5p
2774 3795 hsa-miR-181d 2775 3796 hsa-miR-182-3p 2776 3797
hsa-miR-1825 2777 3798 hsa-miR-182-5p 2778 3799 hsa-miR-1827 2779
3800 hsa-miR-183-3p 2780 3801 hsa-miR-183-5p 2781 3802 hsa-miR-184
2782 3803 hsa-miR-185-3p 2783 3804 hsa-miR-185-5p 2784 3805
hsa-miR-186-3p 2785 3806 hsa-miR-186-5p 2786 3807 hsa-miR-187-3p
2787 3808 hsa-miR-187-5p 2788 3809 hsa-miR-188-3p 2789 3810
hsa-miR-188-5p 2790 3811 hsa-miR-18a-3p 2791 3812 hsa-miR-18a-5p
2792 3813 hsa-miR-18b-3p 2793 3814 hsa-miR-18b-5p 2794 3815
hsa-miR-1908 2795 3816 hsa-miR-1909-3p 2796 3817 hsa-miR-1909-5p
2797 3818 hsa-miR-190a 2798 3819 hsa-miR-190b 2799 3820
hsa-miR-1910 2800 3821 hsa-miR-1911-3p 2801 3822 hsa-miR-1911-5p
2802 3823 hsa-miR-1912 2803 3824 hsa-miR-1913 2804 3825
hsa-miR-191-3p 2805 3826 hsa-miR-1914-3p 2806 3827 hsa-miR-1914-5p
2807 3828 hsa-miR-1915-3p 2808 3829 hsa-miR-1915-5p 2809 3830
hsa-miR-191-5p 2810 3831 hsa-miR-192-3p 2811 3832 hsa-miR-192-5p
2812 3833 hsa-miR-193a-3p 2813 3834 hsa-miR-193a-5p 2814 3835
hsa-miR-193b-3p 2815 3836 hsa-miR-193b-5p 2816 3837 hsa-miR-194-3p
2817 3838 hsa-miR-194-5p 2818 3839 hsa-miR-195-3p 2819 3840
hsa-miR-195-5p 2820 3841 hsa-miR-196a-3p 2821 3842 hsa-miR-196a-5p
2822 3843 hsa-miR-196b-3p 2823 3844 hsa-miR-196b-5p 2824 3845
hsa-miR-1972 2825 3846 hsa-miR-1973 2826 3847 hsa-miR-197-3p 2827
3848 hsa-miR-197-5p 2828 3849 hsa-miR-1976 2829 3850 hsa-miR-198
2830 3851 hsa-miR-199a-3p 2831 3852 hsa-miR-199a-5p 2832 3853
hsa-miR-199b-3p 2833 3854 hsa-miR-199b-5p 2834 3855 hsa-miR-19a-3p
2835 3856 hsa-miR-19a-5p 2836 3857 hsa-miR-19b-1-5p 2837 3858
hsa-miR-19b-2-5p 2838 3859 hsa-miR-19b-3p 2839 3860 hsa-miR-200a-3p
2840 3861 hsa-miR-200a-5p 2841 3862 hsa-miR-200b-3p 2842 3863
hsa-miR-200b-5p 2843 3864 hsa-miR-200c-3p 2844 3865 hsa-miR-200c-5p
2845 3866 hsa-miR-202-3p 2846 3867 hsa-miR-202-5p 2847 3868
hsa-miR-203a 2848 3869 hsa-miR-203b-3p 2849 3870 hsa-miR-203b-5p
2850 3871 hsa-miR-204-3p 2851 3872 hsa-miR-204-5p 2852 3873
hsa-miR-2052 2853 3874 hsa-miR-2053 2854 3875 hsa-miR-205-3p 2855
3876 hsa-miR-2054 2856 3877 hsa-miR-205-5p 2857 3878 hsa-miR-206
2858 3879 hsa-miR-208a 2859 3880 hsa-miR-208b 2860 3881
hsa-miR-20a-3p 2861 3882 hsa-miR-20a-5p 2862 3883 hsa-miR-20b-3p
2863 3884 hsa-miR-20b-5p 2864 3885 hsa-miR-210 2865 3886
hsa-miR-2110 2866 3887 hsa-miR-2113 2867 3888 hsa-miR-211-3p 2868
3889 hsa-miR-2114-3p 2869 3890 hsa-miR-2114-5p 2870 3891
hsa-miR-2115-3p 2871 3892 hsa-miR-2115-5p 2872 3893 hsa-miR-211-5p
2873 3894 hsa-miR-2116-3p 2874 3895 hsa-miR-2116-5p 2875 3896
hsa-miR-2117 2876 3897 hsa-miR-212-3p 2877 3898 hsa-miR-212-5p 2878
3899 hsa-miR-21-3p 2879 3900 hsa-miR-214-3p 2880 3901
hsa-miR-214-5p 2881 3902 hsa-miR-215 2882 3903 hsa-miR-21-5p 2883
3904 hsa-miR-216a-3p 2884 3905 hsa-miR-216a-5p 2885 3906
hsa-miR-216b 2886 3907 hsa-miR-217 2887 3908 hsa-miR-218-1-3p 2888
3909 hsa-miR-218-2-3p 2889 3910 hsa-miR-218-5p 2890 3911
hsa-miR-219-1-3p 2891 3912 hsa-miR-219-2-3p 2892 3913
hsa-miR-219-5p 2893 3914 hsa-miR-221-3p 2894 3915 hsa-miR-221-5p
2895 3916 hsa-miR-222-3p 2896 3917 hsa-miR-222-5p 2897 3918
hsa-miR-223-3p 2898 3919 hsa-miR-223-5p 2899 3920 hsa-miR-22-3p
2900 3921 hsa-miR-224-3p 2901 3922 hsa-miR-224-5p 2902 3923
hsa-miR-22-5p 2903 3924 hsa-miR-2276 2904 3925 hsa-miR-2277-3p 2905
3926 hsa-miR-2277-5p 2906 3927 hsa-miR-2278 2907 3928
hsa-miR-2355-3p 2908 3929 hsa-miR-2355-5p 2909 3930 hsa-miR-2392
2910 3931 hsa-miR-23a-3p 2911 3932 hsa-miR-23a-5p 2912 3933
hsa-miR-23b-3p 2913 3934 hsa-miR-23b-5p 2914 3935 hsa-miR-23c 2915
3936 hsa-miR-24-1-5p 2916 3937 hsa-miR-24-2-5p 2917 3938
hsa-miR-24-3p 2918 3939 hsa-miR-2467-3p 2919 3940 hsa-miR-2467-5p
2920 3941 hsa-miR-25-3p 2921 3942 hsa-miR-25-5p 2922 3943
hsa-miR-2681-3p 2923 3944 hsa-miR-2681-5p 2924 3945 hsa-miR-2682-3p
2925 3946 hsa-miR-2682-5p 2926 3947 hsa-miR-26a-1-3p 2927 3948
hsa-miR-26a-2-3p 2928 3949 hsa-miR-26a-5p 2929 3950 hsa-miR-26b-3p
2930 3951 hsa-miR-26b-5p 2931 3952 hsa-miR-27a-3p 2932 3953
hsa-miR-27a-5p 2933 3954 hsa-miR-27b-3p 2934 3955 hsa-miR-27b-5p
2935 3956 hsa-miR-28-3p 2936 3957 hsa-miR-28-5p 2937 3958
hsa-miR-2861 2938 3959 hsa-miR-2909 2939 3960 hsa-miR-296-3p 2940
3961 hsa-miR-2964a-3p 2941 3962 hsa-miR-2964a-5p 2942 3963
hsa-miR-296-5p 2943 3964 hsa-miR-297 2944 3965 hsa-miR-298 2945
3966 hsa-miR-299-3p 2946 3967 hsa-miR-299-5p 2947 3968
hsa-miR-29a-3p 2948 3969 hsa-miR-29a-5p 2949 3970 hsa-miR-29b-1-5p
2950 3971 hsa-miR-29b-2-5p 2951 3972 hsa-miR-29b-3p 2952 3973
hsa-miR-29c-3p 2953 3974 hsa-miR-29c-5p 2954 3975 hsa-miR-300 2955
3976 hsa-miR-301a-3p 2956 3977 hsa-miR-301a-5p 2957 3978
hsa-miR-301b 2958 3979 hsa-miR-302a-3p 2959 3980 hsa-miR-302a-5p
2960 3981 hsa-miR-302b-3p 2961 3982 hsa-miR-302b-5p 2962 3983
hsa-miR-302c-3p 2963 3984 hsa-miR-302c-5p 2964 3985 hsa-miR-302d-3p
2965 3986 hsa-miR-302d-5p 2966 3987 hsa-miR-302e 2967 3988
hsa-miR-302f 2968 3989 hsa-miR-3064-3p 2969 3990 hsa-miR-3064-5p
2970 3991 hsa-miR-3065-3p 2971 3992 hsa-miR-3065-5p 2972 3993
hsa-miR-3074-3p 2973 3994 hsa-miR-3074-5p 2974 3995 hsa-miR-30a-3p
2975 3996 hsa-miR-30a-5p 2976 3997 hsa-miR-30b-3p 2977 3998
hsa-miR-30b-5p 2978 3999 hsa-miR-30c-1-3p 2979 4000
hsa-miR-30c-2-3p 2980 4001 hsa-miR-30c-5p 2981 4002 hsa-miR-30d-3p
2982 4003 hsa-miR-30d-5p 2983 4004 hsa-miR-30e-3p 2984 4005
hsa-miR-30e-5p 2985 4006 hsa-miR-3115 2986 4007 hsa-miR-3116 2987
4008 hsa-miR-3117-3p 2988 4009 hsa-miR-3117-5p 2989 4010
hsa-miR-3118 2990 4011 hsa-miR-3119 2991 4012 hsa-miR-3120-3p 2992
4013 hsa-miR-3120-5p 2993 4014 hsa-miR-3121-3p 2994 4015
hsa-miR-3121-5p 2995 4016 hsa-miR-3122 2996 4017 hsa-miR-3123 2997
4018 hsa-miR-3124-3p 2998 4019 hsa-miR-3124-5p 2999 4020
hsa-miR-3125 3000 4021 hsa-miR-3126-3p 3001 4022 hsa-miR-3126-5p
3002 4023
hsa-miR-3127-3p 3003 4024 hsa-miR-3127-5p 3004 4025 hsa-miR-3128
3005 4026 hsa-miR-3129-3p 3006 4027 hsa-miR-3129-5p 3007 4028
hsa-miR-3130-3p 3008 4029 hsa-miR-3130-5p 3009 4030 hsa-miR-3131
3010 4031 hsa-miR-3132 3011 4032 hsa-miR-3133 3012 4033
hsa-miR-3134 3013 4034 hsa-miR-3135a 3014 4035 hsa-miR-3135b 3015
4036 hsa-miR-3136-3p 3016 4037 hsa-miR-3136-5p 3017 4038
hsa-miR-3137 3018 4039 hsa-miR-3138 3019 4040 hsa-miR-3139 3020
4041 hsa-miR-31-3p 3021 4042 hsa-miR-3140-3p 3022 4043
hsa-miR-3140-5p 3023 4044 hsa-miR-3141 3024 4045 hsa-miR-3142 3025
4046 hsa-miR-3143 3026 4047 hsa-miR-3144-3p 3027 4048
hsa-miR-3144-5p 3028 4049 hsa-miR-3145-3p 3029 4050 hsa-miR-3145-5p
3030 4051 hsa-miR-3146 3031 4052 hsa-miR-3147 3032 4053
hsa-miR-3148 3033 4054 hsa-miR-3149 3034 4055 hsa-miR-3150a-3p 3035
4056 hsa-miR-3150a-5p 3036 4057 hsa-miR-3150b-3p 3037 4058
hsa-miR-3150b-5p 3038 4059 hsa-miR-3151 3039 4060 hsa-miR-3152-3p
3040 4061 hsa-miR-3152-5p 3041 4062 hsa-miR-3153 3042 4063
hsa-miR-3154 3043 4064 hsa-miR-3155a 3044 4065 hsa-miR-3155b 3045
4066 hsa-miR-3156-3p 3046 4067 hsa-miR-3156-5p 3047 4068
hsa-miR-3157-3p 3048 4069 hsa-miR-3157-5p 3049 4070 hsa-miR-3158-3p
3050 4071 hsa-miR-3158-5p 3051 4072 hsa-miR-3159 3052 4073
hsa-miR-31-5p 3053 4074 hsa-miR-3160-3p 3054 4075 hsa-miR-3160-5p
3055 4076 hsa-miR-3161 3056 4077 hsa-miR-3162-3p 3057 4078
hsa-miR-3162-5p 3058 4079 hsa-miR-3163 3059 4080 hsa-miR-3164 3060
4081 hsa-miR-3165 3061 4082 hsa-miR-3166 3062 4083 hsa-miR-3167
3063 4084 hsa-miR-3168 3064 4085 hsa-miR-3169 3065 4086
hsa-miR-3170 3066 4087 hsa-miR-3171 3067 4088 hsa-miR-3173-3p 3068
4089 hsa-miR-3173-5p 3069 4090 hsa-miR-3174 3070 4091 hsa-miR-3175
3071 4092 hsa-miR-3176 3072 4093 hsa-miR-3177-3p 3073 4094
hsa-miR-3177-5p 3074 4095 hsa-miR-3178 3075 4096 hsa-miR-3179 3076
4097 hsa-miR-3180 3077 4098 hsa-miR-3180-3p 3078 4099
hsa-miR-3180-5p 3079 4100 hsa-miR-3181 3080 4101 hsa-miR-3182 3081
4102 hsa-miR-3183 3082 4103 hsa-miR-3184-3p 3083 4104
hsa-miR-3184-5p 3084 4105 hsa-miR-3185 3085 4106 hsa-miR-3186-3p
3086 4107 hsa-miR-3186-5p 3087 4108 hsa-miR-3187-3p 3088 4109
hsa-miR-3187-5p 3089 4110 hsa-miR-3188 3090 4111 hsa-miR-3189-3p
3091 4112 hsa-miR-3189-5p 3092 4113 hsa-miR-3190-3p 3093 4114
hsa-miR-3190-5p 3094 4115 hsa-miR-3191-3p 3095 4116 hsa-miR-3191-5p
3096 4117 hsa-miR-3192 3097 4118 hsa-miR-3193 3098 4119
hsa-miR-3194-3p 3099 4120 hsa-miR-3194-5p 3100 4121 hsa-miR-3195
3101 4122 hsa-miR-3196 3102 4123 hsa-miR-3197 3103 4124
hsa-miR-3198 3104 4125 hsa-miR-3199 3105 4126 hsa-miR-3200-3p 3106
4127 hsa-miR-3200-5p 3107 4128 hsa-miR-3201 3108 4129 hsa-miR-3202
3109 4130 hsa-miR-320a 3110 4131 hsa-miR-320b 3111 4132
hsa-miR-320c 3112 4133 hsa-miR-320d 3113 4134 hsa-miR-320e 3114
4135 hsa-miR-323a-3p 3115 4136 hsa-miR-323a-5p 3116 4137
hsa-miR-323b-3p 3117 4138 hsa-miR-323b-5p 3118 4139 hsa-miR-32-3p
3119 4140 hsa-miR-324-3p 3120 4141 hsa-miR-324-5p 3121 4142
hsa-miR-325 3122 4143 hsa-miR-32-5p 3123 4144 hsa-miR-326 3124 4145
hsa-miR-328 3125 4146 hsa-miR-329 3126 4147 hsa-miR-330-3p 3127
4148 hsa-miR-330-5p 3128 4149 hsa-miR-331-3p 3129 4150
hsa-miR-331-5p 3130 4151 hsa-miR-335-3p 3131 4152 hsa-miR-335-5p
3132 4153 hsa-miR-337-3p 3133 4154 hsa-miR-337-5p 3134 4155
hsa-miR-338-3p 3135 4156 hsa-miR-338-5p 3136 4157 hsa-miR-339-3p
3137 4158 hsa-miR-339-5p 3138 4159 hsa-miR-33a-3p 3139 4160
hsa-miR-33a-5p 3140 4161 hsa-miR-33b-3p 3141 4162 hsa-miR-33b-5p
3142 4163 hsa-miR-340-3p 3143 4164 hsa-miR-340-5p 3144 4165
hsa-miR-342-3p 3145 4166 hsa-miR-342-5p 3146 4167 hsa-miR-345-3p
3147 4168 hsa-miR-345-5p 3148 4169 hsa-miR-346 3149 4170
hsa-miR-34a-3p 3150 4171 hsa-miR-34a-5p 3151 4172 hsa-miR-34b-3p
3152 4173 hsa-miR-34b-5p 3153 4174 hsa-miR-34c-3p 3154 4175
hsa-miR-34c-5p 3155 4176 hsa-miR-3529-3p 3156 4177 hsa-miR-3529-5p
3157 4178 hsa-miR-3591-3p 3158 4179 hsa-miR-3591-5p 3159 4180
hsa-miR-3605-3p 3160 4181 hsa-miR-3605-5p 3161 4182 hsa-miR-3606-3p
3162 4183 hsa-miR-3606-5p 3163 4184 hsa-miR-3607-3p 3164 4185
hsa-miR-3607-5p 3165 4186 hsa-miR-3609 3166 4187 hsa-miR-3610 3167
4188 hsa-miR-3611 3168 4189 hsa-miR-3612 3169 4190 hsa-miR-3613-3p
3170 4191 hsa-miR-3613-5p 3171 4192 hsa-miR-361-3p 3172 4193
hsa-miR-3614-3p 3173 4194 hsa-miR-3614-5p 3174 4195 hsa-miR-3615
3175 4196 hsa-miR-361-5p 3176 4197 hsa-miR-3616-3p 3177 4198
hsa-miR-3616-5p 3178 4199 hsa-miR-3617-3p 3179 4200 hsa-miR-3617-5p
3180 4201 hsa-miR-3618 3181 4202 hsa-miR-3619-3p 3182 4203
hsa-miR-3619-5p 3183 4204 hsa-miR-3620-3p 3184 4205 hsa-miR-3620-5p
3185 4206 hsa-miR-3621 3186 4207 hsa-miR-3622a-3p 3187 4208
hsa-miR-3622a-5p 3188 4209 hsa-miR-3622b-3p 3189 4210
hsa-miR-3622b-5p 3190 4211 hsa-miR-362-3p 3191 4212 hsa-miR-362-5p
3192 4213 hsa-miR-363-3p 3193 4214 hsa-miR-363-5p 3194 4215
hsa-miR-3646 3195 4216 hsa-miR-3648 3196 4217 hsa-miR-3649 3197
4218 hsa-miR-3650 3198 4219 hsa-miR-3651 3199 4220 hsa-miR-3652
3200 4221 hsa-miR-3653 3201 4222 hsa-miR-3654 3202 4223
hsa-miR-3655 3203 4224 hsa-miR-3656 3204 4225 hsa-miR-3657 3205
4226 hsa-miR-3658 3206 4227 hsa-miR-3659 3207 4228 hsa-miR-365a-3p
3208 4229 hsa-miR-365a-5p 3209 4230 hsa-miR-365b-3p 3210 4231
hsa-miR-365b-5p 3211 4232 hsa-miR-3660 3212 4233 hsa-miR-3661 3213
4234 hsa-miR-3662 3214 4235 hsa-miR-3663-3p 3215 4236
hsa-miR-3663-5p 3216 4237 hsa-miR-3664-3p 3217 4238 hsa-miR-3664-5p
3218 4239 hsa-miR-3665 3219 4240 hsa-miR-3666 3220 4241
hsa-miR-3667-3p 3221 4242 hsa-miR-3667-5p 3222 4243 hsa-miR-3668
3223 4244 hsa-miR-3669 3224 4245 hsa-miR-3670 3225 4246
hsa-miR-3671 3226 4247 hsa-miR-3672 3227 4248 hsa-miR-3673 3228
4249 hsa-miR-367-3p 3229 4250 hsa-miR-3674 3230 4251
hsa-miR-3675-3p 3231 4252 hsa-miR-3675-5p 3232 4253 hsa-miR-367-5p
3233 4254 hsa-miR-3676-3p 3234 4255 hsa-miR-3676-5p 3235 4256
hsa-miR-3677-3p 3236 4257 hsa-miR-3677-5p 3237 4258 hsa-miR-3678-3p
3238 4259 hsa-miR-3678-5p 3239 4260 hsa-miR-3679-3p 3240 4261
hsa-miR-3679-5p 3241 4262 hsa-miR-3680-3p 3242 4263 hsa-miR-3680-5p
3243 4264 hsa-miR-3681-3p 3244 4265 hsa-miR-3681-5p 3245 4266
hsa-miR-3682-3p 3246 4267 hsa-miR-3682-5p 3247 4268 hsa-miR-3683
3248 4269 hsa-miR-3684 3249 4270 hsa-miR-3685 3250 4271
hsa-miR-3686 3251 4272 hsa-miR-3687 3252 4273 hsa-miR-3688-3p 3253
4274
hsa-miR-3688-5p 3254 4275 hsa-miR-3689a-3p 3255 4276
hsa-miR-3689a-5p 3256 4277 hsa-miR-3689b-3p 3257 4278
hsa-miR-3689b-5p 3258 4279 hsa-miR-3689c 3259 4280 hsa-miR-3689d
3260 4281 hsa-miR-3689e 3261 4282 hsa-miR-3689f 3262 4283
hsa-miR-3690 3263 4284 hsa-miR-3691-3p 3264 4285 hsa-miR-3691-5p
3265 4286 hsa-miR-3692-3p 3266 4287 hsa-miR-3692-5p 3267 4288
hsa-miR-369-3p 3268 4289 hsa-miR-369-5p 3269 4290 hsa-miR-370 3270
4291 hsa-miR-3713 3271 4292 hsa-miR-3714 3272 4293 hsa-miR-371a-3p
3273 4294 hsa-miR-371a-5p 3274 4295 hsa-miR-371b-3p 3275 4296
hsa-miR-371b-5p 3276 4297 hsa-miR-372 3277 4298 hsa-miR-373-3p 3278
4299 hsa-miR-373-5p 3279 4300 hsa-miR-374a-3p 3280 4301
hsa-miR-374a-5p 3281 4302 hsa-miR-374b-3p 3282 4303 hsa-miR-374b-5p
3283 4304 hsa-miR-374c-3p 3284 4305 hsa-miR-374c-5p 3285 4306
hsa-miR-375 3286 4307 hsa-miR-376a-2-5p 3287 4308 hsa-miR-376a-3p
3288 4309 hsa-miR-376a-5p 3289 4310 hsa-miR-376b-3p 3290 4311
hsa-miR-376b-5p 3291 4312 hsa-miR-376c-3p 3292 4313 hsa-miR-376c-5p
3293 4314 hsa-miR-377-3p 3294 4315 hsa-miR-377-5p 3295 4316
hsa-miR-378a-3p 3296 4317 hsa-miR-378a-5p 3297 4318 hsa-miR-378b
3298 4319 hsa-miR-378c 3299 4320 hsa-miR-378d 3300 4321
hsa-miR-378e 3301 4322 hsa-miR-378f 3302 4323 hsa-miR-378g 3303
4324 hsa-miR-378h 3304 4325 hsa-miR-378i 3305 4326 hsa-miR-378j
3306 4327 hsa-miR-379-3p 3307 4328 hsa-miR-379-5p 3308 4329
hsa-miR-380-3p 3309 4330 hsa-miR-380-5p 3310 4331 hsa-miR-381-3p
3311 4332 hsa-miR-381-5p 3312 4333 hsa-miR-382-3p 3313 4334
hsa-miR-382-5p 3314 4335 hsa-miR-383 3315 4336 hsa-miR-384 3316
4337 hsa-miR-3907 3317 4338 hsa-miR-3908 3318 4339 hsa-miR-3909
3319 4340 hsa-miR-3910 3320 4341 hsa-miR-3911 3321 4342
hsa-miR-3912 3322 4343 hsa-miR-3913-3p 3323 4344 hsa-miR-3913-5p
3324 4345 hsa-miR-3914 3325 4346 hsa-miR-3915 3326 4347
hsa-miR-3916 3327 4348 hsa-miR-3917 3328 4349 hsa-miR-3918 3329
4350 hsa-miR-3919 3330 4351 hsa-miR-3920 3331 4352 hsa-miR-3921
3332 4353 hsa-miR-3922-3p 3333 4354 hsa-miR-3922-5p 3334 4355
hsa-miR-3923 3335 4356 hsa-miR-3924 3336 4357 hsa-miR-3925-3p 3337
4358 hsa-miR-3925-5p 3338 4359 hsa-miR-3926 3339 4360
hsa-miR-3927-3p 3340 4361 hsa-miR-3927-5p 3341 4362 hsa-miR-3928
3342 4363 hsa-miR-3929 3343 4364 hsa-miR-3934-3p 3344 4365
hsa-miR-3934-5p 3345 4366 hsa-miR-3935 3346 4367 hsa-miR-3936 3347
4368 hsa-miR-3937 3348 4369 hsa-miR-3938 3349 4370 hsa-miR-3939
3350 4371 hsa-miR-3940-3p 3351 4372 hsa-miR-3940-5p 3352 4373
hsa-miR-3941 3353 4374 hsa-miR-3942-3p 3354 4375 hsa-miR-3942-5p
3355 4376 hsa-miR-3943 3356 4377 hsa-miR-3944-3p 3357 4378
hsa-miR-3944-5p 3358 4379 hsa-miR-3945 3359 4380 hsa-miR-3960 3360
4381 hsa-miR-3972 3361 4382 hsa-miR-3973 3362 4383 hsa-miR-3974
3363 4384 hsa-miR-3975 3364 4385 hsa-miR-3976 3365 4386
hsa-miR-3977 3366 4387 hsa-miR-3978 3367 4388 hsa-miR-409-3p 3368
4389 hsa-miR-409-5p 3369 4390 hsa-miR-410 3370 4391 hsa-miR-411-3p
3371 4392 hsa-miR-411-5p 3372 4393 hsa-miR-412 3373 4394
hsa-miR-421 3374 4395 hsa-miR-422a 3375 4396 hsa-miR-423-3p 3376
4397 hsa-miR-423-5p 3377 4398 hsa-miR-424-3p 3378 4399
hsa-miR-424-5p 3379 4400 hsa-miR-4251 3380 4401 hsa-miR-4252 3381
4402 hsa-miR-4253 3382 4403 hsa-miR-425-3p 3383 4404 hsa-miR-4254
3384 4405 hsa-miR-4255 3385 4406 hsa-miR-425-5p 3386 4407
hsa-miR-4256 3387 4408 hsa-miR-4257 3388 4409 hsa-miR-4258 3389
4410 hsa-miR-4259 3390 4411 hsa-miR-4260 3391 4412 hsa-miR-4261
3392 4413 hsa-miR-4262 3393 4414 hsa-miR-4263 3394 4415
hsa-miR-4264 3395 4416 hsa-miR-4265 3396 4417 hsa-miR-4266 3397
4418 hsa-miR-4267 3398 4419 hsa-miR-4268 3399 4420 hsa-miR-4269
3400 4421 hsa-miR-4270 3401 4422 hsa-miR-4271 3402 4423
hsa-miR-4272 3403 4424 hsa-miR-4273 3404 4425 hsa-miR-4274 3405
4426 hsa-miR-4275 3406 4427 hsa-miR-4276 3407 4428 hsa-miR-4277
3408 4429 hsa-miR-4278 3409 4430 hsa-miR-4279 3410 4431
hsa-miR-4280 3411 4432 hsa-miR-4281 3412 4433 hsa-miR-4282 3413
4434 hsa-miR-4283 3414 4435 hsa-miR-4284 3415 4436 hsa-miR-4285
3416 4437 hsa-miR-4286 3417 4438 hsa-miR-4287 3418 4439
hsa-miR-4288 3419 4440 hsa-miR-4289 3420 4441 hsa-miR-429 3421 4442
hsa-miR-4290 3422 4443 hsa-miR-4291 3423 4444 hsa-miR-4292 3424
4445 hsa-miR-4293 3425 4446 hsa-miR-4294 3426 4447 hsa-miR-4295
3427 4448 hsa-miR-4296 3428 4449 hsa-miR-4297 3429 4450
hsa-miR-4298 3430 4451 hsa-miR-4299 3431 4452 hsa-miR-4300 3432
4453 hsa-miR-4301 3433 4454 hsa-miR-4302 3434 4455 hsa-miR-4303
3435 4456 hsa-miR-4304 3436 4457 hsa-miR-4305 3437 4458
hsa-miR-4306 3438 4459 hsa-miR-4307 3439 4460 hsa-miR-4308 3440
4461 hsa-miR-4309 3441 4462 hsa-miR-4310 3442 4463 hsa-miR-4311
3443 4464 hsa-miR-4312 3444 4465 hsa-miR-4313 3445 4466
hsa-miR-431-3p 3446 4467 hsa-miR-4314 3447 4468 hsa-miR-4315 3448
4469 hsa-miR-431-5p 3449 4470 hsa-miR-4316 3450 4471 hsa-miR-4317
3451 4472 hsa-miR-4318 3452 4473 hsa-miR-4319 3453 4474
hsa-miR-4320 3454 4475 hsa-miR-4321 3455 4476 hsa-miR-4322 3456
4477 hsa-miR-4323 3457 4478 hsa-miR-432-3p 3458 4479 hsa-miR-4324
3459 4480 hsa-miR-4325 3460 4481 hsa-miR-432-5p 3461 4482
hsa-miR-4326 3462 4483 hsa-miR-4327 3463 4484 hsa-miR-4328 3464
4485 hsa-miR-4329 3465 4486 hsa-miR-433 3466 4487 hsa-miR-4330 3467
4488 hsa-miR-4417 3468 4489 hsa-miR-4418 3469 4490 hsa-miR-4419a
3470 4491 hsa-miR-4419b 3471 4492 hsa-miR-4420 3472 4493
hsa-miR-4421 3473 4494 hsa-miR-4422 3474 4495 hsa-miR-4423-3p 3475
4496 hsa-miR-4423-5p 3476 4497 hsa-miR-4424 3477 4498 hsa-miR-4425
3478 4499 hsa-miR-4426 3479 4500 hsa-miR-4427 3480 4501
hsa-miR-4428 3481 4502 hsa-miR-4429 3482 4503 hsa-miR-4430 3483
4504 hsa-miR-4431 3484 4505 hsa-miR-4432 3485 4506 hsa-miR-4433-3p
3486 4507 hsa-miR-4433-5p 3487 4508 hsa-miR-4434 3488 4509
hsa-miR-4435 3489 4510 hsa-miR-4436a 3490 4511 hsa-miR-4436b-3p
3491 4512 hsa-miR-4436b-5p 3492 4513 hsa-miR-4437 3493 4514
hsa-miR-4438 3494 4515 hsa-miR-4439 3495 4516 hsa-miR-4440 3496
4517 hsa-miR-4441 3497 4518 hsa-miR-4442 3498 4519 hsa-miR-4443
3499 4520 hsa-miR-4444 3500 4521 hsa-miR-4445-3p 3501 4522
hsa-miR-4445-5p 3502 4523 hsa-miR-4446-3p 3503 4524 hsa-miR-4446-5p
3504 4525
hsa-miR-4447 3505 4526 hsa-miR-4448 3506 4527 hsa-miR-4449 3507
4528 hsa-miR-4450 3508 4529 hsa-miR-4451 3509 4530 hsa-miR-4452
3510 4531 hsa-miR-4453 3511 4532 hsa-miR-4454 3512 4533
hsa-miR-4455 3513 4534 hsa-miR-4456 3514 4535 hsa-miR-4457 3515
4536 hsa-miR-4458 3516 4537 hsa-miR-4459 3517 4538 hsa-miR-4460
3518 4539 hsa-miR-4461 3519 4540 hsa-miR-4462 3520 4541
hsa-miR-4463 3521 4542 hsa-miR-4464 3522 4543 hsa-miR-4465 3523
4544 hsa-miR-4466 3524 4545 hsa-miR-4467 3525 4546 hsa-miR-4468
3526 4547 hsa-miR-4469 3527 4548 hsa-miR-4470 3528 4549
hsa-miR-4471 4550 5571 hsa-miR-4472 4551 5572 hsa-miR-4473 4552
5573 hsa-miR-4474-3p 4553 5574 hsa-miR-4474-5p 4554 5575
hsa-miR-4475 4555 5576 hsa-miR-4476 4556 5577 hsa-miR-4477a 4557
5578 hsa-miR-4477b 4558 5579 hsa-miR-4478 4559 5580 hsa-miR-4479
4560 5581 hsa-miR-448 4561 5582 hsa-miR-4480 4562 5583 hsa-miR-4481
4563 5584 hsa-miR-4482-3p 4564 5585 hsa-miR-4482-5p 4565 5586
hsa-miR-4483 4566 5587 hsa-miR-4484 4567 5588 hsa-miR-4485 4568
5589 hsa-miR-4486 4569 5590 hsa-miR-4487 4570 5591 hsa-miR-4488
4571 5592 hsa-miR-4489 4572 5593 hsa-miR-4490 4573 5594
hsa-miR-4491 4574 5595 hsa-miR-4492 4575 5596 hsa-miR-4493 4576
5597 hsa-miR-4494 4577 5598 hsa-miR-4495 4578 5599 hsa-miR-4496
4579 5600 hsa-miR-4497 4580 5601 hsa-miR-4498 4581 5602
hsa-miR-4499 4582 5603 hsa-miR-449a 4583 5604 hsa-miR-449b-3p 4584
5605 hsa-miR-449b-5p 4585 5606 hsa-miR-449c-3p 4586 5607
hsa-miR-449c-5p 4587 5608 hsa-miR-4500 4588 5609 hsa-miR-4501 4589
5610 hsa-miR-4502 4590 5611 hsa-miR-4503 4591 5612 hsa-miR-4504
4592 5613 hsa-miR-4505 4593 5614 hsa-miR-4506 4594 5615
hsa-miR-4507 4595 5616 hsa-miR-4508 4596 5617 hsa-miR-4509 4597
5618 hsa-miR-450a-3p 4598 5619 hsa-miR-450a-5p 4599 5620
hsa-miR-450b-3p 4600 5621 hsa-miR-450b-5p 4601 5622 hsa-miR-4510
4602 5623 hsa-miR-4511 4603 5624 hsa-miR-4512 4604 5625
hsa-miR-4513 4605 5626 hsa-miR-4514 4606 5627 hsa-miR-4515 4607
5628 hsa-miR-4516 4608 5629 hsa-miR-4517 4609 5630 hsa-miR-4518
4610 5631 hsa-miR-4519 4611 5632 hsa-miR-451a 4612 5633
hsa-miR-451b 4613 5634 hsa-miR-4520a-3p 4614 5635 hsa-miR-4520a-5p
4615 5636 hsa-miR-4520b-3p 4616 5637 hsa-miR-4520b-5p 4617 5638
hsa-miR-4521 4618 5639 hsa-miR-4522 4619 5640 hsa-miR-4523 4620
5641 hsa-miR-452-3p 4621 5642 hsa-miR-4524a-3p 4622 5643
hsa-miR-4524a-5p 4623 5644 hsa-miR-4524b-3p 4624 5645
hsa-miR-4524b-5p 4625 5646 hsa-miR-4525 4626 5647 hsa-miR-452-5p
4627 5648 hsa-miR-4526 4628 5649 hsa-miR-4527 4629 5650
hsa-miR-4528 4630 5651 hsa-miR-4529-3p 4631 5652 hsa-miR-4529-5p
4632 5653 hsa-miR-4530 4633 5654 hsa-miR-4531 4634 5655
hsa-miR-4532 4635 5656 hsa-miR-4533 4636 5657 hsa-miR-4534 4637
5658 hsa-miR-4535 4638 5659 hsa-miR-4536-3p 4639 5660
hsa-miR-4536-5p 4640 5661 hsa-miR-4537 4641 5662 hsa-miR-4538 4642
5663 hsa-miR-4539 4643 5664 hsa-miR-4540 4644 5665 hsa-miR-454-3p
4645 5666 hsa-miR-454-5p 4646 5667 hsa-miR-455-3p 4647 5668
hsa-miR-455-5p 4648 5669 hsa-miR-4632-3p 4649 5670 hsa-miR-4632-5p
4650 5671 hsa-miR-4633-3p 4651 5672 hsa-miR-4633-5p 4652 5673
hsa-miR-4634 4653 5674 hsa-miR-4635 4654 5675 hsa-miR-4636 4655
5676 hsa-miR-4637 4656 5677 hsa-miR-4638-3p 4657 5678
hsa-miR-4638-5p 4658 5679 hsa-miR-4639-3p 4659 5680 hsa-miR-4639-5p
4660 5681 hsa-miR-4640-3p 4661 5682 hsa-miR-4640-5p 4662 5683
hsa-miR-4641 4663 5684 hsa-miR-4642 4664 5685 hsa-miR-4643 4665
5686 hsa-miR-4644 4666 5687 hsa-miR-4645-3p 4667 5688
hsa-miR-4645-5p 4668 5689 hsa-miR-4646-3p 4669 5690 hsa-miR-4646-5p
4670 5691 hsa-miR-4647 4671 5692 hsa-miR-4648 4672 5693
hsa-miR-4649-3p 4673 5694 hsa-miR-4649-5p 4674 5695 hsa-miR-4650-3p
4675 5696 hsa-miR-4650-5p 4676 5697 hsa-miR-4651 4677 5698
hsa-miR-4652-3p 4678 5699 hsa-miR-4652-5p 4679 5700 hsa-miR-4653-3p
4680 5701 hsa-miR-4653-5p 4681 5702 hsa-miR-4654 4682 5703
hsa-miR-4655-3p 4683 5704 hsa-miR-4655-5p 4684 5705 hsa-miR-4656
4685 5706 hsa-miR-4657 4686 5707 hsa-miR-4658 4687 5708
hsa-miR-4659a-3p 4688 5709 hsa-miR-4659a-5p 4689 5710
hsa-miR-4659b-3p 4690 5711 hsa-miR-4659b-5p 4691 5712 hsa-miR-466
4692 5713 hsa-miR-4660 4693 5714 hsa-miR-4661-3p 4694 5715
hsa-miR-4661-5p 4695 5716 hsa-miR-4662a-3p 4696 5717
hsa-miR-4662a-5p 4697 5718 hsa-miR-4662b 4698 5719 hsa-miR-4663
4699 5720 hsa-miR-4664-3p 4700 5721 hsa-miR-4664-5p 4701 5722
hsa-miR-4665-3p 4702 5723 hsa-miR-4665-5p 4703 5724
hsa-miR-4666a-3p 4704 5725 hsa-miR-4666a-5p 4705 5726 hsa-miR-4666b
4706 5727 hsa-miR-4667-3p 4707 5728 hsa-miR-4667-5p 4708 5729
hsa-miR-4668-3p 4709 5730 hsa-miR-4668-5p 4710 5731 hsa-miR-4669
4711 5732 hsa-miR-4670-3p 4712 5733 hsa-miR-4670-5p 4713 5734
hsa-miR-4671-3p 4714 5735 hsa-miR-4671-5p 4715 5736 hsa-miR-4672
4716 5737 hsa-miR-4673 4717 5738 hsa-miR-4674 4718 5739
hsa-miR-4675 4719 5740 hsa-miR-4676-3p 4720 5741 hsa-miR-4676-5p
4721 5742 hsa-miR-4677-3p 4722 5743 hsa-miR-4677-5p 4723 5744
hsa-miR-4678 4724 5745 hsa-miR-4679 4725 5746 hsa-miR-4680-3p 4726
5747 hsa-miR-4680-5p 4727 5748 hsa-miR-4681 4728 5749 hsa-miR-4682
4729 5750 hsa-miR-4683 4730 5751 hsa-miR-4684-3p 4731 5752
hsa-miR-4684-5p 4732 5753 hsa-miR-4685-3p 4733 5754 hsa-miR-4685-5p
4734 5755 hsa-miR-4686 4735 5756 hsa-miR-4687-3p 4736 5757
hsa-miR-4687-5p 4737 5758 hsa-miR-4688 4738 5759 hsa-miR-4689 4739
5760 hsa-miR-4690-3p 4740 5761 hsa-miR-4690-5p 4741 5762
hsa-miR-4691-3p 4742 5763 hsa-miR-4691-5p 4743 5764 hsa-miR-4692
4744 5765 hsa-miR-4693-3p 4745 5766 hsa-miR-4693-5p 4746 5767
hsa-miR-4694-3p 4747 5768 hsa-miR-4694-5p 4748 5769 hsa-miR-4695-3p
4749 5770 hsa-miR-4695-5p 4750 5771 hsa-miR-4696 4751 5772
hsa-miR-4697-3p 4752 5773 hsa-miR-4697-5p 4753 5774 hsa-miR-4698
4754 5775 hsa-miR-4699-3p 4755 5776 hsa-miR-4699-5p 4756 5777
hsa-miR-4700-3p 4757 5778 hsa-miR-4700-5p 4758 5779 hsa-miR-4701-3p
4759 5780 hsa-miR-4701-5p 4760 5781 hsa-miR-4703-3p 4761 5782
hsa-miR-4703-5p 4762 5783 hsa-miR-4704-3p 4763 5784 hsa-miR-4704-5p
4764 5785 hsa-miR-4705 4765 5786 hsa-miR-4706 4766 5787
hsa-miR-4707-3p 4767 5788 hsa-miR-4707-5p 4768 5789 hsa-miR-4708-3p
4769 5790 hsa-miR-4708-5p 4770 5791 hsa-miR-4709-3p 4771 5792
hsa-miR-4709-5p 4772 5793 hsa-miR-4710 4773 5794 hsa-miR-4711-3p
4774 5795 hsa-miR-4711-5p 4775 5796 hsa-miR-4712-3p 4776 5797
hsa-miR-4712-5p 4777 5798 hsa-miR-4713-3p 4778 5799 hsa-miR-4713-5p
4779 5800 hsa-miR-4714-3p 4780 5801 hsa-miR-4714-5p 4781 5802
hsa-miR-4715-3p 4782 5803 hsa-miR-4715-5p 4783 5804 hsa-miR-4716-3p
4784 5805 hsa-miR-4716-5p 4785 5806 hsa-miR-4717-3p 4786 5807
hsa-miR-4717-5p 4787 5808 hsa-miR-4718 4788 5809 hsa-miR-4719 4789
5810 hsa-miR-4720-3p 4790 5811 hsa-miR-4720-5p 4791 5812
hsa-miR-4721 4792 5813 hsa-miR-4722-3p 4793 5814 hsa-miR-4722-5p
4794 5815 hsa-miR-4723-3p 4795 5816 hsa-miR-4723-5p 4796 5817
hsa-miR-4724-3p 4797 5818 hsa-miR-4724-5p 4798 5819 hsa-miR-4725-3p
4799 5820 hsa-miR-4725-5p 4800 5821 hsa-miR-4726-3p 4801 5822
hsa-miR-4726-5p 4802 5823 hsa-miR-4727-3p 4803 5824 hsa-miR-4727-5p
4804 5825 hsa-miR-4728-3p 4805 5826 hsa-miR-4728-5p 4806 5827
hsa-miR-4729 4807 5828 hsa-miR-4730 4808 5829 hsa-miR-4731-3p 4809
5830 hsa-miR-4731-5p 4810 5831 hsa-miR-4732-3p 4811 5832
hsa-miR-4732-5p 4812 5833 hsa-miR-4733-3p 4813 5834 hsa-miR-4733-5p
4814 5835 hsa-miR-4734 4815 5836 hsa-miR-4735-3p 4816 5837
hsa-miR-4735-5p 4817 5838 hsa-miR-4736 4818 5839 hsa-miR-4737 4819
5840 hsa-miR-4738-3p 4820 5841 hsa-miR-4738-5p 4821 5842
hsa-miR-4739 4822 5843 hsa-miR-4740-3p 4823 5844 hsa-miR-4740-5p
4824 5845 hsa-miR-4741 4825 5846 hsa-miR-4742-3p 4826 5847
hsa-miR-4742-5p 4827 5848 hsa-miR-4743-3p 4828 5849 hsa-miR-4743-5p
4829 5850 hsa-miR-4744 4830 5851 hsa-miR-4745-3p 4831 5852
hsa-miR-4745-5p 4832 5853 hsa-miR-4746-3p 4833 5854 hsa-miR-4746-5p
4834 5855 hsa-miR-4747-3p 4835 5856 hsa-miR-4747-5p 4836 5857
hsa-miR-4748 4837 5858 hsa-miR-4749-3p 4838 5859 hsa-miR-4749-5p
4839 5860 hsa-miR-4750-3p 4840 5861 hsa-miR-4750-5p 4841 5862
hsa-miR-4751 4842 5863 hsa-miR-4752 4843 5864 hsa-miR-4753-3p 4844
5865 hsa-miR-4753-5p 4845 5866 hsa-miR-4754 4846 5867
hsa-miR-4755-3p 4847 5868 hsa-miR-4755-5p 4848 5869 hsa-miR-4756-3p
4849 5870 hsa-miR-4756-5p 4850 5871 hsa-miR-4757-3p 4851 5872
hsa-miR-4757-5p 4852 5873 hsa-miR-4758-3p 4853 5874 hsa-miR-4758-5p
4854 5875 hsa-miR-4759 4855 5876 hsa-miR-4760-3p 4856 5877
hsa-miR-4760-5p 4857 5878 hsa-miR-4761-3p 4858 5879 hsa-miR-4761-5p
4859 5880 hsa-miR-4762-3p 4860 5881 hsa-miR-4762-5p 4861 5882
hsa-miR-4763-3p 4862 5883 hsa-miR-4763-5p 4863 5884 hsa-miR-4764-3p
4864 5885 hsa-miR-4764-5p 4865 5886 hsa-miR-4765 4866 5887
hsa-miR-4766-3p 4867 5888 hsa-miR-4766-5p 4868 5889 hsa-miR-4767
4869 5890 hsa-miR-4768-3p 4870 5891 hsa-miR-4768-5p 4871 5892
hsa-miR-4769-3p 4872 5893 hsa-miR-4769-5p 4873 5894 hsa-miR-4770
4874 5895 hsa-miR-4771 4875 5896 hsa-miR-4772-3p 4876 5897
hsa-miR-4772-5p 4877 5898 hsa-miR-4773 4878 5899 hsa-miR-4774-3p
4879 5900 hsa-miR-4774-5p 4880 5901 hsa-miR-4775 4881 5902
hsa-miR-4776-3p 4882 5903 hsa-miR-4776-5p 4883 5904 hsa-miR-4777-3p
4884 5905 hsa-miR-4777-5p 4885 5906 hsa-miR-4778-3p 4886 5907
hsa-miR-4778-5p 4887 5908 hsa-miR-4779 4888 5909 hsa-miR-4780 4889
5910 hsa-miR-4781-3p 4890 5911 hsa-miR-4781-5p 4891 5912
hsa-miR-4782-3p 4892 5913 hsa-miR-4782-5p 4893 5914 hsa-miR-4783-3p
4894 5915 hsa-miR-4783-5p 4895 5916 hsa-miR-4784 4896 5917
hsa-miR-4785 4897 5918 hsa-miR-4786-3p 4898 5919 hsa-miR-4786-5p
4899 5920 hsa-miR-4787-3p 4900 5921 hsa-miR-4787-5p 4901 5922
hsa-miR-4788 4902 5923 hsa-miR-4789-3p 4903 5924 hsa-miR-4789-5p
4904 5925 hsa-miR-4790-3p 4905 5926 hsa-miR-4790-5p 4906 5927
hsa-miR-4791 4907 5928 hsa-miR-4792 4908 5929 hsa-miR-4793-3p 4909
5930 hsa-miR-4793-5p 4910 5931 hsa-miR-4794 4911 5932
hsa-miR-4795-3p 4912 5933 hsa-miR-4795-5p 4913 5934 hsa-miR-4796-3p
4914 5935 hsa-miR-4796-5p 4915 5936 hsa-miR-4797-3p 4916 5937
hsa-miR-4797-5p 4917 5938 hsa-miR-4798-3p 4918 5939 hsa-miR-4798-5p
4919 5940 hsa-miR-4799-3p 4920 5941 hsa-miR-4799-5p 4921 5942
hsa-miR-4800-3p 4922 5943 hsa-miR-4800-5p 4923 5944 hsa-miR-4801
4924 5945 hsa-miR-4802-3p 4925 5946 hsa-miR-4802-5p 4926 5947
hsa-miR-4803 4927 5948 hsa-miR-4804-3p 4928 5949 hsa-miR-4804-5p
4929 5950 hsa-miR-483-3p 4930 5951 hsa-miR-483-5p 4931 5952
hsa-miR-484 4932 5953 hsa-miR-485-3p 4933 5954 hsa-miR-485-5p 4934
5955 hsa-miR-486-3p 4935 5956 hsa-miR-486-5p 4936 5957 hsa-miR-487a
4937 5958 hsa-miR-487b 4938 5959 hsa-miR-488-3p 4939 5960
hsa-miR-488-5p 4940 5961 hsa-miR-489 4941 5962 hsa-miR-490-3p 4942
5963 hsa-miR-490-5p 4943 5964 hsa-miR-491-3p 4944 5965
hsa-miR-491-5p 4945 5966 hsa-miR-492 4946 5967 hsa-miR-493-3p 4947
5968 hsa-miR-493-5p 4948 5969 hsa-miR-494 4949 5970 hsa-miR-495-3p
4950 5971 hsa-miR-495-5p 4951 5972 hsa-miR-496 4952 5973
hsa-miR-497-3p 4953 5974 hsa-miR-497-5p 4954 5975 hsa-miR-498 4955
5976 hsa-miR-4999-3p 4956 5977 hsa-miR-4999-5p 4957 5978
hsa-miR-499a-3p 4958 5979 hsa-miR-499a-5p 4959 5980 hsa-miR-499b-3p
4960 5981 hsa-miR-499b-5p 4961 5982 hsa-miR-5000-3p 4962 5983
hsa-miR-5000-5p 4963 5984 hsa-miR-5001-3p 4964 5985 hsa-miR-5001-5p
4965 5986 hsa-miR-5002-3p 4966 5987 hsa-miR-5002-5p 4967 5988
hsa-miR-5003-3p 4968 5989 hsa-miR-5003-5p 4969 5990 hsa-miR-5004-3p
4970 5991 hsa-miR-5004-5p 4971 5992 hsa-miR-5006-3p 4972 5993
hsa-miR-5006-5p 4973 5994 hsa-miR-5007-3p 4974 5995 hsa-miR-5007-5p
4975 5996 hsa-miR-5008-3p 4976 5997 hsa-miR-5008-5p 4977 5998
hsa-miR-5009-3p 4978 5999 hsa-miR-5009-5p 4979 6000 hsa-miR-500a-3p
4980 6001 hsa-miR-500a-5p 4981 6002 hsa-miR-500b 4982 6003
hsa-miR-5010-3p 4983 6004 hsa-miR-5010-5p 4984 6005 hsa-miR-5011-3p
4985 6006 hsa-miR-5011-5p 4986 6007 hsa-miR-501-3p 4987 6008
hsa-miR-501-5p 4988 6009 hsa-miR-502-3p 4989 6010 hsa-miR-502-5p
4990 6011 hsa-miR-503-3p 4991 6012 hsa-miR-503-5p 4992 6013
hsa-miR-504 4993 6014 hsa-miR-5047 4994 6015 hsa-miR-505-3p 4995
6016 hsa-miR-505-5p 4996 6017 hsa-miR-506-3p 4997 6018
hsa-miR-506-5p 4998 6019 hsa-miR-507 4999 6020 hsa-miR-508-3p 5000
6021 hsa-miR-508-5p 5001 6022 hsa-miR-5087 5002 6023 hsa-miR-5088
5003 6024 hsa-miR-5089-3p 5004 6025 hsa-miR-5089-5p 5005 6026
hsa-miR-5090 5006 6027 hsa-miR-5091 5007 6028 hsa-miR-5092 5008
6029 hsa-miR-5093 5009 6030 hsa-miR-509-3-5p 5010 6031
hsa-miR-509-3p 5011 6032 hsa-miR-5094 5012 6033 hsa-miR-5095 5013
6034 hsa-miR-509-5p 5014 6035 hsa-miR-5096 5015 6036 hsa-miR-510
5016 6037 hsa-miR-5100 5017 6038 hsa-miR-511 5018 6039
hsa-miR-512-3p 5019 6040 hsa-miR-512-5p 5020 6041 hsa-miR-513a-3p
5021 6042 hsa-miR-513a-5p 5022 6043 hsa-miR-513b 5023 6044
hsa-miR-513c-3p 5024 6045 hsa-miR-513c-5p 5025 6046 hsa-miR-514a-3p
5026 6047 hsa-miR-514a-5p 5027 6048
hsa-miR-514b-3p 5028 6049 hsa-miR-514b-5p 5029 6050 hsa-miR-515-3p
5030 6051 hsa-miR-515-5p 5031 6052 hsa-miR-516a-3p 5032 6053
hsa-miR-516a-5p 5033 6054 hsa-miR-516b-3p 5034 6055 hsa-miR-516b-5p
5035 6056 hsa-miR-517-5p 5036 6057 hsa-miR-517a-3p 5037 6058
hsa-miR-517b-3p 5038 6059 hsa-miR-517c-3p 5039 6060 hsa-miR-5186
5040 6061 hsa-miR-5187-3p 5041 6062 hsa-miR-5187-5p 5042 6063
hsa-miR-5188 5043 6064 hsa-miR-5189 5044 6065 hsa-miR-518a-3p 5045
6066 hsa-miR-518a-5p 5046 6067 hsa-miR-518b 5047 6068
hsa-miR-518c-3p 5048 6069 hsa-miR-518c-5p 5049 6070 hsa-miR-518d-3p
5050 6071 hsa-miR-518d-5p 5051 6072 hsa-miR-518e-3p 5052 6073
hsa-miR-518e-5p 5053 6074 hsa-miR-518f-3p 5054 6075 hsa-miR-518f-5p
5055 6076 hsa-miR-5190 5056 6077 hsa-miR-5191 5057 6078
hsa-miR-5192 5058 6079 hsa-miR-5193 5059 6080 hsa-miR-5194 5060
6081 hsa-miR-5195-3p 5061 6082 hsa-miR-5195-5p 5062 6083
hsa-miR-5196-3p 5063 6084 hsa-miR-5196-5p 5064 6085 hsa-miR-5197-3p
5065 6086 hsa-miR-5197-5p 5066 6087 hsa-miR-519a-3p 5067 6088
hsa-miR-519a-5p 5068 6089 hsa-miR-519b-3p 5069 6090 hsa-miR-519b-5p
5070 6091 hsa-miR-519c-3p 5071 6092 hsa-miR-519c-5p 5072 6093
hsa-miR-519d 5073 6094 hsa-miR-519e-3p 5074 6095 hsa-miR-519e-5p
5075 6096 hsa-miR-520a-3p 5076 6097 hsa-miR-520a-5p 5077 6098
hsa-miR-520b 5078 6099 hsa-miR-520c-3p 5079 6100 hsa-miR-520c-5p
5080 6101 hsa-miR-520d-3p 5081 6102 hsa-miR-520d-5p 5082 6103
hsa-miR-520e 5083 6104 hsa-miR-520f 5084 6105 hsa-miR-520g 5085
6106 hsa-miR-520h 5086 6107 hsa-miR-521 5087 6108 hsa-miR-522-3p
5088 6109 hsa-miR-522-5p 5089 6110 hsa-miR-523-3p 5090 6111
hsa-miR-523-5p 5091 6112 hsa-miR-524-3p 5092 6113 hsa-miR-524-5p
5093 6114 hsa-miR-525-3p 5094 6115 hsa-miR-525-5p 5095 6116
hsa-miR-526a 5096 6117 hsa-miR-526b-3p 5097 6118 hsa-miR-526b-5p
5098 6119 hsa-miR-527 5099 6120 hsa-miR-532-3p 5100 6121
hsa-miR-532-5p 5101 6122 hsa-miR-539-3p 5102 6123 hsa-miR-539-5p
5103 6124 hsa-miR-541-3p 5104 6125 hsa-miR-541-5p 5105 6126
hsa-miR-542-3p 5106 6127 hsa-miR-542-5p 5107 6128 hsa-miR-543 5108
6129 hsa-miR-544a 5109 6130 hsa-miR-544b 5110 6131 hsa-miR-545-3p
5111 6132 hsa-miR-545-5p 5112 6133 hsa-miR-548 5113 6134
hsa-miR-548-3p 5114 6135 hsa-miR-548-5p 5115 6136 hsa-miR-548a 5116
6137 hsa-miR-548a-3p 5117 6138 hsa-miR-548a-5p 5118 6139
hsa-miR-548aa 5119 6140 hsa-miR-548ab 5120 6141 hsa-miR-548ac 5121
6142 hsa-miR-548ad 5122 6143 hsa-miR-548ae 5123 6144 hsa-miR-548ag
5124 6145 hsa-miR-548ah-3p 5125 6146 hsa-miR-548ah-5p 5126 6147
hsa-miR-548ai 5127 6148 hsa-miR-548aj-3p 5128 6149 hsa-miR-548aj-5p
5129 6150 hsa-miR-548ak 5130 6151 hsa-miR-548al 5131 6152
hsa-miR-548am-3p 5132 6153 hsa-miR-548am-5p 5133 6154 hsa-miR-548an
5134 6155 hsa-miR-548ao-3p 5135 6156 hsa-miR-548ao-5p 5136 6157
hsa-miR-548ap-3p 5137 6158 hsa-miR-548ap-5p 5138 6159
hsa-miR-548aq-3p 5139 6160 hsa-miR-548aq-5p 5140 6161
hsa-miR-548ar-3p 5141 6162 hsa-miR-548ar-5p 5142 6163
hsa-miR-548as-3p 5143 6164 hsa-miR-548as-5p 5144 6165
hsa-miR-548at-3p 5145 6166 hsa-miR-548at-5p 5146 6167
hsa-miR-548au-3p 5147 6168 hsa-miR-548au-5p 5148 6169
hsa-miR-548av-3p 5149 6170 hsa-miR-548av-5p 5150 6171 hsa-miR-548aw
5151 6172 hsa-miR-548ay-3p 5152 6173 hsa-miR-548ay-5p 5153 6174
hsa-miR-548az-3p 5154 6175 hsa-miR-548az-5p 5155 6176
hsa-miR-548b-3p 5156 6177 hsa-miR-548b-5p 5157 6178 hsa-miR-548c-3p
5158 6179 hsa-miR-548c-5p 5159 6180 hsa-miR-548d-3p 5160 6181
hsa-miR-548d-5p 5161 6182 hsa-miR-548e 5162 6183 hsa-miR-548f 5163
6184 hsa-miR-548g-3p 5164 6185 hsa-miR-548g-5p 5165 6186
hsa-miR-548h-3p 5166 6187 hsa-miR-548h-5p 5167 6188 hsa-miR-548i
5168 6189 hsa-miR-548j 5169 6190 hsa-miR-548k 5170 6191
hsa-miR-548l 5171 6192 hsa-miR-548m 5172 6193 hsa-miR-548n 5173
6194 hsa-miR-548o-3p 5174 6195 hsa-miR-548o-5p 5175 6196
hsa-miR-548p 5176 6197 hsa-miR-548q 5177 6198 hsa-miR-548s 5178
6199 hsa-miR-548t-3p 5179 6200 hsa-miR-548t-5p 5180 6201
hsa-miR-548u 5181 6202 hsa-miR-548w 5182 6203 hsa-miR-548y 5183
6204 hsa-miR-548z 5184 6205 hsa-miR-549a 5185 6206
hsa-miR-550a-3-5p 5186 6207 hsa-miR-550a-3p 5187 6208
hsa-miR-550a-5p 5188 6209 hsa-miR-550b-2-5p 5189 6210
hsa-miR-550b-3p 5190 6211 hsa-miR-551a 5191 6212 hsa-miR-551b-3p
5192 6213 hsa-miR-551b-5p 5193 6214 hsa-miR-552 5194 6215
hsa-miR-553 5195 6216 hsa-miR-554 5196 6217 hsa-miR-555 5197 6218
hsa-miR-556-3p 5198 6219 hsa-miR-556-5p 5199 6220 hsa-miR-557 5200
6221 hsa-miR-5571-3p 5201 6222 hsa-miR-5571-5p 5202 6223
hsa-miR-5572 5203 6224 hsa-miR-5579-3p 5204 6225 hsa-miR-5579-5p
5205 6226 hsa-miR-558 5206 6227 hsa-miR-5580-3p 5207 6228
hsa-miR-5580-5p 5208 6229 hsa-miR-5581-3p 5209 6230 hsa-miR-5581-5p
5210 6231 hsa-miR-5582-3p 5211 6232 hsa-miR-5582-5p 5212 6233
hsa-miR-5583-3p 5213 6234 hsa-miR-5583-5p 5214 6235 hsa-miR-5584-3p
5215 6236 hsa-miR-5584-5p 5216 6237 hsa-miR-5585-3p 5217 6238
hsa-miR-5585-5p 5218 6239 hsa-miR-5586-3p 5219 6240 hsa-miR-5586-5p
5220 6241 hsa-miR-5587-3p 5221 6242 hsa-miR-5587-5p 5222 6243
hsa-miR-5588-3p 5223 6244 hsa-miR-5588-5p 5224 6245 hsa-miR-5589-3p
5225 6246 hsa-miR-5589-5p 5226 6247 hsa-miR-559 5227 6248
hsa-miR-5590-3p 5228 6249 hsa-miR-5590-5p 5229 6250 hsa-miR-5591-3p
5230 6251 hsa-miR-5591-5p 5231 6252 hsa-miR-561-3p 5232 6253
hsa-miR-561-5p 5233 6254 hsa-miR-562 5234 6255 hsa-miR-563 5235
6256 hsa-miR-564 5236 6257 hsa-miR-566 5237 6258 hsa-miR-567 5238
6259 hsa-miR-568 5239 6260 hsa-miR-5680 5240 6261 hsa-miR-5681a
5241 6262 hsa-miR-5681b 5242 6263 hsa-miR-5682 5243 6264
hsa-miR-5683 5244 6265 hsa-miR-5684 5245 6266 hsa-miR-5685 5246
6267 hsa-miR-5686 5247 6268 hsa-miR-5687 5248 6269 hsa-miR-5688
5249 6270 hsa-miR-5689 5250 6271 hsa-miR-569 5251 6272 hsa-miR-5690
5252 6273 hsa-miR-5691 5253 6274 hsa-miR-5692a 5254 6275
hsa-miR-5692b 5255 6276 hsa-miR-5692c 5256 6277 hsa-miR-5693 5257
6278 hsa-miR-5694 5258 6279 hsa-miR-5695 5259 6280 hsa-miR-5696
5260 6281 hsa-miR-5697 5261 6282 hsa-miR-5698 5262 6283
hsa-miR-5699 5263 6284 hsa-miR-5700 5264 6285 hsa-miR-5701 5265
6286 hsa-miR-5702 5266 6287 hsa-miR-5703 5267 6288 hsa-miR-570-3p
5268 6289 hsa-miR-5704 5269 6290 hsa-miR-5705 5270 6291
hsa-miR-570-5p 5271 6292 hsa-miR-5706 5272 6293 hsa-miR-5707 5273
6294 hsa-miR-5708 5274 6295 hsa-miR-571 5275 6296 hsa-miR-572 5276
6297 hsa-miR-573 5277 6298 hsa-miR-5739 5278 6299
hsa-miR-574-3p 5279 6300 hsa-miR-574-5p 5280 6301 hsa-miR-575 5281
6302 hsa-miR-576-3p 5282 6303 hsa-miR-576-5p 5283 6304 hsa-miR-577
5284 6305 hsa-miR-578 5285 6306 hsa-miR-5787 5286 6307 hsa-miR-579
5287 6308 hsa-miR-580 5288 6309 hsa-miR-581 5289 6310
hsa-miR-582-3p 5290 6311 hsa-miR-582-5p 5291 6312 hsa-miR-583 5292
6313 hsa-miR-584-3p 5293 6314 hsa-miR-584-5p 5294 6315 hsa-miR-585
5295 6316 hsa-miR-586 5296 6317 hsa-miR-587 5297 6318 hsa-miR-588
5298 6319 hsa-miR-589-3p 5299 6320 hsa-miR-589-5p 5300 6321
hsa-miR-590-3p 5301 6322 hsa-miR-590-5p 5302 6323 hsa-miR-591 5303
6324 hsa-miR-592 5304 6325 hsa-miR-593-3p 5305 6326 hsa-miR-593-5p
5306 6327 hsa-miR-595 5307 6328 hsa-miR-596 5308 6329 hsa-miR-597
5309 6330 hsa-miR-598 5310 6331 hsa-miR-599 5311 6332 hsa-miR-600
5312 6333 hsa-miR-601 5313 6334 hsa-miR-602 5314 6335 hsa-miR-603
5315 6336 hsa-miR-604 5316 6337 hsa-miR-605 5317 6338 hsa-miR-606
5318 6339 hsa-miR-6068 5319 6340 hsa-miR-6069 5320 6341 hsa-miR-607
5321 6342 hsa-miR-6070 5322 6343 hsa-miR-6071 5323 6344
hsa-miR-6072 5324 6345 hsa-miR-6073 5325 6346 hsa-miR-6074 5326
6347 hsa-miR-6075 5327 6348 hsa-miR-6076 5328 6349 hsa-miR-6077
5329 6350 hsa-miR-6078 5330 6351 hsa-miR-6079 5331 6352 hsa-miR-608
5332 6353 hsa-miR-6080 5333 6354 hsa-miR-6081 5334 6355
hsa-miR-6082 5335 6356 hsa-miR-6083 5336 6357 hsa-miR-6084 5337
6358 hsa-miR-6085 5338 6359 hsa-miR-6086 5339 6360 hsa-miR-6087
5340 6361 hsa-miR-6088 5341 6362 hsa-miR-6089 5342 6363 hsa-miR-609
5343 6364 hsa-miR-6090 5344 6365 hsa-miR-610 5345 6366 hsa-miR-611
5346 6367 hsa-miR-612 5347 6368 hsa-miR-6124 5348 6369 hsa-miR-6125
5349 6370 hsa-miR-6126 5350 6371 hsa-miR-6127 5351 6372
hsa-miR-6128 5352 6373 hsa-miR-6129 5353 6374 hsa-miR-613 5354 6375
hsa-miR-6130 5355 6376 hsa-miR-6131 5356 6377 hsa-miR-6132 5357
6378 hsa-miR-6133 5358 6379 hsa-miR-6134 5359 6380 hsa-miR-614 5360
6381 hsa-miR-615-3p 5361 6382 hsa-miR-615-5p 5362 6383
hsa-miR-616-3p 5363 6384 hsa-miR-6165 5364 6385 hsa-miR-616-5p 5365
6386 hsa-miR-617 5366 6387 hsa-miR-618 5367 6388 hsa-miR-619 5368
6389 hsa-miR-620 5369 6390 hsa-miR-621 5370 6391 hsa-miR-622 5371
6392 hsa-miR-623 5372 6393 hsa-miR-624-3p 5373 6394 hsa-miR-624-5p
5374 6395 hsa-miR-625-3p 5375 6396 hsa-miR-625-5p 5376 6397
hsa-miR-626 5377 6398 hsa-miR-627 5378 6399 hsa-miR-628-3p 5379
6400 hsa-miR-628-5p 5380 6401 hsa-miR-629-3p 5381 6402
hsa-miR-629-5p 5382 6403 hsa-miR-630 5383 6404 hsa-miR-631 5384
6405 hsa-miR-632 5385 6406 hsa-miR-633 5386 6407 hsa-miR-634 5387
6408 hsa-miR-635 5388 6409 hsa-miR-636 5389 6410 hsa-miR-637 5390
6411 hsa-miR-638 5391 6412 hsa-miR-639 5392 6413 hsa-miR-640 5393
6414 hsa-miR-641 5394 6415 hsa-miR-642a-3p 5395 6416
hsa-miR-642a-5p 5396 6417 hsa-miR-642b-3p 5397 6418 hsa-miR-642b-5p
5398 6419 hsa-miR-643 5399 6420 hsa-miR-644a 5400 6421 hsa-miR-645
5401 6422 hsa-miR-646 5402 6423 hsa-miR-647 5403 6424 hsa-miR-648
5404 6425 hsa-miR-649 5405 6426 hsa-miR-6499-3p 5406 6427
hsa-miR-6499-5p 5407 6428 hsa-miR-650 5408 6429 hsa-miR-6500-3p
5409 6430 hsa-miR-6500-5p 5410 6431 hsa-miR-6501-3p 5411 6432
hsa-miR-6501-5p 5412 6433 hsa-miR-6502-3p 5413 6434 hsa-miR-6502-5p
5414 6435 hsa-miR-6503-3p 5415 6436 hsa-miR-6503-5p 5416 6437
hsa-miR-6504-3p 5417 6438 hsa-miR-6504-5p 5418 6439 hsa-miR-6505-3p
5419 6440 hsa-miR-6505-5p 5420 6441 hsa-miR-6506-3p 5421 6442
hsa-miR-6506-5p 5422 6443 hsa-miR-6507-3p 5423 6444 hsa-miR-6507-5p
5424 6445 hsa-miR-6508-3p 5425 6446 hsa-miR-6508-5p 5426 6447
hsa-miR-6509-3p 5427 6448 hsa-miR-6509-5p 5428 6449 hsa-miR-651
5429 6450 hsa-miR-6510-3p 5430 6451 hsa-miR-6510-5p 5431 6452
hsa-miR-6511a-3p 5432 6453 hsa-miR-6511a-5p 5433 6454
hsa-miR-6511b-3p 5434 6455 hsa-miR-6511b-5p 5435 6456
hsa-miR-6512-3p 5436 6457 hsa-miR-6512-5p 5437 6458 hsa-miR-6513-3p
5438 6459 hsa-miR-6513-5p 5439 6460 hsa-miR-6514-3p 5440 6461
hsa-miR-6514-5p 5441 6462 hsa-miR-6515-3p 5442 6463 hsa-miR-6515-5p
5443 6464 hsa-miR-652-3p 5444 6465 hsa-miR-652-5p 5445 6466
hsa-miR-653 5446 6467 hsa-miR-654-3p 5447 6468 hsa-miR-654-5p 5448
6469 hsa-miR-655 5449 6470 hsa-miR-656 5450 6471 hsa-miR-657 5451
6472 hsa-miR-658 5452 6473 hsa-miR-659-3p 5453 6474 hsa-miR-659-5p
5454 6475 hsa-miR-660-3p 5455 6476 hsa-miR-660-5p 5456 6477
hsa-miR-661 5457 6478 hsa-miR-662 5458 6479 hsa-miR-663a 5459 6480
hsa-miR-663b 5460 6481 hsa-miR-664a-3p 5461 6482 hsa-miR-664a-5p
5462 6483 hsa-miR-664b-3p 5463 6484 hsa-miR-664b-5p 5464 6485
hsa-miR-665 5465 6486 hsa-miR-668 5466 6487 hsa-miR-670 5467 6488
hsa-miR-671-3p 5468 6489 hsa-miR-6715a-3p 5469 6490
hsa-miR-6715b-3p 5470 6491 hsa-miR-6715b-5p 5471 6492
hsa-miR-671-5p 5472 6493 hsa-miR-6716-3p 5473 6494 hsa-miR-6716-5p
5474 6495 hsa-miR-6717-5p 5475 6496 hsa-miR-6718-5p 5476 6497
hsa-miR-6719-3p 5477 6498 hsa-miR-6720-3p 5478 6499 hsa-miR-6721-5p
5479 6500 hsa-miR-6722-3p 5480 6501 hsa-miR-6722-5p 5481 6502
hsa-miR-6723-5p 5482 6503 hsa-miR-6724-5p 5483 6504 hsa-miR-675-3p
5484 6505 hsa-miR-675-5p 5485 6506 hsa-miR-676-3p 5486 6507
hsa-miR-676-5p 5487 6508 hsa-miR-708-3p 5488 6509 hsa-miR-708-5p
5489 6510 hsa-miR-711 5490 6511 hsa-miR-7-1-3p 5491 6512
hsa-miR-718 5492 6513 hsa-miR-7-2-3p 5493 6514 hsa-miR-744-3p 5494
6515 hsa-miR-744-5p 5495 6516 hsa-miR-758-3p 5496 6517
hsa-miR-758-5p 5497 6518 hsa-miR-759 5498 6519 hsa-miR-7-5p 5499
6520 hsa-miR-760 5500 6521 hsa-miR-761 5501 6522 hsa-miR-762 5502
6523 hsa-miR-764 5503 6524 hsa-miR-765 5504 6525 hsa-miR-766-3p
5505 6526 hsa-miR-766-5p 5506 6527 hsa-miR-767-3p 5507 6528
hsa-miR-767-5p 5508 6529 hsa-miR-769-3p 5509 6530 hsa-miR-769-5p
5510 6531 hsa-miR-770-5p 5511 6532 hsa-miR-802 5512 6533
hsa-miR-873-3p 5513 6534 hsa-miR-873-5p 5514 6535 hsa-miR-874 5515
6536 hsa-miR-875-3p 5516 6537 hsa-miR-875-5p 5517 6538
hsa-miR-876-3p 5518 6539 hsa-miR-876-5p 5519 6540 hsa-miR-877-3p
5520 6541 hsa-miR-877-5p 5521 6542 hsa-miR-885-3p 5522 6543
hsa-miR-885-5p 5523 6544 hsa-miR-887 5524 6545 hsa-miR-888-3p 5525
6546 hsa-miR-888-5p 5526 6547 hsa-miR-889 5527 6548 hsa-miR-890
5528 6549 hsa-miR-891a 5529 6550
hsa-miR-891b 5530 6551 hsa-miR-892a 5531 6552 hsa-miR-892b 5532
6553 hsa-miR-892c-3p 5533 6554 hsa-miR-892c-5p 5534 6555
hsa-miR-920 5535 6556 hsa-miR-921 5536 6557 hsa-miR-922 5537 6558
hsa-miR-924 5538 6559 hsa-miR-92a-1-5p 5539 6560 hsa-miR-92a-2-5p
5540 6561 hsa-miR-92a-3p 5541 6562 hsa-miR-92b-3p 5542 6563
hsa-miR-92b-5p 5543 6564 hsa-miR-933 5544 6565 hsa-miR-93-3p 5545
6566 hsa-miR-934 5546 6567 hsa-miR-935 5547 6568 hsa-miR-93-5p 5548
6569 hsa-miR-936 5549 6570 hsa-miR-937-3p 5550 6571 hsa-miR-937-5p
5551 6572 hsa-miR-938 5552 6573 hsa-miR-939-3p 5553 6574
hsa-miR-939-5p 5554 6575 hsa-miR-9-3p 5555 6576 hsa-miR-940 5556
6577 hsa-miR-941 5557 6578 hsa-miR-942 5558 6579 hsa-miR-943 5559
6580 hsa-miR-944 5560 6581 hsa-miR-95 5561 6582 hsa-miR-9-5p 5562
6583 hsa-miR-96-3p 5563 6584 hsa-miR-96-5p 5564 6585 hsa-miR-98-3p
5565 6586 hsa-miR-98-5p 5566 6587 hsa-miR-99a-3p 5567 6588
hsa-miR-99a-5p 5568 6589 hsa-miR-99b-3p 5569 6590 hsa-miR-99b-5p
5570 6591
[0336] As shown in Table 10, microRNAs are differentially expressed
in different tissues and cells, and often associated with different
types of dieases (e.g.cancer cells). The decision of removal or
insertion of microRNA binding sites, or any combination, is
dependent on microRNA expression patterns and their profilings in
cancer cells. In Table 10, "HCC" represents hepatocellular
carcinoma, "ALL" stands for acute lymphoblastsic leukemia, "RCC"
stands for renal cell carcinoma, "CLL" stands for chrominc
lymphocytic leukemia and "MALT" stands for mucosa-associated
lymphoid tissue.
TABLE-US-00010 TABLE 10 mirs, tissues/cell expression and diseases
BS mir SEQ Associated Biological microRNA SEQ ID ID Tissues/cells
Disease Function hsa-let-7a-2-3p 2508 3529 Embryonic stem
inflammatory, tumor cells, lung, myeloid various cancers suppressor
cells (lung, cervical, breast, pancreatic, etc) hsa-let-7a-3p 2509
3530 Embryonic stem inflammatory, tumor cells, lung various cancers
suppressor (lung, cervical, breast, pancreatic, etc) hsa-let-7a-5p
2510 3531 Embryonic stem inflammatory, tumor cells, lung various
cancers suppressor (lung, cervical, breast, pancreatic, etc)
hsa-let-7b-3p 2511 3532 epithelial cells, lung cancer, tumor
endothelial cells colorectal cancer, angiogenesis (vascular)
cervical cancer, inflammation and immune response after infection
hsa-let-7b-5p 2512 3533 epithelial cells, cervical cancer, tumor
endothelial cells inflammation and angiogenesis (vascular) immune
response after infection hsa-let-7c 2513 3534 dendritic cells
various cacners tumor (cervical, suppressor, pancreatic, apoptosis
lung, esopphageal, etc) hsa-let-7d-3p 2514 3535 embryonic stem
associated with tumor cells various cancer suppressor cells
hsa-let-7d-5p 2515 3536 embryonic stem associated with tumor cells
various cancer suppressor cells hsa-let-7e-3p 2516 3537 immune
cells various cancer tumor cells, suppressor autoimmunity,
endotoxin tolerance hsa-let-7e-5p 2517 3538 immune cells various
cancer tumor cells suppressor hsa-let-7f-1-3p 2518 3539 immune
cells (T various cancer tumor cells) cells suppressor
hsa-let-7f-2-3p 2519 3540 immune cells (T various cancer tumor
cells) cells suppressor hsa-let-7f-5p 2520 3541 immune cells (T
Various cancer tumor cells) cells suppressor hsa-let-7g-3p 2521
3542 hematopoietic cells, various cancer tumor adipose, smooth
cells (lung, breast, suppressor muscle cells etc) hsa-let-7g-5p
2522 3543 hematopoietic cells, various cancer tumor adipose, smooth
cells (lung, breast, suppressor muscle cells etc) hsa-let-7i-3p
2523 3544 immune cells chronic tumor lymphocyte suppressor leukimia
hsa-let-7i-5p 2524 3545 immune cells chronic tumor lymphocyte
suppressor leukimia hsa-miR-1 2525 3546 muscle, heart angiogenesis,
cell proliferation(myogenesis) hsa-miR-100-3p 2526 3547
hematopoietic cells, gastric cancer, tumor endothelial cells
pancreatic cancer angiogenesis hsa-miR-100-5p 2527 3548
hematopoietic cells, gastric cancer, tumor endothelial cells
pancreatic cancer angiogenesis hsa-miR-101-3p 2528 3549 endothelial
cells various cancers angiogenesis (breast, non-small cell lung,
colon, gastric, pancreatic, bladder, etc); lupus erythematosus
hsa-miR-101-5p 2529 3550 endothelial cells various cancers
angiogenesis (breast, non-small cell lung, colon, gastric,
pancreatic, bladder, etc); lupus erythematosus hsa-miR-103a-2-5p
2530 3551 embryonic stem various cancers oncogene, cell cells, many
(endometrial, growth tissues/cells neuroblastoma, colorectal,
breast, liver, etc) hsa-miR-103a-3p 2531 3552 embryonic stem
various cancers oncogene, cell cells, many (endometrial, growth
tissues/cells neuroblastoma, colorectal, breast, liver, etc)
hsa-miR-103b 2532 3553 Many tissues/cells various cancers oncogene,
cell (endometrial, growth neuroblastoma, colorectal, breast, liver,
etc) hsa-miR-105-3p 2533 3554 pancreatic cells hsa-miR-105-5p 2534
3555 pancreatic cells hsa-miR-106a-3p 2535 3556 osteogenic cells
osteocarcoma, cell other cancers differentiation hsa-miR-106a-5p
2536 3557 osteogenic cells osteocarcoma, cell other cancers
differentiation hsa-miR-106b-3p 2537 3558 embryonic stem various
cancers oncogene cells (non-small lung cancer, gastric cancer, HCC,
gliomas, etc) hsa-miR-106b-5p 2538 3559 embryonic stem various
cancers oncogene cells (non-small lung cancer, gastric cancer, HCC,
gliomas, etc) hsa-miR-107 2539 3560 many tissues, brain breast
cancer, hepatocytes/liver pituitary adenoma, obesity/diabetes
hsa-miR-10a-3p 2540 3561 hematopoeitic cells acute myeoid oncogene,
cell leukemia growth hsa-miR-10a-5p 2541 3562 hematopoeitic cells
acute myeoid oncogene, cell leukemia growth hsa-miR-10b-3p 2542
3563 multiple tissues and various cancers oncogene cells (breast,
ovarian, glioblastoma, pancreatc ductal adenocarcinoma, gastric,
etc) hsa-miR-10b-5p 2543 3564 multiple tissues and various cancers
oncogene cells (breast, ovarian, glioblastoma, pancreatc ductal
adenocarcinoma, gastric, etc) hsa-miR-1178-3p 2544 3565
osteocarcoma hsa-miR-1178-5p 2545 3566 osteocarcoma hsa-miR-1179
2546 3567 osteocarcoma hsa-miR-1180 2547 3568 discovered in
sarcoma, no expression data hsa-miR-1181 2548 3569 downregulated in
ovarian cancer cells, associated with HCV infection in hepatocytes
hsa-miR-1182 2549 3570 placenta hsa-miR-1183 2550 3571 associated
with rectal cancer hsa-miR-1184 2551 3572 Hematopoietic cells
downregulated in oral leukoplakia (OLK) hsa-miR-1185-1-3p 2552 3573
placenta hsa-miR-1185-2-3p 2553 3574 placenta hsa-miR-1185-5p 2554
3575 placenta hsa-miR-1193 2555 3576 melanoma hsa-miR-1197 2556
3577 neublastoma hsa-miR-1200 2557 3578 chronic lynphocytic
leukemia hsa-miR-1202 2558 3579 chronic lynphocytic leukemia,
downregulated in ovarian cancer cells hsa-miR-1203 2559 3580 in the
chromosome 8q24 region, cancer cells hsa-miR-1204 2560 3581 in the
chromosome 8q24 region, cancer cells hsa-miR-1205 2561 3582 in the
chromosome 8q24 region, cancer cells hsa-miR-1206 2562 3583 in the
chromosome 8q24 region, cancer cells hsa-miR-1207-3p 2563 3584 in
the chromosome 8q24 region, cancer cells hsa-miR-1207-5p 2564 3585
in the chromosome 8q24 region, cancer cells hsa-miR-1208 2565 3586
in the chromosome 8q24 region, cancer cells hsa-miR-122-3p 2566
3587 kidney, Renal Cell lipid liver/hepatocytes Carcinoma
metabolism (RCC), cancer cells hsa-miR-1224-3p 2567 3588 Lupus
nephritis hsa-miR-1224-5p 2568 3589 rectal cancer hsa-miR-1225-3p
2569 3590 adrenal pheochromocytomas; upregulated in MITF KnockDown
melanocytes hsa-miR-1225-5p 2570 3591 prostate cancer
hsa-miR-122-5p 2571 3592 liver/hepatocytes cancer cells lipid
metabolism hsa-miR-1226-3p 2572 3593 discovered in a mirtron
screening hsa-miR-1226-5p 2573 3594 discovered in a mirtron
screening hsa-miR-1227-3p 2574 3595 cartilage/chondrocytes
hsa-miR-1227-5p 2575 3596 cartilage/chondrocytes hsa-miR-1228-3p
2576 3597 liver(hepatocytes) Hepatocellular anti-apoptosis
carcinoma(HCC) hsa-miR-1228-5p 2577 3598 liver(hepatocytes)
Hepatocellular anti-apoptosis carcinoma(HCC) hsa-miR-1229-3p 2578
3599 discovered in a mirtron screening hsa-miR-1229-5p 2579 3600
discovered in a mirtron screening hsa-miR-1231 2580 3601 HCC
hsa-miR-1233-1-5p 2581 3602 serum hsa-miR-1233-3p 2582 3603 serum
hsa-miR-1234-3p 2583 3604 discovered in embryonic stem cell
hsa-miR-1234-5p 2584 3605 discovered in embryonic stem cell
hsa-miR-1236-3p 2585 3606 lymphatic target to endothelial cells
VEGFR-3 hsa-miR-1236-5p 2586 3607 lymphatic target to endothelial
cells VEGFR-3 hsa-miR-1237-3p 2587 3608 esophageal cell line
KYSE-150R
hsa-miR-1237-5p 2588 3609 esophageal cell line KYSE-150R
hsa-miR-1238-3p 2589 3610 colorectal cancer hsa-miR-1238-5p 2590
3611 colorectal cancer hsa-miR-1243 2591 3612 discovered in
embryonic stem cells hsa-miR-124-3p 2592 3613 brain, plasma glioma
cell (exosomal) differentiation hsa-miR-1244 2593 3614 discovered
in embryonic stem cells hsa-miR-1245a 2594 3615 discovered in
embryonic stem cells hsa-miR-1245b-3p 2595 3616 discovered in
embryonic stem cells hsa-miR-1245b-5p 2596 3617 discovered in
embryonic stem cells hsa-miR-124-5p 2597 3618 brain, Plasma
upregulated in cell (circulating) heart dysfunction,
differentiation glioma hsa-miR-1246 2598 3619 embryonic stem cells,
epithelial cells hsa-miR-1247-3p 2599 3620 embryoid body cells
hsa-miR-1247-5p 2600 3621 embryoid body cells hsa-miR-1248 2601
3622 component of SnoRNAs hsa-miR-1249 2602 3623 liver(hepatocytes)
hsa-miR-1250 2603 3624 oligodendrocytes hsa-miR-1251 2604 3625
discovered in embryonic stem cells hsa-miR-1252 2605 3626
discovered in embryonic stem cells hsa-miR-1253 2606 3627
discovered in embryonic stem cells hsa-miR-1254 2607 3628 embryonic
stem cells hsa-miR-1255a 2608 3629 discovered in embryonic stem
cells hsa-miR-1255b-2-3p 2609 3630 discovered in embryonic stem
cells hsa-miR-1255b-5p 2610 3631 discovered in embryonic stem cells
hsa-miR-1256 2611 3632 discovered in prostate cancer embryonic stem
cells hsa-miR-1257 2612 3633 discovered in liposarcoma (soft
embryonic stem tissue sarcoma) cells hsa-miR-1258 2613 3634
discovered in breast cancer and embryonic stem lung cancer cells
hsa-miR-125a-3p 2614 3635 brain, various cancer cell proliferation
hematopoietic cells (prostate, HCC, and etc) differentiation
hsa-miR-125a-5p 2615 3636 brain, various cancer cell proliferation
hematopoietic cells (prostate, HCC, and etc) differentiation
hsa-miR-125b-1-3p 2616 3637 hematopoietic cells various cancer
oncogene, cell (monocytes), (prostate, HCC, differentiation
brain(neuron) etc) hsa-miR-125b-2-3p 2617 3638 hematopoietic cells
various cancer oncogene, cell (monocytes), (prostate, HCC,
differentiation brain(neuron) etc) hsa-miR-125b-5p 2618 3639
hematopoietic cells, various cancer oncogene, cell brain (neuron)
(cutaneous T cell differentiation lymphoma, prostate, HCC, etc)
hsa-miR-1260a 2619 3640 periodontal tissue hsa-miR-1260b 2620 3641
periodontal tissue hsa-miR-1261 2621 3642 embryonic stem cells
hsa-miR-1262 2622 3643 embryoid body cells hsa-miR-1263 2623 3644
discovered in embryonic stem cells hsa-miR-126-3p 2624 3645
endothelial B-lieage ALL angiogenesis cells, lung hsa-miR-1264 2625
3646 discovered in embryonic stem cells hsa-miR-1265 2626 3647
discovered in embryonic stem cells hsa-miR-126-5p 2627 3648
endothelial breast cancer, B- angiogenesis cells, lung lieage ALL
hsa-miR-1266 2628 3649 embryonic stem cells hsa-miR-1267 2629 3650
discovered in embryonic stem cells hsa-miR-1268a 2630 3651
embryonic stem cells hsa-miR-1268b 2631 3652 embryonic stem cells
hsa-miR-1269a 2632 3653 embryoid body cells hsa-miR-1269b 2633 3654
embryoid body cells hsa-miR-1270 2634 3655 discovered in embryonic
stem cells hsa-miR-1271-3p 2635 3656 brain Hepatocellular Suppress
GPC-3 carcinoma(HCC) in HCC hsa-miR-1271-5p 2636 3657 brain
Hepatocellular Suppress GPC-3 carcinoma(HCC) in HCC hsa-miR-1272
2637 3658 embryonic stem cells hsa-miR-1273a 2638 3659 discovered
in embryonic stem cells hsa-miR-1273c 2639 3660 colorectal cancer
hsa-miR-1273d 2640 3661 discovered in embryonic stem cells
hsa-miR-1273e 2641 3662 solid tumor cells hsa-miR-1273f 2642 3663
cervical cancer hsa-miR-1273g-3p 2643 3664 cervical cancer
hsa-miR-1273g-5p 2644 3665 cervical cancer hsa-miR-127-3p 2645 3666
lung, placenta hsa-miR-1275 2646 3667 embryonic stem gastric
carcinoma cells hsa-miR-127-5p 2647 3668 lung, placenta(islet)
hsa-miR-1276 2648 3669 discovered in embryonic stem cells
hsa-miR-1277-3p 2649 3670 embryoid body cells hsa-miR-1277-5p 2650
3671 embryoid body cells hsa-miR-1278 2651 3672 discovered in
embryonic stem cells hsa-miR-1279 2652 3673 monocytes hsa-miR-128
2653 3674 glioblast, brain B-lieage ALL target to neurofibrominlin
neuron hsa-miR-1281 2654 3675 muscle invasive bladder cancer
hsa-miR-1282 2655 3676 discovered in embryonic stem cells
hsa-miR-1283 2656 3677 placenta hsa-miR-1284 2657 3678 lung cancer
hsa-miR-1285-3p 2658 3679 various cancer inhibit P53 cells
expression hsa-miR-1285-5p 2659 3680 various cancer inhibit P53
cells expression hsa-miR-1286 2660 3681 smooth muscle esophageal
cancer hsa-miR-1287 2661 3682 embryoid body breast cancer cells
hsa-miR-1288 2662 3683 discovered in embryonic stem cells
hsa-miR-1289 2663 3684 multiple cell types hsa-miR-1290 2664 3685
embryoid body gastric carcinoma cells hsa-miR-1291 2665 3686
hepatocytes component of SnoRNAs hsa-miR-129-1-3p 2666 3687
multiple cell types HCC cancer cells hsa-miR-1292-3p 2667 3688
hsa-miR-129-2-3p 2668 3689 multiple cell types various cancer cells
hsa-miR-1292-5p 2669 3690 hsa-miR-1293 2670 3691 discovered in
embryonic stem cells hsa-miR-1294 2671 3692 discovered in embryonic
stem cells hsa-miR-1295a 2672 3693 tumor cells (follicular
lymphoma) hsa-miR-1295b-3p 2673 3694 tumor cells (follicular
lymphoma) hsa-miR-1295b-5p 2674 3695 tumor cells (follicular
lymphoma) hsa-miR-129-5p 2675 3696 liver(hepatocytes) HCC, thyroid
cell death in cancer cancer cell hsa-miR-1296 2676 3697 breast
cancer hsa-miR-1297 2677 3698 discovered in embryonic stem cells
hsa-miR-1298 2678 3699 hsa-miR-1299 2679 3700 discovered in
embryonic stem cells hsa-miR-1301 2680 3701 breast cancer
hsa-miR-1302 2681 3702 hsa-miR-1303 2682 3703 hepatocyte colorectal
cancer, liver cancer hsa-miR-1304-3p 2683 3704 dental development
hsa-miR-1304-5p 2684 3705 dental development hsa-miR-1305 2685 3706
discovered in embryonic stem cells hsa-miR-1306-3p 2686 3707
discovered in embryonic stem cells hsa-miR-1306-5p 2687 3708
discovered in embryonic stem cells hsa-miR-1307-3p 2688 3709
discovered in embryonic stem cells hsa-miR-1307-5p 2689 3710
discovered in embryonic stem cells hsa-miR-130a-3p 2690 3711 lung,
monocytes, various cancers pro-angiogenic vascular endothelial
(basal cell cells carcinoma, HCC, ovarian, etc), drug resistance
hsa-miR-130a-5p 2691 3712 lung, monocytes, various cancers
pro-angiogenic vascular endothelial (basal cell cells carcinoma,
HCC, ovarian, etc), drug resistance hsa-miR-130b-3p 2692 3713 Lung,
epidermal various cancers cell cells(keratinocytes) (gastric, rena
cell proiferation/senescence carcinoma) hsa-miR-130b-5p 2693 3714
Lung, epidermal various cancers cell cells(keratinocytes) (gastric,
rena cell proiferation/senescence carcinoma) hsa-miR-1321 2694 3715
neuroblastoma hsa-miR-1322 2695 3716 neuroblastoma hsa-miR-1323
2696 3717 placenta neuroblastoma hsa-miR-132-3p 2697 3718
Brain(neuron),
immune cells hsa-miR-1324 2698 3719 neuroblastoma hsa-miR-132-5p
2699 3720 brain(neuron), immune cells hsa-miR-133a 2700 3721
muscle, heart, heart failure, myogenesis epithelial cells
esophageal cancer (lung) hsa-miR-133b 2701 3722 muscle, heart,
heart failure, myogenesis epithelial cells esophageal cancer (lung)
hsa-miR-134 2702 3723 lung (epithelial) non-samll cell lung cancer,
pulmonary embolism hsa-miR-1343 2703 3724 breast cancer cells
hsa-miR-135a-3p 2704 3725 brain, other tissues various cancer tumor
cells (lung, breast, suppressor colorectal, HCC, etc)
hsa-miR-135a-5p 2705 3726 brain, other tissues various cancer tumor
cells (lung, breast, suppressor colorectal, HCC, etc)
hsa-miR-135b-3p 2706 3727 brain, placenta, various cancers other
tissues (gastric, mammary, neuroblastomas, pancreatic, etc)
hsa-miR-135b-5p 2707 3728 brain, placenta, various cancers other
tissues (gastric, mammary, neuroblastomas, pancreatic, etc)
hsa-miR-136-3p 2708 3729 stem cells, placenta glioma tumor
suppressor hsa-miR-136-5p 2709 3730 stem cells, placenta glioma
tumor suppressor hsa-miR-137 2710 3731 brain various cancers
inhibiting (glioblastoma, cancer cell breast, gastric proliferation
and etc), Alzheimer's migration disease hsa-miR-138-1-3p 2711 3732
stem cells, arious cancer cell epidermal cells,
proliferation/senescence cells(keratinocytes) downregulated in HCC
hsa-miR-138-2-3p 2712 3733 stem cells arious cancer cells,
downregulated in HCC hsa-miR-138-5p 2713 3734 stem cells arious
cancer cells, downregulated in HCC hsa-miR-139-3p 2714 3735
hematocytes, brain various cancer repress cancer cells (colorectal,
metastasis gastric, ovarian) hsa-miR-139-5p 2715 3736 hematocytes,
brain various cancer repress cancer cells (colorectal, metastasis
gastric, ovarian) hsa-miR-140-3p 2716 3737 airway smooth Virus
infection, muscle cancers hsa-miR-140-5p 2717 3738 cartilage
csncers (chondrocytes) hsa-miR-141-3p 2718 3739 Many tissues/cells
various cancer cell cells (HCC, differentiation prostate, kidney,
etc) hsa-miR-141-5p 2719 3740 Many tissues/cells various cancer
cell cells (HCC, differentiation prostate, kidney, etc)
hsa-miR-142-3p 2720 3741 meyloid cells, immune hematopoiesis,
response APC cells hsa-miR-142-5p 2721 3742 meyloid cells, immune
hematopoiesis, response APC cells hsa-miR-143-3p 2722 3743 vascular
smooth pre-B-cell acute muscle lymphocytic leukemia, virus
infection hsa-miR-143-5p 2723 3744 vascular smooth virus infection
muscle, T-cells hsa-miR-144-3p 2724 3745 erythroid various cancers
cell (lung, colorectal, differentiation etc) hsa-miR-144-5p 2725
3746 erythroid various cancers cell (lung, colorectal,
differentiation etc) hsa-miR-145-3p 2726 3747 kidney, cartilage,
T-cell lupus tumor vascular smooth suppressor muscle hsa-miR-145-5p
2727 3748 kidney, cartilage, T-cell lupus tumor vascular smooth
suppressor muscle hsa-miR-1468 2728 3749 lung cancer hsa-miR-1469
2729 3750 tumor cell(follicular lymphoma), rectal cancer
hsa-miR-146a-3p 2730 3751 immune cells, various cancers,
hematopoiesis endotoxin tolerance hsa-miR-146a-5p 2731 3752 immune
cells, various cancers, hematopoiesis endotoxin tolerance
hsa-miR-146b-3p 2732 3753 immune cells various cancers
hsa-miR-146b-5p 2733 3754 Embryonic stem various cancers tumor
invation, cells (glioma) migration hsa-miR-1470 2734 3755
hsa-miR-1471 2735 3756 tumor cell(follicular lymphoma), rectal
cancer hsa-miR-147a 2736 3757 Macrophage inflammatory response
hsa-miR-147b 2737 3758 Macrophage inflammatory response
hsa-miR-148a-3p 2738 3759 hematopoietic cells CLL, T-lineage ALL
hsa-miR-148a-5p 2739 3760 hematopoietic cells CLL, T-lineage ALL
hsa-miR-148b-3p 2740 3761 neuron hsa-miR-148b-5p 2741 3762 neuron
hsa-miR-149-3p 2742 3763 heart, brain various cancers (glioma,
colorectal, gastric, etc) hsa-miR-149-5p 2743 3764 heart, brain
various cancers (glioma, colorectal, gastric, etc) hsa-miR-150-3p
2744 3765 hematopoietic cells circulating plasma (lymphoid) (acute
myeloid leukemia) hsa-miR-150-5p 2745 3766 hematopoietic cells
circulating plasma (lymphoid) (acute myeloid leukemia)
hsa-miR-151a-3p 2746 3767 neuron, fetal liver hsa-miR-151a-5p 2747
3768 neuron, fetal liver hsa-miR-151b 2748 3769 immune cells (B-
cells) hsa-miR-152 2749 3770 liver hsa-miR-153 2750 3771 brain
hsa-miR-1537 2751 3772 hsa-miR-1538 2752 3773 blood Cancer cells
hsa-miR-1539 2753 3774 esophageal cell line KYSE-150R
hsa-miR-154-3p 2754 3775 embryonic stem cells hsa-miR-154-5p 2755
3776 embryonic stem cells hsa-miR-155-3p 2756 3777 T/B cells,
various cancers monocytes, breast (CLL, B cell lymphoma, breast,
lung, ovarian, cervical, colorectal, prostate) hsa-miR-155-5p 2757
3778 T/B cells, various cancers monocytes, breast (CLL, B cell
lymphoma, breast, lung, ovarian, cervical, colorectal, prostate)
hsa-miR-1587 2758 3779 identified in B-cells hsa-miR-15a-3p 2759
3780 blood, lymphocyte, cell cycle, hematopoietic proliferation
tissues (spleen) hsa-miR-15a-5p 2760 3781 blood, lymphocyte, cell
cycle, hematopoietic proliferation tissues (spleen) hsa-miR-15b-3p
2761 3782 blood, lymphocyte, cell cycle, hematopoietic
proliferation tissues (spleen) hsa-miR-15b-5p 2762 3783 blood,
lymphocyte, cell cycle, hematopoietic proliferation tissues
(spleen) hsa-miR-16-1-3p 2763 3784 embryonic stem cells, blood,
hematopoietic tissues (spleen) hsa-miR-16-2-3p 2764 3785 blood,
lymphocyte, hematopoietic tissues (spleen) hsa-miR-16-5p 2765 3786
Many tissues, blood hsa-miR-17-3p 2766 3787 embryonic stem tumor
cells, endothelial angiogenesis cells, hsa-miR-17-5p 2767 3788
endothelial cells, tumor kidney, breast; angiogenesis
hsa-miR-181a-2-3p 2768 3789 glioblast, stem cells hsa-miR-181a-3p
2769 3790 glioblast, myeloid cells, Embryonic stem cells
hsa-miR-181a-5p 2770 3791 glioblast, myeloid cells, Embryonic stem
cells hsa-miR-181b-3p 2771 3792 glioblast, cell Embryonic stem
proiferation/senescence cells, epidermal (keratinocytes)
hsa-miR-181b-5p 2772 3793 glioblast, cell Embryonic stem
proiferation/senescence cells, epidermal (keratinocytes)
hsa-miR-181c-3p 2773 3794 brain, stem variou cance cells cell
cells/progenitor (gliobasltoma, differentiation basal cell
carcinoma, prostate) hsa-miR-181c-5p 2774 3795 brain, stem variou
cance cells cell cells/progenitor (gliobasltoma, differentiation
basal cell carcinoma, prostate) hsa-miR-181d 2775 3796 glia cells
hsa-miR-182-3p 2776 3797 immune cells autoimmune immune response
hsa-miR-1825 2777 3798 discovered in a MiRDeep screening
hsa-miR-182-5p 2778 3799 lung, immune cells autoimmune immune
response hsa-miR-1827 2779 3800 small cell lung cancer
hsa-miR-183-3p 2780 3801 brain hsa-miR-183-5p 2781 3802 brain
hsa-miR-184 2782 3803 blood, tongue, pancreas (islet)
hsa-miR-185-3p 2783 3804 hsa-miR-185-5p 2784 3805 hsa-miR-186-3p
2785 3806 osteoblasts, heart various cancer cells hsa-miR-186-5p
2786 3807 osteoblasts, heart various cancer cells hsa-miR-187-3p
2787 3808 thyroid tumor hsa-miR-187-5p 2788 3809 thyroid tumor
hsa-miR-188-3p 2789 3810 irway smooth muscle, central nervous
system hsa-miR-188-5p 2790 3811 irway smooth muscle, central
nervous system hsa-miR-18a-3p 2791 3812 endothelial cells, lung
hsa-miR-18a-5p 2792 3813 endothelial cells, lung hsa-miR-18b-3p
2793 3814 lung hsa-miR-18b-5p 2794 3815 lung hsa-miR-1908 2795 3816
breast cancer hsa-miR-1909-3p 2796 3817 rectal cancer
hsa-miR-1909-5p 2797 3818 rectal cancer hsa-miR-190a 2798 3819
brain hsa-miR-190b 2799 3820 brain hsa-miR-1910 2800 3821 embryonic
stem cells hsa-miR-1911-3p 2801 3822 embryonic stem cells, neural
precursor hsa-miR-1911-5p 2802 3823 embryonic stem cells, neural
precursor hsa-miR-1912 2803 3824 embryonic stem cells, neural
precursor hsa-miR-1913 2804 3825 embryonic stem cells
hsa-miR-191-3p 2805 3826 chroninc lymphocyte leukimia, B- lieage
ALL hsa-miR-1914-3p 2806 3827 embryonic stem cells hsa-miR-1914-5p
2807 3828 embryonic stem cells hsa-miR-1915-3p 2808 3829 embryonic
stem cells hsa-miR-1915-5p 2809 3830 embryonic stem cells
hsa-miR-191-5p 2810 3831 chroninc lymphocyte leukimia, B- lieage
ALL hsa-miR-192-3p 2811 3832 kidney hsa-miR-192-5p 2812 3833 kidney
hsa-miR-193a-3p 2813 3834 many tissues/cells various cancer tumor
cells (lung, suppressor, osteoblastoma, proliferation ALL,
follicular lymphoma, etc) hsa-miR-193a-5p 2814 3835 many
tissues/cells various cancer tumor cells (lung, suppressor,
osteoblastoma, proliferation ALL, follicular lymphoma, etc)
hsa-miR-193b-3p 2815 3836 many tissues/cells, arious cancer tumor
semen cells (prostate, suppressor breast, melanoma, myeloma, non
small cell lung, etc) follicular lymphoma) hsa-miR-193b-5p 2816
3837 many tissues/cells, arious cancer tumor semen cells (prostate,
suppressor breast, melanoma, myeloma, non small cell lung,
etc)follicular lymphoma) hsa-miR-194-3p 2817 3838 kidney, liver
various cancers hsa-miR-194-5p 2818 3839 kidney, liver various
cancers hsa-miR-195-3p 2819 3840 breast, pancreas (islet)
hsa-miR-195-5p 2820 3841 breast, pancreas (islet) hsa-miR-196a-3p
2821 3842 pancreatic various cancer oncogenic, cells, endometrial
cells (pancreatic, tumor tissues, osteosarcoma, suppressor
mesenchymal stem endometrial, cells AML etc) hsa-miR-196a-5p 2822
3843 pancreatic various cancer oncogenic, cells, endometrial cells
(pancreatic, tumor tissues, osteosarcoma, suppressor mesenchymal
stem endometrial, cells AML etc) hsa-miR-196b-3p 2823 3844
endometrial tissues glioblastoma apoptosis hsa-miR-196b-5p 2824
3845 endometrial tissues glioblastoma apoptosis hsa-miR-1972 2825
3846 acute lymphoblastic leukemia hsa-miR-1973 2826 3847 acute
lymphoblastic leukemia hsa-miR-197-3p 2827 3848 blood (myeloid),
various cancers other tissues/cells (thyroid tumor, leukemia, etc)
hsa-miR-197-5p 2828 3849 blood (myeloid), various cancers other
tissues/cells (thyroid tumor, leukemia, etc) hsa-miR-1976 2829 3850
acute lymphoblastic leukemia hsa-miR-198 2830 3851 central nevous
system (CNS) hsa-miR-199a-3p 2831 3852 liver, embryoid body cells,
cardiomyocytes hsa-miR-199a-5p 2832 3853 liver, cardiomyocytes
hsa-miR-199b-3p 2833 3854 liver, osteoblast various cancers
osteogenesis hsa-miR-199b-5p 2834 3855 liver, osteoblast various
cancers osteogenesis hsa-miR-19a-3p 2835 3856 endothelial cells
tumor angiogenesis hsa-miR-19a-5p 2836 3857 endothelial cells tumor
angiogenesis hsa-miR-19b-1-5p 2837 3858 endothelial cells tumor
angiogenesis hsa-miR-19b-2-5p 2838 3859 endothelial cells tumor
angiogenesis hsa-miR-19b-3p 2839 3860 endothelial cells tumor
angiogenesis hsa-miR-200a-3p 2840 3861 epithelial cells, various
cancers tumor many other tissues (breast, cervical, progression and
bladder, etc) metastasis hsa-miR-200a-5p 2841 3862 epithelial
cells, various cancers tumor many other tissues (breast, cervical,
progression and bladder, etc) metastasis hsa-miR-200b-3p 2842 3863
epithelial cells, tumor many other tissues progression and
metastasis hsa-miR-200b-5p 2843 3864 epithelial cells, tumor many
other tissues progression and metastasis hsa-miR-200c-3p 2844 3865
epithelial cells, tumor many other tissues, progression and
embryonic stem metastasis cells hsa-miR-200c-5p 2845 3866
epithelial cells, tumor many other tissues, progression and
embryonic stem metastasis cells hsa-miR-202-3p 2846 3867 blood
lymphomagenesis, other cancers hsa-miR-202-5p 2847 3868 blood
lymphomagenesis, other cancers hsa-miR-203a 2848 3869 skin
(epithelium) psoriasis, autoimmune hsa-miR-203b-3p 2849 3870 skin
specific psoriasis, (epithelium) autoimmune hsa-miR-203b-5p 2850
3871 skin specific psoriasis, (epithelium) autoimmune
hsa-miR-204-3p 2851 3872 adipose, other various cancers tumor
tissues/cells, kidney metastasis hsa-miR-204-5p 2852 3873 adipose,
other various cancers tumor tissues/cells, kidney metastasis
hsa-miR-2052 2853 3874 hsa-miR-2053 2854 3875 hsa-miR-205-3p 2855
3876 blood (plasma) various cancer cells (breast, glioma, melanoma,
endometrial, etc) hsa-miR-2054 2856 3877 hsa-miR-205-5p 2857 3878
blood (plasma) various cancer cells (breast, glioma, melanoma,
endometrial, etc) hsa-miR-206 2858 3879 muscle (cardiac and
myogenesis skeletal) hsa-miR-208a 2859 3880 heart (cardiomyocyte),
cardiac defects muscle hsa-miR-208b 2860 3881 heart
(cardiomyocyte), cardiac defects muscle hsa-miR-20a-3p 2861 3882
endothelial cells, kidney, osteogenic cells hsa-miR-20a-5p 2862
3883 endothelial cells, kidney, osteogenic cells hsa-miR-20b-3p
2863 3884 osteogenic cells hsa-miR-20b-5p 2864 3885 osteogenic
cells hsa-miR-210 2865 3886 kidney, heart, RCC, B-cell angiogenesis
vascular endothelial lymphocytes cells hsa-miR-2110 2866 3887
rectal cancer hsa-miR-2113 2867 3888 embryonic stem cells
hsa-miR-211-3p 2868 3889 melanocytes melanoma and other cancers
hsa-miR-2114-3p 2869 3890 ovary, female reproductuve tract
hsa-miR-2114-5p 2870 3891 ovary, female reproductuve tract
hsa-miR-2115-3p 2871 3892 female reproductive ovarian cancer tract
hsa-miR-2115-5p 2872 3893 female reproductive ovarian cancer tract
hsa-miR-211-5p 2873 3894 melanocytes melanoma and other cancers
hsa-miR-2116-3p 2874 3895 live cancer (hepatocytes) and ovarian
cancer hsa-miR-2116-5p 2875 3896 live cancer (hepatocytes) and
ovarian cancer hsa-miR-2117 2876 3897 ovarian cancer hsa-miR-212-3p
2877 3898 brain (neuron), lymphoma spleen hsa-miR-212-5p 2878 3899
brain (neuron), lymphoma spleen hsa-miR-21-3p 2879 3900 glioblast,
Blood autoimmune, (meyloid cells), heart diseases, liver, vascular
cancers endothelial cells hsa-miR-214-3p 2880 3901 immune cerlls,
varioua cancers immune pancreas (melanoma, response pancreatic,
ovarian) hsa-miR-214-5p 2881 3902 immune cells, varioua cancers
immune pancreas (melanoma, response pancreatic, ovarian)
hsa-miR-215 2882 3903 many tissues/cells various cancers cell cycle
(renal, colon, arrest/p53 osteosarcoma) inducible hsa-miR-21-5p
2883 3904 blood (myeloid autoimmune, cells), liver, heart diseases,
endothelial cells cancers hsa-miR-216a-3p 2884 3905 kidney,
pancreas hsa-miR-216a-5p 2885 3906 kidney, pancreas hsa-miR-216b
2886 3907 cancers senescence hsa-miR-217 2887 3908 endothelial
cells various cancer cells (pancreas, kidney, breast)
hsa-miR-218-1-3p 2888 3909 endothelial cells various cancer cells
(gastric tumor, bladder, cervical, etc) hsa-miR-218-2-3p 2889 3910
various cancer cells (gastric tumor, bladder, cervical, etc)
hsa-miR-218-5p 2890 3911 various cancer cells (gastric tumor,
bladder, cervical, etc) hsa-miR-219-1-3p 2891 3912 brain,
oligodendrocytes hsa-miR-219-2-3p 2892 3913 brain, oligodendrocytes
hsa-miR-219-5p 2893 3914 brain, oligodendrocytes hsa-miR-221-3p
2894 3915 endothelial cells, leukemia and
angiogenesis/vasculogenesis immune cells other cancers
hsa-miR-221-5p 2895 3916 endothelial cells, leukemia and
angiogenesis/vasculogenesis immune cells other cancers
hsa-miR-222-3p 2896 3917 endothelial cells various cancers
angiogenesis
hsa-miR-222-5p 2897 3918 endothelial cells various cancers
angiogenesis hsa-miR-223-3p 2898 3919 meyloid cells leukemia
hsa-miR-223-5p 2899 3920 meyloid cells leukemia hsa-miR-22-3p 2900
3921 many tissues/cells various cancers tumorigenesis
hsa-miR-224-3p 2901 3922 blood (plasma), cancers and ovary
inflammation hsa-miR-224-5p 2902 3923 blood (plasma), cancers and
ovary inflammation hsa-miR-22-5p 2903 3924 many tissues/cells
Various cancers tumorigenesis hsa-miR-2276 2904 3925 breast cancer
hsa-miR-2277-3p 2905 3926 female reproductive tract hsa-miR-2277-5p
2906 3927 female reproductive tract hsa-miR-2278 2907 3928 breast
cancer hsa-miR-2355-3p 2908 3929 embryonic stem cells
hsa-miR-2355-5p 2909 3930 embryonic stem cells hsa-miR-2392 2910
3931 identified in B-cells hsa-miR-23a-3p 2911 3932 brain
(astrocyte), Cancers endothelial cells, blood (erythroid)
hsa-miR-23a-5p 2912 3933 brain (astrocyte), cancers endothelial
cells, blood (erythroid) hsa-miR-23b-3p 2913 3934 blood, meyloid
cancers (renal cells cancer, glioblastoma, prostate, etc) and
autoimmune hsa-miR-23b-5p 2914 3935 blood, meyloid cancers
(glioblastoma, cells prostate, etc) and autoimmune hsa-miR-23c 2915
3936 cervical cancer hsa-miR-24-1-5p 2916 3937 lung, meyloid cells
hsa-miR-24-2-5p 2917 3938 lung, meyloid cells hsa-miR-24-3p 2918
3939 lung, meyloid cells hsa-miR-2467-3p 2919 3940 breast cancer
hsa-miR-2467-5p 2920 3941 breast cancer hsa-miR-25-3p 2921 3942
embryonic stem cells, airway smooth muscle hsa-miR-25-5p 2922 3943
embryonic stem cells, airway smooth muscle hsa-miR-2681-3p 2923
3944 breast cancer hsa-miR-2681-5p 2924 3945 breast cancer
hsa-miR-2682-3p 2925 3946 hsa-miR-2682-5p 2926 3947
hsa-miR-26a-1-3p 2927 3948 embryonic stem CLL and other cell cycle
and cells, blood, other cancers differentiation tissues
hsa-miR-26a-2-3p 2928 3949 blood, other tissues CLL and other cell
cycle and cancers differentiation hsa-miR-26a-5p 2929 3950 blood,
other tissues CLL and other cell cycle and cancers differentiation
hsa-miR-26b-3p 2930 3951 hematopoietic cells hsa-miR-26b-5p 2931
3952 hematopoietic cells hsa-miR-27a-3p 2932 3953 meyloid cells
various cancer cells hsa-miR-27a-5p 2933 3954 meyloid cells various
cancer cells hsa-miR-27b-3p 2934 3955 meyloid cells, various cancer
pro-angiogenic vascular endothelial cells cells hsa-miR-27b-5p 2935
3956 meyloid cells, various cancer pro-angiogenic vascular
endothelial cells cells hsa-miR-28-3p 2936 3957 blood (immune B/T
cell cells) lymphoma hsa-miR-28-5p 2937 3958 blood (immune B/T cell
cells) lymphoma hsa-miR-2861 2938 3959 osteoblasts basal cell
carcinoma hsa-miR-2909 2939 3960 T-Lymphocytes hsa-miR-296-3p 2940
3961 kidney, heart, lung, angiogenesis entothelial cells
hsa-miR-2964a-3p 2941 3962 hsa-miR-2964a-5p 2942 3963
hsa-miR-296-5p 2943 3964 lung, liver, angiogenesis endothelial
cells hsa-miR-297 2944 3965 oocyte and prostate hsa-miR-298 2945
3966 breast cancer hsa-miR-299-3p 2946 3967 myeloid leukaemia,
hepatoma, breast cancer hsa-miR-299-5p 2947 3968 myeloid leukaemia,
hepatoma, breast cancer hsa-miR-29a-3p 2948 3969 immuno system CLL,
other tumor cancers, suppression, neurodegenative immune disease
modulation hsa-miR-29a-5p 2949 3970 immuno system CLL, other tumor
cancers, suppression, neurodegenative immune disease modulation
hsa-miR-29b-1-5p 2950 3971 immuno system CLL, other tumor cancers,
suppression, neurodegenative immune disease modulation
hsa-miR-29b-2-5p 2951 3972 immuno system CLL, other tumor cancers
suppression, immune modulation hsa-miR-29b-3p 2952 3973 immuno
system CLL, other tumor cancers suppression, immune modulation
hsa-miR-29c-3p 2953 3974 immuno system CLL, other tumor cancers
suppression, immune modulation hsa-miR-29c-5p 2954 3975 immuno
system CLL, other tumor cancers suppression, immune modulation
hsa-miR-300 2955 3976 osteoblast Bladder cancer hsa-miR-301a-3p
2956 3977 embryonic stem cells hsa-miR-301a-5p 2957 3978 embryonic
stem cells hsa-miR-301b 2958 3979 esophageal adenocarcinoma,
colonic cancer hsa-miR-302a-3p 2959 3980 embryonic stem lipid
cells, lipid metabolism metabolism hsa-miR-302a-5p 2960 3981
embryonic stem lipid cells, lipid metabolism metabolism
hsa-miR-302b-3p 2961 3982 embryonic stem cells hsa-miR-302b-5p 2962
3983 embryonic stem cells hsa-miR-302c-3p 2963 3984 embryonic stem
cells hsa-miR-302c-5p 2964 3985 embryonic stem cells
hsa-miR-302d-3p 2965 3986 embryonic stem cells hsa-miR-302d-5p 2966
3987 embryonic stem cells hsa-miR-302e 2967 3988 embryoid body
cells hsa-miR-302f 2968 3989 gastric cancer hsa-miR-3064-3p 2969
3990 hsa-miR-3064-5p 2970 3991 hsa-miR-3065-3p 2971 3992
oligodendrocytes anti-virus response hsa-miR-3065-5p 2972 3993
oligodendrocytes solid tumors hsa-miR-3074-3p 2973 3994 various
cancer (melanoma, breast) hsa-miR-3074-5p 2974 3995 various cancer
(melanoma, breast) hsa-miR-30a-3p 2975 3996 kidney, pancreatic
various cancers autophagy cells hsa-miR-30a-5p 2976 3997 CNS
(prefrontal glioma, colon autophagy cortex), other carcinoma
tissues hsa-miR-30b-3p 2977 3998 kidney, adipose, CNS (prefrontal
cortex) hsa-miR-30b-5p 2978 3999 kidney, adipose, CNS (prefrontal
cortex) hsa-miR-30c-1-3p 2979 4000 kidney, adipose, CNS (prefrontal
cortex) hsa-miR-30c-2-3p 2980 4001 kidney, adipose, CNS (prefrontal
cortex) hsa-miR-30c-5p 2981 4002 kidney, adipose, CNS (prefrontal
cortex) hsa-miR-30d-3p 2982 4003 CNS (prefrontal cortex
hsa-miR-30d-5p 2983 4004 CNS (prefrontal cortex, embryoid body
cells hsa-miR-30e-3p 2984 4005 myeloid cells, glia cells
hsa-miR-30e-5p 2985 4006 myeloid cells, glia cells hsa-miR-3115
2986 4007 various cancer (melanoma, breast tumor) hsa-miR-3116 2987
4008 discovered in the melanoma miRNAome hsa-miR-3117-3p 2988 4009
discovered in the melanoma miRNAome hsa-miR-3117-5p 2989 4010
discovered in the melanoma miRNAome hsa-miR-3118 2990 4011
discovered in the melanoma miRNAome hsa-miR-3119 2991 4012
discovered in the melanoma miRNAome hsa-miR-3120-3p 2992 4013
discovered in the breast tumor melanoma miRNAome hsa-miR-3120-5p
2993 4014 discovered in the breast tumor melanoma miRNAome
hsa-miR-3121-3p 2994 4015 discovered in the breast tumor melanoma
miRNAome hsa-miR-3121-5p 2995 4016 discovered in the breast tumor
melanoma miRNAome hsa-miR-3122 2996 4017 discovered in the melanoma
miRNAome hsa-miR-3123 2997 4018 discovered in the melanoma miRNAome
hsa-miR-3124-3p 2998 4019 discovered in the breast tumor melanoma
miRNAome, ovary hsa-miR-3124-5p 2999 4020 discovered in the breast
tumor melanoma miRNAome, ovary hsa-miR-3125 3000 4021 discovered in
the melanoma miRNAome hsa-miR-3126-3p 3001 4022 discovered in the
breast tumor melanoma miRNAome, ovary hsa-miR-3126-5p 3002 4023
discovered in the breast tumor melanoma miRNAome, ovary
hsa-miR-3127-3p 3003 4024 discovered in the breast tumor melanoma
miRNAome hsa-miR-3127-5p 3004 4025 discovered in the breast tumor
melanoma miRNAome hsa-miR-3128 3005 4026 discovered in the breast
tumor melanoma miRNAome
hsa-miR-3129-3p 3006 4027 discovered in the breast tumor melanoma
miRNAome, ovary hsa-miR-3129-5p 3007 4028 discovered in the breast
tumor melanoma miRNAome, ovary hsa-miR-3130-3p 3008 4029 discovered
in the breast tumor melanoma miRNAome, ovary hsa-miR-3130-5p 3009
4030 discovered in the breast tumor melanoma miRNAome, ovary
hsa-miR-3131 3010 4031 discovered in the breast tumor melanoma
miRNAome hsa-miR-3132 3011 4032 discovered in the melanoma miRNAome
hsa-miR-3133 3012 4033 discovered in the melanoma miRNAome
hsa-miR-3134 3013 4034 discovered in the melanoma miRNAome
hsa-miR-3135a 3014 4035 discovered in the melanoma miRNAome
hsa-miR-3135b 3015 4036 discovered in B cells hsa-miR-3136-3p 3016
4037 discovered in the lymphoblastic melanoma leukaemia and
miRNAome breast tumor hsa-miR-3136-5p 3017 4038 discovered in the
lymphoblastic melanoma leukaemia and miRNAome breast tumor
hsa-miR-3137 3018 4039 discovered in the melanoma miRNAome
hsa-miR-3138 3019 4040 discovered in the melanoma miRNAome, ovary
hsa-miR-3139 3020 4041 discovered in the melanoma miRNAome
hsa-miR-31-3p 3021 4042 hsa-miR-3140-3p 3022 4043 discovered in the
lymphoblastic melanoma leukaemia and miRNAome, ovary breast tumor
hsa-miR-3140-5p 3023 4044 discovered in the lymphoblastic melanoma
leukaemia and miRNAome, ovary breast tumor hsa-miR-3141 3024 4045
discovered in the melanoma miRNAome hsa-miR-3142 3025 4046
discovered in the melanoma miRNAome; immune cells hsa-miR-3143 3026
4047 discovered in the breast tumor melanoma miRNAome
hsa-miR-3144-3p 3027 4048 discovered in the melanoma miRNAome,
ovary hsa-miR-3144-5p 3028 4049 discovered in the melanoma
miRNAome, ovary hsa-miR-3145-3p 3029 4050 discovered in the breast
tumor melanoma miRNAome hsa-miR-3145-5p 3030 4051 discovered in the
breast tumor melanoma miRNAome hsa-miR-3146 3031 4052 discovered in
the breast tumor melanoma miRNAome hsa-miR-3147 3032 4053
discovered in the melanoma miRNAome hsa-miR-3148 3033 4054
discovered in the melanoma miRNAome hsa-miR-3149 3034 4055
discovered in the melanoma miRNAome, ovary hsa-miR-3150a-3p 3035
4056 discovered in the breast tumor melanoma miRNAome
hsa-miR-3150a-5p 3036 4057 discovered in the breast tumor melanoma
miRNAome hsa-miR-3150b-3p 3037 4058 discovered in the breast tumor
and melanoma lymphoblastic miRNAome leukaemia hsa-miR-3150b-5p 3038
4059 discovered in the breast tumor and melanoma lymphoblastic
miRNAome leukaemia hsa-miR-3151 3039 4060 discovered in the
lymphoblastic melanoma leukaemia miRNAome hsa-miR-3152-3p 3040 4061
discovered in the breast tumor melanoma miRNAome, ovary
hsa-miR-3152-5p 3041 4062 discovered in the breast tumor melanoma
miRNAome, ovary hsa-miR-3153 3042 4063 discovered in the melanoma
miRNAome hsa-miR-3154 3043 4064 discovered in the lymphoblastic
melanoma leukaemia miRNAome hsa-miR-3155a 3044 4065 discovered in
the melanoma miRNAome hsa-miR-3155b 3045 4066 discovered in B cells
hsa-miR-3156-3p 3046 4067 discovered in the breast tumor melanoma
miRNAome hsa-miR-3156-5p 3047 4068 discovered in the breast tumor
melanoma miRNAome hsa-miR-3157-3p 3048 4069 discovered in the
breast tumor melanoma miRNAome hsa-miR-3157-5p 3049 4070 discovered
in the breast tumor melanoma miRNAome hsa-miR-3158-3p 3050 4071
discovered in the breast tumor melanoma miRNAome, ovary
hsa-miR-3158-5p 3051 4072 discovered in the breast tumor melanoma
miRNAome, ovary hsa-miR-3159 3052 4073 discovered in the melanoma
miRNAome hsa-miR-31-5p 3053 4074 various cancer cells (breast,
lung, prostate) hsa-miR-3160-3p 3054 4075 discovered in the breast
tumor melanoma miRNAome hsa-miR-3160-5p 3055 4076 discovered in the
breast tumor melanoma miRNAome hsa-miR-3161 3056 4077 discovered in
the melanoma miRNAome hsa-miR-3162-3p 3057 4078 discovered in the
breast tumor melanoma miRNAome hsa-miR-3162-5p 3058 4079 discovered
in the breast tumor melanoma miRNAome hsa-miR-3163 3059 4080
discovered in the melanoma miRNAome hsa-miR-3164 3060 4081
discovered in the melanoma miRNAome hsa-miR-3165 3061 4082
discovered in the breast tumor melanoma miRNAome hsa-miR-3166 3062
4083 discovered in the melanoma miRNAome hsa-miR-3167 3063 4084
discovered in the melanoma miRNAome, ovary hsa-miR-3168 3064 4085
discovered in the melanoma miRNAome hsa-miR-3169 3065 4086
discovered in the melanoma miRNAome hsa-miR-3170 3066 4087
discovered in the breast tumor melanoma miRNAome hsa-miR-3171 3067
4088 discovered in the melanoma miRNAome, ovary hsa-miR-3173-3p
3068 4089 discovered in the breast tumor melanoma miRNAome
hsa-miR-3173-5p 3069 4090 discovered in the breast tumor melanoma
miRNAome hsa-miR-3174 3070 4091 discovered in the melanoma miRNAome
hsa-miR-3175 3071 4092 discovered in the breast tumor melanoma
miRNAome, ovary hsa-miR-3176 3072 4093 discovered in the breast
tumor melanoma miRNAome hsa-miR-3177-3p 3073 4094 discovered in the
breast tumor and melanoma lymphoblastic miRNAome leukaemia
hsa-miR-3177-5p 3074 4095 discovered in the breast tumor and
melanoma lymphoblastic miRNAome leukaemia hsa-miR-3178 3075 4096
discovered in the melanoma miRNAome hsa-miR-3179 3076 4097
discovered in the melanoma miRNAome hsa-miR-3180 3077 4098
discovered in the breast tumor melanoma miRNAome, ovary
hsa-miR-3180-3p 3078 4099 discovered in breast tunor
hsa-miR-3180-5p 3079 4100 discovered in breast tumor hsa-miR-3181
3080 4101 discovered in the melanoma miRNAome hsa-miR-3182 3081
4102 discovered in the melanoma miRNAome hsa-miR-3183 3082 4103
discovered in the melanoma miRNAome hsa-miR-3184-3p 3083 4104
discovered in the melanoma miRNAome hsa-miR-3184-5p 3084 4105
discovered in the melanoma miRNAome hsa-miR-3185 3085 4106
discovered in the melanoma miRNAome hsa-miR-3186-3p 3086 4107
discovered in the melanoma miRNAome, ovary hsa-miR-3186-5p 3087
4108 discovered in the melanoma miRNAome, ovary hsa-miR-3187-3p
3088 4109 discovered in the breast tumor melanoma miRNAome
hsa-miR-3187-5p 3089 4110 discovered in the breast tumor melanoma
miRNAome hsa-miR-3188 3090 4111 discovered in the melanoma miRNAome
hsa-miR-3189-3p 3091 4112 discovered in the breast tumor
melanoma miRNAome hsa-miR-3189-5p 3092 4113 discovered in the
breast tumor melanoma miRNAome hsa-miR-3190-3p 3093 4114 discovered
in the lymphoblastic melanoma leukaemia miRNAome hsa-miR-3190-5p
3094 4115 discovered in the lymphoblastic melanoma leukaemia
miRNAome hsa-miR-3191-3p 3095 4116 discovered in the melanoma
miRNAome hsa-miR-3191-5p 3096 4117 discovered in the melanoma
miRNAome hsa-miR-3192 3097 4118 discovered in the breast tumor
melanoma miRNAome hsa-miR-3193 3098 4119 discovered in the melanoma
miRNAome hsa-miR-3194-3p 3099 4120 discovered in the breast tumor
melanoma miRNAome hsa-miR-3194-5p 3100 4121 discovered in the
breast tumor melanoma miRNAome hsa-miR-3195 3101 4122 discovered in
the melanoma miRNAome hsa-miR-3196 3102 4123 basal cell carcinoma
hsa-miR-3197 3103 4124 discovered in the melanoma miRNAome
hsa-miR-3198 3104 4125 discovered in the breast tumor melanoma
miRNAome hsa-miR-3199 3105 4126 discovered in the melanoma miRNAome
hsa-miR-3200-3p 3106 4127 discovered in the breast tumor melanoma
miRNAome, ovary hsa-miR-3200-5p 3107 4128 discovered in the breast
tumor melanoma miRNAome, ovary hsa-miR-3201 3108 4129 discovered in
the melanoma miRNAome, hsa-miR-3202 3109 4130 discovered in the
melanoma miRNAome,epithelial cell BEAS2B hsa-miR-320a 3110 4131
blood, colon cancer heart (myocardiac) cells, heart disease
hsa-miR-320b 3111 4132 central nevous system hsa-miR-320c 3112 4133
chondrocyte cartilage metabolism hsa-miR-320d 3113 4134 cancer stem
cells hsa-miR-320e 3114 4135 neural cells hsa-miR-323a-3p 3115 4136
neurons myeloid leukaemia, mudulla thyroid carcinoma
hsa-miR-323a-5p 3116 4137 neurons myeloid leukaemia, mudulla
thyroid carcinoma hsa-miR-323b-3p 3117 4138 myeloid leukaemia
hsa-miR-323b-5p 3118 4139 myeloid leukaemia hsa-miR-32-3p 3119 4140
blood, glia various cancers (lung, kidney, prostate, etc), virus
infection hsa-miR-324-3p 3120 4141 kidney hsa-miR-324-5p 3121 4142
neurons tumor cells hsa-miR-325 3122 4143 neurons, placenta
hsa-miR-32-5p 3123 4144 blood, glia various cancers (lung, kidney,
prostate, etc), virus infection hsa-miR-326 3124 4145 neurons tumor
cells hsa-miR-328 3125 4146 neuron, blood tumor cells hsa-miR-329
3126 4147 brain and platele hsa-miR-330-3p 3127 4148 various
cancers (prostate, glioblastoma, colorectal) hsa-miR-330-5p 3128
4149 various cancers (prostate, glioblastoma, colorectal)
hsa-miR-331-3p 3129 4150 gastric cancer hsa-miR-331-5p 3130 4151
lymphocytes hsa-miR-335-3p 3131 4152 kidney, breast RCC, multiple
myeloma hsa-miR-335-5p 3132 4153 kidney, breast RCC, multiple
myeloma hsa-miR-337-3p 3133 4154 lung gastric cancer hsa-miR-337-5p
3134 4155 lung hsa-miR-338-3p 3135 4156 epithelial cells, gastric,
rectal oligodendrocytes cancer cells, osteosarcoma hsa-miR-338-5p
3136 4157 oligodendrocytes gastric cancer hsa-miR-339-3p 3137 4158
immune cell hsa-miR-339-5p 3138 4159 immune cell hsa-miR-33a-3p
3139 4160 pancreatic islet, lipid lipid metabolism metabolism
hsa-miR-33a-5p 3140 4161 pancreatic islet, lipid lipid metabolism
metabolism hsa-miR-33b-3p 3141 4162 lipid metabolism lipid
metabolism hsa-miR-33b-5p 3142 4163 lipid metabolism lipid
metabolism hsa-miR-340-3p 3143 4164 various cancers hsa-miR-340-5p
3144 4165 embryoid body cells hsa-miR-342-3p 3145 4166 brain,
circulating multiple plasma myeloma, other cancers hsa-miR-342-5p
3146 4167 circulating plasma multiple myeloma, other cancers
hsa-miR-345-3p 3147 4168 hematopoietic cells follicular lymphoma,
other cancers hsa-miR-345-5p 3148 4169 hematopoietic cells
follicular lymphoma, other cancers hsa-miR-346 3149 4170 immume
cells cancers and autoimmune hsa-miR-34a-3p 3150 4171 breast,
meyloid gastric cancer, tumor cells, ciliated CLL, other
suppressor, p53 epithelial cells inducible hsa-miR-34a-5p 3151 4172
breast, meyloid gastric cancer, tumor cells, ciliated CLL, other
suppressor, p53 epithelial cells inducible hsa-miR-34b-3p 3152 4173
ciliated epithelial various cancers tumor cells suppressor, p53
inducible hsa-miR-34b-5p 3153 4174 ciliated epithelial various
cancers tumor cells suppressor, p53 inducible hsa-miR-34c-3p 3154
4175 ciliated epithelial various cancers tumor cells, placenta
suppressor, p53 inducible hsa-miR-34c-5p 3155 4176 ciliated
epithelial various cancers tumor cells, placenta suppressor, p53
inducible hsa-miR-3529-3p 3156 4177 discovered in breast tumor
hsa-miR-3529-5p 3157 4178 discovered in breast tumor
hsa-miR-3591-3p 3158 4179 discovered in breast tumor
hsa-miR-3591-5p 3159 4180 discovered in breast tumor
hsa-miR-3605-3p 3160 4181 discovered in reprodcutive tracts
hsa-miR-3605-5p 3161 4182 discovered in reprodcutive tracts
hsa-miR-3606-3p 3162 4183 discovered in cervical tumors
hsa-miR-3606-5p 3163 4184 discovered in cervical tumors
hsa-miR-3607-3p 3164 4185 discovered in cervical tumors
hsa-miR-3607-5p 3165 4186 discovered in cervical tumors
hsa-miR-3609 3166 4187 discovered in cervical tumors hsa-miR-3610
3167 4188 discovered in cervical tumors hsa-miR-3611 3168 4189
discovered in cervical tumors hsa-miR-3612 3169 4190 discovered in
cervical tumors hsa-miR-3613-3p 3170 4191 discovered in cervical
tumors hsa-miR-3613-5p 3171 4192 discovered in cervical tumors
hsa-miR-361-3p 3172 4193 blood, endothelial cells hsa-miR-3614-3p
3173 4194 discovered in cervical and breast tumors hsa-miR-3614-5p
3174 4195 discovered in cervical and breast tumors hsa-miR-3615
3175 4196 discovered in cervical tumors hsa-miR-361-5p 3176 4197
endothelial cells hsa-miR-3616-3p 3177 4198 discovered in cervical
tumors hsa-miR-3616-5p 3178 4199 discovered in cervical tumors
hsa-miR-3617-3p 3179 4200 discovered in cervical tumors and
psoriasis hsa-miR-3617-5p 3180 4201 discovered in cervical tumors
and psoriasis hsa-miR-3618 3181 4202 discovered in cervical tumors
hsa-miR-3619-3p 3182 4203 discovered in breast tumors
hsa-miR-3619-5p 3183 4204 discovered in breast tumors
hsa-miR-3620-3p 3184 4205 discovered in cervical tumors
hsa-miR-3620-5p 3185 4206 discovered in cervical tumors
hsa-miR-3621 3186 4207 discovered in cervical tumors
hsa-miR-3622a-3p 3187 4208 discovered in breast tumors
hsa-miR-3622a-5p 3188 4209 discovered in breast tumors
hsa-miR-3622b-3p 3189 4210 discovered in cervical tumors
hsa-miR-3622b-5p 3190 4211 discovered in cervical tumors
hsa-miR-362-3p 3191 4212 melanoma hsa-miR-362-5p 3192 4213 melanoma
hsa-miR-363-3p 3193 4214 kidney stem cell, blood cells
hsa-miR-363-5p 3194 4215 kidney stem cell, blood cells hsa-miR-3646
3195 4216 discovered in solid tumor hsa-miR-3648 3196 4217
discovered in solid tumor hsa-miR-3649 3197 4218 discovered in
solid tumor hsa-miR-3650 3198 4219 discovered in solid tumor
hsa-miR-3651 3199 4220 discovered in solid tumor hsa-miR-3652 3200
4221 discovered in solid tumor hsa-miR-3653 3201 4222 discovered in
solid tumor hsa-miR-3654 3202 4223 discovered in solid tumor
hsa-miR-3655 3203 4224 discovered in solid tumor hsa-miR-3656 3204
4225 discovered in solid tumor hsa-miR-3657 3205 4226 discovered in
solid tumor hsa-miR-3658 3206 4227 discovered in solid tumor
hsa-miR-3659 3207 4228 discovered in breast tumors hsa-miR-365a-3p
3208 4229 various cancer apoptosis cells (Immune cells, lung,
colon, endometriotic) hsa-miR-365a-5p 3209 4230 various cancer
apoptosis cells (Immune cells, lung, colon, endometriotic))
hsa-miR-365b-3p 3210 4231 various cancer apoptosis (retinoblastoma,
colon, endometriotic) hsa-miR-365b-5p 3211 4232 various cancer
apoptosis (colon, endometriotic) hsa-miR-3660 3212 4233 discovered
in breast tumors hsa-miR-3661 3213 4234 discovered in breast tumors
hsa-miR-3662 3214 4235 hsa-miR-3663-3p 3215 4236 hsa-miR-3663-5p
3216 4237 hsa-miR-3664-3p 3217 4238 discovered in breast tumors
hsa-miR-3664-5p 3218 4239 discovered in breast tumors hsa-miR-3665
3219 4240 brain hsa-miR-3666 3220 4241 brain hsa-miR-3667-3p 3221
4242 discovered in peripheral blood hsa-miR-3667-5p 3222 4243
discovered in peripheral blood hsa-miR-3668 3223 4244 discovered in
peripheral blood hsa-miR-3669 3224 4245 discovered in peripheral
blood hsa-miR-3670 3225 4246 discovered in peripheral blood
hsa-miR-3671 3226 4247 discovered in peripheral blood hsa-miR-3672
3227 4248 discovered in peripheral blood hsa-miR-3673 3228 4249
discovered in peripheral blood hsa-miR-367-3p 3229 4250 embryonic
stem reprogramming cells hsa-miR-3674 3230 4251 discovered in
peripheral blood hsa-miR-3675-3p 3231 4252 discovered in peripheral
blood hsa-miR-3675-5p 3232 4253 discovered in peripheral blood
hsa-miR-367-5p 3233 4254 embryonic stem reprogramming cells
hsa-miR-3676-3p 3234 4255 discovered in peripheral blood
hsa-miR-3676-5p 3235 4256 discovered in peripheral blood
hsa-miR-3677-3p 3236 4257 discovered in peripheral blood
hsa-miR-3677-5p 3237 4258 discovered in peripheral blood
hsa-miR-3678-3p 3238 4259 discovered in peripheral blood
hsa-miR-3678-5p 3239 4260 discovered in peripheral blood
hsa-miR-3679-3p 3240 4261 discovered in peripheral blood
hsa-miR-3679-5p 3241 4262 discovered in peripheral blood
hsa-miR-3680-3p 3242 4263 discovered in peripheral blood
hsa-miR-3680-5p 3243 4264 discovered in peripheral blood
hsa-miR-3681-3p 3244 4265 discovered in peripheral blood
hsa-miR-3681-5p 3245 4266 discovered in peripheral blood
hsa-miR-3682-3p 3246 4267 discovered in peripheral blood
hsa-miR-3682-5p 3247 4268 discovered in peripheral blood
hsa-miR-3683 3248 4269 discovered in peripheral blood hsa-miR-3684
3249 4270 discovered in peripheral blood hsa-miR-3685 3250 4271
discovered in peripheral blood hsa-miR-3686 3251 4272 discovered in
peripheral blood hsa-miR-3687 3252 4273 discovered in peripheral
blood hsa-miR-3688-3p 3253 4274 discovered in breast tumor
hsa-miR-3688-5p 3254 4275 discovered in breast tumor
hsa-miR-3689a-3p 3255 4276 discovered in female reproductive tract
hsa-miR-3689a-5p 3256 4277 discovered in female reproductive tract
and peripheral blood hsa-miR-3689b-3p 3257 4278 discovered in
female reproductive tract and peripheral blood hsa-miR-3689b-5p
3258 4279 discovered in female reproductive tract hsa-miR-3689c
3259 4280 discovered in B cells hsa-miR-3689d 3260 4281 discovered
in B cells hsa-miR-3689e 3261 4282 discovered in B cells
hsa-miR-3689f 3262 4283 discovered in B cells hsa-miR-3690 3263
4284 discovered in peripheral blood hsa-miR-3691-3p 3264 4285
discovered in peripheral blood hsa-miR-3691-5p 3265 4286 discovered
in peripheral blood hsa-miR-3692-3p 3266 4287 discovered in
peripheral blood hsa-miR-3692-5p 3267 4288 discovered in peripheral
blood hsa-miR-369-3p 3268 4289 stem cells reprogramming
hsa-miR-369-5p 3269 4290 stem cells reprogramming hsa-miR-370 3270
4291 acute meyloid tumor leukaemia and suppressor, lipid other
cancers metabolism hsa-miR-3713 3271 4292 discovered in
neuroblastoma hsa-miR-3714 3272 4293 discovered in neuroblastoma
hsa-miR-371a-3p 3273 4294 serum hsa-miR-371a-5p 3274 4295 serum
hsa-miR-371b-3p 3275 4296 serum hsa-miR-371b-5p 3276 4297 serum
hsa-miR-372 3277 4298 hematopoietic cells, lung, placental (blood)
hsa-miR-373-3p 3278 4299 breast cancer hsa-miR-373-5p 3279 4300
breast cancer hsa-miR-374a-3p 3280 4301 muscle (myoblasts) breast
and lung myogenic cancer differentiation hsa-miR-374a-5p 3281 4302
muscle (myoblasts) breast and lung myogenic cancer differentiation
hsa-miR-374b-3p 3282 4303 muscle (myoblasts) myogenic
differentiation hsa-miR-374b-5p 3283 4304 muscle (myoblasts)
myogenic differentiation hsa-miR-374c-3p 3284 4305 muscle
(myoblasts) myogenic differentiation hsa-miR-374c-5p 3285 4306
muscle (myoblasts) myogenic differentiation hsa-miR-375 3286 4307
pancreas (islet) hsa-miR-376a-2-5p 3287 4308 regulatory miRs for
hematopoietic cells (erythroid, platelet, lympho) hsa-miR-376a-3p
3288 4309 regulatory miRs for hematopoietic cells (erythroid,
platelet, lympho) hsa-miR-376a-5p 3289 4310 regulatory miRs for
hematopoietic cells (erythroid, platelet, lympho) hsa-miR-376b-3p
3290 4311 blood various cancer autophagy cells hsa-miR-376b-5p 3291
4312 blood various cancer autophagy cells hsa-miR-376c-3p 3292 4313
trophoblast various cancer cell proliferatio cells hsa-miR-376c-5p
3293 4314 trophoblast various cancer cell proliferatio cells
hsa-miR-377-3p 3294 4315 hematopoietic cells hsa-miR-377-5p 3295
4316 hematopoietic cells hsa-miR-378a-3p 3296 4317 ovary, lipid
metabolism hsa-miR-378a-5p 3297 4318 ovary, placenta/trophoblast,
lipid metabolism hsa-miR-378b 3298 4319 lipid metabolism
hsa-miR-378c 3299 4320 lipid metabolism hsa-miR-378d 3300 4321
lipid metabolism hsa-miR-378e 3301 4322 lipid metabolism
hsa-miR-378f 3302 4323 lipid metabolism hsa-miR-378g 3303 4324
lipid metabolism hsa-miR-378h 3304 4325 lipid metabolism
hsa-miR-378i 3305 4326 lipid metabolism hsa-miR-378j 3306 4327
lipid metabolism hsa-miR-379-3p 3307 4328 various cancers (breast,
hepatocytes, colon) hsa-miR-379-5p 3308 4329 various cancers
(breast, hepatocytes, colon) hsa-miR-380-3p 3309 4330 brain
neuroblastoma hsa-miR-380-5p 3310 4331 brain, embryonic
neuroblastoma stem cells hsa-miR-381-3p 3311 4332 chondrogenesis,
lung, brain hsa-miR-381-5p 3312 4333 chondrogenesis, lung, brain
hsa-miR-382-3p 3313 4334 renal epithelial cells hsa-miR-382-5p 3314
4335 renal epithelial cells hsa-miR-383 3315 4336 testes, brain
(medulla) hsa-miR-384 3316 4337 epithelial cells hsa-miR-3907 3317
4338 discovered in female reproductive tract hsa-miR-3908 3318 4339
discovered in female reproductive tract hsa-miR-3909 3319 4340
discovered in female reproductive tract hsa-miR-3910 3320 4341
discovered in female reproductive tract hsa-miR-3911 3321 4342
discovered in breast tumor and female reproductive tract
hsa-miR-3912 3322 4343 discovered in female reproductive tract
hsa-miR-3913-3p 3323 4344 discovered in breast tumor and female
reproductive tract hsa-miR-3913-5p 3324 4345 discovered in breast
tumor and female reproductive tract hsa-miR-3914 3325 4346
discovered in breast
tumor and female reproductive tract hsa-miR-3915 3326 4347
discovered in female reproductive tract hsa-miR-3916 3327 4348
discovered in female reproductive tract hsa-miR-3917 3328 4349
discovered in female reproductive tract hsa-miR-3918 3329 4350
discovered in female reproductive tract hsa-miR-3919 3330 4351
discovered in female reproductive tract hsa-miR-3920 3331 4352
discovered in female reproductive tract hsa-miR-3921 3332 4353
discovered in female reproductive tract hsa-miR-3922-3p 3333 4354
discovered in breast tumor and female reproductive tract
hsa-miR-3922-5p 3334 4355 discovered in breast tumor and female
reproductive tract hsa-miR-3923 3335 4356 discovered in female
reproductive tract hsa-miR-3924 3336 4357 discovered in female
reproductive tract hsa-miR-3925-3p 3337 4358 discovered in breast
tumor and female reproductive tract hsa-miR-3925-5p 3338 4359
discovered in breast tumor and female reproductive tract
hsa-miR-3926 3339 4360 discovered in female reproductive tract
hsa-miR-3927-3p 3340 4361 discovered in female reproductive tract
and psoriasis hsa-miR-3927-5p 3341 4362 discovered in female
reproductive tract and psoriasis hsa-miR-3928 3342 4363 discovered
in female reproductive tract hsa-miR-3929 3343 4364 discovered in
female reproductive tract hsa-miR-3934-3p 3344 4365 discovered in
abnormal skin (psoriasis) hsa-miR-3934-5p 3345 4366 discovered in
abnormal skin (psoriasis) hsa-miR-3935 3346 4367 hsa-miR-3936 3347
4368 discovered in breast tumor and lymphoblastic leukaemia
hsa-miR-3937 3348 4369 hsa-miR-3938 3349 4370 hsa-miR-3939 3350
4371 hsa-miR-3940-3p 3351 4372 discovered in breast tumor
hsa-miR-3940-5p 3352 4373 discovered in breast tumor hsa-miR-3941
3353 4374 hsa-miR-3942-3p 3354 4375 discovered in breast tumor and
lymphoblastic leukaemia hsa-miR-3942-5p 3355 4376 discovered in
breast tumor and lymphoblastic leukaemia hsa-miR-3943 3356 4377
hsa-miR-3944-3p 3357 4378 discovered in breast tumor
hsa-miR-3944-5p 3358 4379 discovered in breast tumor hsa-miR-3945
3359 4380 hsa-miR-3960 3360 4381 osteoblast hsa-miR-3972 3361 4382
discovered in Acute Myeloid Leukaemia hsa-miR-3973 3362 4383
discovered in Acute Myeloid Leukaemia hsa-miR-3974 3363 4384
discovered in Acute Myeloid Leukaemia hsa-miR-3975 3364 4385
discovered in Acute Myeloid Leukaemia hsa-miR-3976 3365 4386
discovered in Acute Myeloid Leukaemia hsa-miR-3977 3366 4387
discovered in Acute Myeloid Leukaemia hsa-miR-3978 3367 4388
discovered in Acute Myeloid Leukaemia hsa-miR-409-3p 3368 4389
gastric cancer hsa-miR-409-5p 3369 4390 gastric cancer hsa-miR-410
3370 4391 brain glioma hsa-miR-411-3p 3371 4392 Glioblastoma others
hsa-miR-411-5p 3372 4393 Glioblastoma others hsa-miR-412 3373 4394
upregulated in lung cancer hsa-miR-421 3374 4395 endothelial cells
gastric cancer, HCC hsa-miR-422a 3375 4396 circulating microRNA (in
plasma) hsa-miR-423-3p 3376 4397 embryonic stem cells
hsa-miR-423-5p 3377 4398 heart, embryonic stem cells hsa-miR-424-3p
3378 4399 endothelial cells various pro-angiogenic cancers (e.g B-
lieage ALL), cardiac diseases hsa-miR-424-5p 3379 4400 endothelial
cells various pro-angiogenic cancers (e.g B- lieage ALL), cardiac
diseases hsa-miR-4251 3380 4401 discovered in embryonic stem cells
and neural precusors hsa-miR-4252 3381 4402 discovered in embryonic
stem cells and neural precusors hsa-miR-4253 3382 4403 discovered
in embryonic stem cells and neural precusors hsa-miR-425-3p 3383
4404 brain ovarian cancer, brain tumor hsa-miR-4254 3384 4405
discovered in embryonic stem cells and neural precusors
hsa-miR-4255 3385 4406 discovered in embryonic stem cells and
neural precusors hsa-miR-425-5p 3386 4407 brain B-lieage ALL, brain
tumor hsa-miR-4256 3387 4408 discovered in embryonic stem cells and
neural precusors hsa-miR-4257 3388 4409 discovered in embryonic
stem cells and neural precusors hsa-miR-4258 3389 4410 discovered
in embryonic stem cells and neural precusors hsa-miR-4259 3390 4411
discovered in embryonic stem cells and neural precusors
hsa-miR-4260 3391 4412 discovered in embryonic stem cells and
neural precusors hsa-miR-4261 3392 4413 discovered in embryonic
stem cells and neural precusors hsa-miR-4262 3393 4414 discovered
in embryonic stem cells and neural precusors hsa-miR-4263 3394 4415
discovered in embryonic stem cells and neural precusors
hsa-miR-4264 3395 4416 discovered in embryonic stem cells and
neural precusors hsa-miR-4265 3396 4417 discovered in embryonic
stem cells and neural precusors hsa-miR-4266 3397 4418 discovered
in embryonic stem cells and neural precusors hsa-miR-4267 3398 4419
discovered in embryonic stem cells and neural precusors
hsa-miR-4268 3399 4420 discovered in embryonic stem cells and
neural precusors hsa-miR-4269 3400 4421 discovered in embryonic
stem cells and neural precusors hsa-miR-4270 3401 4422 discovered
in embryonic stem cells and neural precusors hsa-miR-4271 3402 4423
discovered in embryonic stem cells and neural precusors
hsa-miR-4272 3403 4424 discovered in embryonic stem cells and
neural precusors hsa-miR-4273 3404 4425 hsa-miR-4274 3405 4426
discovered in embryonic stem cells and neural precusors
hsa-miR-4275 3406 4427 discovered in embryonic stem cells and
neural precusors hsa-miR-4276 3407 4428 discovered in embryonic
stem cells and neural precusors hsa-miR-4277 3408 4429 discovered
in embryonic stem cells and neural precusors hsa-miR-4278 3409 4430
discovered in embryonic stem cells and neural precusors
hsa-miR-4279 3410 4431 discovered in embryonic stem cells and
neural precusors hsa-miR-4280 3411 4432 discovered in embryonic
stem cells and neural precusors
hsa-miR-4281 3412 4433 discovered in embryonic stem cells and
neural precusors hsa-miR-4282 3413 4434 discovered in embryonic
stem cells and neural precusors hsa-miR-4283 3414 4435 discovered
in embryonic stem cells and neural precusors hsa-miR-4284 3415 4436
discovered in embryonic stem cells and neural precusors
hsa-miR-4285 3416 4437 discovered in embryonic stem cells and
neural precusors hsa-miR-4286 3417 4438 discovered in embryonic
stem cells and neural precusors hsa-miR-4287 3418 4439 discovered
in embryonic stem cells and neural precusors hsa-miR-4288 3419 4440
discovered in embryonic stem cells and neural precusors
hsa-miR-4289 3420 4441 discovered in embryonic stem cells and
neural precusors hsa-miR-429 3421 4442 Epithelial cells various
cancers (colorectal, endometrial, gastric, ovarian etc)
hsa-miR-4290 3422 4443 discovered in embryonic stem cells and
neural precusors hsa-miR-4291 3423 4444 discovered in embryonic
stem cells and neural precusors hsa-miR-4292 3424 4445 discovered
in embryonic stem cells and neural precusors hsa-miR-4293 3425 4446
discovered in embryonic stem cells and neural precusors
hsa-miR-4294 3426 4447 discovered in embryonic stem cells and
neural precusors hsa-miR-4295 3427 4448 discovered in embryonic
stem cells and neural precusors hsa-miR-4296 3428 4449 discovered
in embryonic stem cells and neural precusors hsa-miR-4297 3429 4450
discovered in embryonic stem cells and neural precusors
hsa-miR-4298 3430 4451 discovered in embryonic stem cells and
neural precusors hsa-miR-4299 3431 4452 discovered in embryonic
stem cells and neural precusors hsa-miR-4300 3432 4453 discovered
in embryonic stem cells and neural precusors hsa-miR-4301 3433 4454
discovered in embryonic stem cells and neural precusors
hsa-miR-4302 3434 4455 discovered in embryonic stem cells and
neural precusors hsa-miR-4303 3435 4456 discovered in embryonic
stem cells and neural precusors hsa-miR-4304 3436 4457 discovered
in embryonic stem cells and neural precusors hsa-miR-4305 3437 4458
discovered in embryonic stem cells and neural precusors
hsa-miR-4306 3438 4459 discovered in embryonic stem cells and
neural precusors hsa-miR-4307 3439 4460 discovered in embryonic
stem cells and neural precusors hsa-miR-4308 3440 4461 discovered
in embryonic stem cells and neural precusors hsa-miR-4309 3441 4462
discovered in embryonic stem cells and neural precusors
hsa-miR-4310 3442 4463 discovered in embryonic stem cells and
neural precusors hsa-miR-4311 3443 4464 discovered in embryonic
stem cells and neural precusors hsa-miR-4312 3444 4465 discovered
in embryonic stem cells and neural precusors hsa-miR-4313 3445 4466
discovered in embryonic stem cells and neural precusors
hsa-miR-431-3p 3446 4467 Cancers (follicular lymphoma) hsa-miR-4314
3447 4468 discovered in embryonic stem cells and neural precusors
hsa-miR-4315 3448 4469 discovered in embryonic stem cells and
neural precusors hsa-miR-431-5p 3449 4470 Cancers (follicular
lymphoma) hsa-miR-4316 3450 4471 discovered in embryonic stem cells
and neural precusors hsa-miR-4317 3451 4472 discovered in embryonic
stem cells and neural precusors hsa-miR-4318 3452 4473 discovered
in embryonic stem cells and neural precusors hsa-miR-4319 3453 4474
discovered in embryonic stem cells and neural precusors
hsa-miR-4320 3454 4475 discovered in embryonic stem cells and
neural precusors hsa-miR-4321 3455 4476 discovered in embryonic
stem cells and neural precusors hsa-miR-4322 3456 4477 discovered
in embryonic stem cells and neural precusors hsa-miR-4323 3457 4478
discovered in embryonic stem cells and neural precusors
hsa-miR-432-3p 3458 4479 myoblast myogenic differentiation
hsa-miR-4324 3459 4480 discovered in embryonic stem cells and
neural precusors hsa-miR-4325 3460 4481 discovered in embryonic
stem cells and neural precusors hsa-miR-432-5p 3461 4482 myoblast
myogenic differentiation hsa-miR-4326 3462 4483 discovered in
embryonic stem cells and neural precusors hsa-miR-4327 3463 4484
discovered in embryonic stem cells and neural precusors
hsa-miR-4328 3464 4485 discovered in embryonic stem cells and
neural precusors hsa-miR-4329 3465 4486 discovered in embryonic
stem cells and neural precusors hsa-miR-433 3466 4487 various
diseases (cancer, Parkinson's, Chondrodysplasia) hsa-miR-4330 3467
4488 discovered in embryonic stem cells and neural precusors
hsa-miR-4417 3468 4489 discovered in B cells hsa-miR-4418 3469 4490
discovered in B cells hsa-miR-4419a 3470 4491 discovered in B cells
hsa-miR-4419b 3471 4492 discovered in B cells hsa-miR-4420 3472
4493 discovered in B cells hsa-miR-4421 3473 4494 discovered in B
cells hsa-miR-4422 3474 4495 discovered in breast tumor and B cells
hsa-miR-4423-3p 3475 4496 discovered in breast tumor, B cells and
skin (psoriasis) hsa-miR-4423-5p 3476 4497 discovered in breast
tumor B cells and skin (psoriasis) hsa-miR-4424 3477 4498
discovered in B cells hsa-miR-4425 3478 4499 discovered in B cells
hsa-miR-4426 3479 4500 discovered in B cells hsa-miR-4427 3480 4501
discovered in B cells hsa-miR-4428 3481 4502 discovered in B cells
hsa-miR-4429 3482 4503 discovered in B cells
hsa-miR-4430 3483 4504 discovered in B cells hsa-miR-4431 3484 4505
discovered in B cells hsa-miR-4432 3485 4506 discovered in B cells
hsa-miR-4433-3p 3486 4507 discovered in B cells hsa-miR-4433-5p
3487 4508 discovered in B cells hsa-miR-4434 3488 4509 discovered
in B cells hsa-miR-4435 3489 4510 discovered in B cells
hsa-miR-4436a 3490 4511 discovered in breast tumor and B cells
hsa-miR-4436b-3p 3491 4512 discovered in breast tumor
hsa-miR-4436b-5p 3492 4513 discovered in breast tumor hsa-miR-4437
3493 4514 discovered in B cells hsa-miR-4438 3494 4515 discovered
in B cells hsa-miR-4439 3495 4516 discovered in B cells
hsa-miR-4440 3496 4517 discovered in B cells hsa-miR-4441 3497 4518
discovered in B cells hsa-miR-4442 3498 4519 discovered in B cells
hsa-miR-4443 3499 4520 discovered in B cells hsa-miR-4444 3500 4521
discovered in B cells hsa-miR-4445-3p 3501 4522 discovered in B
cells hsa-miR-4445-5p 3502 4523 discovered in B cells
hsa-miR-4446-3p 3503 4524 discovered in breast tumor and B cells
hsa-miR-4446-5p 3504 4525 discovered in breast tumor and B cells
hsa-miR-4447 3505 4526 discovered in B cells hsa-miR-4448 3506 4527
discovered in B cells hsa-miR-4449 3507 4528 discovered in B cells
hsa-miR-4450 3508 4529 discovered in B cells hsa-miR-4451 3509 4530
discovered in B cells hsa-miR-4452 3510 4531 discovered in B cells
hsa-miR-4453 3511 4532 discovered in B cells hsa-miR-4454 3512 4533
discovered in B cells hsa-miR-4455 3513 4534 discovered in B cells
hsa-miR-4456 3514 4535 discovered in B cells hsa-miR-4457 3515 4536
discovered in B cells hsa-miR-4458 3516 4537 discovered in B cells
hsa-miR-4459 3517 4538 discovered in B cells hsa-miR-4460 3518 4539
discovered in B cells hsa-miR-4461 3519 4540 discovered in B cells
hsa-miR-4462 3520 4541 discovered in B cells hsa-miR-4463 3521 4542
discovered in B cells hsa-miR-4464 3522 4543 discovered in B cells
hsa-miR-4465 3523 4544 discovered in B cells hsa-miR-4466 3524 4545
discovered in B cells hsa-miR-4467 3525 4546 discovered in breast
tumor and B cells hsa-miR-4468 3526 4547 discovered in B cells
hsa-miR-4469 3527 4548 discovered in breast tumor and B cells
hsa-miR-4470 3528 4549 discovered in B cells hsa-miR-4471 4550 5571
discovered in breast tumor and B cells hsa-miR-4472 4551 5572
discovered in B cells hsa-miR-4473 4552 5573 discovered in B cells
hsa-miR-4474-3p 4553 5574 discovered in breast tumor, lymphoblastic
leukaemia and B cells hsa-miR-4474-5p 4554 5575 discovered in
breast tumor, lymphoblastic leukaemia and B cells hsa-miR-4475 4555
5576 discovered in B cells hsa-miR-4476 4556 5577 discovered in B
cells hsa-miR-4477a 4557 5578 discovered in B cells hsa-miR-4477b
4558 5579 discovered in B cells hsa-miR-4478 4559 5580 discovered
in B cells hsa-miR-4479 4560 5581 discovered in B cells hsa-miR-448
4561 5582 liver (hepatocytes) HCC hsa-miR-4480 4562 5583 discovered
in B cells hsa-miR-4481 4563 5584 discovered in B cells
hsa-miR-4482-3p 4564 5585 discovered in B cells hsa-miR-4482-5p
4565 5586 discovered in B cells hsa-miR-4483 4566 5587 discovered
in B cells hsa-miR-4484 4567 5588 discovered in B cells
hsa-miR-4485 4568 5589 discovered in B cells hsa-miR-4486 4569 5590
discovered in B cells hsa-miR-4487 4570 5591 discovered in B cells
hsa-miR-4488 4571 5592 discovered in B cells hsa-miR-4489 4572 5593
discovered in breast tumor and B cells hsa-miR-4490 4573 5594
discovered in B cells hsa-miR-4491 4574 5595 discovered in B cells
hsa-miR-4492 4575 5596 discovered in B cells hsa-miR-4493 4576 5597
discovered in B cells hsa-miR-4494 4577 5598 discovered in B cells
hsa-miR-4495 4578 5599 discovered in B cells hsa-miR-4496 4579 5600
discovered in B cells hsa-miR-4497 4580 5601 discovered in B cells
hsa-miR-4498 4581 5602 discovered in B cells hsa-miR-4499 4582 5603
discovered in B cells hsa-miR-449a 4583 5604 chondrocytes, ciliated
lung, colonic, cell cycle epithelial cells ovarian cancer
progression and proliferation hsa-miR-449b-3p 4584 5605 ciliated
epithelial various cancer cell cycle cells, other tissues cells
progression and proliferation hsa-miR-449b-5p 4585 5606 ciliated
epithelial various cancer cell cycle cells, other tissues cells
progression and proliferation hsa-miR-449c-3p 4586 5607 epithelial
ovarian cancer cells hsa-miR-449c-5p 4587 5608 epithelial ovarian
cancer cells hsa-miR-4500 4588 5609 discovered in B cells
hsa-miR-4501 4589 5610 discovered in B cells hsa-miR-4502 4590 5611
discovered in B cells hsa-miR-4503 4591 5612 discovered in B cells
hsa-miR-4504 4592 5613 discovered in B cells hsa-miR-4505 4593 5614
discovered in B cells hsa-miR-4506 4594 5615 discovered in B cells
hsa-miR-4507 4595 5616 discovered in B cells hsa-miR-4508 4596 5617
discovered in B cells hsa-miR-4509 4597 5618 discovered in B cells
hsa-miR-450a-3p 4598 5619 hsa-miR-450a-5p 4599 5620 hsa-miR-450b-3p
4600 5621 hsa-miR-450b-5p 4601 5622 hsa-miR-4510 4602 5623
discovered in B cells hsa-miR-4511 4603 5624 discovered in B cells
hsa-miR-4512 4604 5625 discovered in B cells hsa-miR-4513 4605 5626
discovered in B cells hsa-miR-4514 4606 5627 discovered in B cells
hsa-miR-4515 4607 5628 discovered in B cells hsa-miR-4516 4608 5629
discovered in B cells hsa-miR-4517 4609 5630 discovered in B cells
hsa-miR-4518 4610 5631 discovered in B cells hsa-miR-4519 4611 5632
discovered in B cells hsa-miR-451a 4612 5633 heart, central nevous
system, epithelial cells hsa-miR-451b 4613 5634 heart, central
nevous system, epithelial cells hsa-miR-4520a-3p 4614 5635
discovered in breast tumor and B cells, skin (psoriasis)
hsa-miR-4520a-5p 4615 5636 discovered in breast tumor and B cells,
skin (psoriasis) hsa-miR-4520b-3p 4616 5637 discovered in breast
tumor hsa-miR-4520b-5p 4617 5638 discovered in breast tumor
hsa-miR-4521 4618 5639 discovered in B cells hsa-miR-4522 4619 5640
discovered in B cells hsa-miR-4523 4620 5641 discovered in B cells
hsa-miR-452-3p 4621 5642 myoblast bladder cancer and others
hsa-miR-4524a-3p 4622 5643 discovered in breast tumor and B cells,
skin (psoriasis) hsa-miR-4524a-5p 4623 5644 discovered in breast
tumor and B cells, skin (psoriasis) hsa-miR-4524b-3p 4624 5645
discovered in breast
tumor and B cells, skin (psoriasis) hsa-miR-4524b-5p 4625 5646
discovered in breast tumor and B cells, skin (psoriasis)
hsa-miR-4525 4626 5647 discovered in B cells hsa-miR-452-5p 4627
5648 myoblast bladder cancer and others hsa-miR-4526 4628 5649
discovered in breast tumor and B cells hsa-miR-4527 4629 5650
discovered in B cells hsa-miR-4528 4630 5651 discovered in B cells
hsa-miR-4529-3p 4631 5652 discovered in breast tumor and B cells
hsa-miR-4529-5p 4632 5653 discovered in breast tumor and B cells
hsa-miR-4530 4633 5654 discovered in B cells hsa-miR-4531 4634 5655
discovered in B cells hsa-miR-4532 4635 5656 discovered in B cells
hsa-miR-4533 4636 5657 discovered in B cells hsa-miR-4534 4637 5658
discovered in B cells hsa-miR-4535 4638 5659 discovered in B cells
hsa-miR-4536-3p 4639 5660 discovered in B cells hsa-miR-4536-5p
4640 5661 discovered in B cells hsa-miR-4537 4641 5662 discovered
in B cells hsa-miR-4538 4642 5663 discovered in B cells
hsa-miR-4539 4643 5664 discovered in B cells hsa-miR-4540 4644 5665
discovered in B cells hsa-miR-454-3p 4645 5666 embryoid body cells,
central nevous system, monocytes hsa-miR-454-5p 4646 5667 embryoid
body cells, central nevous system, monocytes hsa-miR-455-3p 4647
5668 basal cell carcinoma, other cancers hsa-miR-455-5p 4648 5669
basal cell carcinoma, other cancers hsa-miR-4632-3p 4649 5670
discovred in breast tumor hsa-miR-4632-5p 4650 5671 discovered in
breast tumor hsa-miR-4633-3p 4651 5672 discovered in breast tumor
hsa-miR-4633-5p 4652 5673 discovered in breast tumor hsa-miR-4634
4653 5674 discovered in breast tumor hsa-miR-4635 4654 5675
discovered in breast tumor hsa-miR-4636 4655 5676 discovered in
breast tumor hsa-miR-4637 4656 5677 discovered in breast tumor and
lymphoblastic leukaemia hsa-miR-4638-3p 4657 5678 discovered in
breast tumor hsa-miR-4638-5p 4658 5679 discovered in breast tumor
hsa-miR-4639-3p 4659 5680 discovered in breast tumor
hsa-miR-4639-5p 4660 5681 discovered in breast tumor
hsa-miR-4640-3p 4661 5682 discovered in breast tumor
hsa-miR-4640-5p 4662 5683 discovered in breast tumor hsa-miR-4641
4663 5684 discovered in breast tumor hsa-miR-4642 4664 5685
discovered in breast tumor hsa-miR-4643 4665 5686 discovered in
breast tumor hsa-miR-4644 4666 5687 discovered in breast tumor
hsa-miR-4645-3p 4667 5688 discovered in breast tumor
hsa-miR-4645-5p 4668 5689 discovered in breast tumor
hsa-miR-4646-3p 4669 5690 discovered in breast tumor
hsa-miR-4646-5p 4670 5691 discovered in breast tumor hsa-miR-4647
4671 5692 discovered in breast tumor hsa-miR-4648 4672 5693
discovered in breast tumor hsa-miR-4649-3p 4673 5694 discovered in
breast tumor hsa-miR-4649-5p 4674 5695 discovered in breast tumor
hsa-miR-4650-3p 4675 5696 discovered in breast tumor
hsa-miR-4650-5p 4676 5697 discovered in breast tumor hsa-miR-4651
4677 5698 discovered in breast tumor hsa-miR-4652-3p 4678 5699
discovered in breast tumor hsa-miR-4652-5p 4679 5700 discovered in
breast tumor hsa-miR-4653-3p 4680 5701 discovered in breast tumor
hsa-miR-4653-5p 4681 5702 discovered in breast tumor hsa-miR-4654
4682 5703 discovered in breast tumor hsa-miR-4655-3p 4683 5704
discovered in breast tumor hsa-miR-4655-5p 4684 5705 discovered in
breast tumor hsa-miR-4656 4685 5706 discovered in breast tumor
hsa-miR-4657 4686 5707 discovered in breast tumor hsa-miR-4658 4687
5708 discovered in breast tumor hsa-miR-4659a-3p 4688 5709
discovered in breast tumor hsa-miR-4659a-5p 4689 5710 discovered in
breast tumor hsa-miR-4659b-3p 4690 5711 discovered in breast tumor
hsa-miR-4659b-5p 4691 5712 discovered in breast tumor hsa-miR-466
4692 5713 hsa-miR-4660 4693 5714 discovered in breast tumor
hsa-miR-4661-3p 4694 5715 discovered in breast tumor
hsa-miR-4661-5p 4695 5716 discovered in breast tumor
hsa-miR-4662a-3p 4696 5717 discovered in breast tumor, psoriasis
hsa-miR-4662a-5p 4697 5718 discovered in breast tumor, psoriasis
hsa-miR-4662b 4698 5719 discovered in breast tumor hsa-miR-4663
4699 5720 discovered in breast tumor hsa-miR-4664-3p 4700 5721
discovered in breast tumor hsa-miR-4664-5p 4701 5722 discovered in
breast tumor hsa-miR-4665-3p 4702 5723 discovered in breast tumor
hsa-miR-4665-5p 4703 5724 discovered in breast tumor
hsa-miR-4666a-3p 4704 5725 discovered in breast tumor
hsa-miR-4666a-5p 4705 5726 discovered in breast tumor hsa-miR-4666b
4706 5727 hsa-miR-4667-3p 4707 5728 discovered in breast tumor
hsa-miR-4667-5p 4708 5729 discovered in breast tumor
hsa-miR-4668-3p 4709 5730 discovered in breast tumor
hsa-miR-4668-5p 4710 5731 discovered in breast tumor hsa-miR-4669
4711 5732 discovered in breast tumor hsa-miR-4670-3p 4712 5733
discovered in breast tumor hsa-miR-4670-5p 4713 5734 discovered in
breast tumor hsa-miR-4671-3p 4714 5735 discovered in breast tumor
hsa-miR-4671-5p 4715 5736 discovered in breast tumor hsa-miR-4672
4716 5737 discovered in breast tumor hsa-miR-4673 4717 5738
discovered in breast tumor hsa-miR-4674 4718 5739 discovered in
breast tumor hsa-miR-4675 4719 5740 discovered in breast tumor
hsa-miR-4676-3p 4720 5741 discovered in breast tumor
hsa-miR-4676-5p 4721 5742 discovered in breast tumor
hsa-miR-4677-3p 4722 5743 discovered in breast tumor, psoriasis
hsa-miR-4677-5p 4723 5744 discovered in breast tumor, psoriasis
hsa-miR-4678 4724 5745 discovered in breast tumor hsa-miR-4679 4725
5746 discovered in breast tumor hsa-miR-4680-3p 4726 5747
discovered in breast tumor hsa-miR-4680-5p 4727 5748 discovered in
breast tumor hsa-miR-4681 4728 5749 discovered in breast tumor
hsa-miR-4682 4729 5750 discovered in breast tumor hsa-miR-4683 4730
5751 discovered in breast tumor hsa-miR-4684-3p 4731 5752
discovered in breast tumor hsa-miR-4684-5p 4732 5753 discovered in
breast tumor hsa-miR-4685-3p 4733 5754 discovered in breast tumor
hsa-miR-4685-5p 4734 5755 discovered in breast tumor hsa-miR-4686
4735 5756 discovered in breast tumor hsa-miR-4687-3p 4736 5757
discovered in breast tumor hsa-miR-4687-5p 4737 5758 discovered in
breast tumor hsa-miR-4688 4738 5759 discovered in breast tumor
hsa-miR-4689 4739 5760 discovered in breast tumor hsa-miR-4690-3p
4740 5761 discovered in breast tumor hsa-miR-4690-5p 4741 5762
discovered in breast tumor hsa-miR-4691-3p 4742 5763 discovered in
breast tumor hsa-miR-4691-5p 4743 5764 discovered in breast tumor
hsa-miR-4692 4744 5765 discovered in breast tumor hsa-miR-4693-3p
4745 5766 discovered in breast tumor
hsa-miR-4693-5p 4746 5767 discovered in breast tumor
hsa-miR-4694-3p 4747 5768 discovered in breast tumor
hsa-miR-4694-5p 4748 5769 discovered in breast tumor
hsa-miR-4695-3p 4749 5770 discovered in breast tumor
hsa-miR-4695-5p 4750 5771 discovered in breast tumor hsa-miR-4696
4751 5772 discovered in breast tumor hsa-miR-4697-3p 4752 5773
discovered in breast tumor hsa-miR-4697-5p 4753 5774 discovered in
breast tumor hsa-miR-4698 4754 5775 discovered in breast tumor
hsa-miR-4699-3p 4755 5776 discovered in breast tumor
hsa-miR-4699-5p 4756 5777 discovered in breast tumor
hsa-miR-4700-3p 4757 5778 discovered in breast tumor
hsa-miR-4700-5p 4758 5779 discovered in breast tumor
hsa-miR-4701-3p 4759 5780 discovered in breast tumor
hsa-miR-4701-5p 4760 5781 discovered in breast tumor
hsa-miR-4703-3p 4761 5782 discovered in breast tumor
hsa-miR-4703-5p 4762 5783 discovered in breast tumor
hsa-miR-4704-3p 4763 5784 discovered in breast tumor
hsa-miR-4704-5p 4764 5785 discovered in breast tumor hsa-miR-4705
4765 5786 discovered in breast tumor hsa-miR-4706 4766 5787
discovered in breast tumor hsa-miR-4707-3p 4767 5788 discovered in
breast tumor hsa-miR-4707-5p 4768 5789 discovered in breast tumor
hsa-miR-4708-3p 4769 5790 discovered in breast tumor
hsa-miR-4708-5p 4770 5791 discovered in breast tumor
hsa-miR-4709-3p 4771 5792 discovered in breast tumor
hsa-miR-4709-5p 4772 5793 discovered in breast tumor hsa-miR-4710
4773 5794 discovered in breast tumor hsa-miR-4711-3p 4774 5795
discovered in breast tumor hsa-miR-4711-5p 4775 5796 discovered in
breast tumor hsa-miR-4712-3p 4776 5797 discovered in breast tumor
hsa-miR-4712-5p 4777 5798 discovered in breast tumor
hsa-miR-4713-3p 4778 5799 discovered in breast tumor
hsa-miR-4713-5p 4779 5800 discovered in breast tumor
hsa-miR-4714-3p 4780 5801 discovered in breast tumor
hsa-miR-4714-5p 4781 5802 discovered in breast tumor
hsa-miR-4715-3p 4782 5803 discovered in breast tumor
hsa-miR-4715-5p 4783 5804 discovered in breast tumor
hsa-miR-4716-3p 4784 5805 discovered in breast tumor
hsa-miR-4716-5p 4785 5806 discovered in breast tumor
hsa-miR-4717-3p 4786 5807 discovered in breast tumor
hsa-miR-4717-5p 4787 5808 discovered in breast tumor hsa-miR-4718
4788 5809 discovered in breast tumor hsa-miR-4719 4789 5810
discovered in breast tumor hsa-miR-4720-3p 4790 5811 discovered in
breast tumor hsa-miR-4720-5p 4791 5812 discovered in breast tumor
hsa-miR-4721 4792 5813 discovered in breast tumor hsa-miR-4722-3p
4793 5814 discovered in breast tumor hsa-miR-4722-5p 4794 5815
discovered in breast tumor hsa-miR-4723-3p 4795 5816 discovered in
breast tumor hsa-miR-4723-5p 4796 5817 discovered in breast tumor
hsa-miR-4724-3p 4797 5818 discovered in breast tumor
hsa-miR-4724-5p 4798 5819 discovered in breast tumor
hsa-miR-4725-3p 4799 5820 discovered in breast tumor
hsa-miR-4725-5p 4800 5821 discovered in breast tumor
hsa-miR-4726-3p 4801 5822 discovered in breast tumor
hsa-miR-4726-5p 4802 5823 discovered in breast tumor
hsa-miR-4727-3p 4803 5824 discovered in breast tumor
hsa-miR-4727-5p 4804 5825 discovered in breast tumor
hsa-miR-4728-3p 4805 5826 discovered in breast tumor
hsa-miR-4728-5p 4806 5827 discovered in breast tumor hsa-miR-4729
4807 5828 discovered in breast tumor hsa-miR-4730 4808 5829
discovered in breast tumor hsa-miR-4731-3p 4809 5830 discovered in
breast tumor hsa-miR-4731-5p 4810 5831 discovered in breast tumor
hsa-miR-4732-3p 4811 5832 discovered in breast tumor
hsa-miR-4732-5p 4812 5833 discovered in breast tumor
hsa-miR-4733-3p 4813 5834 discovered in breast tumor
hsa-miR-4733-5p 4814 5835 discovered in breast tumor hsa-miR-4734
4815 5836 discovered in breast tumor hsa-miR-4735-3p 4816 5837
discovered in breast tumor hsa-miR-4735-5p 4817 5838 discovered in
breast tumor hsa-miR-4736 4818 5839 discovered in breast tumor
hsa-miR-4737 4819 5840 discovered in breast tumor hsa-miR-4738-3p
4820 5841 discovered in breast tumor hsa-miR-4738-5p 4821 5842
discovered in breast tumor hsa-miR-4739 4822 5843 discovered in
breast tumor hsa-miR-4740-3p 4823 5844 discovered in breast tumor
hsa-miR-4740-5p 4824 5845 discovered in breast tumor hsa-miR-4741
4825 5846 discovered in breast tumor, psoriasis hsa-miR-4742-3p
4826 5847 discovered in breast tumor, psoriasis hsa-miR-4742-5p
4827 5848 discovered in breast tumor hsa-miR-4743-3p 4828 5849
discovered in breast tumor hsa-miR-4743-5p 4829 5850 discovered in
breast tumor hsa-miR-4744 4830 5851 discovered in breast tumor
hsa-miR-4745-3p 4831 5852 discovered in breast tumor
hsa-miR-4745-5p 4832 5853 discovered in breast tumor
hsa-miR-4746-3p 4833 5854 discovered in breast tumor
hsa-miR-4746-5p 4834 5855 discovered in breast tumor
hsa-miR-4747-3p 4835 5856 discovered in breast tumor
hsa-miR-4747-5p 4836 5857 discovered in breast tumor hsa-miR-4748
4837 5858 discovered in breast tumor hsa-miR-4749-3p 4838 5859
discovered in breast tumor hsa-miR-4749-5p 4839 5860 discovered in
breast tumor hsa-miR-4750-3p 4840 5861 discovered in breast tumor
hsa-miR-4750-5p 4841 5862 discovered in breast tumor hsa-miR-4751
4842 5863 discovered in breast tumor hsa-miR-4752 4843 5864
discovered in breast tumor hsa-miR-4753-3p 4844 5865 discovered in
breast tumor hsa-miR-4753-5p 4845 5866 discovered in breast tumor
hsa-miR-4754 4846 5867 discovered in breast tumor hsa-miR-4755-3p
4847 5868 discovered in breast tumor hsa-miR-4755-5p 4848 5869
discovered in breast tumor hsa-miR-4756-3p 4849 5870 discovered in
breast tumor hsa-miR-4756-5p 4850 5871 discovered in breast tumor
hsa-miR-4757-3p 4851 5872 discovered in breast tumor
hsa-miR-4757-5p 4852 5873 discovered in breast tumor
hsa-miR-4758-3p 4853 5874 discovered in breast tumor
hsa-miR-4758-5p 4854 5875 discovered in breast tumor hsa-miR-4759
4855 5876 discovered in breast tumor hsa-miR-4760-3p 4856 5877
discovered in breast tumor hsa-miR-4760-5p 4857 5878 discovered in
breast tumor hsa-miR-4761-3p 4858 5879 discovered in breast tumor
hsa-miR-4761-5p 4859 5880 discovered in breast tumor
hsa-miR-4762-3p 4860 5881 discovered in breast tumor
hsa-miR-4762-5p 4861 5882 discovered in breast tumor
hsa-miR-4763-3p 4862 5883 discovered in breast tumor
hsa-miR-4763-5p 4863 5884 discovered in breast tumor
hsa-miR-4764-3p 4864 5885 discovered in breast tumor
hsa-miR-4764-5p 4865 5886 discovered in breast tumor hsa-miR-4765
4866 5887 discovered in breast tumor hsa-miR-4766-3p 4867 5888
discovered in breast tumor hsa-miR-4766-5p 4868 5889 discovered in
breast tumor hsa-miR-4767 4869 5890 discovered in breast tumor
hsa-miR-4768-3p 4870 5891 discovered in breast tumor
hsa-miR-4768-5p 4871 5892 discovered in breast
tumor hsa-miR-4769-3p 4872 5893 discovered in breast tumor
hsa-miR-4769-5p 4873 5894 discovered in breast tumor hsa-miR-4770
4874 5895 discovered in breast tumor hsa-miR-4771 4875 5896
discovered in breast tumor hsa-miR-4772-3p 4876 5897 discovered in
breast energy tumor, blood metabolism/ monoclear cells obesity
hsa-miR-4772-5p 4877 5898 discovered in breast energy tumor, blood
metabolism/ monoclear cells obesity hsa-miR-4773 4878 5899
discovered in breast tumor hsa-miR-4774-3p 4879 5900 discovered in
breast tumor and Lymphoblastic leukemia hsa-miR-4774-5p 4880 5901
discovered in breast tumor and Lymphoblastic leukemia hsa-miR-4775
4881 5902 discovered in breast tumor hsa-miR-4776-3p 4882 5903
discovered in breast tumor hsa-miR-4776-5p 4883 5904 discovered in
breast tumor hsa-miR-4777-3p 4884 5905 discovered in breast tumor
hsa-miR-4777-5p 4885 5906 discovered in breast tumor
hsa-miR-4778-3p 4886 5907 discovered in breast tumor
hsa-miR-4778-5p 4887 5908 discovered in breast tumor hsa-miR-4779
4888 5909 discovered in breast tumor hsa-miR-4780 4889 5910
discovered in breast tumor hsa-miR-4781-3p 4890 5911 discovered in
breast tumor hsa-miR-4781-5p 4891 5912 discovered in breast tumor
hsa-miR-4782-3p 4892 5913 discovered in breast tumor
hsa-miR-4782-5p 4893 5914 discovered in breast tumor
hsa-miR-4783-3p 4894 5915 discovered in breast tumor
hsa-miR-4783-5p 4895 5916 discovered in breast tumor hsa-miR-4784
4896 5917 discovered in breast tumor hsa-miR-4785 4897 5918
discovered in breast tumor hsa-miR-4786-3p 4898 5919 discovered in
breast tumor hsa-miR-4786-5p 4899 5920 discovered in breast tumor
hsa-miR-4787-3p 4900 5921 discovered in breast tumor
hsa-miR-4787-5p 4901 5922 discovered in breast tumor hsa-miR-4788
4902 5923 discovered in breast tumor hsa-miR-4789-3p 4903 5924
discovered in breast tumor hsa-miR-4789-5p 4904 5925 discovered in
breast tumor hsa-miR-4790-3p 4905 5926 discovered in breast tumor
hsa-miR-4790-5p 4906 5927 discovered in breast tumor hsa-miR-4791
4907 5928 discovered in breast tumor hsa-miR-4792 4908 5929
discovered in breast tumor hsa-miR-4793-3p 4909 5930 discovered in
breast tumor hsa-miR-4793-5p 4910 5931 discovered in breast tumor
hsa-miR-4794 4911 5932 discovered in breast tumor hsa-miR-4795-3p
4912 5933 discovered in breast tumor hsa-miR-4795-5p 4913 5934
discovered in breast tumor hsa-miR-4796-3p 4914 5935 discovered in
breast tumor hsa-miR-4796-5p 4915 5936 discovered in breast tumor
hsa-miR-4797-3p 4916 5937 discovered in breast tumor
hsa-miR-4797-5p 4917 5938 discovered in breast tumor
hsa-miR-4798-3p 4918 5939 discovered in breast tumor
hsa-miR-4798-5p 4919 5940 discovered in breast tumor
hsa-miR-4799-3p 4920 5941 discovered in breast tumor
hsa-miR-4799-5p 4921 5942 discovered in breast tumor
hsa-miR-4800-3p 4922 5943 discovered in breast tumor
hsa-miR-4800-5p 4923 5944 discovered in breast tumor hsa-miR-4801
4924 5945 discovered in breast tumor hsa-miR-4802-3p 4925 5946
discovered in breast tumor, psoriasis hsa-miR-4802-5p 4926 5947
discovered in breast tumor, psoriasis hsa-miR-4803 4927 5948
discovered in breast tumor hsa-miR-4804-3p 4928 5949 discovered in
breast tumor hsa-miR-4804-5p 4929 5950 discovered in breast tumor
hsa-miR-483-3p 4930 5951 aderonocortical oncogenic carcinoma,
rectal/pancreatic cancer, proliferation of wounded epithelial cells
hsa-miR-483-5p 4931 5952 cartilage aderonocortical angiogenesis
(chondrocyte), fetal carcinoma brain hsa-miR-484 4932 5953
mitochondrial network hsa-miR-485-3p 4933 5954 hsa-miR-485-5p 4934
5955 ovarian epithelial tumor hsa-miR-486-3p 4935 5956 erythroid
cells various cancers hsa-miR-486-5p 4936 5957 stem cells (adipose)
various cancers hsa-miR-487a 4937 5958 laryngeal carcinoma
hsa-miR-487b 4938 5959 neuroblastoma, pulmonary carcinogenesis
hsa-miR-488-3p 4939 5960 prostate cancer, others hsa-miR-488-5p
4940 5961 prostate cancer, others hsa-miR-489 4941 5962 mesenchymal
stem osteogenesis cells hsa-miR-490-3p 4942 5963 neuroblastoma,
terine leiomyoma (ULM)/muscle hsa-miR-490-5p 4943 5964
neuroblastoma, terine leiomyoma (ULM)/muscle hsa-miR-491-3p 4944
5965 various cancers, pro-apoptosis brain disease hsa-miR-491-5p
4945 5966 various cancers, pro-apoptosis brain disease hsa-miR-492
4946 5967 hsa-miR-493-3p 4947 5968 myeloid cells, pancreas (islet)
hsa-miR-493-5p 4948 5969 myeloid cells, pancreas (islet)
hsa-miR-494 4949 5970 epithelial cells various cancers cell cycle
hsa-miR-495-3p 4950 5971 platelet various cancers (gastric, MLL
leukemia, pancreatic etc) and inflammation hsa-miR-495-5p 4951 5972
platelet various cancers (gastric, MLL leukemia, pancreatic etc)
and inflammation hsa-miR-496 4952 5973 Blood hsa-miR-497-3p 4953
5974 various cancers tumor (breast, supressor/pro- colorectal, etc)
apoptosis hsa-miR-497-5p 4954 5975 various cancers tumor (breast,
supressor/pro- colorectal, etc) apoptosis hsa-miR-498 4955 5976
autoimmuno (e.g. rheumatoid arthritis) hsa-miR-4999-3p 4956 5977
hsa-miR-4999-5p 4957 5978 hsa-miR-499a-3p 4958 5979 heart, cardiac
stem cardiovascular cardiomyocyte cells disease differentiation
hsa-miR-499a-5p 4959 5980 heart, cardiac stem cardiovascular
cardiomyocyte cells disease differentiation hsa-miR-499b-3p 4960
5981 heart, cardiac stem cardiovascular cardiomyocyte cells disease
differentiation hsa-miR-499b-5p 4961 5982 heart, cardiac stem
cardiovascular cardiomyocyte cells disease differentiation
hsa-miR-5000-3p 4962 5983 discovered in lymphoblastic leukaemia
hsa-miR-5000-5p 4963 5984 discovered in lymphoblastic leukaemia
hsa-miR-5001-3p 4964 5985 hsa-miR-5001-5p 4965 5986 hsa-miR-5002-3p
4966 5987 hsa-miR-5002-5p 4967 5988 hsa-miR-5003-3p 4968 5989
hsa-miR-5003-5p 4969 5990 hsa-miR-5004-3p 4970 5991 hsa-miR-5004-5p
4971 5992 hsa-miR-5006-3p 4972 5993 discovered in lymphoblastic
leukaemia hsa-miR-5006-5p 4973 5994 discovered in lymphoblastic
leukaemia hsa-miR-5007-3p 4974 5995 hsa-miR-5007-5p 4975 5996
hsa-miR-5008-3p 4976 5997 hsa-miR-5008-5p 4977 5998 hsa-miR-5009-3p
4978 5999 hsa-miR-5009-5p 4979 6000 hsa-miR-500a-3p 4980 6001
hsa-miR-500a-5p 4981 6002 hsa-miR-500b 4982 6003 Blood (plasma)
hsa-miR-5010-3p 4983 6004 abnormal skin (psoriasis) hsa-miR-5010-5p
4984 6005 abnormal skin (psoriasis) hsa-miR-5011-3p 4985 6006
hsa-miR-5011-5p 4986 6007 hsa-miR-501-3p 4987 6008 hsa-miR-501-5p
4988 6009 hsa-miR-502-3p 4989 6010 various cancers (hepatocellular,
ovarian, breast) hsa-miR-502-5p 4990 6011 various cancers
(hepatocellular, ovarian, breast) hsa-miR-503-3p 4991 6012 ovary
hsa-miR-503-5p 4992 6013 ovary hsa-miR-504 4993 6014 glioblastoma
hsa-miR-5047 4994 6015 hsa-miR-505-3p 4995 6016 breast cancer
hsa-miR-505-5p 4996 6017 breast cancer hsa-miR-506-3p 4997 6018
various cancers hsa-miR-506-5p 4998 6019 various cancers
hsa-miR-507 4999 6020 hsa-miR-508-3p 5000 6021 renal cell carcinoma
hsa-miR-508-5p 5001 6022 endothelial progenitor cells (EPCs)
hsa-miR-5087 5002 6023 hsa-miR-5088 5003 6024 hsa-miR-5089-3p 5004
6025 hsa-miR-5089-5p 5005 6026 hsa-miR-5090 5006 6027 hsa-miR-5091
5007 6028 hsa-miR-5092 5008 6029 hsa-miR-5093 5009 6030
hsa-miR-509-3-5p 5010 6031 testis hsa-miR-509-3p 5011 6032 renal
cell carcinoma, brain disease hsa-miR-5094 5012 6033 hsa-miR-5095
5013 6034 cervical cancer hsa-miR-509-5p 5014 6035 metabolic
syndrome, brain disease hsa-miR-5096 5015 6036 cervical cance
hsa-miR-510 5016 6037 brain hsa-miR-5100 5017 6038 discoverd in
Salivary gland hsa-miR-511 5018 6039 dendritic cells and
macrophages hsa-miR-512-3p 5019 6040 embryonic stem cells, placenta
hsa-miR-512-5p 5020 6041 embryonic stem cells, placenta,
hsa-miR-513a-3p 5021 6042 lung carcinoma hsa-miR-513a-5p 5022 6043
endothelial cells hsa-miR-513b 5023 6044 follicular lymphoma
hsa-miR-513c-3p 5024 6045 hsa-miR-513c-5p 5025 6046 hsa-miR-514a-3p
5026 6047 hsa-miR-514a-5p 5027 6048 hsa-miR-514b-3p 5028 6049
various cancer cells hsa-miR-514b-5p 5029 6050 various cancer cells
hsa-miR-515-3p 5030 6051 hsa-miR-515-5p 5031 6052 placenta
hsa-miR-516a-3p 5032 6053 frontal cortex hsa-miR-516a-5p 5033 6054
placenta hsa-miR-516b-3p 5034 6055 hsa-miR-516b-5p 5035 6056
hsa-miR-517-5p 5036 6057 placenta hsa-miR-517a-3p 5037 6058
placenta hsa-miR-517b-3p 5038 6059 placenta hsa-miR-517c-3p 5039
6060 placenta hsa-miR-5186 5040 6061 discovered in lymphoblastic
leukaemia hsa-miR-5187-3p 5041 6062 discovered in lymphoblastic
leukaemia, skin (psoriasis) hsa-miR-5187-5p 5042 6063 discovered in
lymphoblastic leukaemia, skin (psoriasis) hsa-miR-5188 5043 6064
discovered in lymphoblastic leukaemia hsa-miR-5189 5044 6065
discovered in lymphoblastic leukaemia hsa-miR-518a-3p 5045 6066 HCC
hsa-miR-518a-5p 5046 6067 various cancer cells hsa-miR-518b 5047
6068 placenta HCC cell cycle progression hsa-miR-518c-3p 5048 6069
placenta hsa-miR-518c-5p 5049 6070 placenta hsa-miR-518d-3p 5050
6071 hsa-miR-518d-5p 5051 6072 hsa-miR-518e-3p 5052 6073 HCC cell
cycle progression hsa-miR-518e-5p 5053 6074 HCC cell cycle
progression hsa-miR-518f-3p 5054 6075 placenta hsa-miR-518f-5p 5055
6076 placenta hsa-miR-5190 5056 6077 discovered in lymphoblastic
leukaemia hsa-miR-5191 5057 6078 discovered in lymphoblastic
leukaemia hsa-miR-5192 5058 6079 discovered in lymphoblastic
leukaemia hsa-miR-5193 5059 6080 discovered in lymphoblastic
leukaemia hsa-miR-5194 5060 6081 discovered in lymphoblastic
leukaemia hsa-miR-5195-3p 5061 6082 discovered in lymphoblastic
leukaemia hsa-miR-5195-5p 5062 6083 discovered in lymphoblastic
leukaemia hsa-miR-5196-3p 5063 6084 discovered in lymphoblastic
leukaemia hsa-miR-5196-5p 5064 6085 discovered in lymphoblastic
leukaemia hsa-miR-5197-3p 5065 6086 discovered in lymphoblastic
leukaemia hsa-miR-5197-5p 5066 6087 discovered in lymphoblastic
leukaemia hsa-miR-519a-3p 5067 6088 placenta HCC hsa-miR-519a-5p
5068 6089 placenta HCC hsa-miR-519b-3p 5069 6090 breast cancer
hsa-miR-519b-5p 5070 6091 breast cancer hsa-miR-519c-3p 5071 6092
hsa-miR-519c-5p 5072 6093 hsa-miR-519d 5073 6094 placenta
hsa-miR-519e-3p 5074 6095 placenta hsa-miR-519e-5p 5075 6096
placenta hsa-miR-520a-3p 5076 6097 placenta hsa-miR-520a-5p 5077
6098 placenta hsa-miR-520b 5078 6099 breast cancer hsa-miR-520c-3p
5079 6100 gastric cancer, breast tumor hsa-miR-520c-5p 5080 6101
breast tumor hsa-miR-520d-3p 5081 6102 various cancer cells
hsa-miR-520d-5p 5082 6103 various cancer cells hsa-miR-520e 5083
6104 hepatoma tomor suppressor hsa-miR-520f 5084 6105 breast cancer
hsa-miR-520g 5085 6106 HCC, bladder cancer, breast cancer
hsa-miR-520h 5086 6107 placental specific hsa-miR-521 5087 6108
prostate cancer hsa-miR-522-3p 5088 6109 HCC hsa-miR-522-5p 5089
6110 HCC hsa-miR-523-3p 5090 6111 hsa-miR-523-5p 5091 6112
hsa-miR-524-3p 5092 6113 colon cancer stem cells hsa-miR-524-5p
5093 6114 placental specific gliomas hsa-miR-525-3p 5094 6115
placental specific HCC hsa-miR-525-5p 5095 6116 placental specific
hsa-miR-526a 5096 6117 placental specific hsa-miR-526b-3p 5097 6118
placental specific hsa-miR-526b-5p 5098 6119 placental specific
hsa-miR-527 5099 6120 hsa-miR-532-3p 5100 6121 ALL hsa-miR-532-5p
5101 6122 ALL hsa-miR-539-3p 5102 6123 hsa-miR-539-5p 5103 6124
hsa-miR-541-3p 5104 6125 hsa-miR-541-5p 5105 6126 hsa-miR-542-3p
5106 6127 monocytes hsa-miR-542-5p 5107 6128 basal cell carcinoma,
neuroblastoma hsa-miR-543 5108 6129 hsa-miR-544a 5109 6130
osteocarcoma hsa-miR-544b 5110 6131 osteocarcoma hsa-miR-545-3p
5111 6132 hsa-miR-545-5p 5112 6133 rectal cancer hsa-miR-548 5113
6134 hsa-miR-548-3p 5114 6135 hsa-miR-548-5p 5115 6136 hsa-miR-548a
5116 6137 identified in colorectal microRNAome hsa-miR-548a-3p 5117
6138 identified in colorectal microRNAome hsa-miR-548a-5p 5118 6139
identified in colorectal microRNAome hsa-miR-548aa 5119 6140
identified in cervical tumor hsa-miR-548ab 5120 6141 discovered in
B- cells hsa-miR-548ac 5121 6142 discovered in B- cells
hsa-miR-548ad 5122 6143 discovered in B- cells hsa-miR-548ae 5123
6144 discovered in B- cells hsa-miR-548ag 5124 6145 discovered in
B- cells hsa-miR-548ah-3p 5125 6146 discovered in B- cells
hsa-miR-548ah-5p 5126 6147 discovered in B- cells hsa-miR-548ai
5127 6148 discovered in B- cells hsa-miR-548aj-3p 5128 6149
discovered in B- cells hsa-miR-548aj-5p 5129 6150 discovered in B-
cells hsa-miR-548ak 5130 6151 discovered in B- cells hsa-miR-548al
5131 6152 discovered in B- cells hsa-miR-548am-3p 5132 6153
discovered in B- cells hsa-miR-548am-5p 5133 6154 discovered in B-
cells hsa-miR-548an 5134 6155 discovered in B- cells
hsa-miR-548ao-3p 5135 6156 hsa-miR-548ao-5p 5136 6157
hsa-miR-548ap-3p 5137 6158 hsa-miR-548ap-5p 5138 6159
hsa-miR-548aq-3p 5139 6160 hsa-miR-548aq-5p 5140 6161
hsa-miR-548ar-3p 5141 6162 hsa-miR-548ar-5p 5142 6163
hsa-miR-548as-3p 5143 6164 hsa-miR-548as-5p 5144 6165
hsa-miR-548at-3p 5145 6166 prostate cancer hsa-miR-548at-5p 5146
6167 prostate cancer hsa-miR-548au-3p 5147 6168 hsa-miR-548au-5p
5148 6169 hsa-miR-548av-3p 5149 6170 hsa-miR-548av-5p 5150 6171
hsa-miR-548aw 5151 6172 prostate cancer hsa-miR-548ay-3p 5152 6173
discovered in abnormal skin (psoriasis) hsa-miR-548ay-5p 5153 6174
discovered in abnormal skin (psoriasis) hsa-miR-548az-3p 5154 6175
discovered in abnormal skin (psoriasis) hsa-miR-548az-5p 5155 6176
discovered in abnormal skin (psoriasis) hsa-miR-548b-3p 5156 6177
identified in colorectal microRNAome
hsa-miR-548b-5p 5157 6178 immune cells, frontal cortex
hsa-miR-548c-3p 5158 6179 identified in colorectal microRNAome
hsa-miR-548c-5p 5159 6180 immune cells, frontal cortex
hsa-miR-548d-3p 5160 6181 identified in colorectal microRNAome
hsa-miR-548d-5p 5161 6182 identified in colorectal microRNAome
hsa-miR-548e 5162 6183 embryonic stem cells hsa-miR-548f 5163 6184
embryonic stem cells hsa-miR-548g-3p 5164 6185 embryonic stem cells
hsa-miR-548g-5p 5165 6186 embryonic stem cells hsa-miR-548h-3p 5166
6187 embryonic stem cells hsa-miR-548h-5p 5167 6188 embryonic stem
cells hsa-miR-548i 5168 6189 embryonic stem cells, immune cells
hsa-miR-548j 5169 6190 immune cells hsa-miR-548k 5170 6191
embryonic stem cells hsa-miR-5481 5171 6192 embryonic stem cells
hsa-miR-548m 5172 6193 embryonic stem cells hsa-miR-548n 5173 6194
embryonic stem cells, immune cells hsa-miR-548o-3p 5174 6195
embryonic stem cells hsa-miR-548o-5p 5175 6196 embryonic stem cells
hsa-miR-548p 5176 6197 embryonic stem cells hsa-miR-548q 5177 6198
ovarian cancer cells hsa-miR-548s 5178 6199 discovered in the
melanoma MicroRNAome hsa-miR-548t-3p 5179 6200 discovered in the
melanoma MicroRNAome hsa-miR-548t-5p 5180 6201 discovered in the
melanoma MicroRNAome hsa-miR-548u 5181 6202 discovered in the
melanoma MicroRNAome hsa-miR-548w 5182 6203 discovered in the
melanoma MicroRNAome hsa-miR-548y 5183 6204 hsa-miR-548z 5184 6205
discovered in cervical tumor hsa-miR-549a 5185 6206 discovered in a
colorectal MicroRNAome hsa-miR-550a-3-5p 5186 6207 Hepatocellular
Carcinoma hsa-miR-550a-3p 5187 6208 Hepatocellular Carcinoma
hsa-miR-550a-5p 5188 6209 Hepatocellular Carcinoma
hsa-miR-550b-2-5p 5189 6210 discovered in cervical tumor
hsa-miR-550b-3p 5190 6211 discovered in cervical tumor hsa-miR-551a
5191 6212 gastric cancer hsa-miR-551b-3p 5192 6213 hepatocytes
hsa-miR-551b-5p 5193 6214 hepatocytes hsa-miR-552 5194 6215
discovered in a colorectal MicroRNAome hsa-miR-553 5195 6216
discovered in a colorectal MicroRNAome hsa-miR-554 5196 6217
discovered in a colorectal MicroRNAome hsa-miR-555 5197 6218
discovered in a colorectal MicroRNAome hsa-miR-556-3p 5198 6219
discovered in a colorectal MicroRNAome hsa-miR-556-5p 5199 6220
discovered in a colorectal MicroRNAome hsa-miR-557 5200 6221 liver
(hepatocytes) hsa-miR-5571-3p 5201 6222 discoveredd in Salivary
gland hsa-miR-5571-5p 5202 6223 discoveredd in Salivary gland
hsa-miR-5572 5203 6224 discoveredd in Salivary gland
hsa-miR-5579-3p 5204 6225 hsa-miR-5579-5p 5205 6226 hsa-miR-558
5206 6227 neuroblastoma hsa-miR-5580-3p 5207 6228 hsa-miR-5580-5p
5208 6229 hsa-miR-5581-3p 5209 6230 hsa-miR-5581-5p 5210 6231
hsa-miR-5582-3p 5211 6232 hsa-miR-5582-5p 5212 6233 hsa-miR-5583-3p
5213 6234 hsa-miR-5583-5p 5214 6235 hsa-miR-5584-3p 5215 6236
hsa-miR-5584-5p 5216 6237 hsa-miR-5585-3p 5217 6238 hsa-miR-5585-5p
5218 6239 hsa-miR-5586-3p 5219 6240 hsa-miR-5586-5p 5220 6241
hsa-miR-5587-3p 5221 6242 hsa-miR-5587-5p 5222 6243 hsa-miR-5588-3p
5223 6244 hsa-miR-5588-5p 5224 6245 hsa-miR-5589-3p 5225 6246
hsa-miR-5589-5p 5226 6247 hsa-miR-559 5227 6248 hsa-miR-5590-3p
5228 6249 hsa-miR-5590-5p 5229 6250 hsa-miR-5591-3p 5230 6251
hsa-miR-5591-5p 5231 6252 hsa-miR-561-3p 5232 6253 multiple myeloma
hsa-miR-561-5p 5233 6254 multiple myeloma hsa-miR-562 5234 6255
hsa-miR-563 5235 6256 discovered in a colorectal MicroRNAome
hsa-miR-564 5236 6257 Chronic myeloid leukemia hsa-miR-566 5237
6258 MALT lymphoma/lymphocyte hsa-miR-567 5238 6259 colorectal
cancer hsa-miR-568 5239 6260 discovered in a colorectal MicroRNAome
hsa-miR-5680 5240 6261 Associated with metastatic prostate cancer
hsa-miR-5681a 5241 6262 Associated with metastatic prostate cancer
hsa-miR-5681b 5242 6263 Associated with metastatic prostate cancer
hsa-miR-5682 5243 6264 Associated with metastatic prostate cancer
hsa-miR-5683 5244 6265 Associated with metastatic prostate cancer
hsa-miR-5684 5245 6266 Associated with metastatic prostate cancer
hsa-miR-5685 5246 6267 Associated with metastatic prostate cancer
hsa-miR-5686 5247 6268 Associated with metastatic prostate cancer
hsa-miR-5687 5248 6269 Associated with metastatic prostate cancer
hsa-miR-5688 5249 6270 Associated with metastatic prostate cancer
hsa-miR-5689 5250 6271 Associated with metastatic prostate cancer
hsa-miR-569 5251 6272 hsa-miR-5690 5252 6273 Associated with
metastatic prostate cancer hsa-miR-5691 5253 6274 Associated with
metastatic prostate cancer hsa-miR-5692a 5254 6275 Associated with
metastatic prostate cancer hsa-miR-5692b 5255 6276 Associated with
metastatic prostate cancer hsa-miR-5692c 5256 6277 Associated with
metastatic prostate cancer hsa-miR-5693 5257 6278 Associated with
metastatic prostate cancer hsa-miR-5694 5258 6279 Associated with
metastatic prostate cancer hsa-miR-5695 5259 6280 Associated with
metastatic prostate cancer hsa-miR-5696 5260 6281 Associated with
metastatic prostate cancer hsa-miR-5697 5261 6282 Associated with
metastatic prostate cancer hsa-miR-5698 5262 6283 Associated with
metastatic prostate cancer hsa-miR-5699 5263 6284 Associated with
metastatic prostate cancer hsa-miR-5700 5264 6285 Associated with
metastatic prostate cancer hsa-miR-5701 5265 6286 Associated with
metastatic prostate cancer hsa-miR-5702 5266 6287 Associated with
metastatic prostate cancer hsa-miR-5703 5267 6288 Associated with
metastatic prostate cancer hsa-miR-570-3p 5268 6289 follicular
lymphoma hsa-miR-5704 5269 6290 Associated with metastatic prostate
cancer hsa-miR-5705 5270 6291 Associated with metastatic prostate
cancer hsa-miR-570-5p 5271 6292 follicular lymphoma hsa-miR-5706
5272 6293 Associated with metastatic prostate cancer hsa-miR-5707
5273 6294 Associated with metastatic prostate cancer hsa-miR-5708
5274 6295 Associated with metastatic prostate cancer hsa-miR-571
5275 6296 frontal cortex hsa-miR-572 5276 6297 circulating basal
cell microRNA (in carcinoma plasma) hsa-miR-573 5277 6298
discovered in the
colorectal MicroRNAome hsa-miR-5739 5278 6299 endothelial cells
hsa-miR-574-3p 5279 6300 blood (myeloid follicular cells) lymphoma
hsa-miR-574-5p 5280 6301 semen hsa-miR-575 5281 6302 gastric cancer
hsa-miR-576-3p 5282 6303 discovered in a colorectal MicroRNAome
hsa-miR-576-5p 5283 6304 cartilage/ chondrocyte hsa-miR-577 5284
6305 discovered in a colorectal MicroRNAome hsa-miR-578 5285 6306
discovered in a colorectal MicroRNAome hsa-miR-5787 5286 6307
fibroblast hsa-miR-579 5287 6308 hsa-miR-580 5288 6309 breast
cancer hsa-miR-581 5289 6310 liver (hepatocytes) hsa-miR-582-3p
5290 6311 cartilage/chondrocyte bladder cancer hsa-miR-582-5p 5291
6312 bladder cancer hsa-miR-583 5292 6313 rectal cancer cells
hsa-miR-584-3p 5293 6314 tumor cells (follicular lymphoma, rectal
cancer cells) hsa-miR-584-5p 5294 6315 tumor cells (follicular
lymphoma, rectal cancer cells) hsa-miR-585 5295 6316 oral squamous
cell carcinoma hsa-miR-586 5296 6317 discovered in a colorectal
MicroRNAome hsa-miR-587 5297 6318 discovered in a colorectal
MicroRNAome hsa-miR-588 5298 6319 discovered in a colorectal
MicroRNAome hsa-miR-589-3p 5299 6320 mesothelial cells
hsa-miR-589-5p 5300 6321 mesothelial cells hsa-miR-590-3p 5301 6322
cardiomyocytes Cell cycle progression hsa-miR-590-5p 5302 6323
cardiomyocytes Cell cycle progression hsa-miR-591 5303 6324
neuroblastoma hsa-miR-592 5304 6325 hepatocellular carcinoma
hsa-miR-593-3p 5305 6326 esophageal cancer hsa-miR-593-5p 5306 6327
esophageal cancer hsa-miR-595 5307 6328 heart failure hsa-miR-596
5308 6329 ependymoma, cancers hsa-miR-597 5309 6330 discovered in a
colorectal MicroRNAome hsa-miR-598 5310 6331 Blood (lymphocytes)
hsa-miR-599 5311 6332 Multiple sclerosis hsa-miR-600 5312 6333
discovered in a colorectal MicroRNAome hsa-miR-601 5313 6334
various cancers (colonrectal, gastric) hsa-miR-602 5314 6335 oocyte
hsa-miR-603 5315 6336 hsa-miR-604 5316 6337 discovered in a
colorectal MicroRNAome hsa-miR-605 5317 6338 discovered in a
colorectal MicroRNAome hsa-miR-606 5318 6339 discovered in a
colorectal MicroRNAome hsa-miR-6068 5319 6340 discovered in
endothelial cells hsa-miR-6069 5320 6341 discovered in endothelial
cells hsa-miR-607 5321 6342 discovered in a colorectal MicroRNAome
hsa-miR-6070 5322 6343 discovered in a colorectal MicroRNAome
hsa-miR-6071 5323 6344 discovered in endothelial cells hsa-miR-6072
5324 6345 discovered in endothelial cells hsa-miR-6073 5325 6346
discovered in endothelial cells hsa-miR-6074 5326 6347 discovered
in endothelial cells hsa-miR-6075 5327 6348 discovered in
endothelial cells hsa-miR-6076 5328 6349 discovered in endothelial
cells hsa-miR-6077 5329 6350 discovered in endothelial cells
hsa-miR-6078 5330 6351 discovered in endothelial cells hsa-miR-6079
5331 6352 discovered in endothelial cells hsa-miR-608 5332 6353
various cancers hsa-miR-6080 5333 6354 discovered in endothelial
cells hsa-miR-6081 5334 6355 discovered in endothelial cells
hsa-miR-6082 5335 6356 discovered in endothelial cells hsa-miR-6083
5336 6357 discovered in endothelial cells hsa-miR-6084 5337 6358
discovered in endothelial cells hsa-miR-6085 5338 6359 discovered
in endothelial cells hsa-miR-6086 5339 6360 embryonic stem cells
hsa-miR-6087 5340 6361 embryonic stem cells hsa-miR-6088 5341 6362
embryonic stem cells hsa-miR-6089 5342 6363 embryonic stem cells
hsa-miR-609 5343 6364 discovered in a colorectal MicroRNAome
hsa-miR-6090 5344 6365 embryonic stem cells hsa-miR-610 5345 6366
gastric cancer hsa-miR-611 5346 6367 Renal cell carcinoma
hsa-miR-612 5347 6368 AM leukemia hsa-miR-6124 5348 6369
hsa-miR-6125 5349 6370 hsa-miR-6126 5350 6371 hsa-miR-6127 5351
6372 hsa-miR-6128 5352 6373 hsa-miR-6129 5353 6374 hsa-miR-613 5354
6375 lipid metabollism hsa-miR-6130 5355 6376 hsa-miR-6131 5356
6377 hsa-miR-6132 5357 6378 hsa-miR-6133 5358 6379 hsa-miR-6134
5359 6380 hsa-miR-614 5360 6381 circulating micrRNAs (in Plasma)
hsa-miR-615-3p 5361 6382 hsa-miR-615-5p 5362 6383 hsa-miR-616-3p
5363 6384 prostate cancer hsa-miR-6165 5364 6385 Pro-apoptotic
factor hsa-miR-616-5p 5365 6386 prostate cancer hsa-miR-617 5366
6387 hsa-miR-618 5367 6388 hsa-miR-619 5368 6389 discovered in a
colorectal MicroRNAome hsa-miR-620 5369 6390 discovered in a
colorectal MicroRNAome hsa-miR-621 5370 6391 hsa-miR-622 5371 6392
hsa-miR-623 5372 6393 hsa-miR-624-3p 5373 6394 chondrocyte
hsa-miR-624-5p 5374 6395 chondrocyte hsa-miR-625-3p 5375 6396 liver
(hepatocytes), circulating various cancers (blood) hsa-miR-625-5p
5376 6397 liver (hepatocytes), circulating various cancers (blood)
hsa-miR-626 5377 6398 discovered in the colorectal MicroRNAome
hsa-miR-627 5378 6399 colorectal cancer hsa-miR-628-3p 5379 6400
neuroblastoma hsa-miR-628-5p 5380 6401 neuroblastoma hsa-miR-629-3p
5381 6402 B-lineage ALL, T cell lupus, RCC/kidney hsa-miR-629-5p
5382 6403 B-lineage ALL, T cell lupus, RCC/kidney hsa-miR-630 5383
6404 chondrocytes rectal cancer hsa-miR-631 5384 6405 discovered in
the colorectal MicroRNAom hsa-miR-632 5385 6406 myelodysplastic
syndromes hsa-miR-633 5386 6407 multiple sclerosis hsa-miR-634 5387
6408 cartilage/ chondrocyte hsa-miR-635 5388 6409 discovered in the
colorectal MicroRNAome hsa-miR-636 5389 6410 myelodysplastic
syndromes hsa-miR-637 5390 6411 discovered in the colorectal
MicroRNAome hsa-miR-638 5391 6412 Lupus nephritis, basal cell
carcinoma hsa-miR-639 5392 6413 discovered in the colorectal
MicroRNAome hsa-miR-640 5393 6414 Chronic lymphocytic leukemia
hsa-miR-641 5394 6415 cartilage/ chondrocyte hsa-miR-642a-3p 5395
6416 adipocyte hsa-miR-642a-5p 5396 6417 discovered in the
colorectal MicroRNAome hsa-miR-642b-3p 5397 6418 discovered in a
cervial tumo hsa-miR-642b-5p 5398 6419 discovered in a cervial tumo
hsa-miR-643 5399 6420 discovered in the colorectal MicroRNAome
hsa-miR-644a 5400 6421 hsa-miR-645 5401 6422 ovarian cancer
hsa-miR-646 5402 6423 hsa-miR-647 5403 6424 prostate and lung
cancer hsa-miR-648 5404 6425 circulating micrRNAs (in Plasma)
hsa-miR-649 5405 6426 Serum hsa-miR-6499-3p 5406 6427 discovered in
abnormal skin (psoriasis) hsa-miR-6499-5p 5407 6428 discovered in
abnormal skin (psoriasis) hsa-miR-650 5408 6429 melanoma
hsa-miR-6500-3p 5409 6430 discovered in abnormal skin (psoriasis)
hsa-miR-6500-5p 5410 6431 discovered in abnormal skin (psoriasis)
hsa-miR-6501-3p 5411 6432 discovered in
abnormal skin (psoriasis) hsa-miR-6501-5p 5412 6433 discovered in
abnormal skin (psoriasis) hsa-miR-6502-3p 5413 6434 discovered in
abnormal skin (psoriasis) hsa-miR-6502-5p 5414 6435 discovered in
abnormal skin (psoriasis) hsa-miR-6503-3p 5415 6436 discovered in
abnormal skin (psoriasis) hsa-miR-6503-5p 5416 6437 discovered in
abnormal skin (psoriasis) hsa-miR-6504-3p 5417 6438 discovered in
abnormal skin (psoriasis) hsa-miR-6504-5p 5418 6439 discovered in
abnormal skin (psoriasis) hsa-miR-6505-3p 5419 6440 discovered in
abnormal skin (psoriasis) hsa-miR-6505-5p 5420 6441 discovered in
abnormal skin (psoriasis) hsa-miR-6506-3p 5421 6442 discovered in
abnormal skin (psoriasis) hsa-miR-6506-5p 5422 6443 discovered in
abnormal skin (psoriasis) hsa-miR-6507-3p 5423 6444 discovered in
abnormal skin (psoriasis) hsa-miR-6507-5p 5424 6445 discovered in
abnormal skin (psoriasis) hsa-miR-6508-3p 5425 6446 discovered in
abnormal skin (psoriasis) hsa-miR-6508-5p 5426 6447 discovered in
abnormal skin (psoriasis) hsa-miR-6509-3p 5427 6448 discovered in
abnormal skin (psoriasis) hsa-miR-6509-5p 5428 6449 discovered in
abnormal skin (psoriasis) hsa-miR-651 5429 6450 discovered in the
lung cancer colorectal MicroRNAome hsa-miR-6510-3p 5430 6451
discovered in abnormal skin (psoriasis) hsa-miR-6510-5p 5431 6452
discovered in abnormal skin (psoriasis) hsa-miR-6511a-3p 5432 6453
discovered in abnormal skin (psoriasis) and epididymis
hsa-miR-6511a-5p 5433 6454 discovered in abnormal skin (psoriasis)
and epididymis hsa-miR-6511b-3p 5434 6455 discovered in epididymis
hsa-miR-6511b-5p 5435 6456 discovered in epididymis hsa-miR-6512-3p
5436 6457 discovered in abnormal skin (psoriasis) hsa-miR-6512-5p
5437 6458 discovered in abnormal skin (psoriasis) hsa-miR-6513-3p
5438 6459 discovered in abnormal skin (psoriasis) hsa-miR-6513-5p
5439 6460 discovered in abnormal skin (psoriasis) hsa-miR-6514-3p
5440 6461 discovered in abnormal skin (psoriasis) hsa-miR-6514-5p
5441 6462 discovered in abnormal skin (psoriasis) hsa-miR-6515-3p
5442 6463 discovered in abnormal skin (psoriasis) and epididymis
hsa-miR-6515-5p 5443 6464 discovered in abnormal skin (psoriasis)
and epididymis hsa-miR-652-3p 5444 6465 rectal cancer cells
hsa-miR-652-5p 5445 6466 rectal cancer cells hsa-miR-653 5446 6467
Discovered in the colorectal MicroRNAome hsa-miR-654-3p 5447 6468
Discovered in the colorectal MicroRNAome hsa-miR-654-5p 5448 6469
bone marrow prostate cancer hsa-miR-655 5449 6470 hsa-miR-656 5450
6471 various cancers hsa-miR-657 5451 6472 oligodendrocytes
diabetes hsa-miR-658 5452 6473 gastric cancer hsa-miR-659-3p 5453
6474 myoblast hsa-miR-659-5p 5454 6475 myoblast hsa-miR-660-3p 5455
6476 myoblast hsa-miR-660-5p 5456 6477 myoblast hsa-miR-661 5457
6478 breast cancer hsa-miR-662 5458 6479 endothelial progenitor
cells, oocytes hsa-miR-663a 5459 6480 follicular lymphoma, Lupus
nephritis hsa-miR-663b 5460 6481 follicular lymphoma, Lupus
nephritis hsa-miR-664a-3p 5461 6482 embryonic stem component of
cells SnoRNAs hsa-miR-664a-5p 5462 6483 embryonic stem component of
cells SnoRNAs hsa-miR-664b-3p 5463 6484 embryonic stem component of
cells SnoRNAs hsa-miR-664b-5p 5464 6485 embryonic stem component of
cells SnoRNAs hsa-miR-665 5465 6486 breast cancer hsa-miR-668 5466
6487 keratinocytes senescence hsa-miR-670 5467 6488 hsa-miR-671-3p
5468 6489 hsa-miR-6715a-3p 5469 6490 discovered in epididymis
hsa-miR-6715b-3p 5470 6491 discovered in epididymis
hsa-miR-6715b-5p 5471 6492 discovered in epididymis hsa-miR-671-5p
5472 6493 rectal cancer, prolactinomas hsa-miR-6716-3p 5473 6494
discovered in epididymis hsa-miR-6716-5p 5474 6495 discovered in
epididymis hsa-miR-6717-5p 5475 6496 discovered in epididymis
hsa-miR-6718-5p 5476 6497 discovered in epididymis hsa-miR-6719-3p
5477 6498 discovered in epididymis hsa-miR-6720-3p 5478 6499
discovered in epididymis hsa-miR-6721-5p 5479 6500 discovered in
epididymis hsa-miR-6722-3p 5480 6501 discovered in epididymis
hsa-miR-6722-5p 5481 6502 discovered in epididymis hsa-miR-6723-5p
5482 6503 discovered in epididymis hsa-miR-6724-5p 5483 6504
discovered in epididymis hsa-miR-675-3p 5484 6505 adrenocortical
tumor hsa-miR-675-5p 5485 6506 adrenocortical tumor hsa-miR-676-3p
5486 6507 discovered in female reproductive tract hsa-miR-676-5p
5487 6508 discovered in female reproductive tract hsa-miR-708-3p
5488 6509 Various cancers (lung, bladder, pancreatic, ALL)
hsa-miR-708-5p 5489 6510 Various cancers (lung, bladder,
pancreatic, ALL) hsa-miR-711 5490 6511 cutaneous T-cell lymphomas
hsa-miR-7-1-3p 5491 6512 Glioblast, brain, prancreas hsa-miR-718
5492 6513 blood hsa-miR-7-2-3p 5493 6514 brain, pancreas
hsa-miR-744-3p 5494 6515 heart hsa-miR-744-5p 5495 6516 embryonic
stem cells, heart hsa-miR-758-3p 5496 6517 cholesterol regulation
and brain hsa-miR-758-5p 5497 6518 cholesterol regulation and brain
hsa-miR-759 5498 6519 hsa-miR-7-5p 5499 6520 brain hsa-miR-760 5500
6521 colonrectal and breast cancer hsa-miR-761 5501 6522
hsa-miR-762 5502 6523 corneal epithelial cells hsa-miR-764 5503
6524 osteoblast hsa-miR-765 5504 6525 rectal cancer hsa-miR-766-3p
5505 6526 embryonic stem cells hsa-miR-766-5p 5506 6527 embryonic
stem cells hsa-miR-767-3p 5507 6528 / hsa-miR-767-5p 5508 6529 /
hsa-miR-769-3p 5509 6530 hsa-miR-769-5p 5510 6531 hsa-miR-770-5p
5511 6532 hsa-miR-802 5512 6533 brain, epithelial down symdrome
cells, hepatocytes hsa-miR-873-3p 5513 6534 hsa-miR-873-5p 5514
6535 hsa-miR-874 5515 6536 cervical cancer, lung cancer, carcinoma
hsa-miR-875-3p 5516 6537 hsa-miR-875-5p 5517 6538 hsa-miR-876-3p
5518 6539 hsa-miR-876-5p 5519 6540 hsa-miR-877-3p 5520 6541
hsa-miR-877-5p 5521 6542 hsa-miR-885-3p 5522 6543 embryonic stem
cells hsa-miR-885-5p 5523 6544 embryonic stem cells hsa-miR-887
5524 6545 hsa-miR-888-3p 5525 6546 hsa-miR-888-5p 5526 6547
hsa-miR-889 5527 6548 hsa-miR-890 5528 6549 epididymis hsa-miR-891a
5529 6550 epididymis osteosarcoma hsa-miR-891b 5530 6551 epididymis
hsa-miR-892a 5531 6552 epididymis hsa-miR-892b 5532 6553 epididymis
hsa-miR-892c-3p 5533 6554 discovered in epididymis hsa-miR-892c-5p
5534 6555 discovered in epididymis hsa-miR-920 5535 6556 human
testis hsa-miR-921 5536 6557 human testis muscle invasive bladder
cancer hsa-miR-922 5537 6558 human testis, multiple sclerosis,
neuronal tissues Alcoholic liver
disease hsa-miR-924 5538 6559 human testis hsa-miR-92a-1-5p 5539
6560 endothelial cells hsa-miR-92a-2-5p 5540 6561 endothelial cells
hsa-miR-92a-3p 5541 6562 endothelial cells, CNS hsa-miR-92b-3p 5542
6563 endothelial cells, heart hsa-miR-92b-5p 5543 6564 endothelial
cells, heart hsa-miR-933 5544 6565 discovered in cervical cancer
hsa-miR-93-3p 5545 6566 embryonic stem basal cell cells carcinoma
hsa-miR-934 5546 6567 discovered in cervical cancer hsa-miR-935
5547 6568 blood monoclear energy cells metabolism/ obesity,
medullablastoma/ neural stem cells hsa-miR-93-5p 5548 6569
embryonic stem cells hsa-miR-936 5549 6570 skin hsa-miR-937-3p 5550
6571 cervical cancer hsa-miR-937-5p 5551 6572 cervical cancer
hsa-miR-938 5552 6573 Various cancer cells hsa-miR-939-3p 5553 6574
hepatocytes hsa-miR-939-5p 5554 6575 hepatocytes hsa-miR-9-3p 5555
6576 brain Cancers and brain diseases hsa-miR-940 5556 6577
identified in Cervical cancer hsa-miR-941 5557 6578 Embryonic stem
cells hsa-miR-942 5558 6579 lung cancer hsa-miR-943 5559 6580
identified in Cervical cancer hsa-miR-944 5560 6581 various cancers
(cervical, pancreatic, colonrectal) hsa-miR-95 5561 6582 various
cancers (pancreatic, glioblastoma, colorectal etc) hsa-miR-9-5p
5562 6583 brain Cancers and brain disease hsa-miR-96-3p 5563 6584
stem cells various cancers (prostate, lymphoma, HCC, etc) and
inflammation hsa-miR-96-5p 5564 6585 stem cells various cancers
(prostate, lymphoma, HCC, etc) and inflammation hsa-miR-98-3p 5565
6586 various cancer apoptosis cells hsa-miR-98-5p 5566 6587 various
cancer apoptosis cells hsa-miR-99a-3p 5567 6588 hemapoietic cells
hsa-miR-99a-5p 5568 6589 hemapoietic cells hsa-miR-99b-3p 5569 6590
hemapoietic cells, embryonic stem cells hsa-miR-99b-5p 5570 6591
hemapoietic cells, embryonic stem cells
[0337] MicroRNAs that are enriched in specific types of immune
cells are listed in Table 11. Furthermore, novel miroRNAs are
discovered in the immune cells in the art through micro-array
hybridization and microtome analysis (Jima D D et al, Blood, 2010,
116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, the
content of each of which is incorporated herein by reference in its
entirety). In Table 11, "HCC" represents hepatocellular carcinoma,
"ALL" stands for acute lymphoblastsic leukemia and "CLL" stands for
chrominc lymphocytic leukemia.
TABLE-US-00011 TABLE 11 microRNAs in immune cells mir BS
tissues/cells SEQ SEQ with biological microRNA ID ID MicroRNAs
associated diseases functions/targets hsa-let-7a-2-3p 2508 3529
embryonic stem inflammatory, tumor cells, lung, various cancers
suppressor, myeloid cells (lung, cervical, target to c-myc breast,
pancreatic, etc) hsa-let-7a-3p 2509 3530 embryonic stem
inflammatory, tumor cell, lung, various cancers suppressor, myeloid
cells (lung, cervical, target to c-myc breast, pancreatic, etc)
hsa-let-7a-5p 2510 3531 embryonic stem inflammatory, tumor cells,
lung, various cancers suppressor, myeloid cells (lung, cervical,
target to c-myc breast, pancreatic, etc) hsa-let-7c 2513 3534
dendritic cells various cacners tumor (cervical, pancreatic,
suppressor lung, esopphageal, apoptosis etc) (target to BCL- x1)
hsa-let-7e-3p 2516 3537 immune cells various cancer cells, tumor
autoimmunity suppressor TLR signal pathway in endotoxin tolerance
hsa-let-7e-5p 2517 3538 immune cells associated with tumor various
cancer cells suppressor hsa-let-7f-1-3p 2518 3539 immune cells (T
associated with tumor cells) various cancer cells suppressor
hsa-let-7f-2-3p 2519 3540 immune cells (T associated with tumor
cells) various cancer cells suppressor hsa-let-7f-5p 2520 3541
immune cells (T associated with tumor cells) various cancer cells
suppressor hsa-let-7g-3p 2521 3542 hematopoietic various cancer
cells tumor cells, adipose, (lung, breast, etc) suppressor smooth
muscle (target to cells NFkB, LOX1) hsa-let-7g-5p 2522 3543
hematopoietic various cancer cells tumor cells, adipose, (lung,
breast, etc) suppressor smooth muscle (target to cells NFkB, LOX1)
hsa-let-7i-3p 2523 3544 immune cells chronic lymphocyte tumor
leukimia suppressor hsa-let-7i-5p 2524 3545 immune cells chronic
lymphocyte tumor leukimia suppressor hsa-miR-10a-3p 2530 3551
hematopoeitic acute myeoid oncogene, cell cells leukemia growth
hsa-miR-10a-5p 2541 3562 hematopoietic acute myeloid oncogene, cell
cells leukemia growth hsa-miR-1184 2551 3572 Hematopoietic
downregulated in predited in the cells oral leukoplakia intron 22
of F8 (OLK) gene hsa-miR-125b-1- 2616 3637 hematopoietic various
cancer oncogene, cell 3p cells (ALL, prostate, differentiation
(monocytes), HCC, etc); TLR brain (neuron) signal pathway in
endotoxin tolerance hsa-miR-125b-2- 2617 3638 hematopoietic various
cancer oncogene cell 3p cells (ALL, prostate, differentiation
(monocytes), HCC etc); TLR brain (neuron) signal pathway in
endotoxin tolerance hsa-miR-125b- 2618 3639 hematopoietic various
cancer oncogene cell 5p cells, brain (Cutaneous T cell
differentiation (neuron) lymphomas, prostate, HCC, etc); TLR signal
pathway in endotoxin tolerance hsa-miR-1279 2652 3673 monocytes
hsa-miR-130a-3p 2690 3711 lung, monocytes, various cancers
pro-angiogenic vascular (basal cell endothelial cells carcinoma,
HCC, ovarian, etc), drug resistance hsa-miR-130a-5p 2691 3712 lung,
monocytes, various cancers pro-angiogenic vasscular (basal cell
endothelial cells carcinoma, HCC, ovarian, etc), drug resistance
hsa-miR-132-3p 2697 3718 brain(neuron), immune cells hsa-miR-132-5p
2699 3720 brain(neuron), immune cells hsa-miR-142-3p 2720 3741
meyloid cells, tumor hematopoiesis, suppressor, APC cells immune
response hsa-miR-142-5p 2721 3742 meyloid cells, immune
hematopoiesis, response APC cells hsa-miR-143-5p 2723 3744 vascular
smooth increased in serum muscle, T-cells after virus infection
hsa-miR-146a-3p 2730 3751 immune cells, associated with
hematopoiesis, cartilage, CLL, TLR signal pathway in endotoxin
tolerance hsa-miR-146a-5p 2731 3752 immune cells, associated with
hematopoiesis, CLL, TLR signal cartilage, pathway in endotoxin
tolerance hsa-miR-146b- 2732 3753 immune cells cancers (thyroid
immune 3p carcimona) response hsa-miR-146b- 2733 3754 embryoid body
thyroid cancer, tumor invation, 5p cells associated with CLL
migration hsa-miR-147a 2736 3757 Macrophage inflammatory response
hsa-miR-147b 2737 3758 Macrophage inflammatory response
hsa-miR-148a-3p 2738 3759 hematopoietic associated with cells CLL,
T-lineage ALL hsa-miR-148a-5p 2739 3760 hematopoietic associated
with cells CLL, T-lineage ALL hsa-miR-150-3p 2744 3765 hematopoitic
circulating plasma cells (lymphoid) (acute myeloid leukemia)
hsa-miR-150-5p 2745 3766 hematopoitic circulating plasma cells
(lymphoid) (acute myeloid leukemia) hsa-miR-151b 2748 3769 immune
cells (B- cells) hsa-miR-155-3p 2756 3777 T/B cells, associated
with monocytes, breast CLL, TLR signal pathway in endotoxin
tolerance; upregulated in B cell lymphoma (CLL) and other cancers
(breast, lung, ovarian, cervical, colorectal, prostate)
hsa-miR-155-5p 2757 3778 T/B cells, associated with CLL, monocytes,
breast TLR signal pathway in endotoxin tolerance, upregulated in B
cell lymphoma (CLL) and other cancers (breast, lung, ovarian,
cervical, colorectal, prostate) hsa-miR-15a-3p 2759 3780 blood,
chronic lymphocytic lymphocyte, leukemia hematopoietic tissues
(spleen) hsa-miR-15a-5p 2760 3781 blood, chronic lymphoytic
lymphocyte, leukemia hematopoietic tissues (spleen) hsa-miR-15b-3p
2761 3782 blood, cell cycle, lymphocyte, proliferation
hematopoietic tissues (spleen) hsa-miR-15b-5p 2762 3783 blood, cell
cycle, lymphocyte, proliferation hematopoietic tissues (spleen)
hsa-miR-16-1-3p 2763 3784 embryonic stem chronic lymphocytic cells,
blood, leukemia hematopoietic tissues (spleen) hsa-miR-16-2-3p 2764
3785 blood, lymphocyte, hematopoietic tissues (spleen)
hsa-miR-16-5p 2765 3786 blood, lymphocyte, hematopoietic tissues
hsa-miR-181a-3p 2769 3790 glioblast, myeloid cells, Embryonic stem
cells hsa-miR-181a-5p 2770 3791 glioblast, myeloid cells, Embryonic
stem cells hsa-miR-182-3p 2776 3797 immune cells colonrectal
cancer, immune autoimmne response hsa-miR-182-5p 2778 3799 lung,
immune autoimmune immune cells response hsa-miR-197-3p 2827 3848
blood (myeloid), various cancers other tissues (thyroid tumor,
leukemia, etc) hsa-miR-197-5p 2828 3849 blood (myeloid), various
cancers other tissues (thyroid tumor, leukemia, etc) hsa-miR-21-3p
2879 3099 glioblast, Blood autoimmune, heart (meyloid cells),
diseases, cancers liver, vascular endothelial cells hsa-miR-214-3p
2880 3901 immune cells, varioua cancers immune pancreas (melanoma,
response pancreatic, ovarian) hsa-miR-214-5p 2881 3902 immune
cells, varioua cancers immune pancreas (melanoma, response
pancreatic, ovarian) hsa-miR-21-5p 2883 3904 blood (myeloid
autoimmune, heart cells), liver, diseases, cancers endothelial
cells hsa-miR-221-3p 2894 3915 endothelial cells, breast
angiogenesis/vasculogenesis immune cells cancer, upregulated in
thyroid cell transformation induced by HMGA1, TLR signal pathway in
endotoxin tolerance, upregulated in T cell ALL hsa-miR-221-5p 2895
3916 endothelial breast angiogenesis/vasculogenesis cells, immune
cancer, upregulated cells in thyroid cell transformation induced by
HMGA1, TLR signal pathway in endotoxin tolerance, upregulated in T
cell ALL hsa-miR-223-3p 2898 3919 meyloid cells associated with CLL
hsa-miR-223-5p 2899 3920 meyloid cells associated with CLL
hsa-miR-23b-3p 2913 3934 blood, myeloid cancers (renal cells
cancer, glioblastoma, prostate, etc) and autoimmune hsa-miR-23b-5p
2914 3935 blood, myeloid cancers(glioblastoma, cells prostate, etc)
and autoimmune hsa-miR-24-1-5p 2916 3937 lung, myeloid cells
hsa-miR-24-2-5p 2917 3938 lung, myeloid cells hsa-miR-24-3p 2918
3939 lung, myeloid cells
hsa-miR-26a-1- 2927 3948 embryonic stem chronic lymphocyte cell
cycle and 3p cells, blood (T leukemia and other differentiation
cells) cancers hsa-miR-26a-2- 2928 3949 blood (Tcells), chronic
lymphocyte cell cycle and 3p other tissues leukemia and other
differentiation cancers hsa-miR-26a-5p 2929 3950 blood (Tcells),
chronic lymphocyte cell cycle and other tissues leukemia and other
differentiation cancers hsa-miR-26b-3p 2930 3951 hematopoietic
cells hsa-miR-26b-5p 2931 3952 hematopoietic cells hsa-miR-27a-3p
2932 3953 myeloid cells various cancer cells hsa-miR-27a-5p 2933
3954 myeloid cells various cancer cells hsa-miR-27b-3p 2934 3955
myeloid cells, various cancer cells pro-angiogenic vascular
endothelial cells hsa-miR-28-3p 2936 3957 blood(immune B/T cell
lymphoma cells) hsa-miR-28-5p 2937 3958 blood(immune B/T cell
lymphoma cells) hsa-miR-2909 2939 3960 T-Lymphocytes hsa-miR-29a-3p
2948 3969 immuno system, various cancers, tumor colonrectun
neurodegenative suppression, disease immune modulation (mir-29
family) hsa-miR-29a-5p 2949 3970 immuno system, various cancers,
adaptive colonrectun neurodegenative immunity disease
hsa-miR-29b-1- 2950 3971 immuno system associated with adaptive 5p
CLL, other cancers, immunity neurodegenative disease hsa-miR-29b-2-
2951 3972 immuno system associated with adaptive 5p CLL, other
cancers, immunity hsa-miR-29b-3p 2952 3973 immuno system associated
with adaptive CLL, other cancers immunity hsa-miR-29c-3p 2953 3974
immuno system associated with adaptive CLL, other cancers immunity
hsa-miR-29c-5p 2954 3975 immuno system associated with adaptive
CLL, other cancers immunity hsa-miR-30e-3p 2984 4005 myeloid cells,
glia cells hsa-miR-30e-5p 2985 4006 myeloid cells, glia cells
hsa-miR-331-5p 3130 4151 lymphocytes hsa-miR-339-3p 3137 4158
immune cells hsa-miR-339-5p 3138 4159 immune cells hsa-miR-345-3p
3147 4168 hematopoietic increased in cells follicular lymphoma(53),
other cancers hsa-miR-345-5p 3148 4169 hematopoietic increased in
cells follicular lymphoma(53) hsa-miR-346 3149 4170 immume cells
cancers and autoimmune hsa-miR-34a-3p 3150 4171 breast, myeloid
gastric cancer, tumor cells, ciliated CLL, other suppressor, p53
epithelial cells inducible hsa-miR-34a-5p 3151 4172 breast, myeloid
gastric cancer, tumor cells, ciliated CLL, other suppressor, p53
epithelial cells inducible hsa-miR-363-3p 3193 4214 kidney stem
cell, blood cells hsa-miR-363-5p 3194 4215 kidney stem cell, blood
cells hsa-miR-372 3277 4298 hematopoietic cells, lung, placental
(blood) hsa-miR-377-3p 3294 4315 hematopoietic cells hsa-miR-377-5p
3295 4316 hematopoietic cells hsa-miR-493-3p 4947 5968 myeloid
cells, pancreas (islet) hsa-miR-493-5p 4948 5969 myeloid cells,
pancreas (islet) hsa-miR-542-3p 5106 6127 monocytes targets to
survivin, introduce growth arrest hsa-miR-548b- 5157 6178 immune
cells 5p frontal cortex hsa-miR-548c-5p 5159 6180 immune cells
frontal cortex hsa-miR-548i 5168 6189 embryonic stem cells (41),
immune cells hsa-miR-548j 5169 6190 immune cells hsa-miR-548n 5173
6194 embryonic stem cells, immune cells hsa-miR-574-3p 5279 6300
blood (myeloid increased in cells) follicular lymphoma(53)
hsa-miR-598 5310 6331 in blood lymphocytes (PBL) hsa-miR-935 5547
6568 identified in associated with human cervical energy cancer
metabolism/obesity, blood medullablastoma/neural mononuclear stem
cells cells hsa-miR-99a-3p 5567 6588 hemapoietic cells
hsa-miR-99a-5p 5568 6589 hemapoietic cells, plasma (exosome)
hsa-miR-99b-3p 5569 6590 hemapoietic cells, Embryonic stem cells,
hsa-miR-99b-5p 5570 6591 hemapoietic cells, Embryonic stem cells,
plasma(exosome)
III. Modifications
[0338] Herein, in a signal-sensor polynucleotide (such as a primary
construct or a mRNA molecule), the terms "modification" or, as
appropriate, "modified" refer to modification with respect to A, G,
U or C ribonucleotides. Generally, herein, these terms are not
intended to refer to the ribonucleotide modifications in naturally
occurring 5'-terminal mRNA cap moieties. In a polypeptide, the term
"modification" refers to a modification as compared to the
canonical set of 20 amino acids.
[0339] The modifications may be various distinct modifications. In
some embodiments, the coding region, the flanking regions and/or
the terminal regions may contain one, two, or more (optionally
different) nucleoside or nucleotide modifications. In some
embodiments, a modified signal-sensor polynucleotide, primary
construct, or mmRNA introduced to a cell may exhibit reduced
degradation in the cell, as compared to an unmodified signal-sensor
polynucleotide, primary construct, or mmRNA.
[0340] The signal-sensor polynucleotides, primary constructs, and
mmRNA can include any useful modification, such as to the sugar,
the nucleobase, or the internucleoside linkage (e.g. to a linking
phosphate/to a phosphodiester linkage/to the phosphodiester
backbone). One or more atoms of a pyrimidine nucleobase may be
replaced or substituted with optionally substituted amino,
optionally substituted thiol, optionally substituted alkyl (e.g.,
methyl or ethyl), or halo (e.g., chloro or fluoro). In certain
embodiments, modifications (e.g., one or more modifications) are
present in each of the sugar and the internucleoside linkage.
Modifications according to the present invention may be
modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids
(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),
peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or
hybrids thereof). Additional modifications are described
herein.
[0341] As described herein, in some embodiments, the signal-sensor
polynucleotides, primary constructs, and mmRNA of the invention do
not substantially induce an innate immune response of a cell into
which the mRNA is introduced. Featues of an induced innate immune
response include 1) increased expression of pro-inflammatory
cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc,
and/or 3) termination or reduction in protein translation. In other
embodiments, an immune response is induced.
[0342] In certain embodiments, it may desirable to intracellularly
degrade a modified nucleic acid molecule introduced into the cell.
For example, degradation of a modified nucleic acid molecule may be
preferable if precise timing of protein production is desired.
Thus, in some embodiments, the invention provides a modified
nucleic acid molecule containing a degradation domain, which is
capable of being acted on in a directed manner within a cell.
[0343] In another aspect, the present disclosure provides
signal-sensor polynucleotides comprising a nucleoside or nucleotide
that can disrupt the binding of a major groove interacting, e.g.
binding, partner with the polynucleotide (e.g., where the modified
nucleotide has decreased binding affinity to major groove
interacting partner, as compared to an unmodified nucleotide).
[0344] The signal-sensor polynucleotides, primary constructs, and
mmRNA can optionally include other agents (e.g., RNAi-inducing
agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs,
ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix
formation, aptamers, vectors, etc.). In some embodiments, the
signal-sensor polynucleotides, primary constructs, or mmRNA may
include one or more messenger RNAs (mRNAs) and one or more modified
nucleoside or nucleotides (e.g., mmRNA molecules). Details for
these signal-sensor polynucleotides, primary constructs, and mmRNA
follow.
Signal-Sensor Polynucleotides and Primary Constructs
[0345] The signal-sensor polynucleotides, primary constructs, and
mmRNA of the invention includes a first region of linked
nucleosides encoding an oncology-related polypeptide of interest, a
first flanking region located at the 5' terminus of the first
region, and a second flanking region located at the 3' terminus of
the first region.
[0346] In some embodiments, the signal-sensor polynucleotide,
primary construct, or mmRNA are constructed according to the
methods and modifications of International Application
PCT/US12/058519 filed Oct. 3, 2012 (M9), the contents of which are
incorporated herein by reference in their entirety.
[0347] The signal-sensor polynucleotides, primary constructs, and
mmRNA can optionally include 5' and/or 3' flanking regions, which
are described herein.
Signal-Sensor Modified RNA (mmRNA) Molecules
[0348] The present invention also includes the building blocks,
e.g., modified ribonucleosides, modified ribonucleotides, of
modified signal-sensor mRNA (mmRNA) molecules. For example, these
building blocks can be useful for preparing the signal-sensor
polynucleotides, primary constructs, or mmRNA of the invention.
Such building blocks are taught in co-pending International
Application PCT/US12/058519 filed Oct. 3, 2012 (M9), the contents
of which are incorporated herein by reference in their
entirety.
Modifications on the Nucleobase
[0349] The present disclosure provides for modified nucleosides and
nucleotides. As described herein "nucleoside" is defined as a
compound containing a sugar molecule (e.g., a pentose or ribose) or
a derivative thereof in combination with an organic base (e.g., a
purine or pyrimidine) or a derivative thereof (also referred to
herein as "nucleobase"). As described herein, "nucleotide" is
defined as a nucleoside including a phosphate group. In some
embodiments, the nucleosides and nucleotides described herein are
generally chemically modified on the major groove face. Exemplary
non-limiting modifications include an amino group, a thiol group,
an alkyl group, a halo group, or any described herein. The modified
nucleotides may by synthesized by any useful method, as described
herein (e.g., chemically, enzymatically, or recombinantly to
include one or more modified or non-natural nucleosides).
[0350] The modified nucleosides and nucleotides can include a
modified nucleobase. Examples of nucleobases found in RNA include,
but are not limited to, adenine, guanine, cytosine, and uracil.
Examples of nucleobase found in DNA include, but are not limited
to, adenine, guanine, cytosine, and thymine. These nucleobases can
be modified or wholly replaced to provide signal-sensor
polynucleotides, primary constructs, or mmRNA molecules having
enhanced properties. For example, the nucleosides and nucleotides
described herein can be chemically modified. In some embodiments,
chemical modifications can include an amino group, a thiol group,
an alkyl group, or a halo group.
Modifications on the Internucleoside Linkage
[0351] The modified nucleotides, which may be incorporated into a
signal-sensor polynucleotide, primary construct, or mmRNA molecule,
can be modified on the internucleoside linkage (e.g., phosphate
backbone). Herein, in the context of the polynucleotide backbone,
the phrases "phosphate" and "phosphodiester" are used
interchangeably. Backbone phosphate groups can be modified by
replacing one or more of the oxygen atoms with a different
substituent. Further, the modified nucleosides and nucleotides can
include the wholesale replacement of an unmodified phosphate moiety
with another internucleoside linkage as described herein. Examples
of modified phosphate groups include, but are not limited to,
phosphorothioate, phosphoroselenates, boranophosphates,
boranophosphate esters, hydrogen phosphonates, phosphoramidates,
phosphorodiamidates, alkyl or aryl phosphonates, and
phosphotriesters. Phosphorodithioates have both non-linking oxygens
replaced by sulfur. The phosphate linker can also be modified by
the replacement of a linking oxygen with nitrogen (bridged
phosphoramidates), sulfur (bridged phosphorothioates), and carbon
(bridged methylene-phosphonates).
[0352] The .alpha.-thio substituted phosphate moiety is provided to
confer stability to RNA and DNA polymers through the unnatural
phosphorothioate backbone linkages. Phosphorothioate DNA and RNA
have increased nuclease resistance and subsequently a longer
half-life in a cellular environment. Phosphorothioate linked
signal-sensor polynucleotides, primary constructs, or mmRNA
molecules are expected to also reduce the innate immune response
through weaker binding/activation of cellular innate immune
molecules.
[0353] In specific embodiments, a modified nucleoside includes an
alpha-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine,
5'-O-(1-thiophosphate)-cytidine (.alpha.-thio-cytidine),
5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine,
or 5'-O-(1-thiophosphate)-pseudouridine).
[0354] Other internucleoside linkages that may be employed
according to the present invention, including internucleoside
linkages which do not contain a phosphorous atom, are described
herein below.
Combinations of Modified Sugars, Nucleobases, and Internucleoside
Linkages
[0355] The signal-sensor polynucleotides, primary constructs, and
mmRNA of the invention can include a combination of modifications
to the sugar, the nucleobase, and/or the internucleoside linkage.
These combinations can include any one or more modifications
described herein or in International Application PCT/US12/058519
filed Oct. 3, 2012 (M9), the contents of which are incorporated
herein by reference in their entirety.
Synthesis of Signal-Sensor Primary Constructs, and mmRNA
Molecules
[0356] The signal-sensor polypeptides, primary constructs, and
mmRNA molecules for use in accordance with the invention may be
prepared according to any useful technique, as described herein.
The modified nucleosides and nucleotides used in the synthesis of
signal-sensor polynucleotides, primary constructs, and mmRNA
molecules disclosed herein can be prepared from readily available
starting materials using the following general methods and
procedures. Where typical or preferred process conditions (e.g.,
reaction temperatures, times, mole ratios of reactants, solvents,
pressures, etc.) are provided, a skilled artisan would be able to
optimize and develop additional process conditions. Optimum
reaction conditions may vary with the particular reactants or
solvent used, but such conditions can be determined by one skilled
in the art by routine optimization procedures.
[0357] The processes described herein can be monitored according to
any suitable method known in the art. For example, product
formation can be monitored by spectroscopic means, such as nuclear
magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C)
infrared spectroscopy, spectrophotometry (e.g., UV-visible), or
mass spectrometry, or by chromatography such as high performance
liquid chromatography (HPLC) or thin layer chromatography.
[0358] Preparation of signal-sensor polynucleotides, primary
constructs, and mmRNA molecules of the present invention can
involve the protection and deprotection of various chemical groups.
The need for protection and deprotection, and the selection of
appropriate protecting groups can be readily determined by one
skilled in the art. The chemistry of protecting groups can be
found, for example, in Greene, et al., Protective Groups in Organic
Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated
herein by reference in its entirety.
[0359] The reactions of the processes described herein can be
carried out in suitable solvents, which can be readily selected by
one of skill in the art of organic synthesis. Suitable solvents can
be substantially nonreactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, i.e., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected.
[0360] Resolution of racemic mixtures of modified nucleosides and
nucleotides (e.g., mmRNA molecules) can be carried out by any of
numerous methods known in the art. An example method includes
fractional recrystallization using a "chiral resolving acid" which
is an optically active, salt-forming organic acid. Suitable
resolving agents for fractional recrystallization methods are, for
example, optically active acids, such as the D and L forms of
tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,
mandelic acid, malic acid, lactic acid or the various optically
active camphorsulfonic acids. Resolution of racemic mixtures can
also be carried out by elution on a column packed with an optically
active resolving agent (e.g., dinitrobenzoylphenylglycine).
Suitable elution solvent composition can be determined by one
skilled in the art.
[0361] Modified nucleosides and nucleotides (e.g., building block
molecules) can be prepared according to the synthetic methods
described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009);
Purmal et al., Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et
al., Biochemistry, 1(4): 563-568 (1962); and Xu et al.,
Tetrahedron, 48(9): 1729-1740 (1992), each of which are
incorporated by reference in their entirety.
[0362] The signal-sensor polynucleotides, primary constructs, and
mmRNA of the invention may or may not be uniformly modified along
the entire length of the molecule. For example, one or more or all
types of nucleotide (e.g., purine or pyrimidine, or any one or more
or all of A, G, U, C) may or may not be uniformly modified in a
polynucleotide of the invention, or in a given predetermined
sequence region thereof (e.g. one or more of the sequence regions
represented in FIG. 1). In some embodiments, all nucleotides X in a
signal-sensor polynucleotide of the invention (or in a given
sequence region thereof) are modified, wherein X may any one of
nucleotides A, G, U, C, or any one of the combinations A+G, A+U,
A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
[0363] Different sugar modifications, nucleotide modifications,
and/or internucleoside linkages (e.g., backbone structures) may
exist at various positions in the signal-sensor polynucleotide,
primary construct, or mmRNA. One of ordinary skill in the art will
appreciate that the nucleotide analogs or other modification(s) may
be located at any position(s) of a signal-sensor polynucleotide,
primary construct, or mmRNA such that the function of the
signal-sensor polynucleotide, primary construct, or mmRNA is not
substantially decreased. A modification may also be a 5' or 3'
terminal modification. The signal-sensor polynucleotide, primary
construct, or mmRNA may contain from about 1% to about 100%
modified nucleotides (either in relation to overall nucleotide
content, or in relation to one or more types of nucleotide, i.e.
any one or more of A, G, U or C) or any intervening percentage
(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to
60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to
95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to
60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to
95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20%
to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20%
to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from
50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,
from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to
100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90%
to 95%, from 90% to 100%, and from 95% to 100%).
[0364] In some embodiments, the signal-sensor polynucleotide,
primary construct, or mmRNA includes a modified pyrimidine (e.g., a
modified uracil/uridine/U or modified cytosine/cytidine/C). In some
embodiments, the uracil or uridine (generally: U) in the
signal-sensor polynucleotide, primary construct, or mmRNA molecule
may be replaced with from about 1% to about 100% of a modified
uracil or modified uridine (e.g., from 1% to 20%, from 1% to 25%,
from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,
from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%,
from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%,
from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to
25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to
80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50%
to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50%
to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from
70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%,
from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%
to 100% of a modified uracil or modified uridine). The modified
uracil or uridine can be replaced by a compound having a single
unique structure or by a plurality of compounds having different
structures (e.g., 2, 3, 4 or more unique structures, as described
herein). In some embodiments, the cytosine or cytidine (generally:
C) in the signal-sensor polynucleotide, primary construct, or mmRNA
molecule may be replaced with from about 1% to about 100% of a
modified cytosine or modified cytidine (e.g., from 1% to 20%, from
1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1%
to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10%
to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10%
to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from
20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from
20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%,
from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%,
from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to
90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80%
to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and
from 95% to 100% of a modified cytosine or modified cytidine). The
modified cytosine or cytidine can be replaced by a compound having
a single unique structure or by a plurality of compounds having
different structures (e.g., 2, 3, 4 or more unique structures, as
described herein).
Combinations of Nucleotides
[0365] Further examples of modified nucleotides and modified
nucleotide combinations are provided in International Application
PCT/US12/058519 filed Oct. 3, 2012 (M9) the contents of which are
incorporated herein by reference in their entirety.
[0366] In some embodiments, at least 25% of the cytidines are
replaced (e.g., at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or about 100%).
[0367] In some embodiments, at least 25% of the uracils are
replaced (e.g., at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or about 100%).
[0368] In some embodiments, at least 25% of the cytidines are
replaced, and at least 25% of the uracils are replaced (e.g., at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or about 100%).
IV. Pharmaceutical Compositions
Formulation, Administration, Delivery and Dosing
[0369] The present invention provides signal-sensor
polynucleotides, primary constructs and mmRNA compositions and
complexes in combination with one or more pharmaceutically
acceptable excipients. Pharmaceutical compositions may optionally
comprise one or more additional active substances, e.g.
therapeutically and/or prophylactically active substances. General
considerations in the formulation and/or manufacture of
pharmaceutical agents may be found, for example, in Remington: The
Science and Practice of Pharmacy 21.sup.st ed., Lippincott Williams
& Wilkins, 2005 (incorporated herein by reference).
[0370] In some embodiments, compositions are administered to
humans, human patients or subjects. For the purposes of the present
disclosure, the phrase "active ingredient" generally refers to
signal-sensor polynucleotides, primary constructs and mmRNA to be
delivered as described herein.
[0371] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by the skilled artisan that such compositions
are generally suitable for administration to any other animal,
e.g., to non-human animals, e.g. non-human mammals. Modification of
pharmaceutical compositions suitable for administration to humans
in order to render the compositions suitable for administration to
various animals is well understood, and the ordinarily skilled
veterinary pharmacologist can design and/or perform such
modification with merely ordinary, if any, experimentation.
Subjects to which administration of the pharmaceutical compositions
is contemplated include, but are not limited to, humans and/or
other primates; mammals, including commercially relevant mammals
such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;
and/or birds, including commercially relevant birds such as
poultry, chickens, ducks, geese, and/or turkeys.
[0372] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient into association
with an excipient and/or one or more other accessory ingredients,
and then, if necessary and/or desirable, dividing, shaping and/or
packaging the product into a desired single- or multi-dose
unit.
[0373] A pharmaceutical composition in accordance with the
invention may be prepared, packaged, and/or sold in bulk, as a
single unit dose, and/or as a plurality of single unit doses. As
used herein, a "unit dose" is discrete amount of the pharmaceutical
composition comprising a predetermined amount of the active
ingredient. The amount of the active ingredient is generally equal
to the dosage of the active ingredient which would be administered
to a subject and/or a convenient fraction of such a dosage such as,
for example, one-half or one-third of such a dosage.
[0374] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
invention will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100%, e.g.,
between 0.5 and 50%, between 1-30%, between 5-80%, at least 80%
(w/w) active ingredient.
Formulations
[0375] The signal-sensor polynucleotide, primary construct, and
mmRNA of the invention can be formulated using one or more
excipients to: (1) increase stability; (2) increase cell
transfection; (3) permit the sustained or delayed release (e.g.,
from a depot formulation of the signal-sensor polynucleotide,
primary construct, or mmRNA); (4) alter the biodistribution (e.g.,
target the polynucleotide, primary construct, or mmRNA to specific
tissues or cell types); (5) increase the translation of encoded
protein in vivo; and/or (6) alter the release profile of encoded
protein in vivo. In addition to traditional excipients such as any
and all solvents, dispersion media, diluents, or other liquid
vehicles, dispersion or suspension aids, surface active agents,
isotonic agents, thickening or emulsifying agents, preservatives,
excipients of the present invention can include, without
limitation, lipidoids, liposomes, lipid nanoparticles, polymers,
lipoplexes, core-shell nanoparticles, peptides, proteins, cells
transfected with signal-sensor polynucleotide, primary construct,
or mmRNA (e.g., for transplantation into a subject), hyaluronidase,
nanoparticle mimics and combinations thereof. Further, the
signal-sensor polynucleotide, primary construct, or mmRNA of the
present invention may be formulated using self-assembled nucleic
acid nanoparticles.
[0376] Accordingly, the formulations of the invention can include
one or more excipients, each in an amount that together increases
the stability of the signal-sensor polynucleotide, primary
construct, or mmRNA, increases cell transfection by the
signal-sensor polynucleotide, primary construct, or mmRNA,
increases the expression of polynucleotide, primary construct, or
mmRNA encoded protein, and/or alters the release profile of
signal-sensor polynucleotide, primary construct, or mmRNA encoded
proteins. Further, the primary construct and mmRNA of the present
invention may be formulated using self-assembled nucleic acid
nanoparticles.
[0377] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of associating the active ingredient with an
excipient and/or one or more other accessory ingredients.
[0378] A pharmaceutical composition in accordance with the present
disclosure may be prepared, packaged, and/or sold in bulk, as a
single unit dose, and/or as a plurality of single unit doses. As
used herein, a "unit dose" refers to a discrete amount of the
pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient may
generally be equal to the dosage of the active ingredient which
would be administered to a subject and/or a convenient fraction of
such a dosage including, but not limited to, one-half or one-third
of such a dosage.
[0379] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
present disclosure may vary, depending upon the identity, size,
and/or condition of the subject being treated and further depending
upon the route by which the composition is to be administered. For
example, the composition may comprise between 0.1% and 99% (w/w) of
the active ingredient.
[0380] In some embodiments, the formulations described herein may
contain at least one signal-sensor mmRNA. As a non-limiting
example, the formulations may contain 1, 2, 3, 4 or 5 signal-sensor
mmRNA. In one embodiment the formulation may contain modified mRNA
encoding proteins selected from categories such as, proteins. In
one embodiment, the formulation contains at least three
signal-sensor modified mRNA encoding oncology-related proteins. In
one embodiment, the formulation contains at least five
signal-sensor modified mRNA encoding oncology-related proteins.
[0381] Pharmaceutical formulations may additionally comprise a
pharmaceutically acceptable excipient, which, as used herein,
includes, but is not limited to, any and all solvents, dispersion
media, diluents, or other liquid vehicles, dispersion or suspension
aids, surface active agents, isotonic agents, thickening or
emulsifying agents, preservatives, and the like, as suited to the
particular dosage form desired. Various excipients for formulating
pharmaceutical compositions and techniques for preparing the
composition are known in the art (see Remington: The Science and
Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro, Lippincott,
Williams & Wilkins, Baltimore, Md., 2006; incorporated herein
by reference). The use of a conventional excipient medium may be
contemplated within the scope of the present disclosure, except
insofar as any conventional excipient medium may be incompatible
with a substance or its derivatives, such as by producing any
undesirable biological effect or otherwise interacting in a
deleterious manner with any other component(s) of the
pharmaceutical composition.
[0382] In some embodiments, the particle size of the lipid
nanoparticle may be increased and/or decreased. The change in
particle size may be able to help counter biological reaction such
as, but not limited to, inflammation or may increase the biological
effect of the signal-sensor modified mRNA delivered to mammals.
[0383] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inert diluents, surface active agents and/or
emulsifiers, preservatives, buffering agents, lubricating agents,
and/or oils. Such excipients may optionally be included in the
pharmaceutical formulations of the invention.
[0384] Pharmaceutical compositions of the present invention may
comprise at least one adjuvant which may be a chemo-adjuvant.
Non-limiting examples of chemo-adjuvants and delivery systems which
comprises a chemo-adjuvant are described in International Patent
Publication No. WO2013134349, the contents of which is herein
incorporated by reference in its entirety. The chemo-adjuvant may
be bonded to, non-covalently bonded to or encapsulated within a
delivery vehicle described herein.
Lipidoids
[0385] The synthesis of lipidoids has been extensively described
and formulations containing these compounds are particularly suited
for delivery of signal-sensor polynucleotides, primary constructs
or mmRNA (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454;
Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat
Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA.
2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011
108:12996-3001; all of which are incorporated herein in their
entireties).
[0386] While these lipidoids have been used to effectively deliver
double stranded small interfering RNA molecules in rodents and
non-human primates (see Akinc et al., Nat Biotechnol. 2008
26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008
105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et
al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Leuschner et al.,
Nat Biotechnol. 2011 29:1005-1010; all of which is incorporated
herein in their entirety), the present disclosure describes their
formulation and use in delivering single stranded signal-sensor
polynucleotides, primary constructs, or mmRNA. Complexes, micelles,
liposomes or particles can be prepared containing these lipidoids
and therefore, can result in an effective delivery of the
signal-sensor polynucleotide, primary construct, or mmRNA, as
judged by the production of an encoded protein, following the
injection of a lipidoid formulation via localized and/or systemic
routes of administration. Lipidoid complexes of signal-sensor
polynucleotides, primary constructs, or mmRNA can be administered
by various means including, but not limited to, intravenous,
intramuscular, or subcutaneous routes.
[0387] In vivo delivery of nucleic acids may be affected by many
parameters, including, but not limited to, the formulation
composition, nature of particle PEGylation, degree of loading,
oligonucleotide to lipid ratio, and biophysical parameters such as
particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein
incorporated by reference in its entirety). As an example, small
changes in the anchor chain length of poly(ethylene glycol) (PEG)
lipids may result in significant effects on in vivo efficacy.
Formulations with the different lipidoids, including, but not
limited to penta[3-(1-laurylaminopropionyl)]-triethylenetetramine
hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al.,
Analytical Biochemistry, 401:61 (2010)), C12-200 (including
derivatives and variants), and MD1, can be tested for in vivo
activity.
[0388] The lipidoid referred to herein as "98N12-5" is disclosed by
Akinc et al., Mol Ther. 2009 17:872-879 and is incorporated by
reference in its entirety.
[0389] The lipidoid referred to herein as "C12-200" is disclosed by
Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and
Huang, Molecular Therapy. 2010 669-670; both of which are herein
incorporated by reference in their entirety. The lipidoid
formulations can include particles comprising either 3 or 4 or more
components in addition to signal-sensor polynucleotide, primary
construct, or mmRNA. As an example, formulations with certain
lipidoids, include, but are not limited to, 98N12-5 and may contain
42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chain length).
As another example, formulations with certain lipidoids, include,
but are not limited to, C12-200 and may contain 50% lipidoid, 10%
disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5%
PEG-DMG.
[0390] Combinations of different lipidoids may be used to improve
the efficacy of signal-sensor polynucleotide, primary construct, or
mmRNA directed protein production as the lipidoids may be able to
increase cell transfection by the signal-sensor polynucleotide,
primary construct, or mmRNA; and/or increase the translation of
encoded oncology-related protein (see Whitehead et al., Mol. Ther.
2011, 19:1688-1694, herein incorporated by reference in its
entirety).
[0391] In some embodiments, the particle size of the lipid
nanoparticle may be increased and/or decreased. The change in
particle size may be able to help counter biological reaction such
as, but not limited to, inflammation or may increase the biological
effect of, the signal-sensor polynucleotide, primary construct, or
mmRNA delivered to subjects.
Liposomes, Lipoplexes, and Lipid Nanoparticles
[0392] The signal-sensor polynucleotide, primary construct, and
mmRNA of the invention can be formulated using one or more
liposomes, lipoplexes, or lipid nanoparticles. In one embodiment,
pharmaceutical compositions of signal-sensor polynucleotide,
primary construct, or mmRNA include liposomes. Liposomes are
artificially-prepared vesicles which may primarily be composed of a
lipid bilayer and may be used as a delivery vehicle for the
administration of nutrients and pharmaceutical formulations.
Liposomes can be of different sizes such as, but not limited to, a
multilamellar vesicle (MLV) which may be hundreds of nanometers in
diameter and may contain a series of concentric bilayers separated
by narrow aqueous compartments, a small unicellular vesicle (SUV)
which may be smaller than 50 nm in diameter, and a large
unilamellar vesicle (LUV) which may be between 50 and 500 nm in
diameter. Liposome design may include, but is not limited to,
opsonins or ligands in order to improve the attachment of liposomes
to unhealthy tissue or to activate events such as, but not limited
to, endocytosis. Liposomes may contain a low or a high pH in order
to improve the delivery of the pharmaceutical formulations.
[0393] The formation of liposomes may depend on the physicochemical
characteristics such as, but not limited to, the pharmaceutical
formulation entrapped and the liposomal ingredients, the nature of
the medium in which the lipid vesicles are dispersed, the effective
concentration of the entrapped substance and its potential
toxicity, any additional processes involved during the application
and/or delivery of the vesicles, the optimization size,
polydispersity and the shelf-life of the vesicles for the intended
application, and the batch-to-batch reproducibility and possibility
of large-scale production of safe and efficient liposomal
products.
[0394] In one embodiment, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA)
liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.),
1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by
reference in its entirety) and liposomes which may deliver small
molecule drugs such as, but not limited to, DOXIL.RTM. from Janssen
Biotech, Inc. (Horsham, Pa.).
[0395] In one embodiment, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from the synthesis of stabilized plasmid-lipid particles
(SPLP) or stabilized nucleic acid lipid particle (SNALP) that have
been previously described and shown to be suitable for
oligonucleotide delivery in vitro and in vivo (see Wheeler et al.
Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999
6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et
al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature.
2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;
Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin
Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008
19:125-132; all of which are incorporated herein in their
entireties.) The original manufacture method by Wheeler et al. was
a detergent dialysis method, which was later improved by Jeffs et
al. and is referred to as the spontaneous vesicle formation method.
The liposome formulations are composed of 3 to 4 lipid components
in addition to the signal-sensor polynucleotide, primary construct,
or mmRNA. As an example a liposome can contain, but is not limited
to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10%
PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),
as described by Jeffs et al. As another example, certain liposome
formulations may contain, but are not limited to, 48% cholesterol,
20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic
lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA),
DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane
(DLenDMA), as described by Heyes et al.
[0396] In one embodiment, pharmaceutical compositions may include
liposomes which may be formed to deliver signal-sensor mmRNA which
may encode at least one immunogen. The mmRNA may be encapsulated by
the liposome and/or it may be contained in an aqueous core which
may then be encapsulated by the liposome (see International Pub.
Nos. WO2012031046, WO2012031043, WO201203091 and WO2012006378
herein incorporated by reference in their entireties). In another
embodiment, the signal-sensor mmRNA which may encode an immunogen
may be formulated in a cationic oil-in-water emulstion where the
emulsion particle comprises an oil core and a cationic lipid which
can interact with the signal-sensor mmRNA anchoring the molecule to
the emulsion particle (see International Pub. No. WO2012006380). In
yet another embodiment, the lipid formulation may include at least
cationic lipid, a lipid which may enhance transfection and a least
one lipid which contains a hydrophilic head group linked to a lipid
moiety (International Pub. No. WO2011076807 and U.S. Pub. No.
20110200582; herein incorporated by reference in their entireties).
In another embodiment, the signal-sensor polynucleotides, primary
constructs and/or mmRNA encoding an immunogen may be formulated in
a lipid vesicle which may have crosslinks between functionalized
lipid bilayers (see U.S. Pub. No. 20120177724, herein incorporated
by reference in its entirety).
[0397] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may be formulated in a lipid
vesicle which may have crosslinks between functionalized lipid
bilayers.
[0398] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may be formulated in a
lipid-polycation complex. The formation of the lipid-polycation
complex may be accomplished by methods known in the art and/or as
described in U.S. Pub. No. 20120178702, herein incorporated by
reference in its entirety. As a non-limiting example, the
polycation may include a cationic peptide or a polypeptide such as,
but not limited to, polylysine, polyornithine and/or polyarginine.
In another embodiment, the signal-sensor polynucleotides, primary
constructs and/or mmRNA may be formulated in a lipid-polycation
complex which may further include a neutral lipid such as, but not
limited to, cholesterol or dioleoyl phosphatidylethanolamine
(DOPE).
[0399] The liposome formulation may be influenced by, but not
limited to, the selection of the cationic lipid component, the
degree of cationic lipid saturation, the nature of the PEGylation,
ratio of all components and biophysical parameters such as size. In
one example by Semple et al. (Semple et al. Nature Biotech. 2010
28:172-176), the liposome formulation was composed of 57.1%
cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%
cholesterol, and 1.4% PEG-c-DMA. As another example, changing the
composition of the cationic lipid could more effectively deliver
siRNA to various antigen presenting cells (Basha et al. Mol Ther.
2011 19:2186-2200; herein incorporated by reference in its
entirety).
[0400] In some embodiments, the ratio of PEG in the LNP
formulations may be increased or decreased and/or the carbon chain
length of the PEG lipid may be modified from C14 to C18 to alter
the pharmacokinetics and/or biodistribution of the LNP
formulations. As a non-limiting example, LNP formulations may
contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to
the cationic lipid, DSPC and cholesterol. In another embodiment the
PEG-c-DOMG may be replaced with a PEG lipid such as, but not
limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol,
methoxypolyethylene glycol) or PEG-DPG
(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The
cationic lipid may be selected from any lipid known in the art such
as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and
DLin-KC2-DMA.
[0401] In one embodiment, the LNP formulations of the signal-sensor
polynucleotides, primary constructs and/or mmRNA may contain
PEG-c-DOMG 3% lipid molar ratio. In another embodiment, the LNP
formulations of the signal-sensor polynucleotides, primary
constructs and/or mmRNA may contain PEG-c-DOMG 1.5% lipid molar
ratio.
[0402] In one embodiment, the pharmaceutical compositions of the
signal-sensor polynucleotides, primary constructs and/or mmRNA may
include at least one of the PEGylated lipids described in
International Publication No. 2012099755, herein incorporated by
reference.
[0403] In one embodiment, the pharmaceutical compositions may be
formulated in liposomes such as, but not limited to, DiLa2
liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES.RTM. (Marina
Biotech, Bothell, Wash.), neutral DOPC
(1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,
siRNA delivery for ovarian cancer (Landen et al. Cancer Biology
& Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes
(Quiet Therapeutics, Israel).
[0404] In some embodiments the liposome may be a liposomal
nanostructure which has been formulated for treatment of cancers
and other diseases or to control the cholesterol metabolism in
cells. The liposome nanostructure may also comprise a scavenger
receptor type B-1 (SR-B1) in order to kill cancer cells.
Non-limiting examples of liposomal nanostructures, which may be
used with the signal-sensor polynucleotides described herein, are
described in International Publication No. WO2013126776, the
contents of which are herein incorporated by reference in its
entirety.
[0405] In one embodiment, the liposomes described herein may
comprise at least one immunomodulator such as, but not limited to,
cytokines Formulations and methods of using the liposomes
comprising at least one immunomodulator are described in
International Publication No WO2013129935 and WO2013129936, the
contents of each of which are herein incorporated by reference in
their entirety. As a non-limiting example, the liposomes comprising
at least one immunomodulator may be used in the treatment of
cancer. The liposomes comprising an immunomodulator may comprise a
signal-sensor polynucleotide described herein. As a non-limiting
example, the liposome comprising an immunomodulator may be used in
a combination with at least one antibody such as the particulate or
vesicular immunomodulators described in International Publication
No WO2013129936, the contents of which are herein incorporated by
reference in its entirety.
[0406] Lipid nanoparticle formulations may be improved by replacing
the cationic lipid with a biodegradable cationic lipid which is
known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable
cationic lipids, such as, but not limited to, DLinDMA,
DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in
plasma and tissues over time and may be a potential source of
toxicity. The rapid metabolism of the rapidly eliminated lipids can
improve the tolerability and therapeutic index of the lipid
nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10
mg/kg dose in rat. Inclusion of an enzymatically degraded ester
linkage can improve the degradation and metabolism profile of the
cationic component, while still maintaining the activity of the
reLNP formulation. The ester linkage can be internally located
within the lipid chain or it may be terminally located at the
terminal end of the lipid chain. The internal ester linkage may
replace any carbon in the lipid chain.
[0407] In one embodiment, the internal ester linkage may be located
on either side of the saturated carbon.
[0408] In one embodiment, an immune response may be elicited by
delivering a lipid nanoparticle which may include a nanospecies, a
polymer and an immunogen. (U.S. Publication No. 20120189700 and
International Publication No. WO2012099805; herein incorporated by
reference in their entireties). The polymer may encapsulate the
nanospecies or partially encapsulate the nanospecies. The immunogen
may be a recombinant oncology-related protein, a signal-sensor
modified RNA and/or a primary construct described herein. In one
embodiment, the lipid nanoparticle may be formulated for use in a
vaccine such as, but not limited to, against a pathogen.
[0409] Lipid nanoparticles may be engineered to alter the surface
properties of particles so the lipid nanoparticles may penetrate
the mucosal barrier. Mucus is located on mucosal tissue such as,
but not limted to, oral (e.g., the buccal and esophageal membranes
and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach,
small intestine, large intestine, colon, rectum), nasal,
respiratory (e.g., nasal, pharyngeal, tracheal and bronchial
membranes), genital (e.g., vaginal, cervical and urethral
membranes). Nanoparticles larger than 10-200 nm which are preferred
for higher drug encapsulation efficiency and the ability to provide
the sustained delivery of a wide array of drugs have been thought
to be too large to rapidly diffuse through mucosal barriers. Mucus
is continuously secreted, shed, discarded or digested and recycled
so most of the trapped particles may be removed from the mucosla
tissue within seconds or within a few hours. Large polymeric
nanoparticles (200 nm-500 nm in diameter) which have been coated
densely with a low molecular weight polyethylene glycol (PEG)
diffused through mucus only 4 to 6-fold lower than the same
particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487;
Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; herein
incorporated by reference in their entirety). The transport of
nanoparticles may be determined using rates of permeation and/or
fluorescent microscopy techniques including, but not limited to,
fluorescence recovery after photobleaching (FRAP) and high
resolution multiple particle tracking (MPT).
[0410] The lipid nanoparticle engineered to penetrate mucus may
comprise a polymeric material (i.e. a polymeric core) and/or a
polymer-vitamin conjugate and/or a tri-block co-polymer. The
polymeric material may include, but is not limited to, polyamines,
polyethers, polyamides, polyesters, polycarbamates, polyureas,
polycarbonates, poly(styrenes), polyimides, polysulfones,
polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines,
polyisocyanates, polyacrylates, polymethacrylates,
polyacrylonitriles, and polyarylates. The polymeric material may be
biodegradable and/or biocompatible. Non-limiting examples of
specific polymers include poly(caprolactone) (PCL), ethylene vinyl
acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid)
(PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic
acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA),
poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),
poly(D,L-lactide-co-caprolactone),
poly(D,L-lactide-co-caprolactone-co-glycolide),
poly(D,L-lactide-co-PEO-co-D,L-lactide),
poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,
polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate
(HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy
acids), polyanhydrides, polyorthoesters, poly(ester amides),
polyamides, poly(ester ethers), polycarbonates, polyalkylenes such
as polyethylene and polypropylene, polyalkylene glycols such as
poly(ethylene glycol) (PEG), polyalkylene oxides (PEO),
polyalkylene terephthalates such as poly(ethylene terephthalate),
polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such
as poly(vinyl acetate), polyvinyl halides such as poly(vinyl
chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene
(PS), polyurethanes, derivatized celluloses such as alkyl
celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, hydroxypropylcellulose,
carboxymethylcellulose, polymers of acrylic acids, such as
poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),
poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate),
poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),
poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl
acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),
poly(octadecyl acrylate) and copolymers and mixtures thereof,
polydioxanone and its copolymers, polyhydroxyalkanoates,
polypropylene fumarate, polyoxymethylene, poloxamers,
poly(ortho)esters, poly(butyric acid), poly(valeric acid),
poly(lactide-co-caprolactone), and trimethylene carbonate,
polyvinylpyrrolidone. The lipid nanoparticle may be coated or
associated with a co-polymer such as, but not limited to, a block
co-polymer, and (poly(ethylene glycol))-(poly(propylene
oxide))-(poly(ethylene glycol)) triblock copolymer (see US
Publication 20120121718 and US Publication 20100003337; herein
incorporated by reference in their entireties). The co-polymer may
be a polymer that is generally regarded as safe (GRAS) and the
formation of the lipid nanoparticle may be in such a way that no
new chemical entities are created. For example, the lipid
nanoparticle may comprise poloxamers coating PLGA nanoparticles
without forming new chemical entities which are still able to
rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed.
2011 50:2597-2600; herein incorporated by reference in its
entirety).
[0411] The vitamin of the polymer-vitamin conjugate may be vitamin
E. The vitamin portion of the conjugate may be substituted with
other suitable components such as, but not limited to, vitamin A,
vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a
hydrophobic component of other surfactants (e.g., sterol chains,
fatty acids, hydrocarbon chains and alkylene oxide chains).
[0412] The lipid nanoparticle engineered to penetrate mucus may
include surface altering agents such as, but not limited to,
signal-sensor mmRNA, anionic protein (e.g., bovine serum albumin),
surfactants (e.g., cationic surfactants such as for example
dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives
(e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin,
polyethylene glycol and poloxamer), mucolytic agents (e.g.,
N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,
acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna,
ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,
gelsolin, thymosin (34 dornase alfa, neltenexine, erdosteine) and
various DNases including rhDNase. The surface altering agent may be
embedded or enmeshed in the particle's surface or disposed (e.g.,
by coating, adsorption, covalent linkage, or other process) on the
surface of the lipid nanoparticle. (see US Publication 20100215580
and US Publication 20080166414; herein incorporated by reference in
their entireties).
[0413] The mucus penetrating lipid nanoparticles may comprise at
least one signal-sensor mmRNA described herein. The signal-sensor
mmRNA may be encapsulated in the lipid nanoparticle and/or disposed
on the surface of the paricle. The signal-sensor mmRNA may be
covalently coupled to the lipid nanoparticle. Formulations of mucus
penetrating lipid nanoparticles may comprise a plurality of
nanoparticles. Further, the formulations may contain particles
which may interact with the mucus and alter the structural and/or
adhesive properties of the surrounding mucus to decrease
mucoadhesion which may increase the delivery of the mucus
penetrating lipid nanoparticles to the mucosal tissue.
[0414] Lipid nanoparticles may be engineered to alter the surface
properties of particles so the lipid nanoparticles may penetrate
the mucosal barrier. Mucus is located on mucosal tissue such as,
but not limted to, oral (e.g., the buccal and esophageal membranes
and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach,
small intestine, large intestine, colon, rectum), nasal,
respiratory (e.g., nasal, pharyngeal, tracheal and bronchial
membranes), genital (e.g., vaginal, cervical and urethral
membranes). Nanoparticles larger than 10-200 nm which are preferred
for higher drug encapsulation efficiency and the ability to provide
the sustained delivery of a wide array of drugs have been thought
to be too large to rapidly diffuse through mucosal barriers. Mucus
is continuously secreted, shed, discarded or digested and recycled
so most of the trapped particles may be removed from the mucosla
tissue within seconds or within a few hours. Large polymeric
nanoparticles (200 nm-500 nm in diameter) which have been coated
densely with a low molecular weight polyethylene glycol (PEG)
diffused through mucus only 4 to 6-fold lower than the same
particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487;
Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; herein
incorporated by reference in their entirety). The transport of
nanoparticles may be determined using rates of permeation and/or
fluorescent microscopy techniques including, but not limited to,
fluorescence recovery after photobleaching (FRAP) and high
resolution multiple particle tracking (MPT).
[0415] The lipid nanoparticle engineered to penetrate mucus may
comprise a polymeric material (i.e. a polymeric core) and/or a
polymer-vitamin conjugate and/or a tri-block co-polymer. The
polymeric material may including, but is not limited to,
polyamines, polyethers, polyamides, polyesters, polycarbamates,
polyureas, polycarbonates, poly(styrenes), polyimides,
polysulfones, polyurethanes, polyacetylenes, polyethylenes,
polyethyeneimines, polyisocyanates, polyacrylates,
polymethacrylates, polyacrylonitriles, and polyarylates. The
polymeric material may be biodegradable and/or biocompatible.
Non-limiting examples of specific polymers include
poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA),
poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic
acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),
poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)
(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),
poly(D,L-lactide-co-caprolactone-co-glycolide),
poly(D,L-lactide-co-PEO-co-D,L-lactide),
poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,
polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate
(HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy
acids), polyanhydrides, polyorthoesters, poly(ester amides),
polyamides, poly(ester ethers), polycarbonates, polyalkylenes such
as polyethylene and polypropylene, polyalkylene glycols such as
poly(ethylene glycol) (PEG), polyalkylene oxides (PEO),
polyalkylene terephthalates such as poly(ethylene terephthalate),
polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such
as poly(vinyl acetate), polyvinyl halides such as poly(vinyl
chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene
(PS), polyurethanes, derivatized celluloses such as alkyl
celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, hydroxypropylcellulose,
carboxymethylcellulose, polymers of acrylic acids, such as
poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),
poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate),
poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),
poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl
acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),
poly(octadecyl acrylate) and copolymers and mixtures thereof,
polydioxanone and its copolymers, polyhydroxyalkanoates,
polypropylene fumarate, polyoxymethylene, poloxamers,
poly(ortho)esters, poly(butyric acid), poly(valeric acid),
poly(lactide-co-caprolactone), and trimethylene carbonate,
polyvinylpyrrolidone. The lipid nanoparticle may be coated or
associated with a co-polymer such as, but not limited to, a block
co-polymer, and (poly(ethylene glycol))-(poly(propylene
oxide))-(poly(ethylene glycol)) triblock copolymer (see US
Publication 20120121718 and US Publication 20100003337; herein
incorporated by reference in their entireties).
[0416] The vitamin of the polymer-vitamin conjugate may be vitamin
E. The vitamin portion of the conjugate may be substituted with
other suitable components such as, but not limited to, vitamin A,
vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a
hydrophobic component of other surfactants (e.g., sterol chains,
fatty acids, hydrocarbon chains and alkylene oxide chains).
[0417] The lipid nanoparticle engineered to penetrate mucus may
include surface altering agents such as, but not limited to, mmRNA,
anionic protein (e.g., bovine serum albumin), surfactants (e.g.,
cationic surfactants such as for example
dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives
(e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin,
polyethylene glycol and poloxamer), mucolytic agents (e.g.,
N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,
acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna,
ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,
gelsolin, thymosin (34 dornase alfa, neltenexine, erdosteine) and
various DNases including rhDNase. The surface altering agent may be
embedded or enmeshed in the particle's surface or disposed (e.g.,
by coating, adsorption, covalent linkage, or other process) on the
surface of the lipid nanoparticle. (see US Publication 20100215580
and US Publication 20080166414; herein incorporated by reference in
their entireties).
[0418] The mucus penetrating lipid nanoparticles may comprise at
least one signal-sensor polynucleotide, primary construct, or mmRNA
described herein. The signal-sensor polynucleotide, primary
construct, or mmRNA may be encapsulated in the lipid nanoparticle
and/or disposed on the surface of the paricle. The signal-sensor
polynucleotide, primary construct, or mmRNA may be covalently
coupled to the lipid nanoparticle. Formulations of mucus
penetrating lipid nanoparticles may comprise a plurality of
nanoparticles. Further, the formulations may contain particles
which may interact with the mucus and alter the structural and/or
adhesive properties of the surrounding mucus to decrease
mucoadhesion which may increase the delivery of the mucus
penetrating lipid nanoparticles to the mucosal tissue.
[0419] In one embodiment, the nanoparticle may be for a dual
modality therapy such as described by Mieszawska et al.
(Bioconjugate Chemistry, 2013, 24 (9), pp 1429-1434; the contents
of which is herein incorporated by reference in its entirety)
comprising at least one therapeutic agent (e.g., a signal-sequence
polynucleotide described herein). The therapeutic agent or agents
formulated in the lipid nanoparticle may be an anti-angiogenic and
a cytotoxic agent (see e.g., the polymer-lipid nanoparticles taught
by Mieszawska et al. Bioconjugate Chemistry, 2013, 24 (9), pp
1429-1434; the contents of which is herein incorporated by
reference in its entirety).
[0420] In another embodiment, the nanoparticle may comprise a LyP-1
peptide such as the nanocarrier composition described in
International Patent Publication No. WO2013100869, the contents of
which are herein incorporated by reference in its entirety. The
LyP-1 peptide may be contained in the nanoparticles disclosed
herein, or may be a conjugate, derivative, analogue or pegylated
form of the peptide. In one embodiment, a nanoparticle comprising
the LyP-1 peptide may comprise a signal-sensor polynucleotide and
may be used for cancer treatment and/or imaging.
[0421] In one embodiment, the signal-sensor polynucleotide, primary
construct, or mmRNA is formulated as a lipoplex, such as, without
limitation, the ATUPLEX.TM. system, the DACC system, the DBTC
system and other siRNA-lipoplex technology from Silence
Therapeutics (London, United Kingdom), STEMFECT.TM. from
STEMGENT.RTM. (Cambridge, Mass.), and polyethylenimine (PEI) or
protamine-based targeted and non-targeted delivery of nucleic acids
acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al.
Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther
2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;
Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et
al. Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 2009
32:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo
Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J.
Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,
23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6;
104:4095-4100; deFougerolles Hum Gene Ther. 2008 19:125-132; all of
which are incorporated herein by reference in its entirety).
[0422] In one embodiment such formulations may also be constructed
or compositions altered such that they passively or actively are
directed to different cell types in vivo, including but not limited
to hepatocytes, immune cells, tumor cells, endothelial cells,
antigen presenting cells, and leukocytes (Akinc et al. Mol Ther.
2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717;
Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al.,
Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006
13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier
et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol.
Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv.
2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and
Lieberman, Gene Ther. 2011 18:1127-1133; all of which are
incorporated herein by reference in its entirety). One example of
passive targeting of formulations to liver cells includes the
DLin-DMA, DLin-KC2-DMA and MC3-based lipid nanoparticle
formulations which have been shown to bind to apolipoprotein E and
promote binding and uptake of these formulations into hepatocytes
in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein
incorporated by reference in its entirety). Formulations can also
be selectively targeted through expression of different ligands on
their surface as exemplified by, but not limited by, folate,
transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted
approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011
8:197-206; Musacchio and Torchilin, Front Biosci. 2011
16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et
al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,
Biomacromolecules. 2011 12:2708-2714 Zhao et al., Expert Opin Drug
Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364;
Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et
al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control
Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007
104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;
Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat
Biotechnol. 2005 23:709-717; Peer et al., Science. 2008
319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all
of which are incorporated herein by reference in its entirety).
[0423] In one embodiment, the signal-sensor polynucleotide, primary
construct, or mmRNA is formulated as a solid lipid nanoparticle. A
solid lipid nanoparticle (SLN) may be spherical with an average
diameter between 10 to 1000 nm. SLN possess a solid lipid core
matrix that can solubilize lipophilic molecules and may be
stabilized with surfactants and/or emulsifiers. In a further
embodiment, the lipid nanoparticle may be a self-assembly
lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2
(8), pp 1696-1702; herein incorporated by reference in its
entirety).
[0424] Liposomes, lipoplexes, or lipid nanoparticles may be used to
improve the efficacy of signal-sensor polynucleotide, primary
construct, or mmRNA directed protein production as these
formulations may be able to increase cell transfection by the
signal-sensor polynucleotide, primary construct, or mmRNA; and/or
increase the translation of encoded protein. One such example
involves the use of lipid encapsulation to enable the effective
systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther.
2007 15:713-720; herein incorporated by reference in its entirety).
The liposomes, lipoplexes, or lipid nanoparticles may also be used
to increase the stability of the signal-sensor polynucleotide,
primary construct, or mmRNA.
Polymers, Biodegradable Nanoparticles, and Core-Shell
Nanoparticles
[0425] The signal-sensor polynucleotide, primary construct, and
mmRNA of the invention can be formulated using natural and/or
synthetic polymers. Non-limiting examples of polymers which may be
used for delivery include, but are not limited to, Dynamic
POLYCONJUGATE.TM. formulations from MIRUS.RTM. Bio (Madison, Wis.)
and Roche Madison (Madison, Wis.), PHASERX.TM. polymer formulations
such as, without limitation, SMARTT POLYMER TECHNOLOGY.TM.
(Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN.RTM. adjuvant
from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando
Pharmaceuticals (Pasadena, Calif.), dendrimers and
poly(lactic-co-glycolic acid) (PLGA) polymers. RONDEL.TM.
(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead
Research Corporation, Pasadena, Calif.) and pH responsive co-block
polymers such as, but not limited to, PHASERX.TM. (Seattle,
Wash.).
[0426] A non-limiting example of PLGA formulations include, but are
not limited to, PLGA injectable depots (e.g., ELIGARD.RTM. which is
formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and
the remainder being aqueous solvent and leuprolide. Once injected,
the PLGA and leuprolide peptide precipitates into the subcutaneous
space).
[0427] Many of these polymer approaches have demonstrated efficacy
in delivering oligonucleotides in vivo into the cell cytoplasm
(reviewed in deFougerolles Hum Gene Ther. 2008 19:125-132; herein
incorporated by reference in its entirety). Two polymer approaches
that have yielded robust in vivo delivery of nucleic acids, in this
case with small interfering RNA (siRNA), are dynamic polyconjugates
and cyclodextrin-based nanoparticles. The first of these delivery
approaches uses dynamic polyconjugates and has been shown in vivo
in mice to effectively deliver siRNA and silence endogenous target
mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007
104:12982-12887). This particular approach is a multicomponent
polymer system whose key features include a membrane-active polymer
to which nucleic acid, in this case siRNA, is covalently coupled
via a disulfide bond and where both PEG (for charge masking) and
N-acetylgalactosamine (for hepatocyte targeting) groups are linked
via pH-sensitive bonds (Rozema et al., Proc Natl Acad Sci USA. 2007
104:12982-12887). On binding to the hepatocyte and entry into the
endosome, the polymer complex disassembles in the low-pH
environment, with the polymer exposing its positive charge, leading
to endosomal escape and cytoplasmic release of the siRNA from the
polymer. Through replacement of the N-acetylgalactosamine group
with a mannose group, it was shown one could alter targeting from
asialoglycoprotein receptor-expressing hepatocytes to sinusoidal
endothelium and Kupffer cells. Another polymer approach involves
using transferrin-targeted cyclodextrin-containing polycation
nanoparticles. These nanoparticles have demonstrated targeted
silencing of the EWS-FLI1 gene product in transferrin
receptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et
al., Cancer Res. 2005 65: 8984-8982) and siRNA formulated in these
nanoparticles was well tolerated in non-human primates (Heidel et
al., Proc Natl Acad Sci USA 2007 104:5715-21). Both of these
delivery strategies incorporate rational approaches using both
targeted delivery and endosomal escape mechanisms.
[0428] The polymer formulation can permit the sustained or delayed
release of signal-sensor polynucleotide, primary construct, or
mmRNA (e.g., following intramuscular or subcutaneous injection).
The altered release profile for the signal-sensor polynucleotide,
primary construct, or mmRNA can result in, for example, translation
of an encoded protein over an extended period of time. The polymer
formulation may also be used to increase the stability of the
signal-sensor polynucleotide, primary construct, or mmRNA.
Biodegradable polymers have been previously used to protect nucleic
acids other than mmRNA from degradation and been shown to result in
sustained release of payloads in vivo (Rozema et al., Proc Natl
Acad Sci USA. 2007 104:12982-12887; Sullivan et al., Expert Opin
Drug Deliv. 2010 7:1433-1446; Convertine et al., Biomacromolecules.
2010 Oct. 1; Chu et al., Acc Chem Res. 2012 Jan. 13; Manganiello et
al., Biomaterials. 2012 33:2301-2309; Benoit et al.,
Biomacromolecules. 2011 12:2708-2714; Singha et al., Nucleic Acid
Ther. 2011 2:133-147; deFougerolles Hum Gene Ther. 2008 19:125-132;
Schaffert and Wagner, Gene Ther. 2008 16:1131-1138; Chaturvedi et
al., Expert Opin Drug Deliv. 2011 8:1455-1468; Davis, Mol Pharm.
2009 6:659-668; Davis, Nature 2010 464:1067-1070; herein
incorporated by reference in its entirety).
[0429] In one embodiment, the pharmaceutical compositions may be
sustained release formulations. In a further embodiment, the
sustained release formulations may be for subcutaneous delivery.
Sustained release formulations may include, but are not limited to,
PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer,
GELSITE.RTM. (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX.RTM.
(Halozyme Therapeutics, San Diego Calif.), surgical sealants such
as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.). TISSELL.RTM.
(Baxter International, Inc Deerfield, Ill.), PEG-based sealants,
and COSEAL.RTM. (Baxter International, Inc Deerfield, Ill.).
[0430] As a non-limiting example modified mRNA may be formulated in
PLGA microspheres by preparing the PLGA microspheres with tunable
release rates (e.g., days and weeks) and encapsulating the
signal-sensor modified mRNA in the PLGA microspheres while
maintaining the integrity of the signal-sensor modified mRNA during
the encapsulation process. EVAc are non-biodegradeable,
biocompatible polymers which are used extensively in pre-clinical
sustained release implant applications (e.g., extended release
products Ocusert a pilocarpine ophthalmic insert for glaucoma or
progestasert a sustained release progesterone intrauterine deivce;
transdermal delivery systems Testoderm, Duragesic and Selegiline;
catheters). Poloxamer F-407 NF is a hydrophilic, non-ionic
surfactant triblock copolymer of
polyoxyethylene-polyoxypropylene-polyoxyethylene having a low
viscosity at temperatures less than 5.degree. C. and forms a solid
gel at temperatures greater than 15.degree. C. PEG-based surgical
sealants comprise two synthetic PEG components mixed in a delivery
device which can be prepared in one minute, seals in 3 minutes and
is reabsorbed within 30 days. GELSITE.RTM. and natural polymers are
capable of in-situ gelation at the site of administration. They
have been shown to interact with protein and peptide therapeutic
candidates through ionic ineraction to provide a stabilizing
effect.
[0431] Polymer formulations can also be selectively targeted
through expression of different ligands as exemplified by, but not
limited by, folate, transferrin, and N-acetylgalactosamine (GalNAc)
(Benoit et al., Biomacromolecules. 2011 12:2708-2714; Rozema et
al., Proc Natl Acad Sci USA. 2007 104:12982-12887; Davis, Mol
Pharm. 2009 6:659-668; Davis, Nature 2010 464:1067-1070; herein
incorporated by reference in its entirety).
[0432] The signal-sensor mmRNA of the invention may be formulated
with or in a polymeric compound. The polymer may include at least
one polymer such as, but not limited to, polyethylene glycol (PEG),
poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,
biodegradable cationic lipopolymer, polyethyleneimine (PEI),
cross-linked branched poly(alkylene imines), a polyamine
derivative, a modified poloxamer, a biodegradable polymer,
biodegradable block copolymer, biodegradable random copolymer,
biodegradable polyester copolymer, biodegradable polyester block
copolymer, biodegradable polyester block random copolymer, linear
biodegradable copolymer, poly[.alpha.-(4-aminobutyl)-L-glycolic
acid) (PAGA), biodegradable cross-linked cationic multi-block
copolymers or combinations thereof.
[0433] As a non-limiting example, the signal-sensor mmRNA of the
invention may be formulated with the polymeric compound of PEG
grafted with PLL as described in U.S. Pat. No. 6,177,274 herein
incorporated by reference in its entirety. The formulation may be
used for transfecting cells in vitro or for in vivo delivery of the
signal-sensor mmRNA. In another example, the signal-sensor mmRNA
may be suspended in a solution or medium with a cationic polymer,
in a dry pharmaceutical composition or in a solution that is
capable of being dried as described in U.S. Pub. Nos. 20090042829
and 20090042825 each of which are herein incorporated by reference
in their entireties.
[0434] A polyamine derivative may be used to deliver nucleic acids
or to treat and/or prevent a disease or to be included in an
implantable or injectable device (U.S. Pub. No. 20100260817 herein
incorporated by reference in its entirety). As a non-limiting
example, a pharmaceutical composition may include the signal-sensor
mmRNA and the polyamine derivative described in U.S. Pub. No.
20100260817 (the contents of which are incorporated herein by
reference in its entirety.
[0435] For example, the signal-sensor mmRNA of the invention may be
formulated in a pharmaceutical compound including a poly(alkylene
imine), a biodegradable cationic lipopolymer, a biodegradable block
copolymer, a biodegradable polymer, or a biodegradable random
copolymer, a biodegradable polyester block copolymer, a
biodegradable polyester polymer, a biodegradable polyester random
copolymer, a linear biodegradable copolymer, PAGA, a biodegradable
cross-linked cationic multi-block copolymer or combinations
thereof. The biodegradable cationic lipopolymer may be made my
methods known in the art and/or described in U.S. Pat. No.
6,696,038, U.S. App. Nos. 20030073619 and 20040142474 which is
herein incorporated by reference in their entireties. The
poly(alkylene imine) may be made using methods known in the art
and/or as described in U.S. Pub. No. 20100004315, herein
incorporated by reference in its entirety. The biodegradabale
polymer, biodegradable block copolymer, the biodegradable random
copolymer, biodegradable polyester block copolymer, biodegradable
polyester polymer, or biodegradable polyester random copolymer may
be made using methods known in the art and/or as described in U.S.
Pat. Nos. 6,517,869 and 6,267,987, the contents of which are each
incorporated herein by reference in its entirety. The linear
biodegradable copolymer may be made using methods known in the art
and/or as described in U.S. Pat. No. 6,652,886. The PAGA polymer
may be made using methods known in the art and/or as described in
U.S. Pat. No. 6,217,912 herein incorporated by reference in its
entirety. The PAGA polymer may be copolymerized to form a copolymer
or block copolymer with polymers such as but not limited to,
poly-L-lysine, polyargine, polyornithine, histones, avidin,
protamines, polylactides and poly(lactide-co-glycolides). The
biodegradable cross-linked cationic multi-block copolymers may be
made my methods known in the art and/or as described in U.S. Pat.
No. 8,057,821 or U.S. Pub. No. 2012009145 herein incorporated by
reference in their entireties. For example, the multi-block
copolymers may be synthesized using linear polyethyleneimine (LPEI)
blocks which have distinct patterns as compared to branched
polyethyleneimines. Further, the composition or pharmaceutical
composition may be made by the methods known in the art, described
herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat.
Nos. 6,267,987 and 6,217,912 herein incorporated by reference in
their entireties.
[0436] As described in U.S. Pub. No. 20100004313, herein
incorporated by reference in its entirety, a gene delivery
composition may include a nucleotide sequence and a poloxamer. For
example, the signal-sensor mmRNA of the present inveition may be
used in a gene delivery composition with the poloxamer described in
U.S. Pub. No. 20100004313.
[0437] In one embodiment, the polymer formulation of the present
invention may be stabilized by contacting the polymer formulation,
which may include a cationic carrier, with a cationic lipopolymer
which may be covalently linked to cholesterol and polyethylene
glycol groups. The polymer formulation may be contacted with a
cationic lipopolymer using the methods described in U.S. Pub. No.
20090042829 herein incorporated by reference in its entirety. The
cationic carrier may include, but is not limited to,
polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine),
polypropylenimine, aminoglycoside-polyamine,
dideoxy-diamino-b-cyclodextrin, spermine, spermidine,
poly(2-dimethylamino)ethyl methacrylate, poly(lysine),
poly(histidine), poly(arginine), cationized gelatin, dendrimers,
chitosan, 1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP),
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA),
1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium
chloride (DOTIM),
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-pr-
opanaminium trifluoroacetate (DOSPA),
3B--[N--(N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol
Hydrochloride (DC-Cholesterol HCl) diheptadecylamidoglycyl
spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide
(DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl
ammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride
DODAC) and combinations thereof.
[0438] The signal-sensor polynucleotide, primary construct, and
mmRNA of the invention can also be formulated as a nanoparticle
using a combination of polymers, lipids, and/or other biodegradable
agents, such as, but not limited to, calcium phosphate. Components
may be combined in a core-shell, hybrid, and/or layer-by-layer
architecture, to allow for fine-tuning of the nanoparticle so to
delivery of the signal-sensor polynucleotide, primary construct and
mmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796;
Fuller et al., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv
Drug Deliv Rev. 2011 63:748-761; Endres et al., Biomaterials. 2011
32:7721-7731; Su et al., Mol Pharm. 2011 Jun. 6; 8(3):774-87;
herein incorporated by reference in its entirety).
[0439] Biodegradable calcium phosphate nanoparticles in combination
with lipids and/or polymers have been shown to deliver
signal-sensor polynucleotides, primary constructs and mmRNA in
vivo. In one embodiment, a lipid coated calcium phosphate
nanoparticle, which may also contain a targeting ligand such as
anisamide, may be used to deliver the signal-sensor polynucleotide,
primary construct and mmRNA of the present invention. For example,
to effectively deliver siRNA in a mouse metastatic lung model a
lipid coated calcium phosphate nanoparticle was used (Li et al., J
Contr Rel. 2010 142: 416-421; Li et al., J Contr Rel. 2012
158:108-114; Yang et al., Mol Ther. 2012 20:609-615). This delivery
system combines both a targeted nanoparticle and a component to
enhance the endosomal escape, calcium phosphate, in order to
improve delivery of the siRNA.
[0440] In one embodiment, calcium phosphate with a PEG-polyanion
block copolymer may be used to deliver signal-sensor
polynucleotides, primary constructs and mmRNA (Kazikawa et al., J
Contr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006
111:368-370).
[0441] In one embodiment, a PEG-charge-conversional polymer
(Pitella et al., Biomaterials. 2011 32:3106-3114) may be used to
form a nanoparticle to deliver the signal-sensor polynucleotides,
primary constructs and mmRNA of the present invention. The
PEG-charge-conversional polymer may improve upon the PEG-polyanion
block copolymers by being cleaved into a polycation at acidic pH,
thus enhancing endosomal escape.
[0442] The use of core-shell nanoparticles has additionally focused
on a high-throughput approach to synthesize cationic cross-linked
nanogel cores and various shells (Siegwart et al., Proc Natl Acad
Sci USA. 2011 108:12996-13001). The complexation, delivery, and
internalization of the polymeric nanoparticles can be precisely
controlled by altering the chemical composition in both the core
and shell components of the nanoparticle. For example, the
core-shell nanoparticles may efficiently deliver siRNA to mouse
hepatocytes after they covalently attach cholesterol to the
nanoparticle.
[0443] In one embodiment, a hollow lipid core comprising a middle
PLGA layer and an outer neutral lipid layer containg PEG may be
used to delivery of the signal-sensor polynucleotide, primary
construct and mmRNA of the present invention. As a non-limiting
example, in mice bearing a luciferease-expressing tumor, it was
determined that the lipid-polymer-lipid hybrid nanoparticle
significantly suppressed luciferase expression, as compared to a
conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011
50:7027-7031).
Peptides and Proteins
[0444] The signal-sensor polynucleotide, primary construct, and
mmRNA of the invention can be formulated with peptides and/or
proteins in order to increase transfection of cells by the
polynucleotide, primary construct, or mmRNA. In one embodiment,
peptides such as, but not limited to, cell penetrating peptides and
proteins and peptides that enable intracellular delivery may be
used to deliver pharmaceutical formulations. A non-limiting example
of a cell penetrating peptide which may be used with the
pharmaceutical formulations of the present invention includes a
cell-penetrating peptide sequence attached to polycations that
facilitates delivery to the intracellular space, e.g., HIV-derived
TAT peptide, penetratins, transportans, or hCT derived
cell-penetrating peptides (see, e.g., Caron et al., Mol. Ther.
3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and
Applications (CRC Press, Boca Raton Fla., 2002); El-Andaloussi et
al., Curr. Pharm. Des. 11(28):3597-611 (2003); and Deshayes et al.,
Cell. Mol. Life Sci. 62(16):1839-49 (2005), all of which are
incorporated herein by reference). The compositions can also be
formulated to include a cell penetrating agent, e.g., liposomes,
which enhance delivery of the compositions to the intracellular
space. signal-sensor polynucleotides, primary constructs, and mmRNA
of the invention may be complexed to peptides and/or proteins such
as, but not limited to, peptides and/or proteins from Aileron
Therapeutics (Cambridge, Mass.) and Permeon Biologics (Cambridge,
Mass.) in order to enable intracellular delivery (Cronican et al.,
ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl.
Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 2009
73:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all
of which are herein incorporated by reference in its entirety).
[0445] In one embodiment, the cell-penetrating polypeptide may
comprise a first domain and a second domain. The first domain may
comprise a supercharged polypeptide. The second domain may comprise
a protein-binding partner. As used herein, "protein-binding
partner" includes, but are not limited to, antibodies and
functional fragments thereof, scaffold proteins, or peptides. The
cell-penetrating polypeptide may further comprise an intracellular
binding partner for the protein-binding partner. The
cell-penetrating polypeptide may be capable of being secreted from
a cell where the signal-sensor polynucleotide, primary construct,
or mmRNA may be introduced.
[0446] Formulations of the including peptides or proteins may be
used to increase cell transfection by the signal-sensor
polynucleotide, primary construct, or mmRNA, alter the
biodistribution of the signal-sensor polynucleotide, primary
construct, or mmRNA (e.g., by targeting specific tissues or cell
types), and/or increase the translation of encoded protein.
Cells
[0447] The signal-sensor polynucleotide, primary construct, and
mmRNA of the invention can be transfected ex vivo into cells, which
are subsequently transplanted into a subject. As non-limiting
examples, the pharmaceutical compositions may include red blood
cells to deliver modified RNA to liver and myeloid cells, virosomes
to deliver modified RNA in virus-like particles (VLPs), and
electroporated cells such as, but not limited to, from MAXCYTE.RTM.
(Gaithersburg, Md.) and from ERYTECH.RTM. (Lyon, France) to deliver
modified RNA. Examples of use of red blood cells, viral particles
and electroporated cells to deliver payloads other than mmRNA have
been documented (Godfrin et al., Expert Opin Biol Ther. 2012
12:127-133; Fang et al., Expert Opin Biol Ther. 2012 12:385-389; Hu
et al., Proc Natl Acad Sci USA. 2011 108:10980-10985; Lund et al.,
Pharm Res. 2010 27:400-420; Huckriede et al., J Liposome Res. 2007;
17:39-47; Cusi, Hum Vaccin. 2006 2:1-7; de Jonge et al., Gene Ther.
2006 13:400-411; all of which are herein incorporated by reference
in its entirety).
[0448] Cell-based formulations of the signal-sensor polynucleotide,
primary construct, and mmRNA of the invention may be used to ensure
cell transfection (e.g., in the cellular carrier), alter the
biodistribution of the signal-sensor polynucleotide, primary
construct, or mmRNA (e.g., by targeting the cell carrier to
specific tissues or cell types), and/or increase the translation of
encoded oncology-related protein.
[0449] A variety of methods are known in the art and suitable for
introduction of nucleic acid into a cell, including viral and
non-viral mediated techniques. Examples of typical non-viral
mediated techniques include, but are not limited to,
electroporation, calcium phosphate mediated transfer,
nucleofection, sonoporation, heat shock, magnetofection, liposome
mediated transfer, microinjection, microproj ectile mediated
transfer (nanoparticles), cationic polymer mediated transfer
(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the
like) or cell fusion.
[0450] The technique of sonoporation, or cellular sonication, is
the use of sound (e.g., ultrasonic frequencies) for modifying the
permeability of the cell plasma membrane. Sonoporation methods are
known to those in the art and are used to deliver nucleic acids in
vivo (Yoon and Park, Expert Opin Drug Deliv. 2010 7:321-330;
Postema and Gilja, Curr Pharm Biotechnol. 2007 8:355-361; Newman
and Bettinger, Gene Ther. 2007 14:465-475; all herein incorporated
by reference in their entirety). Sonoporation methods are known in
the art and are also taught for example as it relates to bacteria
in US Patent Publication 20100196983 and as it relates to other
cell types in, for example, US Patent Publication 20100009424, each
of which are incorporated herein by reference in their
entirety.
[0451] Electroporation techniques are also well known in the art
and are used to deliver nucleic acids in vivo and clinically (Andre
et al., Curr Gene Ther. 2010 10:267-280; Chiarella et al., Curr
Gene Ther. 2010 10:281-286; Hojman, Curr Gene Ther. 2010
10:128-138; all herein incorporated by reference in their
entirety). In one embodiment, signal-sensor polynucleotides,
primary constructs or mmRNA may be delivered by electroporation as
described in Example 12.
Hyaluronidase
[0452] The intramuscular or subcutaneous localized injection of
signal-sensor polynucleotide, primary construct, or mmRNA of the
invention can include hyaluronidase, which catalyzes the hydrolysis
of hyaluronan. By catalyzing the hydrolysis of hyaluronan, a
constituent of the interstitial barrier, hyaluronidase lowers the
viscosity of hyaluronan, thereby increasing tissue permeability
(Frost, Expert Opin. Drug Deliv. (2007) 4:427-440; herein
incorporated by reference in its entirety). It is useful to speed
their dispersion and systemic distribution of encoded proteins
produced by transfected cells. Alternatively, the hyaluronidase can
be used to increase the number of cells exposed to a signal-sensor
polynucleotide, primary construct, or mmRNA of the invention
administered intramuscularly or subcutaneously.
Nanoparticle Mimics
[0453] The signal-sensor polynucleotide, primary construct or mmRNA
of the invention may be encapsulated within and/or absorbed to a
nanoparticle mimic. A nanoparticle mimic can mimic the delivery
function organisms or particles such as, but not limited to,
pathogens, viruses, bacteria, fungus, parasites, prions and cells.
As a non-limiting example the signal-sensor polynucleotide, primary
construct or mmRNA of the invention may be encapsulated in a
non-viron particle which can mimic the delivery function of a virus
(see International Pub. No. WO2012006376 herein incorporated by
reference in its entirety).
Nanotubes
[0454] The signal-sensor polynucleotides, primary constructs or
mmRNA of the invention can be attached or otherwise bound to at
least one nanotube such as, but not limited to, rosette nanotubes,
rosette nanotubes having twin bases with a linker, carbon nanotubes
and/or single-walled carbon nanotubes, The signal-sensor
polynucleotides, primary constructs or mmRNA may be bound to the
nanotubes through forces such as, but not limited to, steric,
ionic, covalent and/or other forces.
[0455] In one embodiment, the nanotube can release one or more
signal-sensor polynucleotides, primary constructs or mmRNA into
cells. The size and/or the surface structure of at least one
nanotube may be altered so as to govern the interaction of the
nanotubes within the body and/or to attach or bind to the
signal-sensor polynucleotides, primary constructs or mmRNA
disclosed herein. In one embodiment, the building block and/or the
functional groups attached to the building block of the at least
one nanotube may be altered to adjust the dimensions and/or
properties of the nanotube. As a non-limiting example, the length
of the nanotubes may be altered to hinder the nanotubes from
passing through the holes in the walls of normal blood vessels but
still small enough to pass through the larger holes in the blood
vessels of tumor tissue.
[0456] In one embodiment, at least one nanotube may also be coated
with delivery enhancing compounds including polymers, such as, but
not limited to, polyethylene glycol. In another embodiment, at
least one nanotube and/or the signal-sensor polynucleotides,
primary constructs or mmRNA may be mixed with pharmaceutically
acceptable excipients and/or delivery vehicles.
[0457] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA are attached and/or otherwise bound to
at least one rosette nanotube. The rosette nanotubes may be formed
by a process known in the art and/or by the process described in
International Publication No. WO2012094304, herein incorporated by
reference in its entirety. At least one signal-sensor
polynucleotide, primary construct and/or mmRNA may be attached
and/or otherwise bound to at least one rosette nanotube by a
process as described in International Publication No. WO2012094304,
herein incorporated by reference in its entirety, where rosette
nanotubes or modules forming rosette nanotubes are mixed in aqueous
media with at least one signal-sensor polynucleotide, primary
construct and/or mmRNA under conditions which may cause at least
one signal-sensor polynucleotide, primary construct or mmRNA to
attach or otherwise bind to the rosette nanotubes.
Conjugates
[0458] The signal-sensor polynucleotides, primary constructs, and
mmRNA of the invention include conjugates, such as a
polynucleotide, primary construct, or mmRNA covalently linked to a
carrier or targeting group, or including two encoding regions that
together produce a fusion protein (e.g., bearing a targeting group
and therapeutic protein or peptide).
[0459] The conjugates of the invention include a naturally
occurring substance, such as a protein (e.g., human serum albumin
(HSA), low-density lipoprotein (LDL), high-density lipoprotein
(HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan,
chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a
lipid. The ligand may also be a recombinant or synthetic molecule,
such as a synthetic polymer, e.g., a synthetic polyamino acid, an
oligonucleotide (e.g. an aptamer). Examples of polyamino acids
include polyamino acid is a polylysine (PLL), poly L-aspartic acid,
poly L-glutamic acid, styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, or polyphosphazine. Example of polyamines include:
polyethylenimine, polylysine (PLL), spermine, spermidine,
polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine, arginine, amidine, protamine, cationic lipid,
cationic porphyrin, quaternary salt of a polyamine, or an alpha
helical peptide.
[0460] Representative U.S. patents that teach the preparation of
polynucleotide conjugates, particularly to RNA, include, but are
not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;
4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;
4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;
5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;
5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928
and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;
6,900,297; 7,037,646; each of which is herein incorporated by
reference in their entirety.
[0461] In one embodiment, the conjugate of the present invention
may function as a carrier for the signal-sensor mmRNA of the
present invention. The conjugate may comprise a cationic polymer
such as, but not limited to, polyamine, polylysine,
polyalkylenimine, and polyethylenimine which may be grafted to with
poly(ethylene glycol). As a non-limiting example, the conjugate may
be similar to the polymeric conjugate and the method of
synthesizing the polymeric conjugate described in U.S. Pat. No.
6,586,524 herein incorporated by reference in its entirety.
[0462] The conjugates can also include targeting groups, e.g., a
cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid
or protein, e.g., an antibody, that binds to a specified cell type
such as a kidney cell. A targeting group can be a thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin
carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent
galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,
vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an
aptamer.
[0463] Targeting groups can be proteins, e.g., glycoproteins, or
peptides, e.g., molecules having a specific affinity for a
co-ligand, or antibodies e.g., an antibody, that binds to a
specified cell type such as a cancer cell, endothelial cell, or
bone cell. Targeting groups may also include hormones and hormone
receptors. They can also include non-peptidic species, such as
lipids, lectins, carbohydrates, vitamins, cofactors, multivalent
lactose, multivalent galactose, N-acetyl-galactosamine,
N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or
aptamers. The ligand can be, for example, a lipopolysaccharide, or
an activator of p38 MAP kinase.
[0464] The targeting group can be any ligand that is capable of
targeting a specific receptor. Examples include, without
limitation, folate, GalNAc, galactose, mannose, mannose-6P,
apatamers, integrin receptor ligands, chemokine receptor ligands,
transferrin, biotin, serotonin receptor ligands, PSMA, endothelin,
GCPII, somatostatin, LDL, and HDL ligands. In particular
embodiments, the targeting group is an aptamer. The aptamer can be
unmodified or have any combination of modifications disclosed
herein.
[0465] In one embodiment, pharmaceutical compositions of the
present invention may include chemical modifications such as, but
not limited to, modifications similar to locked nucleic acids.
[0466] Representative U.S. Patents that teach the preparation of
locked nucleic acid (LNA) such as those from Santaris, include, but
are not limited to, the following: U.S. Pat. Nos. 6,268,490;
6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and
7,399,845, each of which is herein incorporated by reference in its
entirety.
[0467] Representative U.S. patents that teach the preparation of
PNA compounds include, but are not limited to, U.S. Pat. Nos.
5,539,082; 5,714,331; and 5,719,262, each of which is herein
incorporated by reference. Further teaching of PNA compounds can be
found, for example, in Nielsen et al., Science, 1991, 254,
1497-1500.
[0468] Some embodiments featured in the invention include
signal-sensor polynucleotides, primary constructs or mmRNA with
phosphorothioate backbones and oligonucleosides with other modified
backbones, and in particular --CH.sub.2--NH--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as
--O--P(O).sub.2--O--CH.sub.2--] of the above-referenced U.S. Pat.
No. 5,489,677, and the amide backbones of the above-referenced U.S.
Pat. No. 5,602,240. In some embodiments, the polynucletotides
featured herein have morpholino backbone structures of the
above-referenced U.S. Pat. No. 5,034,506.
[0469] Modifications at the 2' position may also aid in delivery.
Preferably, modifications at the 2' position are not located in a
polypeptide-coding sequence, i.e., not in a translatable region.
Modifications at the 2' position may be located in a 5'UTR, a 3'UTR
and/or a tailing region. Modifications at the 2' position can
include one of the following at the 2' position: H (i.e.,
2'-deoxy); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or
N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Exemplary
suitable modifications include O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2)..sub.nOH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
I(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. In other embodiments, the signal-sensor
polynucleotides, primary constructs or mmRNA include one of the
following at the 2' position: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties, or a group for improving the
pharmacodynamic properties, and other substituents having similar
properties. In some embodiments, the modification includes a
2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary
modification is 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples herein below, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples herein below. Other modifications include 2'-methoxy
(2'-OCH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions,
particularly the 3' position of the sugar on the 3' terminal
nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5'
terminal nucleotide. signal-sensor polynucleotides of the invention
may also have sugar mimetics such as cyclobutyl moieties in place
of the pentofuranosyl sugar. Representative U.S. patents that teach
the preparation of such modified sugar structures include, but are
not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;
5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each
of which is herein incorporated by reference.
[0470] In still other embodiments, the signal-sensor
polynucleotide, primary construct, or mmRNA is covalently
conjugated to a cell penetrating polypeptide. The cell-penetrating
peptide may also include a signal peptide sequence. The conjugates
of the invention can be designed to have increased stability;
increased cell transfection; and/or altered the biodistribution
(e.g., targeted to specific tissues or cell types).
Self-Assembled Nucleic Acid Nanoparticles
[0471] Self-assembled nanoparticles have a well-defined size which
may be precisely controlled as the nucleic acid strands may be
easily reprogrammable. For example, the optimal particle size for a
cancer-targeting nanodelivery carrier is 20-100 nm as a diameter
greater than 20 nm avoids renal clearance and enhances delivery to
certain tumors through enhanced permeability and retention effect.
Using self-assembled nucleic acid nanoparticles a single uniform
population in size and shape having a precisely controlled spatial
orientation and density of cancer-targeting ligands for enhanced
delivery. As a non-limiting example, oligonucleotide nanoparticles
were prepared using programmable self-assembly of short DNA
fragments and therapeutic siRNAs. These nanoparticles are
molecularly identical with controllable particle size and target
ligand location and density. The DNA fragments and siRNAs
self-assembled into a one-step reaction to generate DNA/siRNA
tetrahedral nanoparticles for targeted in vivo delivery. (Lee et
al., Nature Nanotechnology 2012 7:389-393).
Excipients
[0472] Pharmaceutical formulations may additionally comprise a
pharmaceutically acceptable excipient, which, as used herein,
includes any and all solvents, dispersion media, diluents, or other
liquid vehicles, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's The Science and
Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro (Lippincott,
Williams & Wilkins, Baltimore, Md., 2006; incorporated herein
by reference) discloses various excipients used in formulating
pharmaceutical compositions and known techniques for the
preparation thereof. Except insofar as any conventional excipient
medium is incompatible with a substance or its derivatives, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutical composition, its use is contemplated to be
within the scope of this invention.
[0473] In some embodiments, a pharmaceutically acceptable excipient
is at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% pure. In some embodiments, an excipient is approved
for use in humans and for veterinary use. In some embodiments, an
excipient is approved by United States Food and Drug
Administration. In some embodiments, an excipient is pharmaceutical
grade. In some embodiments, an excipient meets the standards of the
United States Pharmacopoeia (USP), the European Pharmacopoeia (EP),
the British Pharmacopoeia, and/or the International
Pharmacopoeia.
[0474] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inert diluents, dispersing and/or granulating agents,
surface active agents and/or emulsifiers, disintegrating agents,
binding agents, preservatives, buffering agents, lubricating
agents, and/or oils. Such excipients may optionally be included in
pharmaceutical compositions.
[0475] Exemplary diluents include, but are not limited to, calcium
carbonate, sodium carbonate, calcium phosphate, dicalcium
phosphate, calcium sulfate, calcium hydrogen phosphate, sodium
phosphate lactose, sucrose, cellulose, microcrystalline cellulose,
kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch,
cornstarch, powdered sugar, etc., and/or combinations thereof.
[0476] Exemplary granulating and/or dispersing agents include, but
are not limited to, potato starch, corn starch, tapioca starch,
sodium starch glycolate, clays, alginic acid, guar gum, citrus
pulp, agar, bentonite, cellulose and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(VEEGUM.RTM.), sodium lauryl sulfate, quaternary ammonium
compounds, etc., and/or combinations thereof.
[0477] Exemplary surface active agents and/or emulsifiers include,
but are not limited to, natural emulsifiers (e.g. acacia, agar,
alginic acid, sodium alginate, tragacanth, chondrux, cholesterol,
xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol,
wax, and lecithin), colloidal clays (e.g. bentonite [aluminum
silicate] and VEEGUM.RTM. [magnesium aluminum silicate]), long
chain amino acid derivatives, high molecular weight alcohols (e.g.
stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin
monostearate, ethylene glycol distearate, glyceryl monostearate,
and propylene glycol monostearate, polyvinyl alcohol), carbomers
(e.g. carboxy polymethylene, polyacrylic acid, acrylic acid
polymer, and carboxyvinyl polymer), carrageenan, cellulosic
derivatives (e.g. carboxymethylcellulose sodium, powdered
cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty
acid esters (e.g. polyoxyethylene sorbitan monolaurate
[TWEEN.RTM.20], polyoxyethylene sorbitan [TWEENn.RTM.60],
polyoxyethylene sorbitan monooleate [TWEEN.RTM.80], sorbitan
monopalmitate [SPAN.RTM.40], sorbitan monostearate [Span.RTM.60],
sorbitan tristearate [Span.RTM.65], glyceryl monooleate, sorbitan
monooleate [SPAN.RTM.80]), polyoxyethylene esters (e.g.
polyoxyethylene monostearate [MYRJ.RTM.45], polyoxyethylene
hydrogenated castor oil, polyethoxylated castor oil,
polyoxymethylene stearate, and SOLUTOL.RTM.), sucrose fatty acid
esters, polyethylene glycol fatty acid esters (e.g.
CREMOPHOR.RTM.), polyoxyethylene ethers, (e.g. polyoxyethylene
lauryl ether [BRIJ.degree. 30]), poly(vinyl-pyrrolidone),
diethylene glycol monolaurate, triethanolamine oleate, sodium
oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate,
sodium lauryl sulfate, PLUORINC.RTM.F 68, POLOXAMER.RTM. 188,
cetrimonium bromide, cetylpyridinium chloride, benzalkonium
chloride, docusate sodium, etc. and/or combinations thereof.
[0478] Exemplary binding agents include, but are not limited to,
starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g.
sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol,
mannitol); natural and synthetic gums (e.g. acacia, sodium
alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage
of isapol husks, carboxymethylcellulose, methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,
hydroxypropyl methylcellulose, microcrystalline cellulose,
cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum
silicate (VEEGUM.RTM.), and larch arabogalactan); alginates;
polyethylene oxide; polyethylene glycol; inorganic calcium salts;
silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and
combinations thereof
[0479] Exemplary preservatives may include, but are not limited to,
antioxidants, chelating agents, antimicrobial preservatives,
antifungal preservatives, alcohol preservatives, acidic
preservatives, and/or other preservatives. Exemplary antioxidants
include, but are not limited to, alpha tocopherol, ascorbic acid,
acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and/or sodium sulfite. Exemplary chelating
agents include ethylenediaminetetraacetic acid (EDTA), citric acid
monohydrate, disodium edetate, dipotassium edetate, edetic acid,
fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric
acid, and/or trisodium edetate. Exemplary antimicrobial
preservatives include, but are not limited to, benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and/or thimerosal.
Exemplary antifungal preservatives include, but are not limited to,
butyl paraben, methyl paraben, ethyl paraben, propyl paraben,
benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium
sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
Exemplary alcohol preservatives include, but are not limited to,
ethanol, polyethylene glycol, phenol, phenolic compounds,
bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl
alcohol. Exemplary acidic preservatives include, but are not
limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric
acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid,
and/or phytic acid. Other preservatives include, but are not
limited to, tocopherol, tocopherol acetate, deteroxime mesylate,
cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened
(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl
ether sulfate (SLES), sodium bisulfite, sodium metabisulfite,
potassium sulfite, potassium metabisulfite, GLYDANT PLUS.RTM.,
PHENONIP.RTM., methylparaben, GERMALL.RTM.115, GERMABEN.RTM.II,
NEOLONE.TM., KATHON.TM., and/or EUXYL.RTM..
[0480] Exemplary buffering agents include, but are not limited to,
citrate buffer solutions, acetate buffer solutions, phosphate
buffer solutions, ammonium chloride, calcium carbonate, calcium
chloride, calcium citrate, calcium glubionate, calcium gluceptate,
calcium gluconate, D-gluconic acid, calcium glycerophosphate,
calcium lactate, propanoic acid, calcium levulinate, pentanoic
acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium
phosphate, calcium hydroxide phosphate, potassium acetate,
potassium chloride, potassium gluconate, potassium mixtures,
dibasic potassium phosphate, monobasic potassium phosphate,
potassium phosphate mixtures, sodium acetate, sodium bicarbonate,
sodium chloride, sodium citrate, sodium lactate, dibasic sodium
phosphate, monobasic sodium phosphate, sodium phosphate mixtures,
tromethamine, magnesium hydroxide, aluminum hydroxide, alginic
acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl
alcohol, etc., and/or combinations thereof
[0481] Exemplary lubricating agents include, but are not limited
to, magnesium stearate, calcium stearate, stearic acid, silica,
talc, malt, glyceryl behanate, hydrogenated vegetable oils,
polyethylene glycol, sodium benzoate, sodium acetate, sodium
chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate,
etc., and combinations thereof
[0482] Exemplary oils include, but are not limited to, almond,
apricot kernel, avocado, babassu, bergamot, black current seed,
borage, cade, camomile, canola, caraway, carnauba, castor,
cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton
seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol,
gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba,
kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary oils include, but are not limited
to, butyl stearate, caprylic triglyceride, capric triglyceride,
cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl
myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone
oil, and/or combinations thereof.
[0483] Excipients such as cocoa butter and suppository waxes,
coloring agents, coating agents, sweetening, flavoring, and/or
perfuming agents can be present in the composition, according to
the judgment of the formulator.
Delivery
[0484] The present disclosure encompasses the delivery of
signal-sensor polynucleotides, primary constructs or mmRNA for any
of therapeutic, pharmaceutical, diagnostic or imaging by any
appropriate route taking into consideration likely advances in the
sciences of drug delivery. Delivery may be naked or formulated.
Naked Delivery
[0485] The signal-sensor polynucleotides, primary constructs or
mmRNA of the present invention may be delivered to a cell naked. As
used herein in, "naked" refers to delivering signal-sensor
polynucleotides, primary constructs or mmRNA free from agents which
promote transfection. For example, the polynucleotides, primary
constructs or mmRNA delivered to the cell may contain no
modifications. The naked signal-sensor polynucleotides, primary
constructs or mmRNA may be delivered to the cell using routes of
administration known in the art and described herein.
Formulated Delivery
[0486] The signal-sensor polynucleotides, primary constructs or
mmRNA of the present invention may be formulated, using the methods
described herein. The formulations may contain signal-sensor
polynucleotides, primary constructs or mmRNA which may be modified
and/or unmodified. The formulations may further include, but are
not limited to, cell penetration agents, a pharmaceutically
acceptable carrier, a delivery agent, a bioerodible or
biocompatible polymer, a solvent, and a sustained-release delivery
depot. The formulated signal-sensor polynucleotides, primary
constructs or mmRNA may be delivered to the cell using routes of
administration known in the art and described herein.
[0487] The compositions may also be formulated for direct delivery
to an organ or tissue in any of several ways in the art including,
but not limited to, direct soaking or bathing, via a catheter, by
gels, powder, ointments, creams, gels, lotions, and/or drops, by
using substrates such as fabric or biodegradable materials coated
or impregnated with the compositions, and the like.
Administration
[0488] The signal-sensor polynucleotides, primary constructs or
mmRNA of the present invention may be administered by any route
which results in a therapeutically effective outcome. These
include, but are not limited to enteral, gastroenteral, epidural,
oral, transdermal, epidural (peridural), intracerebral (into the
cerebrum), intracerebroventricular (into the cerebral ventricles),
epicutaneous (application onto the skin), intradermal, (into the
skin itself), subcutaneous (under the skin), nasal administration
(through the nose), intravenous (into a vein), intraarterial (into
an artery), intramuscular (into a muscle), intracardiac (into the
heart), intraosseous infusion (into the bone marrow), intrathecal
(into the spinal canal), intraperitoneal, (infusion or injection
into the peritoneum), intravesical infusion, intravitreal, (through
the eye), intracavernous injection, (into the base of the penis),
intravaginal administration, intrauterine, extra-amniotic
administration, transdermal (diffusion through the intact skin for
systemic distribution), transmucosal (diffusion through a mucous
membrane), insufflation (snorting), sublingual, sublabial, enema,
eye drops (onto the conjunctiva), or in ear drops. In specific
embodiments, compositions may be administered in a way which allows
them cross the blood-brain barrier, vascular barrier, or other
epithelial barrier. Non-limiting routes of administration for the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention are described below.
Parenteral and Injectable Administration
[0489] Liquid dosage forms for oral and parenteral administration
include, but are not limited to, pharmaceutically acceptable
emulsions, microemulsions, solutions, suspensions, syrups, and/or
elixirs. In addition to active ingredients, liquid dosage forms may
comprise inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, oral compositions can include adjuvants
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, and/or perfuming agents. In certain
embodiments for parenteral administration, compositions are mixed
with solubilizing agents such as CREMOPHOR.RTM., alcohols, oils,
modified oils, glycols, polysorbates, cyclodextrins, polymers,
and/or combinations thereof.
[0490] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing agents, wetting agents,
and/or suspending agents. Sterile injectable preparations may be
sterile injectable solutions, suspensions, and/or emulsions in
nontoxic parenterally acceptable diluents and/or solvents, for
example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P., and isotonic sodium chloride solution. Sterile,
fixed oils are conventionally employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. Fatty acids such as
oleic acid can be used in the preparation of injectables.
[0491] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, and/or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0492] In order to prolong the effect of an active ingredient, it
is often desirable to slow the absorption of the active ingredient
from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution
which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered
drug form is accomplished by dissolving or suspending the drug in
an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the drug in biodegradable polymers such
as polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body
tissues.
Rectal and Vaginal Administration
[0493] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing
compositions with suitable non-irritating excipients such as cocoa
butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore
melt in the rectum or vaginal cavity and release the active
ingredient.
Oral Administration
[0494] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
an active ingredient is mixed with at least one inert,
pharmaceutically acceptable excipient such as sodium citrate or
dicalcium phosphate and/or fillers or extenders (e.g. starches,
lactose, sucrose, glucose, mannitol, and silicic acid), binders
(e.g. carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.
glycerol), disintegrating agents (e.g. agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate), solution retarding agents (e.g. paraffin),
absorption accelerators (e.g. quaternary ammonium compounds),
wetting agents (e.g. cetyl alcohol and glycerol monostearate),
absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g.
talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate), and mixtures thereof. In the case
of capsules, tablets and pills, the dosage form may comprise
buffering agents.
Topical or Transdermal Administration
[0495] As described herein, compositions containing the
signal-sensor polynucleotides, primary constructs or mmRNA of the
invention may be formulated for administration topically. The skin
may be an ideal target site for delivery as it is readily
accessible. Gene expression may be restricted not only to the skin,
potentially avoiding nonspecific toxicity, but also to specific
layers and cell types within the skin.
[0496] The site of cutaneous expression of the delivered
compositions will depend on the route of nucleic acid delivery.
Three routes are commonly considered to deliver signal-sensor
polynucleotides, primary constructs or mmRNA to the skin: (i)
topical application (e.g. for local/regional treatment and/or
oncology-related applications); (ii) intradermal injection (e.g.
for local/regional treatment and/or oncology-related applications);
and (iii) systemic delivery (e.g. for treatment of dermatologic
diseases that affect both cutaneous and extracutaneous regions).
Signal-sensor polynucleotides, primary constructs or mmRNA can be
delivered to the skin by several different approaches known in the
art. Most topical delivery approaches have been shown to work for
delivery of DNA, such as but not limited to, topical application of
non-cationic liposome--DNA complex, cationic liposome--DNA complex,
particle-mediated (gene gun), puncture-mediated gene transfections,
and viral delivery approaches. After delivery of the nucleic acid,
gene products have been detected in a number of different skin cell
types, including, but not limited to, basal keratinocytes,
sebaceous gland cells, dermal fibroblasts and dermal
macrophages.
[0497] In one embodiment, the invention provides for a variety of
dressings (e.g., wound dressings) or bandages (e.g., adhesive
bandages) for conveniently and/or effectively carrying out methods
of the present invention. Typically dressing or bandages may
comprise sufficient amounts of pharmaceutical compositions and/or
signal-sensor polynucleotides, primary constructs or mmRNA
described herein to allow a user to perform multiple treatments of
a subject(s).
[0498] In one embodiment, the invention provides for the
signal-sensor polynucleotides, primary constructs or mmRNA
compositions to be delivered in more than one injection.
[0499] In one embodiment, before topical and/or transdermal
administration at least one area of tissue, such as skin, may be
subjected to a device and/or solution which may increase
permeability. In one embodiment, the tissue may be subjected to an
abrasion device to increase the permeability of the skin (see U.S.
Patent Publication No. 20080275468, herein incorporated by
reference in its entirety). In another embodiment, the tissue may
be subjected to an ultrasound enhancement device. An ultrasound
enhancement device may include, but is not limited to, the devices
described in U.S. Publication No. 20040236268 and U.S. Pat. Nos.
6,491,657 and 6,234,990; herein incorporated by reference in their
entireties. Methods of enhancing the permeability of tissue are
described in U.S. Publication Nos. 20040171980 and 20040236268 and
U.S. Pat. No. 6,190,315; herein incorporated by reference in their
entireties.
[0500] In one embodiment, a device may be used to increase
permeability of tissue before delivering formulations of modified
mRNA described herein. The permeability of skin may be measured by
methods known in the art and/or described in U.S. Pat. No.
6,190,315, herein incorporated by reference in its entirety. As a
non-limiting example, a modified mRNA formulation may be delivered
by the drug delivery methods described in U.S. Pat. No. 6,190,315,
herein incorporated by reference in its entirety.
[0501] In another non-limiting example tissue may be treated with a
eutectic mixture of local anesthetics (EMLA) cream before, during
and/or after the tissue may be subjected to a device which may
increase permeability. Katz et al. (Anesth Analg (2004); 98:371-76;
herein incorporated by reference in its entirety) showed that using
the EMLA cream in combination with a low energy, an onset of
superficial cutaneous analgesia was seen as fast as 5 minutes after
a pretreatment with a low energy ultrasound.
[0502] In one embodiment, enhancers may be applied to the tissue
before, during, and/or after the tissue has been treated to
increase permeability. Enhancers include, but are not limited to,
transport enhancers, physical enhancers, and cavitation enhancers.
Non-limiting examples of enhancers are described in U.S. Pat. No.
6,190,315, herein incorporated by reference in its entirety.
[0503] In one embodiment, a device may be used to increase
permeability of tissue before delivering formulations of modified
mRNA described herein, which may further contain a substance that
invokes an immune response. In another non-limiting example, a
formulation containing a substance to invoke an immune response may
be delivered by the methods described in U.S. Publication Nos.
20040171980 and 20040236268; herein incorporated by reference in
their entireties.
[0504] Dosage forms for topical and/or transdermal administration
of a composition may include ointments, pastes, creams, lotions,
gels, foams, powders, solutions, sprays, inhalants and/or patches.
Generally, an active ingredient is admixed under sterile conditions
with a pharmaceutically acceptable excipient and/or any needed
preservatives and/or buffers as may be required. Additionally, the
present invention contemplates the use of transdermal patches,
which often have the added advantage of providing controlled
delivery of a compound to the body. Such dosage forms may be
prepared, for example, by dissolving and/or dispensing the compound
in the proper medium. Alternatively or additionally, rate may be
controlled by either providing a rate controlling membrane and/or
by dispersing the compound in a polymer matrix and/or gel.
[0505] Formulations suitable for topical administration include,
but are not limited to, liquid and/or semi liquid preparations such
as liniments, lotions, oil in water and/or water in oil emulsions
such as creams, ointments and/or pastes, and/or solutions and/or
suspensions.
[0506] Topically-administrable formulations may, for example,
comprise from about 0.1% to about 10% (w/w) active ingredient,
although the concentration of active ingredient may be as high as
the solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
Penetration Enhancers
[0507] In one embodiment, the signal-sensor polynucleotides,
primary construct and mmRNA of present invention may use various
penetration enhancers to deliver the signal-sensor polynucleotides,
primary construct and mmRNA to at least one area associated with
one or more hyperproliferative diseases, disorders or conditions.
Most drugs are present in solution in both ionized and nonionized
forms. However, usually only lipid soluble or lipophilic drugs
readily cross cell membranes. It has been discovered that even
non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is treated with a penetration enhancer. In addition to
aiding the diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs.
[0508] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.
92). Each of the above mentioned classes of penetration enhancers
are described below in greater detail. Combinations of penetration
enhancer may also be encompassed by the scope of the present
invention, for example, fatty acids/salts in combination with bile
acids/salts. Other non-limiting examples of combinations of
penetration enhancers include the combination of sodium salt of
lauric acid, capric acid and UDCA.
Surfactants
[0509] In connection with the present invention, surfactants (or
"surface-active agents") are chemical entities which, when
dissolved in an aqueous solution, reduce the surface tension of the
solution or the interfacial tension between the aqueous solution
and another liquid, with the result that absorption of the
signal-sensor polynucleotides, primary constructs and mmRNA through
the mucosa is enhanced. In addition to bile salts and fatty acids,
these penetration enhancers include, for example, sodium lauryl
sulfate, polyoxyethylene-9-lauryl ether and
polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92); and
perfluorochemical emulsions, such as FC-43 (Takahashi et al., J.
Pharm. Pharmacol., 1988, 40, 252).
Fatty Acids
[0510] Various fatty acids and their derivatives which act as
penetration enhancers include, but are not limited to, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-C.sub.10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems,
1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651-654).
Bile Salts
[0511] The physiological role of bile includes the facilitation of
dispersion and absorption of lipids and fat-soluble vitamins
(Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill,
New York, 1996, pp. 934-935). Various natural bile salts, and their
synthetic derivatives, act as penetration enhancers. Thus the term
"bile salts" includes any of the naturally occurring components of
bile as well as any of their synthetic derivatives. The bile salts
of the invention include, but are not limited to, cholic acid (or
its pharmaceutically acceptable sodium salt, sodium cholate),
dehydrocholic acid (sodium dehydrocholate), deoxycholic acid
(sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
Chelating Agents
[0512] Chelating agents, as used in connection with the present
invention, can be defined as compounds that remove metallic ions
from solution by forming complexes therewith, with the result that
absorption of signal-sensor polynucleotides, primary construct and
mmRNA through the mucosa is enhanced. With regards to their use as
penetration enhancers in the present invention, chelating agents
have the added advantage of also serving as DNase inhibitors, as
most characterized DNA nucleases require a divalent metal ion for
catalysis and are thus inhibited by chelating agents (Jarrett, J.
Chromatogr., 1993, 618, 315-339). Chelating agents of the invention
include but are not limited to disodium ethylenediaminetetraacetate
(EDTA), citric acid, salicylates (e.g., sodium salicylate,
5-methoxysalicylate and homovanilate), N-acyl derivatives of
collagen, laureth-9 and N-amino acyl derivatives of beta-diketones
(enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier
Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel.,
1990, 14, 43-51).
Non-Chelating Non-Surfactants
[0513] As used herein, non-chelating non-surfactant penetration
enhancing compounds can be defined as compounds that demonstrate
insignificant activity as chelating agents or as surfactants but
that nonetheless enhance absorption of signal-sensor
polynucleotides, primary construct and mmRNA through the alimentary
mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33). This class of penetration enhancers
include, but are not limited to, unsaturated cyclic ureas, 1-alkyl-
and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical
Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and
non-steroidal anti-inflammatory agents such as diclofenac sodium,
indomethacin and phenylbutazone (Yamashita et al., J. Pharm.
Pharmacol., 1987, 39, 621-626).
[0514] Agents that enhance uptake of signal-sensor polynucleotides,
primary construct and mmRNA at the cellular level may also be added
to the pharmaceutical and other compositions of the present
invention. For example, cationic lipids, such as lipofectin
(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol
derivatives, and polycationic molecules, such as polylysine (Lollo
et al., PCT Application WO 97/30731), are also known to enhance the
cellular uptake of signal-sensor polynucleotides, primary construct
and mmRNA.
[0515] Other agents may be utilized to enhance the penetration of
the administered signal-sensor polynucleotides, primary construct
and mmRNA, including glycols such as ethylene glycol and propylene
glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as
limonene and menthone.
Depot Administration
[0516] As described herein, in some embodiments, the composition is
formulated in depots for extended release. Generally, a specific
organ or tissue (a "target tissue") is targeted for
administration.
[0517] In some aspects of the invention, the signal-sensor
polynucleotides, primary constructs or mmRNA are spatially retained
within or proximal to a target tissue. Provided are method of
providing a composition to a target tissue of a mammalian subject
by contacting the target tissue (which contains one or more target
cells) with the composition under conditions such that the
composition, in particular the nucleic acid component(s) of the
composition, is substantially retained in the target tissue,
meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95,
96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the
composition is retained in the target tissue. Advantageously,
retention is determined by measuring the amount of the nucleic acid
present in the composition that enters one or more target cells.
For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90,
95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the
nucleic acids administered to the subject are present
intracellularly at a period of time following administration. For
example, intramuscular injection to a mammalian subject is
performed using an aqueous composition containing a ribonucleic
acid and a transfection reagent, and retention of the composition
is determined by measuring the amount of the ribonucleic acid
present in the muscle cells.
[0518] Aspects of the invention are directed to methods of
providing a composition to a target tissue of a mammalian subject,
by contacting the target tissue (containing one or more target
cells) with the composition under conditions such that the
composition is substantially retained in the target tissue. The
composition contains an effective amount of a signal-sensor
polynucleotides, primary constructs or mmRNA such that the
polypeptide of interest is produced in at least one target cell.
The compositions generally contain a cell penetration agent,
although "naked" nucleic acid (such as nucleic acids without a cell
penetration agent or other agent) is also contemplated, and a
pharmaceutically acceptable carrier.
[0519] In some circumstances, the amount of an oncology-related
protein produced by cells in a tissue is desirably increased.
Preferably, this increase in oncology-related protein production is
spatially restricted to cells within the target tissue. Thus,
provided are methods of increasing production of an
oncology-related protein of interest in a tissue of a mammalian
subject. A composition is provided that contains signal-sensor
polynucleotides, primary constructs or mmRNA characterized in that
a unit quantity of composition has been determined to produce the
polypeptide of interest in a substantial percentage of cells
contained within a predetermined volume of the target tissue.
[0520] In some embodiments, the composition includes a plurality of
different signal-sensor polynucleotides, primary constructs or
mmRNA, where one or more than one of the signal-sensor
polynucleotides, primary constructs or mmRNA encodes an
oncology-related polypeptide of interest. Optionally, the
composition also contains a cell penetration agent to assist in the
intracellular delivery of the composition. A determination is made
of the dose of the composition required to produce the
oncology-related polypeptide of interest in a substantial
percentage of cells contained within the predetermined volume of
the target tissue (generally, without inducing significant
production of the oncology-related polypeptide of interest in
tissue adjacent to the predetermined volume, or distally to the
target tissue). Subsequent to this determination, the determined
dose is introduced directly into the tissue of the mammalian
subject.
[0521] In one embodiment, the invention provides for the
signal-sensor polynucleotides, primary constructs or mmRNA to be
delivered in more than one injection or by split dose
injections.
[0522] In one embodiment, the invention may be retained near target
tissue using a small disposable drug reservoir or patch pump.
Non-limiting examples of patch pumps include those manufactured
and/or sold by BD.RTM. (Franklin Lakes, N.J.), Insulet Corporation
(Bedford, Mass.), SteadyMed Therapeutics (San Francisco, Calif.),
Medtronic (Minneapolis, Minn.), UniLife (York, Pa.), Valeritas
(Bridgewater, N.J.), and SpringLeaf Therapeutics (Boston,
Mass.).
Pulmonary Administration
[0523] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for pulmonary administration
via the buccal cavity. Such a formulation may comprise dry
particles which comprise the active ingredient and which have a
diameter in the range from about 0.5 nm to about 7 nm or from about
1 nm to about 6 nm. Such compositions are suitably in the form of
dry powders for administration using a device comprising a dry
powder reservoir to which a stream of propellant may be directed to
disperse the powder and/or using a self propelling solvent/powder
dispensing container such as a device comprising the active
ingredient dissolved and/or suspended in a low-boiling propellant
in a sealed container. Such powders comprise particles wherein at
least 98% of the particles by weight have a diameter greater than
0.5 nm and at least 95% of the particles by number have a diameter
less than 7 nm. Alternatively, at least 95% of the particles by
weight have a diameter greater than 1 nm and at least 90% of the
particles by number have a diameter less than 6 nm. Dry powder
compositions may include a solid fine powder diluent such as sugar
and are conveniently provided in a unit dose form.
[0524] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50% to 99.9%
(w/w) of the composition, and active ingredient may constitute 0.1%
to 20% (w/w) of the composition. A propellant may further comprise
additional ingredients such as a liquid non-ionic and/or solid
anionic surfactant and/or a solid diluent (which may have a
particle size of the same order as particles comprising the active
ingredient).
[0525] Pharmaceutical compositions formulated for pulmonary
delivery may provide an active ingredient in the form of droplets
of a solution and/or suspension. Such formulations may be prepared,
packaged, and/or sold as aqueous and/or dilute alcoholic solutions
and/or suspensions, optionally sterile, comprising active
ingredient, and may conveniently be administered using any
nebulization and/or atomization device. Such formulations may
further comprise one or more additional ingredients including, but
not limited to, a flavoring agent such as saccharin sodium, a
volatile oil, a buffering agent, a surface active agent, and/or a
preservative such as methylhydroxybenzoate. Droplets provided by
this route of administration may have an average diameter in the
range from about 0.1 nm to about 200 nm.
Intranasal, Nasal and Buccal Administration
[0526] Formulations described herein as being useful for pulmonary
delivery are useful for intranasal delivery of a pharmaceutical
composition. Another formulation suitable for intranasal
administration is a coarse powder comprising the active ingredient
and having an average particle from about 0.2 .mu.m to 500 .mu.m.
Such a formulation is administered in the manner in which snuff is
taken, i.e. by rapid inhalation through the nasal passage from a
container of the powder held close to the nose.
[0527] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of active ingredient, and may comprise one or more of
the additional ingredients described herein. A pharmaceutical
composition may be prepared, packaged, and/or sold in a formulation
suitable for buccal administration. Such formulations may, for
example, be in the form of tablets and/or lozenges made using
conventional methods, and may, for example, 0.1% to 20% (w/w)
active ingredient, the balance comprising an orally dissolvable
and/or degradable composition and, optionally, one or more of the
additional ingredients described herein. Alternately, formulations
suitable for buccal administration may comprise a powder and/or an
aerosolized and/or atomized solution and/or suspension comprising
active ingredient. Such powdered, aerosolized, and/or aerosolized
formulations, when dispersed, may have an average particle and/or
droplet size in the range from about 0.1 nm to about 200 nm, and
may further comprise one or more of any additional ingredients
described herein.
Ophthalmic Administration
[0528] A pharmaceutical composition may be prepared, packaged,
and/or sold in a formulation suitable for ophthalmic
administration. Such formulations may, for example, be in the form
of eye drops including, for example, a 0.1/1.0% (w/w) solution
and/or suspension of the active ingredient in an aqueous or oily
liquid excipient. Such drops may further comprise buffering agents,
salts, and/or one or more other of any additional ingredients
described herein. Other ophthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form and/or in a liposomal preparation. Ear
drops and/or eye drops are contemplated as being within the scope
of this invention.
Payload Administration: Detectable Agents and Therapeutic
Agents
[0529] The signal-sensor polynucleotides, primary constructs or
mmRNA described herein can be used in a number of different
scenarios in which delivery of a substance (the "payload") to a
biological target is desired, for example delivery of detectable
substances for detection of the target, or delivery of a
therapeutic agent. Detection methods can include, but are not
limited to, both imaging in vitro and in vivo imaging methods,
e.g., immunohistochemistry, bioluminescence imaging (BLI), Magnetic
Resonance Imaging (MRI), positron emission tomography (PET),
electron microscopy, X-ray computed tomography, Raman imaging,
optical coherence tomography, absorption imaging, thermal imaging,
fluorescence reflectance imaging, fluorescence microscopy,
fluorescence molecular tomographic imaging, nuclear magnetic
resonance imaging, X-ray imaging, ultrasound imaging, photoacoustic
imaging, lab assays, or in any situation where
tagging/staining/imaging is required.
[0530] The signal-sensor polynucleotides, primary constructs or
mmRNA can be designed to include both a linker and a payload in any
useful orientation. For example, a linker having two ends is used
to attach one end to the payload and the other end to the
nucleobase, such as at the C-7 or C-8 positions of the
deaza-adenosine or deaza-guanosine or to the N-3 or C-5 positions
of cytosine or uracil. The signal-sensor polynucleotide of the
invention can include more than one payload (e.g., a label and a
transcription inhibitor), as well as a cleavable linker. In one
embodiment, the modified nucleotide is a modified 7-deaza-adenosine
triphosphate, where one end of a cleavable linker is attached to
the C7 position of 7-deaza-adenine, the other end of the linker is
attached to an inhibitor (e.g., to the C5 position of the
nucleobase on a cytidine), and a label (e.g., Cy5) is attached to
the center of the linker (see, e.g., compound 1 of A*pCp C5 Parg
Capless in FIG. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304,
incorporated herein by reference). Upon incorporation of the
modified 7-deaza-adenosine triphosphate to an encoding region, the
resulting signal-sensor polynucleotide having a cleavable linker
attached to a label and an inhibitor (e.g., a polymerase
inhibitor). Upon cleavage of the linker (e.g., with reductive
conditions to reduce a linker having a cleavable disulfide moiety),
the label and inhibitor are released. Additional linkers and
payloads (e.g., therapeutic agents, detectable labels, and cell
penetrating payloads) are described herein.
[0531] For example, the signal-sensor polynucleotides, primary
constructs or mmRNA described herein can be used in reprogramming
induced pluripotent stem cells (iPS cells), which can directly
track cells that are transfected compared to total cells in the
cluster. In another example, a drug that may be attached to the
signal-sensor polynucleotides, primary constructs or mmRNA via a
linker and may be fluorescently labeled can be used to track the
drug in vivo, e.g. intracellularly. Other examples include, but are
not limited to, the use of signal-sensor polynucleotides, primary
constructs or mmRNA in reversible drug delivery into cells.
[0532] The signal-sensor polynucleotides, primary constructs or
mmRNA described herein can be used in intracellular targeting of a
payload, e.g., detectable or therapeutic agent, to specific
organelle. Exemplary intracellular targets can include, but are not
limited to, the nuclear localization for advanced mRNA processing,
or a nuclear localization sequence (NLS) linked to the mRNA
containing an inhibitor.
[0533] In addition, the signal-sensor polynucleotides, primary
constructs or mmRNA described herein can be used to deliver
therapeutic agents to cells or tissues, e.g., in living animals.
For example, the signal-sensor polynucleotides, primary constructs
or mmRNA described herein can be used to deliver highly polar
chemotherapeutics agents to kill cancer cells. The signal-sensor
polynucleotides, primary constructs or mmRNA attached to the
therapeutic agent through a linker can facilitate member permeation
allowing the therapeutic agent to travel into a cell to reach an
intracellular target.
[0534] In another example, the signal-sensor polynucleotides,
primary constructs or mmRNA can be attached to the polynucleotides,
primary constructs or mmRNA a viral inhibitory peptide (VIP)
through a cleavable linker. The cleavable linker can release the
VIP and dye into the cell. In another example, the signal-sensor
polynucleotides, primary constructs or mmRNA can be attached
through the linker to an ADP-ribosylate, which is responsible for
the actions of some bacterial toxins, such as cholera toxin,
diphtheria toxin, and pertussis toxin. These toxin proteins are
ADP-ribosyltransferases that modify target proteins in human cells.
For example, cholera toxin ADP-ribosylates G proteins modifies
human cells by causing massive fluid secretion from the lining of
the small intestine, which results in life-threatening
diarrhea.
[0535] In some embodiments, the payload may be a therapeutic agent
such as a cytotoxin, radioactive ion, chemotherapeutic, or other
therapeutic agent. A cytotoxin or cytotoxic agent includes any
agent that may be detrimental to cells. Examples include, but are
not limited to, taxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, teniposide, vincristine,
vinblastine, colchicine, doxorubicin, daunorubicin,
dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol
(see U.S. Pat. No. 5,208,020 incorporated herein in its entirety),
rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092, 5,585,499, and
5,846,545, all of which are incorporated herein by reference), and
analogs or homologs thereof. Radioactive ions include, but are not
limited to iodine (e.g., iodine 125 or iodine 131), strontium 89,
phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium
90, samarium 153, and praseodymium. Other therapeutic agents
include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065),
melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide,
busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine, vinblastine, taxol and maytansinoids).
[0536] In some embodiments, the payload may be a detectable agent,
such as various organic small molecules, inorganic compounds,
nanoparticles, enzymes or enzyme substrates, fluorescent materials,
luminescent materials (e.g., luminol), bioluminescent materials
(e.g., luciferase, luciferin, and aequorin), chemiluminescent
materials, radioactive materials (e.g., .sup.18F, .sup.67 Ga,
.sup.81mKr, .sup.82 Rb, .sup.111In, .sup.123I, .sup.133Xe,
.sup.201Tl, .sup.125I, .sup.35S, .sup.14C, .sup.3H, or .sup.99mTc
(e.g., as pertechnetate (technetate(VII), TcO.sub.4.sup.-)), and
contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium
(e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron
oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and
ultrasmall superparamagnetic iron oxide (USPIO)), manganese
chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media
(iohexol), microbubbles, or perfluorocarbons). Such
optically-detectable labels include for example, without
limitation, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic
acid; acridine and derivatives (e.g., acridine and acridine
isothiocyanate); 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
(EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5
disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide;
BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120), and
7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5'
5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives (e.g., eosin and eosin
isothiocyanate); erythrosin and derivatives (e.g., erythrosin B and
erythrosin isothiocyanate); ethidium; fluorescein and derivatives
(e.g., 5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate
(QFITC or XRITC), and fluorescamine);
2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-yl-
idene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]-
ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indolium
hydroxide, inner salt, compound with n,n-diethylethanamine (1:1)
(IR144);
5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene-
]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethyl
benzothiazolium perchlorate (IR140); Malachite Green
isothiocyanate; 4-methylumbelliferone orthocresolphthalein;
nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin;
o-phthaldialdehyde; pyrene and derivatives (e.g., pyrene, pyrene
butyrate, and succinimidyl 1-pyrene); butyrate quantum dots;
Reactive Red 4 (CIBACRON.TM. Brilliant Red 3B-A); rhodamine and
derivatives (e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative
of sulforhodamine 101 (Texas Red),
N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl
rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC));
riboflavin; rosolic acid; terbium chelate derivatives; Cyanine-3
(Cy3); Cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD
700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and
naphthalo cyanine.
[0537] In some embodiments, the detectable agent may be a
non-detectable pre-cursor that becomes detectable upon activation
(e.g., fluorogenic tetrazine-fluorophore constructs (e.g.,
tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or
tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents
(e.g., PROSENSE.RTM. (VisEn Medical))). In vitro assays in which
the enzyme labeled compositions can be used include, but are not
limited to, enzyme linked immunosorbent assays (ELISAs),
immunoprecipitation assays, immunofluorescence, enzyme immunoassays
(EIA), radioimmunoassays (RIA), and Western blot analysis.
Combinations
[0538] The signal-sensor polynucleotides, primary constructs or
mmRNA may be used in combination with one or more other
therapeutic, prophylactic, diagnostic, or imaging agents. By "in
combination with," it is not intended to imply that the agents must
be administered at the same time and/or formulated for delivery
together, although these methods of delivery are within the scope
of the present disclosure. Compositions can be administered
concurrently with, prior to, or subsequent to, one or more other
desired therapeutics or medical procedures. In general, each agent
will be administered at a dose and/or on a time schedule determined
for that agent. In some embodiments, the present disclosure
encompasses the delivery of pharmaceutical, prophylactic,
diagnostic, or imaging compositions in combination with agents that
may improve their bioavailability, reduce and/or modify their
metabolism, inhibit their excretion, and/or modify their
distribution within the body. As a non-limiting example, the
signal-sensor nucleic acids or mmRNA may be used in combination
with a pharmaceutical agent for the treatment of cancer or to
control hyperproliferative cells. In U.S. Pat. No. 7,964,571,
herein incorporated by reference in its entirety, a combination
therapy for the treatment of solid primary or metastasized tumor is
described using a pharmaceutical composition including a DNA
plasmid encoding for interleukin-12 with a lipopolymer and also
administering at least one anticancer agent or chemotherapeutic.
Further, the signal-sensor nucleic acids and mmRNA of the present
invention that encodes anti-proliferative molecules may be in a
pharmaceutical composition with a lipopolymer (see e.g., U.S. Pub.
No. 20110218231, herein incorporated by reference in its entirety,
claiming a pharmaceutical composition comprising a DNA plasmid
encoding an anti-proliferative molecule and a lipopolymer) which
may be administered with at least one chemotherapeutic or
anticancer agent.
Dosing
[0539] The present invention provides methods comprising
administering modified mRNAs and their encoded proteins or
complexes in accordance with the invention to a subject in need
thereof. Nucleic acids, proteins or complexes, or pharmaceutical,
imaging, diagnostic, or prophylactic compositions thereof, may be
administered to a subject using any amount and any route of
administration effective for preventing, treating, diagnosing, or
imaging a disease, disorder, and/or condition (e.g., a disease,
disorder, and/or condition relating to working memory deficits).
The exact amount required will vary from subject to subject,
depending on the species, age, and general condition of the
subject, the severity of the disease, the particular composition,
its mode of administration, its mode of activity, and the like.
Compositions in accordance with the invention are typically
formulated in dosage unit form for ease of administration and
uniformity of dosage. It will be understood, however, that the
total daily usage of the compositions of the present invention may
be decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective,
prophylactically effective, or appropriate imaging dose level for
any particular patient will depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed;
and like factors well known in the medical arts.
[0540] In certain embodiments, compositions in accordance with the
present invention may be administered at dosage levels sufficient
to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about
0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about
0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about
0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50
mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg
to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from
about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about
25 mg/kg, of subject body weight per day, one or more times a day,
to obtain the desired therapeutic, diagnostic, prophylactic, or
imaging effect. The desired dosage may be delivered three times a
day, two times a day, once a day, every other day, every third day,
every week, every two weeks, every three weeks, or every four
weeks. In certain embodiments, the desired dosage may be delivered
using multiple administrations (e.g., two, three, four, five, six,
seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or
more administrations).
[0541] According to the present invention, it has been discovered
that administration of mmRNA in split-dose regimens produce higher
levels of proteins in mammalian subjects. As used herein, a "split
dose" is the division of single unit dose or total daily dose into
two or more doses, e.g, two or more administrations of the single
unit dose. As used herein, a "single unit dose" is a dose of any
therapeutic administed in one dose/at one time/single route/single
point of contact, i.e., single administration event. As used
herein, a "total daily dose" is an amount given or prescribed in 24
hr period. It may be administered as a single unit dose. In one
embodiment, the mmRNA of the present invention are administed to a
subject in split doses. The mmRNA may be formulated in buffer only
or in a formulation described herein.
Dosage Forms
[0542] A pharmaceutical composition described herein can be
formulated into a dosage form described herein, such as a topical,
intranasal, intratracheal, or injectable (e.g., intravenous,
intraocular, intravitreal, intramuscular, intracardiac,
intraperitoneal, subcutaneous).
Liquid Dosage Forms
[0543] Liquid dosage forms for parenteral administration include,
but are not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups, and/or elixirs. In
addition to active ingredients, liquid dosage forms may comprise
inert diluents commonly used in the art including, but not limited
to, water or other solvents, solubilizing agents and emulsifiers
such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl
acetate, benzyl alcohol, benzyl benzoate, propylene glycol,
1,3-butylene glycol, dimethylformamide, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),
glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and
fatty acid esters of sorbitan, and mixtures thereof. In certain
embodiments for parenteral administration, compositions may be
mixed with solubilizing agents such as CREMOPHOR.RTM., alcohols,
oils, modified oils, glycols, polysorbates, cyclodextrins,
polymers, and/or combinations thereof.
Injectable
[0544] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art and may include suitable dispersing agents, wetting
agents, and/or suspending agents. Sterile injectable preparations
may be sterile injectable solutions, suspensions, and/or emulsions
in nontoxic parenterally acceptable diluents and/or solvents, for
example, a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed include, but are not
limited to, are water, Ringer's solution, U.S.P., and isotonic
sodium chloride solution. Sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or
diglycerides. Fatty acids such as oleic acid can be used in the
preparation of injectables.
[0545] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, and/or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0546] In order to prolong the effect of an active ingredient, it
may be desirable to slow the absorption of the active ingredient
from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the signal-sensor polynucleotide, primary construct
or mmRNA then depends upon its rate of dissolution which, in turn,
may depend upon crystal size and crystalline form. Alternatively,
delayed absorption of a parenterally administered signal-sensor
polynucleotide, primary construct or mmRNA may be accomplished by
dissolving or suspending the signal-sensor polynucleotide, primary
construct or mmRNA in an oil vehicle. Injectable depot forms are
made by forming microencapsule matrices of the signal-sensor
polynucleotide, primary construct or mmRNA in biodegradable
polymers such as polylactide-polyglycolide. Depending upon the
ratio of the signal-sensor polynucleotide, primary construct or
mmRNA to polymer and the nature of the particular polymer employed,
the rate of signal-sensor polynucleotide, primary construct or
mmRNA release can be controlled. Examples of other biodegradable
polymers include, but are not limited to, poly(orthoesters) and
poly(anhydrides). Depot injectable formulations may be prepared by
entrapping the signal-sensor polynucleotide, primary construct or
mmRNA in liposomes or microemulsions which are compatible with body
tissues.
Pulmonary
[0547] Formulations described herein as being useful for pulmonary
delivery may also be use for intranasal delivery of a
pharmaceutical composition. Another formulation suitable for
intranasal administration may be a coarse powder comprising the
active ingredient and having an average particle from about 0.2
.mu.m to 500 .mu.m. Such a formulation may be administered in the
manner in which snuff is taken, i.e. by rapid inhalation through
the nasal passage from a container of the powder held close to the
nose.
[0548] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of active ingredient, and may comprise one or more of
the additional ingredients described herein. A pharmaceutical
composition may be prepared, packaged, and/or sold in a formulation
suitable for buccal administration. Such formulations may, for
example, be in the form of tablets and/or lozenges made using
conventional methods, and may, for example, contain about 0.1% to
20% (w/w) active ingredient, where the balance may comprise an
orally dissolvable and/or degradable composition and, optionally,
one or more of the additional ingredients described herein.
Alternately, formulations suitable for buccal administration may
comprise a powder and/or an aerosolized and/or atomized solution
and/or suspension comprising active ingredient. Such powdered,
aerosolized, and/or aerosolized formulations, when dispersed, may
have an average particle and/or droplet size in the range from
about 0.1 nm to about 200 nm, and may further comprise one or more
of any additional ingredients described herein.
[0549] General considerations in the formulation and/or manufacture
of pharmaceutical agents may be found, for example, in Remington:
The Science and Practice of Pharmacy 21.sup.st ed., Lippincott
Williams & Wilkins, 2005 (incorporated herein by
reference).
Coatings or Shells
[0550] Solid dosage forms of tablets, dragees, capsules, pills, and
granules can be prepared with coatings and shells such as enteric
coatings and other coatings well known in the pharmaceutical
formulating art. They may optionally comprise opacifying agents and
can be of a composition that they release the active ingredient(s)
only, or preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
which can be used include polymeric substances and waxes. Solid
compositions of a similar type may be employed as fillers in soft
and hard-filled gelatin capsules using such excipients as lactose
or milk sugar as well as high molecular weight polyethylene glycols
and the like.
Properties of Pharmaceutical Compositions
[0551] The pharmaceutical compositions described herein can be
characterized by one or more of bioavailability, therapeutic window
and/or volume of distribution.
Bioavailability
[0552] The signal-sensor polynucleotides, primary constructs or
mmRNA, when formulated into a composition with a delivery agent as
described herein, can exhibit an increase in bioavailability as
compared to a composition lacking a delivery agent as described
herein. As used herein, the term "bioavailability" refers to the
systemic availability of a given amount of signal-sensor
polynucleotides, primary constructs or mmRNA administered to a
mammal. Bioavailability can be assessed by measuring the area under
the curve (AUC) or the maximum serum or plasma concentration
(C.sub.max) of the unchanged form of a compound following
administration of the compound to a mammal. AUC is a determination
of the area under the curve plotting the serum or plasma
concentration of a compound along the ordinate (Y-axis) against
time along the abscissa (X-axis). Generally, the AUC for a
particular compound can be calculated using methods known to those
of ordinary skill in the art and as described in G. S. Banker,
Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,
Marcel Dekker, New York, Inc., 1996, herein incorporated by
reference.
[0553] The C.sub.max value is the maximum concentration of the
compound achieved in the serum or plasma of a mammal following
administration of the compound to the mammal. The C.sub.max value
of a particular compound can be measured using methods known to
those of ordinary skill in the art. The phrases "increasing
bioavailability" or "improving the pharmacokinetics," as used
herein mean that the systemic availability of a first signal-sensor
polynucleotide, primary construct or mmRNA, measured as AUC,
C.sub.max, or C.sub.min in a mammal is greater, when
co-administered with a delivery agent as described herein, than
when such co-administration does not take place. In some
embodiments, the bioavailability of the signal-sensor
polynucleotide, primary construct or mmRNA can increase by at least
about 2%, at least about 5%, at least about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, or about
100%.
Therapeutic Window
[0554] The signal-sensor polynucleotides, primary constructs or
mmRNA, when formulated into a composition with a delivery agent as
described herein, can exhibit an increase in the therapeutic window
of the administered signal-sensor polynucleotide, primary construct
or mmRNA composition as compared to the therapeutic window of the
administered signal-sensor polynucleotide, primary construct or
mmRNA composition lacking a delivery agent as described herein. As
used herein "therapeutic window" refers to the range of plasma
concentrations, or the range of levels of therapeutically active
substance at the site of action, with a high probability of
eliciting a therapeutic effect. In some embodiments, the
therapeutic window of the signal-sensor polynucleotide, primary
construct or mmRNA when co-administered with a delivery agent as
described herein can increase by at least about 2%, at least about
5%, at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or about 100%.
Volume of Distribution
[0555] The signal-sensor polynucleotides, primary constructs or
mmRNA, when formulated into a composition with a delivery agent as
described herein, can exhibit an improved volume of distribution
(V.sub.dist), e.g., reduced or targeted, relative to a composition
lacking a delivery agent as described herein. The volume of
distribution (V.sub.dist) relates the amount of the drug in the
body to the concentration of the drug in the blood or plasma. As
used herein, the term "volume of distribution" refers to the fluid
volume that would be required to contain the total amount of the
drug in the body at the same concentration as in the blood or
plasma: V.sub.dist equals the amount of drug in the
body/concentration of drug in blood or plasma. For example, for a
10 mg dose and a plasma concentration of 10 mg/L, the volume of
distribution would be 1 liter. The volume of distribution reflects
the extent to which the drug is present in the extravascular
tissue. A large volume of distribution reflects the tendency of a
compound to bind to the tissue components compared with plasma
protein binding. In a clinical setting, V.sub.dist can be used to
determine a loading dose to achieve a steady state concentration.
In some embodiments, the volume of distribution of the
signal-sensor polynucleotide, primary construct or mmRNA when
co-administered with a delivery agent as described herein can
decrease at least about 2%, at least about 5%, at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%.
Biological Effect
[0556] In one embodiment, the biological effect of the
signal-sensor modified mRNA delivered to the animals may be
categorized by analyzing the protein expression in the animals. The
protein expression may be determined from analyzing a biological
sample collected from a mammal administered the signal-sensor
modified mRNA of the present invention. In one embodiment, the
expression protein encoded by the signal-sensor modified mRNA
administered to the mammal of at least 50 pg/ml may be preferred.
For example, a protein expression of 50-200 pg/ml for the protein
encoded by the signal-sensor modified mRNA delivered to the mammal
may be seen as a therapeutically effective amount of protein in the
mammal.
Detection of Modified Nucleic Acids by Mass Spectrometry
[0557] Mass spectrometry (MS) is an analytical technique that can
provide structural and molecular mass/concentration information on
molecules after their conversion to ions. The molecules are first
ionized to acquire positive or negative charges and then they
travel through the mass analyzer to arrive at different areas of
the detector according to their mass/charge (m/z) ratio.
[0558] Mass spectrometry is performed using a mass spectrometer
which includes an ion source for ionizing the fractionated sample
and creating charged molecules for further analysis. For example
ionization of the sample may be performed by electrospray
ionization (ESI), atmospheric pressure chemical ionization (APCI),
photoionization, electron ionization, fast atom bombardment
(FAB)/liquid secondary ionization (LSIMS), matrix assisted laser
desorption/ionization (MALDI), field ionization, field desorption,
thermospray/plasmaspray ionization, and particle beam ionization.
The skilled artisan will understand that the choice of ionization
method can be determined based on the analyte to be measured, type
of sample, the type of detector, the choice of positive versus
negative mode, etc.
[0559] After the sample has been ionized, the positively charged or
negatively charged ions thereby created may be analyzed to
determine a mass-to-charge ratio (i.e., m/z). Suitable analyzers
for determining mass-to-charge ratios include quadropole analyzers,
ion traps analyzers, and time-of-flight analyzers. The ions may be
detected using several detection modes. For example, selected ions
may be detected (i.e., using a selective ion monitoring mode
(SIM)), or alternatively, ions may be detected using a scanning
mode, e.g., multiple reaction monitoring (MRM) or selected reaction
monitoring (SRM).
[0560] Liquid chromatography-multiple reaction monitoring
(LC-MS/MRM) coupled with stable isotope labeled dilution of peptide
standards has been shown to be an effective method for protein
verification (e.g., Keshishian et al., Mol Cell Proteomics 2009 8:
2339-2349; Kuhn et al., Clin Chem 2009 55:1108-1117; Lopez et al.,
Clin Chem 2010 56:281-290). Unlike untargeted mass spectrometry
frequently used in biomarker discovery studies, targeted MS methods
are peptide sequence--based modes of MS that focus the full
analytical capacity of the instrument on tens to hundreds of
selected peptides in a complex mixture. By restricting detection
and fragmentation to only those peptides derived from proteins of
interest, sensitivity and reproducibility are improved dramatically
compared to discovery-mode MS methods. This method of mass
spectrometry-based multiple reaction monitoring (MRM) quantitation
of proteins can dramatically impact the discovery and quantitation
of biomarkers via rapid, targeted, multiplexed protein expression
profiling of clinical samples.
[0561] In one embodiment, a biological sample which may contain at
least one protein encoded by at least one modified mRNA of the
present invention may be analyzed by the method of MRM-MS. The
quantification of the biological sample may further include, but is
not limited to, isotopically labeled peptides or proteins as
internal standards.
[0562] According to the present invention, the biological sample,
once obtained from the subject, may be subjected to enzyme
digestion. As used herein, the term "digest" means to break apart
into shorter peptides. As used herein, the phrase "treating a
sample to digest proteins" means manipulating a sample in such a
way as to break down proteins in a sample. These enzymes include,
but are not limited to, trypsin, endoproteinase Glu-C and
chymotrypsin. In one embodiment, a biological sample which may
contain at least one protein encoded by at least one modified mRNA
of the present invention may be digested using enzymes.
[0563] In one embodiment, a biological sample which may contain
protein encoded by modified mRNA of the present invention may be
analyzed for protein using electrospray ionization. Electrospray
ionization (ESI) mass spectrometry (ESIMS) uses electrical energy
to aid in the transfer of ions from the solution to the gaseous
phase before they are analyzed by mass spectrometry. Samples may be
analyzed using methods known in the art (e.g., Ho et al., Clin
Biochem Rev. 2003 24(1):3-12). The ionic species contained in
solution may be transferred into the gas phase by dispersing a fine
spray of charge droplets, evaporating the solvent and ejecting the
ions from the charged droplets to generate a mist of highly charged
droplets. The mist of highly charged droplets may be analyzed using
at least 1, at least 2, at least 3 or at least 4 mass analyzers
such as, but not limited to, a quadropole mass analyzer. Further,
the mass spectrometry method may include a purification step. As a
non-limiting example, the first quadrapole may be set to select a
single m/z ratio so it may filter out other molecular ions having a
different m/z ratio which may eliminate complicated and
time-consuming sample purification procedures prior to MS
analysis.
[0564] In one embodiment, a biological sample which may contain
protein encoded by modified mRNA of the present invention may be
analyzed for protein in a tandem ESIMS system (e.g., MS/MS). As
non-limiting examples, the droplets may be analyzed using a product
scan (or daughter scan) a precursor scan (parent scan) a neutral
loss or a multiple reaction monitoring.
[0565] In one embodiment, a biological sample which may contain
protein encoded by modified mRNA of the present invention may be
analyzed using matrix-assisted laser desorption/ionization (MALDI)
mass spectrometry (MALDIMS). MALDI provides for the nondestructive
vaporization and ionization of both large and small molecules, such
as proteins. In MALDI analysis, the analyte is first
co-crystallized with a large molar excess of a matrix compound,
which may also include, but is not limited to, an ultraviolet
absorbing weak organic acid. Non-limiting examples of matrices used
in MALDI are .alpha.-cyano-4-hydroxycinnamic acid,
3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.
Laser radiation of the analyte-matrix mixture may result in the
vaporization of the matrix and the analyte. The laser induced
desorption provides high ion yields of the intact analyte and
allows for measurement of compounds with high accuracy. Samples may
be analyzed using methods known in the art (e.g., Lewis, Wei and
Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894). As
non-limiting examples, mass analyzers used in the MALDI analysis
may include a linear time-of-flight (TOF), a TOF reflectron or a
Fourier transform mass analyzer.
[0566] In one embodiment, the analyte-matrix mixture may be formed
using the dried-droplet method. A biologic sample is mixed with a
matrix to create a saturated matrix solution where the
matrix-to-sample ratio is approximately 5000:1. An aliquot
(approximately 0.5-2.0 uL) of the saturated matrix solution is then
allowed to dry to form the analyte-matrix mixture.
[0567] In one embodiment, the analyte-matrix mixture may be formed
using the thin-layer method. A matrix homogeneous film is first
formed and then the sample is then applied and may be absorbed by
the matrix to form the analyte-matrix mixture.
[0568] In one embodiment, the analyte-matrix mixture may be formed
using the thick-layer method. A matrix homogeneous film is formed
with a nitro-cellulose matrix additive. Once the uniform
nitro-cellulose matrix layer is obtained the sample is applied and
absorbed into the matrix to form the analyte-matrix mixture.
[0569] In one embodiment, the analyte-matrix mixture may be formed
using the sandwich method. A thin layer of matrix crystals is
prepared as in the thin-layer method followed by the addition of
droplets of aqueous trifluoroacetic acid, the sample and matrix.
The sample is then absorbed into the matrix to form the
analyte-matrix mixture.
V. Uses of Signal-Sensor Polynucleotides, Primary constructs and
mmRNA of the Invention
[0570] The signal-sensor polynucleotides, primary constructs and
mmRNA of the present invention are designed, in preferred
embodiments, to provide for avoidance or evasion of deleterious
bio-responses such as the immune response and/or degradation
pathways, overcoming the threshold of expression and/or improving
protein production capacity, improved expression rates or
translation efficiency, improved drug or protein half life and/or
protein concentrations, optimized protein localization, to improve
one or more of the stability and/or clearance in tissues, receptor
uptake and/or kinetics, cellular access by the compositions,
engagement with translational machinery, secretion efficiency (when
applicable), accessibility to circulation, and/or modulation of a
cell's status, function and/or activity.
Therapeutics
Therapeutic Agents
[0571] The signal-sensor polynucleotides, primary constructs or
mmRNA of the present invention, such as modified nucleic acids and
modified RNAs, and the proteins translated from them described
herein can be used as therapeutic or prophylactic agents. They are
provided for use in medicine. For example, signal-sensor
polynucleotide, primary construct or mmRNA described herein can be
administered to a subject, wherein the signal-sensor
polynucleotide, primary construct or mmRNA is translated in vivo to
produce a therapeutic or prophylactic oncology-related polypeptide
in the subject. Provided are compositions, methods, kits, and
reagents for diagnosis, treatment or prevention of a disease or
condition in humans and other mammals. The active therapeutic
agents of the invention include signal-sensor polynucleotides,
primary constructs or mmRNA, cells containing polynucleotides,
primary constructs or mmRNA or polypeptides translated from the
signal-sensor polynucleotides, primary constructs or mmRNA.
[0572] In certain embodiments, provided herein are combination
therapeutics containing one or more signal-sensor polynucleotide,
primary construct or mmRNA containing translatable regions that
encode for a protein or proteins that boost a mammalian subject's
immunity along with a protein that induces antibody-dependent
cellular toxicity.
[0573] Provided herein are methods of inducing translation of a
recombinant polypeptide in a cell population using the
signal-sensor polynucleotide, primary construct or mmRNA described
herein. Such translation can be in vivo, ex vivo, in culture, or in
vitro. The cell population is contacted with an effective amount of
a composition containing the signal-sensor nucleic acid that has at
least one nucleoside modification, and a translatable region
encoding the recombinant oncology-related polypeptide. The
population is contacted under conditions such that the
signal-sensor nucleic acid is localized into one or more cells of
the cell population and the recombinant oncology-related
polypeptide is translated in the cell from the signal-sensor
nucleic acid.
[0574] An "effective amount" of the composition is provided based,
at least in part, on the target tissue, target cell type, means of
administration, physical characteristics of the nucleic acid (e.g.,
size, and extent of modified nucleosides), and other determinants.
In general, an effective amount of the composition provides
efficient protein production in the cell, preferably more efficient
than a composition containing a corresponding unmodified nucleic
acid. Increased efficiency may be demonstrated by increased cell
transfection (i.e., the percentage of cells transfected with the
nucleic acid), increased protein translation from the nucleic acid,
decreased nucleic acid degradation (as demonstrated, e.g., by
increased duration of protein translation from a modified nucleic
acid), or reduced innate immune response of the host cell.
[0575] Aspects of the invention are directed to methods of inducing
in vivo translation of a recombinant polypeptide in a mammalian
subject in need thereof. Therein, an effective amount of a
composition containing a nucleic acid that has at least one
structural or chemical modification and a translatable region
encoding the recombinant polypeptide is administered to the subject
using the delivery methods described herein. The nucleic acid is
provided in an amount and under other conditions such that the
nucleic acid is localized into a cell of the subject and the
recombinant polypeptide is translated in the cell from the nucleic
acid. The cell in which the nucleic acid is localized, or the
tissue in which the cell is present, may be targeted with one or
more than one rounds of nucleic acid administration.
[0576] In certain embodiments, the administered signal-sensor
polynucleotide, primary construct or mmRNA directs production of
one or more recombinant polypeptides that provide a functional
activity which is substantially absent in the cell, tissue or
organism in which the recombinant oncology-related polypeptide is
translated. For example, the missing functional activity may be
enzymatic, structural, or gene regulatory in nature. In related
embodiments, the administere signal-sensor polynucleotide, primary
construct or mmRNA directs production of one or more recombinant
oncology-related polypeptides that increases (e.g.,
synergistically) a functional activity which is present but
substantially deficient in the cell in which the recombinant
oncology-related polypeptide is translated.
[0577] In other embodiments, the administere signal-sensor
polynucleotide, primary construct or mmRNA directs production of
one or more recombinant polypeptides that replace an
oncology-related polypeptide (or multiple oncology-related
polypeptides) that is substantially absent in the cell in which the
recombinant oncology-related polypeptide is translated. Such
absence may be due to genetic mutation of the encoding gene or
regulatory pathway thereof. In some embodiments, the recombinant
oncology-related polypeptide increases the level of an endogenous
oncology-related protein in the cell to a desirable level; such an
increase may bring the level of the endogenous oncology-related
protein from a subnormal level to a normal level or from a normal
level to a super-normal level.
[0578] Alternatively, the recombinant oncology-related polypeptide
functions to antagonize the activity of an endogenous protein
present in, on the surface of, or secreted from the cell. Usually,
the activity of the endogenous oncology-related protein is
deleterious to the subject; for example, due to mutation of the
endogenous oncology-related protein resulting in altered activity
or localization. Additionally, the recombinant oncology-related
polypeptide antagonizes, directly or indirectly, the activity of a
biological moiety present in, on the surface of, or secreted from
the cell. Examples of antagonized biological moieties include
lipids (e.g., cholesterol), a lipoprotein (e.g., low density
lipoprotein), a nucleic acid, a carbohydrate, a protein toxin such
as shiga and tetanus toxins, or a small molecule toxin such as
botulinum, cholera, and diphtheria toxins. Additionally, the
antagonized biological molecule may be an endogenous protein that
exhibits an undesirable activity, such as a cytotoxic or cytostatic
activity.
[0579] The recombinant oncology-related proteins described herein
may be engineered for localization within the cell, potentially
within a specific compartment such as the nucleus, or are
engineered for secretion from the cell or translocation to the
plasma membrane of the cell.
[0580] In some embodiments, modified signal-sensor mRNAs and their
encoded oncology-related polypeptides in accordance with the
present invention may be used for treatment of any of a variety of
diseases, disorders, and/or conditions described herein.
Oncology-Related Applications
[0581] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may be used in the treatment,
management, characterization and/or diagnosis of cancer, a
cancer-related and/or a cancer treatment-related disorder, side
effect and/or condition. Such disease, disorders and conditions
include, but are not limited to, adrenal cortical cancer, advanced
cancer, anal cancer, aplastic anemia, bileduct cancer, bladder
cancer, bone cancer, bone metastasis, brain tumors, brain cancer,
breast cancer, childhood cancer, cancer of unknown primary origin,
Castleman disease, cervical cancer, colon/rectal cancer,
endometrial cancer, esophagus cancer, Ewing family of tumors, eye
cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal
carcinoid tumors, gastrointestinal stromal tumors, gestational
trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell
carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic
leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myeloid leukemia, chronic myelomonocytic leukemia, liver
cancer, non-small cell lung cancer, small cell lung cancer, lung
carcinoid tumor, lymphoma of the skin, malignant mesothelioma,
multiple myeloma, myelodysplastic syndrome, nasal cavity and
paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,
non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer,
pituitary tumors, prostate cancer, retinoblastoma,
rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft
tissue, basal and squamous cell skin cancer, melanoma, small
intestine cancer, stomach cancer, testicular cancer, thymus cancer,
thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom macroglobulinemia, Wilms tumor.
[0582] In another embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may be used in the treating,
managing or manipulating at least one cancer-related or cancer
treatment-related disorder, side effect or condition such as chemo
brain, peripheral neuropathy, fatigue, depression, nausea and
vomiting, pain, anemia, lymphedema, infections, second cancers
caused by cancer treatment, sexual side effects, reduced fertility
or infertility, ostomies, insomnia and hair loss.
[0583] In one embodiment, the signal-sensor polynucleotides,
primary constructs and/or mmRNA may be used to reduce the effect of
at least one symptom of cancer in a subject. The symptom may
include, but is not limited to, weakness, aches and pains, fever,
fatigue, weight loss, blood clots, increased blood calcium levels,
low white blood cell count, short of breath, dizziness, headaches,
hyperpigmentation, jaundice, erthema, pruritis, excessive hair
growth, change in bowel habits, change in bladder function,
long-lasting sores, white patches inside the mouth, white spots on
the tongue, unusual bleeding or discharge, thickening or lump on
parts of the body, indigestion, trouble swallowing, changes in
warts or moles, change in new skin and nagging cough or
hoarseness.
[0584] In one embodiment, the signal-sensor polynucleotides may be
investigated in any number of cancer or normal cell lines.
Non-limiting examples of cell lines which may be useful in these
investigations include those from ATCC (Manassas, Va.) including
MRC-5, A549, T84, NCI-H2126 [H2126], NCI-H1688 [H1688], WI-38,
WI-38 VA-13 subline 2RA, WI-26 VA4, C3A [HepG2/C3A, derivative of
Hep G2 (ATCC HB-8065)], THLE-3, H69AR, NCI-H292 [H292], CFPAC-1,
NTERA-2 cl.D1 [NT2/D1], DMS 79, DMS 53, DMS 153, DMS 114,
MSTO-211H, SW 1573 [SW-1573, SW1573], SW 1271 [SW-1271, SW1271],
SHP-77, SNU-398, SNU-449, SNU-182, SNU-475, SNU-387, SNU-423, NL20,
NL20-TA [NL20T-A], THLE-2, HBE135-E6E7, HCC827, HCC4006, NCI-H23
[H23], NCI-H1299, NCI-H187 [H187], NCI-H358 [H-358, H358], NCI-H378
[H378], NCI-H522 [H522], NCI-H526 [H526], NCI-H727 [H727], NCI-H810
[H810], NCI-H889 [H889], NCI-H1155 [H1155], NCI-H1404 [H1404],
NCI-N87 [N87], NCI-H196 [H196], NCI-H211 [H211], NCI-H220 [H220],
NCI-H250 [H250], NCI-H524 [H524], NCI-H647 [H647], NCI-H650 [H650],
NCI-H711 [H711], NCI-H719 [H719], NCI-H740 [H740], NCI-H748 [H748],
NCI-H774 [H774], NCI-H838 [H838], NCI-H841 [H841], NCI-H847 [H847],
NCI-H865 [H865], NCI-H920 [H920], NCI-H1048 [H1048], NCI-H1092
[H1092], NCI-H1105 [H1105], NCI-H1184 [H1184], NCI-H1238 [H1238],
NCI-H1341 [H1341], NCI-H1385 [H1385], NCI-H1417 [H1417], NCI-H1435
[H1435], NCI-H1436 [H1436], NCI-H1437 [H1437], NCI-H1522 [H1522],
NCI-H1563 [H1563], NCI-H1568 [H1568], NCI-H1573 [H1573], NCI-H1581
[H1581], NCI-H1618 [H1618], NCI-H1623 [H1623], NCI-H1650 [H-1650,
H1650], NCI-H1651 [H1651], NCI-H1666 [H-1666, H1666], NCI-H1672
[H1672], NCI-H1693 [H1693], NCI-H1694 [H1694], NCI-H1703 [H1703],
NCI-H1734 [H-1734, H1734], NCI-H1755 [H1755], NCI-H1755 [H1755],
NCI-H1770 [H1770], NCI-H1793 [H1793], NCI-H1836 [H1836], NCI-H1838
[H1838], NCI-H1869 [H1869], NCI-H1876 [H1876], NCI-H1882 [H1882],
NCI-H1915 [H1915], NCI-H1930 [H1930], NCI-H1944 [H1944], NCI-H1975
[H-1975, H1975], NCI-H1993 [H1993], NCI-H2023 [H2023], NCI-H2029
[H2029], NCI-H2030 [H2030], NCI-H2066 [H2066], NCI-H2073 [H2073],
NCI-H2081 [H2081], NCI-H2085 [H2085], NCI-H2087 [H2087], NCI-H2106
[H2106], NCI-H2110 [H2110], NCI-H2135 [H2135], NCI-H2141 [H2141],
NCI-H2171 [H2171], NCI-H2172 [H2172], NCI-H2195 [H2195], NCI-H2196
[H2196], NCI-H2198 [H2198], NCI-H2227 [H2227], NCI-H2228 [H2228],
NCI-H2286 [H2286], NCI-H2291 [H2291], NCI-H2330 [H2330], NCI-H2342
[H2342], NCI-H2347 [H2347], NCI-H2405 [H2405], NCI-H2444 [H2444],
UMC-11, NCI-H64 [H64], NCI-H735 [H735], NCI-H735 [H735], NCI-H1963
[H1963], NCI-H2107 [H2107], NCI-H2108 [H2108], NCI-H2122 [H2122],
Hs 573.T, Hs 573.Lu, PLC/PRF/5, BEAS-2B, Hep G2, Tera-1, Tera-2,
NCI-H69 [H69], NCI-H128 [H128], ChaGo-K-1, NCI-H446 [H446],
NCI-H209 [H209], NCI-H146 [H146], NCI-H441 [H441], NCI-H82 [H82],
NCI-H460 [H460], NCI-H596 [H596], NCI-H676B [H676B], NCI-H345
[H345], NCI-H820 [H820], NCI-H520 [H520], NCI-H661 [H661],
NCI-H510A [H510A, NCI-H510], SK-HEP-1, A-427, Calu-1, Calu-3,
Calu-6, SK-LU-1, SK-MES-1, SW 900 [SW-900, SW900], Malme-3M, and
Capan-1.
[0585] In one embodiment, the signal-sensor polynucleotides
described herein may be investigated in human lung adenocarcinoma.
As a non-limiting example, a signal-sensor polynucleotide encoding
constitutively active caspase 3 fully modified with
5-methylcytidine and 1-methylpseudouridine or fully modified with
1-methylpseudouridine may be delivered to cultured human lung
adenocarcinoma A549 cells (see e.g., the experiment outlined in
Example 53). As another non-limiting example, a signal-sensor
polynucleotide encoding constitutively active caspase 6 fully
modified with 5-methylcytidine and 1-methylpseudouridine or fully
modified with 1-methylpseudouridine may be delivered to cultured
human lung adenocarcinoma A549 cells (see e.g., the experiment
outlined in Example 53).
[0586] In another embodiment, the signal-sensor polynucleotides
described herein may be investigated in human hepatocellular
carcinoma. As a non-limiting example, a signal-sensor
polynucleotide encoding constitutively active caspase 3 fully
modified with 5-methylcytidine and 1-methylpseudouridine or fully
modified with 1-methylpseudouridine may be delivered to human
hepatocellular carcinoma Hep3B cells (see e.g., the experiment
outlined in Example 54).
[0587] In one embodiment, the signal-sensor polynucleotides may be
investigated in an animal model. As a non-limiting example, the
animal model may be for lung cancer such as the lung cancer model
of Fukazawa et al (Anticancer Research, 2010; 30: 4193-4200) where
a congenic mouse is created by crossing a ubiquitously expressing
dominant negative Myc (Omomyc) mouse with a KRAS mutation-positive
lung cancer model mouse. In the presence of Omomyc, lung tumors
caused by the expression of mutated KRAS regresses in the congenic
mouse, indicating that Omomyc caused tumor cell death of KRAS
mutation-positive lung cancer.
[0588] As another non-limiting example, Human lung cancer
xenografts are also prepared by the method of Fukazawa where human
lung cancer xenografts are established in 4-week-old female BALB/C
nude mice (Charles River Laboratories Japan, Kanagawa, Japan) by
subcutaneous inoculation of 4.times.106 A549 cells into the dorsal
flank. The mice are randomly assigned into six groups (n=6/group).
After the tumors reach a diameter of about 0.5 cm (approximately 6
days after tumor inoculations), each group of mice are injected
with 100 .mu.l solution containing PBS, 5.times.1010 vp of control
or signal-sensor polynucleotide into the dorsalflank tumor for the
selected dosing regimen. Animals are then observed closely and
survival studiesor other analyses are performed.
[0589] In one embodiment, the signal-sensor polynucleotides may be
investigated in a transgenic animal model. As a non-limiting
example, the transgenic animal model is a LSL-KRAS.sup.G12D: TRE
Omomyc:CMV rtTA triple transgenic model which involves the use of
an adenovirus expressing Cre recombinase which is administered via
inhalation to induce oncogene expression via excision of the floxed
STOP codon, and ubiquitous Omomyc expression is controlled via
doxycycline. The model is reported in Soucek et al. (Nature, 1-5
(2008)). As another non-limiting example, the mice of Soucek may be
crossed with the LSLKRAS.sup.G12D single transgenic mice (Jackson
Laboratories) and may be used for inhalation delivered or otherwise
lung-delivered studies of signal-sensor polynucleotides expressing
MYC inhibitor D or other oncology related polypeptide described
herein.
[0590] In another embodiment, the signal-sensor polynucleotides may
be investigated in a mouse-in-mouse model such as, but not limited
to a model which is akin to the p53-/-:c-Myc overexpressing HCC
model of Zender (Cell. 2006 Jun. 30; 125(7): 1253-1267).
[0591] In one embodiment, the signal-sensor polynucleotides may be
investigated in a Nongermline genetically engineered mouse model
(NGEMM). As a non-limiting example, the design of mouse-in-mouse
model may involve starting with the WT or tumor suppressor deleted
(such as p53-/-) 129 Sv/Ev Mm ES cell clone; introduction of liver
activated protein (LAP) promoter directed tetracycline
transactivator (tTA) and tetO-luciferase for liver specific
imaging; freezing the resulting LAP-tTA: tetO-luciferase clones to
be used for c-Myc as well as other liver relevant programs
oncogene; adding tetO driven oncogene, e.g. tetOcMyc; Freeze
resulting LAP-tTA: tetO-luciferase: tetO-MYC clones; injecting
resulting ES clones into C57B1/6 blastocytes and implant in pseudo
pregnant mothers whereby the resulting chimeric animals are the
tumor model upon removal of doxycycline (i.e. Tet-Off). The type of
model will ideally evince inducible nodules of c-Myc-driven,
luciferase-expressing HCC surrounded by normal hepatocytes.
[0592] In another embodiment, the signal-sensor polynucleotides may
be investigated in Orthotopic HCC models using the HEP3B cell lines
in mice (Crown Bio).
[0593] As a non-limiting example, any of the animal models
described above may be used to investigate a signal-sensor
polynucleotide encoding MYC inhibitor D. The study may also include
a signal-sensor polynucleotide encoding a negative control such as,
but not limited to, an untranslatable mRNA for MYC inhibitor D and
a vehicle only delivery. The animal may be evaluated for gene
expression, tumor status and/or for any of the hallmarks that are
generally associated with cancer phenotypes or genotypes.
[0594] As another non-limiting example, any of the animal models
described above may be used to investigate a signal-sensor
polynucleotide encoding dominant negative hTERT. The study may also
include a signal-sensor polynucleotide encoding a negative control
such as, but not limited to, an untranslatable mRNA for dominant
negative hTERT and a vehicle only delivery. The animal may be
evaluated for gene expression, tumor status and/or for any of the
hallmarks that are generally associated with cancer phenotypes or
genotypes.
[0595] As another non-limiting example, any of the animal models
described above may be used to investigate a signal-sensor
polynucleotide encoding dominant negative survivin. The study may
also include a signal-sensor polynucleotide encoding a negative
control such as, but not limited to, an untranslatable mRNA for
dominant negative survivin and a vehicle only delivery. The animal
may be evaluated for gene expression, tumor status and/or for any
of the hallmarks that are generally associated with cancer
phenotypes or genotypes.
[0596] In one embodiment, signal-sensor polynucleotides may include
at least one miRNA-binding site in the 3'UTR in order to direct
cytotoxic or cytoprotective mRNA therapeutics to specific cells
such as, but not limited to, normal and/or cancerous cells in an
animal model described herein. As a non-limiting example, a strong
apoptotic signal and at least one miR-122a binding site is encoded
by the signal-sensor polynucleotide where the at least one miR-122a
binding site is located in the 3'UTR. As another non-limiting
example, apoptosis inducing factor short isoform (AIFsh) and at
least one miR-122a binding site is encoded by the signal-sensor
polynucleotide where the at least one miR-122a binding site is
located in the 3'UTR. As yet another non-limiting example,
constitutively active (C.A.) caspase 6 and at least one miR-122a
binding site is encoded by the signal-sensor polynucleotide where
the at least one miR-122a binding site is located in the 3'UTR. As
another non-limiting example, HSV1-tk and at least one miR-122a
binding site is encoded by the signal-sensor polynucleotide where
the at least one miR-122a binding site is located in the 3'UTR.
[0597] In another embodiment, signal-sensor polynucleotides may
include three miRNA-binding sites in the 3'UTR in order to direct
cytotoxic or cytoprotective mRNA therapeutics to specific cells
such as, but not limited to, normal and/or cancerous cells in an
animal model described herein. As a non-limiting example, a strong
apoptotic signal and three miR-122a binding sites are encoded by
the signal-sensor polynucleotide where the three miR-122a binding
sites are located in the 3'UTR. As another non-limiting example,
apoptosis inducing factor short isoform (AIFsh) and three miR-122a
binding sites are encoded by the signal-sensor polynucleotide where
the three miR-122a binding sites are located in the 3'UTR. As yet
another non-limiting example, constitutively active (C.A.) caspase
6 and three miR-122a binding sites are encoded by the signal-sensor
polynucleotide where the three miR-122a binding sites are located
in the 3'UTR. As another non-limiting example, HSV1-tk and and
three miR-122a binding sites are encoded by the signal-sensor
polynucleotide where the three miR-122a binding sites are located
in the 3'UTR.
Common Categories of Cancer
Brain Cancer
[0598] Brain cancer is the growth of abnormal cells in the tissues
of the brain usually related to the growth of malignant brain
tumors. Brain tumors grow and press on the nearby areas of the
brain which can stop that part of the brain from working the way it
should. Brain cancer rarely spreads into other tissues outside of
the brain. The grade of tumor, based on how abnormal the cancer
cells look under a microscope, may be used to tell the difference
between slow- and fast-growing tumors. Grade I tumors grow slowly,
rarely spreads into nearby tissues, has cells that look like normal
cells and the entire tumor may be removable by surgery. Grade II
tumors also grow slowly but may spread into nearby tissue and may
recur. Grade III tumors grow quickly, is likely to spread into
nearby tissue and the tumor cells look very different from normal
cells. Grade IV, high-grade, grows and spreads very quickly and
there may be areas of dead cells in the tumor. Symptoms of brain
cancer may include, but are not limited to, morning headache or
headache that goes away after vomiting, frequent nausea and
vomiting, vision, hearing, and speech problems, loss of balance and
trouble walking, weakness on one side of the body, unusual
sleepiness or change in activity level, unusual changes in
personality or behavior, seizures.
[0599] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with brain cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
polynucleotides, primary constructs or mmRNA of the present
invention may be used to reduce, eliminate, or prevent tumor growth
in a subject who has been diagnosed or may be diagnosed with brain
cancer by administering to said subject an isolated polynucleotide
encoding an oncology-related polypeptide of interest. In one
embodiment, the signal-sensor polynucleotides, primary constructs
or mmRNA of the present invention may be used to reduce and/or
ameliorate at least one symptom of cancer in a subject who has been
diagnosed or may be diagnosed with brain cancer by administering to
said subject an isolated polynucleotide encoding an
oncology-related polypeptide of interest.
Breast Cancer
[0600] Breast cancer forms in the tissues of the breast, of both
men and women, such as, but not limited to, the ducts and the
lobules. The most common type of breast cancer is ductal carcinoma
which begins in the cells of the ducts. Lobular cancer, which
begins in the lobes or lobules, is often found in both breasts. An
uncommon type of breast cancer, inflammatory breast cancer, causes
the breast to be warm, red and swollen. Hereditary breast cancer
makes up approximately 5-10% of all breast cancer and altered genes
are common in some ethnic groups making that enthic group more
susceptible to breast cancer. Symptoms of breast cancer include,
but are not limited to, a lumpm or thickening in or near the breast
or in the underarm area, change in the size or shape of the breast,
dimple or puckering in the skin of the breast, inward turned nipple
of the breast, fluid from the nipple which is not breast milk,
scaly, red or swollen skin on the breast, nipple, or areola, and
dimples in the breast that look like the skin of orange (peau
d'orange).
[0601] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with breast cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
polynucleotides, primary constructs or mmRNA of the present
invention may be used to reduce, eliminate, or prevent tumor growth
in a subject who has been diagnosed or may be diagnosed with breast
cancer by administering to said subject an isolated polynucleotide
encoding an oncology-related polypeptide of interest. In one
embodiment, the signal-sensor polynucleotides, primary constructs
or mmRNA of the present invention may be used to reduce and/or
ameliorate at least one symptom of cancer in a subject who has been
diagnosed or may be diagnosed with breast cancer by administering
to said subject an isolated polynucleotide encoding an
oncology-related polypeptide of interest.
Cervical Cancer
[0602] Cervical cancer forms in the tissues of the cervic and is
usually slow-growing. The cause of cervical cancer usually related
to the human papillomavirus (HPV) infection. Although cervical
cancer may not not show any signs, possible symptoms may include,
but are not limited to, vaignal bleeding, unusal vaginal discharge,
pelvic pain and pain during sexual intercourse.
[0603] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with cervical cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with cervical cancer by administering to said subject an
isolated polynucleotide encoding an oncology-related polypeptide of
interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
reduce and/or ameliorate at least one symptom of cancer in a
subject who has been diagnosed or may be diagnosed with cervical
cancer by administering to said subject an isolated polynucleotide
encoding an oncology-related polypeptide of interest.
Esophageal Cancer
[0604] Esophageal cancer is cancer that forms in the tissues lining
the esophagus. There are two common types of esophageal cancer
which are named for the type of cells that become malignant.
Squamous cell carcinoma is cancer that forms in the thin, flat
cells lining the esophagus (also called epidermoid carcinoma).
Cancer that begins in the glandular (secretory) cells which produce
and release fluids such as mucus is called adneocarcinoma. Common
symptoms associated with esophageal cancer include, but are not
limited to, painful or difficult swallowing, weight loss, pain
behind the breastbone, hoarseness and cough, and indigestion and
heartburn.
[0605] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with esophageal cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with esophageal cancer by administering to said subject
an isolated polynucleotide encoding an oncology-related polypeptide
of interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
reduce and/or ameliorate at least one symptom of cancer in a
subject who has been diagnosed or may be diagnosed with esophageal
cancer by administering to said subject an isolated polynucleotide
encoding an oncology-related polypeptide.
Familial Cancer Syndrome
[0606] Familial cancer syndrome describes the genetic
predisposition of a subject to develop cancer. 5-10% of all cancers
are hereditary and are passed on through specific in specific genes
passed from one blood relative to another. Subjects that inherit
one of these gene changes may have a higher likelihood of
developing cancer within their lifetime. Familial cancer syndrome
includes disorder such as, but not limited to, Ataxia
Telangiectasia, Basal Cell Nevus Syndrome, Nevoid Basal Cell
Carcinoma Syndrome, Gorlin Syndrome, Beck-with Wiedemann Syndrome,
Birt-Hogg-Dube Syndrome, Bloom Syndrome, hereditary breast and/or
ovarian cancer, Carney Complex, Types I and II, Familial Chordoma,
Colon Cancer, Hereditary Nonpolyposis-Lynch Syndrome, Costello
Syndrome, Facio-Cutaneous-Skeletal Syndrome, Cowden Syndrome,
Dyskeratosis Congenita, Tylosis with Esophaeal Cancer, Keratosis
Palmaris et Plantaris with Esophageal Cancer, Howel-Evans Syndrome,
Herediatary Multiple Exostosis, Fanconi Anemia, Hereditary Diffuse
Gastric Cancer, Gastrointestinal Stromal Tumor, Multiple
Gastrointestinal Stromal Tumor, Familial Hyperparathyroidism, Acute
Myeloid Leukemia, Familial Leukemia, Chronic Lymphocytic Leukemia,
Li-Fraumeni Syndrome, Hodgkin Lymphoma, Non-Hodgkin Lymphoma,
Hereditary Multiple Melanoma, Mosaic Varigated Aneuploidy, Multple
Endocrine Neoplasia Type 1, Type 2A and 2B, Familial Medullary
Thyroid Cancer, Familial Mulitple Myeloma, Hereditary
Neuroblastoma, Neurofibromatosis Type 1 and 2, Nijmegen Breakage
Syndrome, Hereditary Pancreatic Cancer, Hereditary Paraganglioma,
Peutz-Jeghers Syndrome, Familial Adenomatous Polyposis, Familial
Juvenile Polyposis, MYH-Associated Polyposis, Hereditary Prostate
Cancer, Hereditary Renal Cell Carcinoma with Multiple Cutaneous and
Uterine Leiomyomas, Hereditary Renal Cell Carcinoma, Hereditary
Papillary Renal Cell Carcinoma, Rhabdoid Predisposition Syndrome,
Rothmund-Thomson Syndrome, Simpson-Golabi-Behmel Syndrome, Familial
Testicular Germ Cell Tumor, Familial Non-medullary Thyroid
Carcinoma, Tuberous Sclerosis Complex, von Hippel-Lindau Syndrome,
Familial Waldenstrom Macroglobulinemia, Werner Syndrome, Familial
Wilms Tumor and Xeroderma Pigmentosum.
[0607] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with Familial cancer syndrome by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with Familial cancer syndrome by administering to said
subject an isolated polynucleotide encoding an oncology-related
polypeptide of interest. In one embodiment, the signal-sensor
polynucleotides, primary constructs or mmRNA of the present
invention may be used to reduce and/or ameliorate at least one
symptom of cancer in a subject who has been diagnosed or may be
diagnosed with Familial cancer syndrome by administering to said
subject an isolated polynucleotide encoding an oncology-related
polypeptide of interest.
Leukemia
[0608] Leukemia is a form of cancer that starts in blood-forming
tissue such as the bone marrow which can cause a large number of
blood cells to be produced and enter the blood stream. Leukemia can
also spread to the central nervous system and cause brain and
spinal cord cancer. Types of leukemia include, but are not limited
to, adult acute lymphoblastic, childhood acute lymphoblastic, aduct
acute myeloid, chronic lymphocytic, chronic myelogenous and hairy
cell. Non-limiting examples of symptoms of leukemia include
weakness or feeling tired, fever, easy bruising or bleeding,
petechiae, shortness of breath, weight loss or loss of appetite,
pain in the bones or stomach, pain or feeling of fullness below the
ribs, and painless lumps in the neck, underarm, stomach or
groin.
[0609] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with leukemia by administering
to said subject an isolated polynucleotide encoding an
oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with leukemia by administering to said subject an
isolated polynucleotide encoding an oncology-related polypeptide of
interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
reduce and/or ameliorate at least one symptom of cancer in a
subject who has been diagnosed or may be diagnosed with leukemia by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest.
Liver Cancer
[0610] There are two types of liver cancer, primary liver cancer
which forms in the tissue of the liver and secondary liver cancer,
or metastatic liver cancer, that spreads to the liver from another
part of the body. Possible symptoms of liver canver include, but
are not limited to, a hard lump on the right side just below the
rib cage, discomfort in the upper abdomen on the right side, pain
around the right shoulder blade, unexplained weight loss, jaundice,
unusual tiredness, nausea and loss of appetide.
[0611] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with liver cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with liver cancer by administering to said subject an
isolated polynucleotide encoding an oncology-related polypeptide of
interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
reduce and/or ameliorate at least one symptom of cancer in a
subject who has been diagnosed or may be diagnosed with liver
cancer by administering to said subject an isolated polynucleotide
encoding an oncology-related polypeptide of interest.
Hepatocellular Carcinoma
[0612] The c-myc protein is a multifunctional bHLHZip transcription
factor with critical roles in normal cellular processes and
aberrantly regulated in the majority of human cancers. c-, N- and
L-Myc are family members that can dimerize with partners such as
Max, Mad and Miz-1. The protein is implicated in the
transactivation and repression of a vast number of proposed
transcriptional targets and recent work has demonstrated a role for
Myc as a "transcriptional amplifier" of otherwise transactivated
genes in developing cancers. It has a well established function in
cancer cell proliferation, growth, biosynthetic metabolism,
ribogenesis and translation and possibly a non-redundant node
through whichoncogenic signals must navigate.
[0613] MYC inhibitor D (also known as Omomyc) is a unique
dominant-negative 90 a.a. protein comprised of the human c-Myc
oligomerization domain with 4 introduced mutations E57T, E64I,
R70Q, R71N (Soucek et al., Oncogene, 1998; 17, 2463-2472).
Importantly, it exhibits selectivity in binding and inhibitory
capability: binding c-Myc, N-Myc, Max and Miz-1. It also prevents
E-box mediated transactivation while retaining Miz-1 directed
transrepression. The therapeutic potential of MYC inhibitor D has
been specifically exhibited in vivo where transgenic expression of
OMOMYC blocked MycERTAM induced keratinocyte proliferation (Soucek
et al., CDD 2004; 11, 1038-1045); transgenic Omomyc prevented the
establishment and induced the regression of forming and mature lung
tumors, respectively, in the LSL-KrasGl2D mouse model with
reversible toxicity (Soucek et al., Nature 2008, 455, 679-683);
transgenic Omomyc prevents tumor formation and regresses
established tumors in the RIP 1-TAG2 model of pancreatic
neuroendocrine cancer with controllable side effects, and further
shows a role for cancer cell Myc in the maintenance of a permissive
tumor microenvironment (Sodir et al., Genes and Development 2011,
25, 907-916); and it was reported "that Omomyc induces cell death
of KRAS-mutated human lung adenocarcinoma A549 cells in vitro and
in vivo" (Fukazawa et al., Anticancer Res, 2010, 30,
4193-4200).
[0614] Although it stands to reason that the inhibition of
oncogenic c-Myc via the directed expression of MYC inhibitor D
might prove to be an effective therapy in at least a subset of
HCCs, proof of concept in HCC remains to be demonstrated.
[0615] In some embodiments, the present invention includes
signal-sensor polynucleotides encoding MYC inhibitor D as the
oncology-related polypeptide; with or without a sensor sequence for
the treatment of hepatocellular carcinoma (HCC). The studies of HCC
may be performed in any of the subclasses of HCC cell lines as
described by Hoshida et al (Cancer Research 2009; 69: 7385-7392).
These include S2 cells wich have higher TGF-beta and WNT signaling
and demonstrate and associated with a greater risk of early
recurrence, S2 which exhibit increased myc and AKT expression and
the highest level of alpha feto-protein or S3 which retain the
hepatocyte like phenotype. S1 and S2 types have also been shown to
exhibit increased E2F1 and decreased p53 expression; while S2 alone
has shown decreased levels of interferon. S1 cell lines include
SNU-387, SNU-423, SNU-449, SNU-475, SNU-182, SK-Hepl, HLE, HLF, and
Focus, whereas S2 cell lines include Huh-1, Huh-6, Huh-7, HepG2,
Hep3B, Hep3B-TR, Hep40, and PLC/PRF/5 cells.
Lung Cancer
[0616] Lung cancer forms in the tissues of the lung usually in the
cells lining the air passages and is classified as either small
cell lung cancer or non-small cell lung cancer. There are two types
of small cell lung cancer, small cell carcinoma and combined small
cell carcinoma. The types of on-small cell lung cancer are squamous
cell carcinoma (cancer begins in the squamous cells), large cell
carcinoma (cancer may begin in several types of cells) and
adenocarcinoma (cancer begins in the cells that line the alveoli
and in cells that make mucus). Symptoms of lung cancer include, but
are not limited to, chest discomfort or pain, cough that does not
go away or gets worse over time, trouble breathing, wheezing, blood
in the sputum, hoarseness, loss of appetite, weight loss for no
known reason, feeling very tired, trouble swallowing and swelling
in the face and/or veins in the neck.
[0617] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with lung cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with lung cancer by administering to said subject an
isolated polynucleotide encoding a polypeptide of interest. In one
embodiment, the signal-sensor polynucleotides, primary constructs
or mmRNA of the present invention may be used to reduce and/or
ameliorate at least one symptom of cancer in a subject who has been
diagnosed or may be diagnosed with lung cancer by administering to
said subject an isolated polynucleotide encoding an
oncology-related polypeptide of interest.
Lymphoma
[0618] Lymphoma is cancer that beings in the cells of the immune
system. Subjects who have Hodgkin lymphoma have a cell called
Reed-Sternberg cell and non-Hodgkin lymphoma includes a large group
of cancers of immune system cells. Examples of Lymphoma include,
but are not limited to, painless, swollen lymph nodes in the neck,
underarm or groin, fever for no known reason, drenching night
sweats, weight loss for no known reason, itchy skin and
fatigue.
[0619] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with lymphoma by administering
to said subject an isolated polynucleotide encoding a polypeptide
of interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
reduce, eliminate, or prevent tumor growth in a subject who has
been diagnosed or may be diagnosed with lymphoma by administering
to said subject an isolated polynucleotide encoding a polypeptide
of interest. In one embodiment, the polynucleotides, primary
constructs or mmRNA of the present invention may be used to reduce
and/or ameliorate at least one symptom of cancer in a subject who
has been diagnosed or may be diagnosed with lymphoma by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest.
Ovarian Cancer
[0620] Ovarian cancer is cancer which forms in the tissues of the
ovary which are either ovarian epithelial carcinomas (begins on the
surface of the ovary) or malignant germ cell tumors (cancer that
begins in the egg cells). Symptoms of ovarian cancer include, but
are not limited to, pain or swelling in the abdomen, pain in the
pelvis, gastrointestinal problems such as gas, bloating, or
constipation and vaginal bleeding after menopause.
[0621] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with ovarian cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or signal-sensor
mmRNA of the present invention may be used to reduce, eliminate, or
prevent tumor growth in a subject who has been diagnosed or may be
diagnosed with ovarian cancer by administering to said subject an
isolated polynucleotide encoding an oncology-related polypeptide of
interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or signal-sensor mmRNA of the present invention
may be used to reduce and/or ameliorate at least one symptom of
cancer in a subject who has been diagnosed or may be diagnosed with
ovarian cancer by administering to said subject an isolated
polynucleotide encoding an oncology-related polypeptide of
interest.
Prostate Cancer
[0622] Prostate that forms in the tissue of the prostate mainly
affects older men. Non-limiting examples of prostate cancer
include, but are not limited to, weak or interrupted flow of urine,
frequent urination, trouble urinating, pain or burning during
urination, blood in the urine or semen, pain in the back, hips or
pelvis that does not go away and painful ejaculation.
[0623] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with prostate cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with prostate cancer by administering to said subject an
isolated polynucleotide encoding an oncology-related polypeptide of
interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
reduce and/or ameliorate at least one symptom of cancer in a
subject who has been diagnosed or may be diagnosed with prostate
cancer by administering to said subject an isolated polynucleotide
encoding an oncology-related polypeptide of interest.
Testicular Cancer
[0624] Testicular cancer forms in the tissues of one or both
testicles and is most common in young or middle-aged men. Most
testicular cancers being in germ cells and are called testicular
germ cell tumors. There are two types of testicular germ cell
tumors called seminomas and nonseminomas. Common symptoms of
testicular cancer include, but are not limited to, a painless lump
or swelling in either testicle, change in how the testicle feels,
dull ache in the lower abdomen or the groin, sudden build-up of
fluid in the scrotum and pain or discomfort in a testicle or in the
scrotum.
[0625] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with testicular cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-related polypeptide of interest. In one embodiment, the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with testicular cancer by administering to said subject
an isolated signal-sensor polynucleotide encoding an
oncology-related polypeptide of interest. In one embodiment, the
polynucleotides, primary constructs or mmRNA of the present
invention may be used to reduce and/or ameliorate at least one
symptom of cancer in a subject who has been diagnosed or may be
diagnosed with testicular cancer by administering to said subject
an isolated polynucleotide encoding an oncology-related polypeptide
of interest.
Throat Cancer
[0626] Throat cancer forms in the tissues of the pharynx and
includes cancer of the nasopharynx (nasopharyngeal cancer),
oropharynx (oropharyngeal cancer), hypopharynx (hypopharyngeal
cancer), and larynx (laryngeal cancer). Common symptoms of throat
cancer include, but are not limited to, a sore throat that does not
go away, ear pain, lump in the neck, painful or difficulty
swallowing, change or hoarseness in the voice, trouble breathing or
speaking, nosebleeds, trouble hearing, pain or ringing in the ear,
headaches, dull pain behind the breast bone, cough and weight loss
for no reason.
[0627] In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
treat a disease, disorder and/or condition in a subject who has
been diagnosed or may be diagnosed with throat cancer by
administering to said subject an isolated polynucleotide encoding
an oncology-relateda polypeptide of interest. In one embodiment,
the signal-sensor polynucleotides, primary constructs or mmRNA of
the present invention may be used to reduce, eliminate, or prevent
tumor growth in a subject who has been diagnosed or may be
diagnosed with throat cancer by administering to said subject an
isolated polynucleotide encoding an oncology-related polypeptide of
interest. In one embodiment, the signal-sensor polynucleotides,
primary constructs or mmRNA of the present invention may be used to
reduce and/or ameliorate at least one symptom of cancer in a
subject who has been diagnosed or may be diagnosed with throat
cancer by administering to said subject an isolated polynucleotide
encoding an oncology-related polypeptide of interest.
Inhibition of Hypoxia-Inducible Factors (HIFs)
[0628] Hypoxia-inducible factors (HIFs) control cellular adaptation
to oxygen deprivation. Cancer cells engage HIFs to sustain their
growth in adverse conditions, thus promoting a cellular
reprograming that includes metabolism, proliferation, survival and
mobility. HIFs overexpression in human cancer biopsies correlates
with high metastasis and mortality.
[0629] HIFs regulate genes related to metabolism such as GLUT1,
GLUT3, ALDOA, ENO1, GAPDH, HK1, HK2, PFKL, PGK1, PKM2, LDHA,
proliferation such as IGF-2, TGFA, VEGFA, survival such as TERT,
NANOG, OCT4 and cell migration-invasion such as ZEB1, ZEB2, SNAI2,
MMP14, MMP9, AMF, MET, PTHrP. (Keith, et al Nat Rev Cancer 2012;
12:9-22).
[0630] In one embodiment, one or more signal-sensor polynucleotides
may be administered to the cancer cell to investigate the
destabilization of cancer, The selection of the sequence, dose or
administrative route is optionally informed by diagnostic
evaluation of the cell, tumor, tissue or organism including, but
not limited to, expression profiling of the cancer, metabolic
evaluation (hypoxic, acidotic), apoptotic vs. survival profiling,
cell cycle vs. senescent profiling, immune sensitivities, and/or
evaluation of stromal factors.
[0631] In one embodiment, the signal-sensor polynucleotides may
encode either or both of the oncology related polypeptides, CITED4
and SHARP1. The signal-sensor polynucleotides are then administered
where the administration of either or both results in the
inhibition of the transcriptome of HIF-1alpha in cancer cells.
Suppression of HIF1-alpha gene regulated expression occurs upon
administration with higher suppression when both polynucleotides
are administered together. Reporter constructs such as luciferase
under HIF1-alpha are used in the manner similar to the methods
disclosed in van de Sluis et al, (J Clin Invest. 2010;
120(6):2119-2130). It is known that both CITED4 and SHARP 1
expression results in decreased HIF1-alpha and concomitant
reduction in HIF1-alpha regulated gene expression. Cell death
and/or proliferation may also be evaluated in order to determine
the effectiveness of the signal-sensor polynucleotide.
[0632] In another embodiment, additional experiments can be
conducted using a cancer cell line where CITED4 and SHARP1 are
themselves down regulated either under hypoxic conditions. A
positive result woudl demonstrate that specifically targeting the
metabolic profile (in this case hypoxic-adaptations of CITED4 and
SHAPR1) with replacement of native proteins via signal-sensor
polynucleotides can directly impact the transcriptome and survival
advantage of cancer cells with this profile. Further, the data
could show that the relative impact of signal-sensor polynucleotide
vs. vehicle under hypoxic conditions was more significant for
cancer cells than for normal cells. (i.e., the cancer cells have a
disproportionate survival advantage based on their CITED4+SHARP1
down regulation) that makes them more sensitive to the replacement
of this protein then a normal cell is to overproduction of it. It
is understood that a cancer cell will likely be experiencing
hypoxic conditions and that a normal cell under normoxic conditions
might be able to tolerate CITED4 and SHARP1 over expression because
the normal cell is not dependent on HIF1alpha transctiptome for
survival advantage.
[0633] In one embodiment, in vivo experiments are performed
according to the design of the in vitro experiments where the
animal model is one evincing metastasis in the cancer setting
because HIF-1alpha appears to confer the largest portion of its
advantage in metastasis. Animals are administered the signal-sensor
polynucleotide compared to no treatment or a control
polynucleotide. Animal cells, tissues and/or organs are then
evaluated for alterations in gene expression profiles or
transcriptome levels.
Titration Between Cofactors
[0634] Experiments may be conducted in order to titrate the binding
affinity between two cofactors. As used herein, the term "titrate"
refers to a method whereby one or more factors are introduced
systematically (such as at increasing levels or wherein the one or
more factors are systematically modified) to a solution, scenario
or series thereof in order to assess a property of interest. In
this embodiment, the property of interest is the binding affinity
between two cofactors. In one embodiment, constructs encoding the
two cofactors are obtained and/or synthesized and a series of
mutant constructs are prepared and/or synthesized. Mutant
constructs encode cofactor mutants that may include truncated
mutants (mutant proteins lacking one or more amino acids from
either the N- or C-terminal domains), mutants with regional
deletions [proteins wherein internal regions (comprising one or
more amino acids) of the protein are absent], mutants with single
amino acid substitutions (wherein a normally expressed amino acid
is replaced with an alternative amino acid), mutants with one or
more additional amino acids added internally or at either terminus,
mutants with regional substitutions [proteins wherein internal
regions (comprising one or more amino acids) of the protein are
substituted with alternative regions (comprising one or more amino
acids) and/or combinations of any of these. Mutant constructs are
mutated randomly or subjected to targeted mutation based on
existing knowledge of the molecular interactions necessary for
binding between the two cofactors being investigated.
[0635] In some embodiments, a series of mutant proteins are
designed such that the mutations follow a progressive pattern along
the polypeptide chain. Such series may allow for a better
understanding of a particular aspect or feature of the interaction
between cofactors. A mutant series may include, for example, the
production of a series of mutants, each with a single amino acid
substitution, wherein each mutant has a different amino acid along
it's polypeptide sequence mutated (e.g. alanine is substituted,
thereby eliminating the influence of an amino acid side chain at
each position). In another example, a series of mutants are
designed such that the mutants in the series comprise truncations
of increasing size. In another example, amino acids capable of
being post-translationally modified (e.g. phosphorylated,
acetylated, ubiquitinated, glycosylated, etc.) in a similar manner
may be mutated along the polypeptide sequence in a series of
mutants.
[0636] For titration experiments with mutant cofactors, a baseline
affinity between the two cofactors is established by combining both
cofactors under conditions favorable for binding and the binding
affinity between the cofactors is assayed. Binding affinity may be
assessed using any of a variety of methods known in the art. Such
methods may include, but are not limited to Western blot analysis,
immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),
fluorescence resonance energy transfer (FRET), fluorescence
recovery after photobleaching (FRAP), fluorescence polarization
technologies and/or surface plasmon resonance (SPR) based
technologies. For titration, according to one method, a mutant
series of one or both cofactors are combined with the two unmutated
cofactors (to allow for binding competition between the wild type
and mutated proteins). Changes in affinity between the two
cofactors in the presence of increasing concentrations of different
mutants are assessed and compared and/or plotted against the
specific mutations present in the series of mutants that are
competing for binding. Alternatively, mutant cofactors in a series
are individually combined with a corresponding unmutated binding
partner and assessed for binding affinity. Increasing
concentrations of the wild type cofactor (corresponding to the
mutant cofactor) are introduced and changes in binding between the
mutant cofactors and the corresponding unmutated binding partner
are assessed. Comparisons are made between the resulting binding
curves and the binding curves of other mutants tested.
[0637] In some embodiments, titration of the binding affinity
between two cofactors is assessed in the presence or absence of
increasing concentrations of a third factor. Such a third factor
may be an inhibitor or activator of binding between the two
cofactors. A series of mutants, as described above, may be
generated for a third factor and such a series may be used in
titration experiments to assess the effect of mutations on binding
between the two cofactors.
[0638] Information obtained from titration experiments may be used
to design modified mRNA molecules to encode factors that modulate
the interaction between cofactors.
[0639] In some embodiments, titration experiments are carried out
wherein the binding affinity between HIF1 subunits (HIF1-alpha,
HIF2-alpha and ARNT) and/or mutated HIF1 subunits and/or other
proteins that interact with HIF1 is assessed. Titration experiments
may utilize mutant series generated using constructs for one or
more of HIF1-alpha, HIF2-alpha, ARNT and/or a third interacting
factor. In some embodiments, a mutant series is generated for
HIF1-alpha. HIF1-alpha and HIF2-alpha are hyrdroxylated by HIF
hydroxylase enzymes under normal levels of oxygen in the cell,
facilitating degredation and/or blocking transcriptional activity.
Hyrdorxylation decreases as oxygen levels drop, allowing HIF1-alpha
and/or HIF2-alpha to associate with their cofactor, ARNT leading to
elevated expression of genes comprising HIF-response elements
(HREs) (Keith, B. et al., HIF1.alpha. and HIF2.alpha.: sibling
rivalry in hypoxic tumour growth and progression. Nat Rev Cancer.
2011 Dec. 15; 12(1):9-22). In one embodiment, HIF1-alpha mutant
series are generated wherein mutations in the series progressively
eliminate one or more hydroxylation sites along the polypeptide
chain (including, but not limited to proline 402, proline 564
and/or asparagine 803), thereby modulating stability and/or
transcriptional activity in mutant versions of HIF1-alpha. In
another embodiment, an alternative cofactor, HIF2-alpha is used to
generate a mutant series. Such a mutant series may progressively
eliminate one or more hydroxylation sites along the polypeptide
chain (including, but not limited to proline 405, proline 531
and/or asparagine 847), thereby modulating stability and/or
transcriptional activity in mutant versions of HIF2-alpha. In
another embodiment, HIF1-alpha and/or HIF2-alpha mutant series are
generated that progressively mutate regions necessary for
interaction with ARNT, thereby creating mutants with altered
abilities to bind ARNT and modulate HIF-dependent gene expression.
In another embodiment, ARNT mutant series are generated that
progressively mutate regions necessary for interactions with other
HIF subunits, thereby creating mutants with altered abilities to
bind HIF subunits and modulate HIF-dependent gene expression.
[0640] In some embodiments, mutant series are generated for Von
Hippel-Landau tumor suppressor protein (pVHL). This protein binds
hydroxylated HIF1-alpha and HIF2-alpha, facilitating their
ubiquitination and degradation. In one embodiment, mutant series
are generated that progressively mutate regions necessary for
interaction with HIF1 subunits, thereby creating mutants with
altered abilities to bind HIF1 subunits and modulate HIF-dependent
gene expression.
[0641] Non-limiting examples of transcript and polypeptide
sequences which may be used for the titration experiments are shown
in Table 27 (transcript) and Table 28 (polypeptide).
VI. Kits and Devices
Kits
[0642] The invention provides a variety of kits for conveniently
and/or effectively carrying out methods of the present invention.
Typically kits will comprise sufficient amounts and/or numbers of
components to allow a user to perform multiple treatments of a
subject(s) and/or to perform multiple experiments.
[0643] In one aspect, the present invention provides kits
comprising the molecules (signal-sensor polynucleotides, primary
constructs or mmRNA) of the invention. In one embodiment, the kit
comprises one or more functional antibodies or function fragments
thereof
[0644] Said kits can be for oncology-related protein production,
comprising a first signal-sensor polynucleotide, primary construct
or mmRNA comprising a translatable region. The kit may further
comprise packaging and instructions and/or a delivery agent to form
a formulation composition. The delivery agent may comprise a
saline, a buffered solution, a lipidoid or any delivery agent
disclosed herein.
[0645] In one embodiment, the buffer solution may include sodium
chloride, calcium chloride, phosphate and/or EDTA. In another
embodiment, the buffer solution may include, but is not limited to,
saline, saline with 2 mM calcium, 5% sucrose, 5% sucrose with 2 mM
calcium, 5% Mannitol, 5% Mannitol with 2 mM calcium, Ringer's
lactate, sodium chloride, sodium chloride with 2 mM calcium. In a
further embodiment, the buffer solutions may be precipitated or it
may be lyophilized. The amount of each component may be varied to
enable consistent, reproducible higher concentration saline or
simple buffer formulations. The components may also be varied in
order to increase the stability of modified RNA in the buffer
solution over a period of time and/or under a variety of
conditions. In one aspect, the present invention provides kits for
oncology-related protein production, comprising: signal-sensor
polynucleotide, primary construct or mmRNA comprising a
translatable region, provided in an amount effective to produce a
desired amount of an oncology-related protein encoded by the
translatable region when introduced into a target cell; a second
signal-sensor polynucleotide comprising an inhibitory nucleic acid,
provided in an amount effective to substantially inhibit the innate
immune response of the cell; and packaging and instructions.
[0646] In one aspect, the present invention provides kits for
oncology-related protein production, comprising signal-sensor
polynucleotide, primary construct or mmRNA comprising a
translatable region, wherein the signal-sensor polynucleotide
exhibits reduced degradation by a cellular nuclease, and packaging
and instructions.
[0647] In one aspect, the present invention provides kits for
oncology-related protein production, comprising signal-sensor
polynucleotide, primary construct or mmRNA comprising a
translatable region, wherein the polynucleotide exhibits reduced
degradation by a cellular nuclease, and a mammalian cell suitable
for translation of the translatable region of the first nucleic
acid.
Devices
[0648] The present invention provides for devices which may
incorporate signal-sensor polynucleotides, primary constructs or
mmRNA that encode polypeptides of interest. These devices contain
in a stable formulation the reagents to synthesize a signal-sensor
polynucleotide in a formulation available to be immediately
delivered to a subject in need thereof, such as a human
patient.
[0649] In some embodiments the device is self-contained, and is
optionally capable of wireless remote access to obtain instructions
for synthesis and/or analysis of the generated signal-sensor
polynucleotide, primary construct or mmRNA. The device is capable
of mobile synthesis of at least one signal-sensor polynucleotide,
primary construct or mmRNA and preferably an unlimited number of
different signal-sensor polynucleotides, primary constructs or
mmRNA. In certain embodiments, the device is capable of being
transported by one or a small number of individuals. In other
embodiments, the device is scaled to fit on a benchtop or desk. In
other embodiments, the device is scaled to fit into a suitcase,
backpack or similarly sized object. In another embodiment, the
device may be a point of care or handheld device. In further
embodiments, the device is scaled to fit into a vehicle, such as a
car, truck or ambulance, or a military vehicle such as a tank or
personnel carrier. The information necessary to generate a modified
signal-sensor mRNA encoding oncology-related polypeptide of
interest is present within a computer readable medium present in
the device.
[0650] In one embodiment, a device may be used to assess levels of
an oncology-related protein which has been administered in the form
of signal-sensor polynucleotide, primary construct or mmRNA. The
device may comprise a blood, urine or other biofluidic test.
[0651] In some embodiments, the device is capable of communication
(e.g., wireless communication) with a database of nucleic acid and
polypeptide sequences which may be signal-sensor nucleic acid and
oncology-related polypeptide seqeunces. The device contains at
least one sample block for insertion of one or more sample vessels.
Such sample vessels are capable of accepting in liquid or other
form any number of materials such as template DNA, nucleotides,
enzymes, buffers, and other reagents. The sample vessels are also
capable of being heated and cooled by contact with the sample
block. The sample block is generally in communication with a device
base with one or more electronic control units for the at least one
sample block. The sample block preferably contains a heating
module, such heating molecule capable of heating and/or cooling the
sample vessels and contents thereof to temperatures between about
-20 C and above +100 C. The device base is in communication with a
voltage supply such as a battery or external voltage supply. The
device also contains means for storing and distributing the
materials for RNA synthesis.
[0652] Optionally, the sample block contains a module for
separating the synthesized nucleic acids. Alternatively, the device
contains a separation module operably linked to the sample block.
Preferably the device contains a means for analysis of the
synthesized nucleic acid. Such analysis includes sequence identity
(demonstrated such as by hybridization), absence of non-desired
sequences, measurement of integrity of synthesized mRNA (such has
by microfluidic viscometry combined with spectrophotometry), and
concentration and/or potency of modified RNA (such as by
spectrophotometry).
[0653] In certain embodiments, the device is combined with a means
for detection of pathogens present in a biological material
obtained from a subject, e.g., the IBIS PLEX-ID system (Abbott,
Abbott Park, IL) for microbial identification.
[0654] Suitable devices for use in delivering intradermal
pharmaceutical compositions described herein include short needle
devices such as those described in U.S. Pat. Nos. 4,886,499;
5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496;
and 5,417,662. Intradermal compositions may be administered by
devices which limit the effective penetration length of a needle
into the skin, such as those described in PCT publication WO
99/34850 and functional equivalents thereof. Jet injection devices
which deliver liquid compositions to the dermis via a liquid jet
injector and/or via a needle which pierces the stratum corneum and
produces a jet which reaches the dermis are suitable. Jet injection
devices are described, for example, in U.S. Pat. Nos. 5,480,381;
5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;
5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;
5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460;
and PCT publications WO 97/37705 and WO 97/13537. Ballistic
powder/particle delivery devices which use compressed gas to
accelerate vaccine in powder form through the outer layers of the
skin to the dermis are suitable. Alternatively or additionally,
conventional syringes may be used in the classical mantoux method
of intradermal administration.
[0655] In some embodiments, the device may be a pump or comprise a
catheter for administration of compounds or compositions of the
invention across the blood brain barrier. Such devices include but
are not limited to a pressurized olfactory delivery device,
iontophoresis devices, multi-layered microfluidic devices, and the
like. Such devices may be portable or stationary. They may be
implantable or externally tethered to the body or combinations
thereof.
[0656] Devices for administration may be employed to deliver the
signal-sensor polynucleotides, primary constructs or mmRNA of the
present invention according to single, multi- or split-dosing
regimens taught herein. Such devices are described below.
[0657] Method and devices known in the art for multi-administration
to cells, organs and tissues are contemplated for use in
conjunction with the methods and compositions disclosed herein as
embodiments of the present invention. These include, for example,
those methods and devices having multiple needles, hybrid devices
employing for example lumens or catheters as well as devices
utilizing heat, electric current or radiation driven
mechanisms.
[0658] According to the present invention, these
multi-administration devices may be utilized to deliver the single,
multi- or split doses contemplated herein.
[0659] A method for delivering therapeutic agents to a solid tissue
has been described by Bahrami et al. and is taught for example in
US Patent Publication 20110230839, the contents of which are
incorporated herein by reference in their entirety. According to
Bahrami, an array of needles is incorporated into a device which
delivers a substantially equal amount of fluid at any location in
said solid tissue along each needle's length.
[0660] A device for delivery of biological material across the
biological tissue has been described by Kodgule et al. and is
taught for example in US Patent Publication 20110172610, the
contents of which are incorporated herein by reference in their
entirety. According to Kodgule, multiple hollow micro-needles made
of one or more metals and having outer diameters from about 200
microns to about 350 microns and lengths of at least 100 microns
are incorporated into the device which delivers peptides, proteins,
carbohydrates, nucleic acid molecules, lipids and other
pharmaceutically active ingredients or combinations thereof.
[0661] A delivery probe for delivering a therapeutic agent to a
tissue has been described by Gunday et al. and is taught for
example in US Patent Publication 20110270184, the contents of which
are incorporated herein by reference in their entirety. According
to Gunday, multiple needles are incorporated into the device which
moves the attached capsules between an activated position and an
inactivated position to force the agent out of the capsules through
the needles.
[0662] A multiple-injection medical apparatus has been described by
Assaf and is taught for example in US Patent Publication
20110218497, the contents of which are incorporated herein by
reference in their entirety. According to Assaf, multiple needles
are incorporated into the device which has a chamber connected to
one or more of said needles and a means for continuously refilling
the chamber with the medical fluid after each injection.
[0663] In one embodiment, the signal-sensor polynucleotide, primary
construct, or mmRNA is administered subcutaneously or
intramuscularly via at least 3 needles to three different,
optionally adjacent, sites simultaneously, or within a 60 minutes
period (e.g., administration to 4, 5, 6, 7, 8, 9, or 10 sites
simultaneously or within a 60 minute period). The split doses can
be administered simultaneously to adjacent tissue using the devices
described in U.S. Patent Publication Nos. 20110230839 and
20110218497, each of which is incorporated herein by reference.
[0664] An at least partially implantable system for injecting a
substance into a patient's body, in particular a penis erection
stimulation system has been described by Forsell and is taught for
example in US Patent Publication 20110196198, the contents of which
are incorporated herein by reference in their entirety. According
to Forsell, multiple needles are incorporated into the device which
is implanted along with one or more housings adjacent the patient's
left and right corpora cavernosa. A reservoir and a pump are also
implanted to supply drugs through the needles.
[0665] A method for the transdermal delivery of a therapeutic
effective amount of iron has been described by Berenson and is
taught for example in US Patent Publication 20100130910, the
contents of which are incorporated herein by reference in their
entirety. According to Berenson, multiple needles may be used to
create multiple micro channels in stratum corneum to enhance
transdermal delivery of the ionic iron on an iontophoretic
patch.
[0666] A method for delivery of biological material across the
biological tissue has been described by Kodgule et al and is taught
for example in US Patent Publication 20110196308, the contents of
which are incorporated herein by reference in their entirety.
According to Kodgule, multiple biodegradable microneedles
containing a therapeutic active ingredient are incorporated in a
device which delivers proteins, carbohydrates, nucleic acid
molecules, lipids and other pharmaceutically active ingredients or
combinations thereof
[0667] A transdermal patch comprising a botulinum toxin composition
has been described by Donovan and is taught for example in US
Patent Publication 20080220020, the contents of which are
incorporated herein by reference in their entirety. According to
Donovan, multiple needles are incorporated into the patch which
delivers botulinum toxin under stratum corneum through said needles
which project through the stratum corneum of the skin without
rupturing a blood vessel.
[0668] A small, disposable drug reservoir, or patch pump, which can
hold approximately 0.2 to 15 mL of liquid formulations can be
placed on the skin and deliver the formulation continuously
subcutaneously using a small bore needed (e.g., 26 to 34 gauge). As
non-limiting examples, the patch pump may be 50 mm by 76 mm by 20
mm spring loaded having a 30 to 34 gauge needle (BD.TM.
Microinfuser, Franklin Lakes N.J.), 41 mm by 62 mm by 17 mm with a
2 mL reservoir used for drug delivery such as insulin
(OMNIPOD.RTM., Insulet Corporation Bedford, Mass.), or 43-60 mm
diameter, 10 mm thick with a 0.5 to 10 mL reservoir
(PATCHPUMP.RTM., SteadyMed Therapeutics, San Francisco, Calif.).
Further, the patch pump may be battery powered and/or
rechargeable.
[0669] A cryoprobe for administration of an active agent to a
location of cryogenic treatment has been described by Toubia and is
taught for example in US Patent Publication 20080140061, the
contents of which are incorporated herein by reference in their
entirety. According to Toubia, multiple needles are incorporated
into the probe which receives the active agent into a chamber and
administers the agent to the tissue.
[0670] A method for treating or preventing inflammation or
promoting healthy joints has been described by Stock et al and is
taught for example in US Patent Publication 20090155186, the
contents of which are incorporated herein by reference in their
entirety. According to Stock, multiple needles are incorporated in
a device which administers compositions containing signal
transduction modulator compounds.
[0671] A multi-site injection system has been described by Kimmell
et al. and is taught for example in US Patent Publication
20100256594, the contents of which are incorporated herein by
reference in their entirety. According to Kimmell, multiple needles
are incorporated into a device which delivers a medication into a
stratum corneum through the needles.
[0672] A method for delivering interferons to the intradermal
compartment has been described by Dekker et al. and is taught for
example in US Patent Publication 20050181033, the contents of which
are incorporated herein by reference in their entirety. According
to Dekker, multiple needles having an outlet with an exposed height
between 0 and 1 mm are incorporated into a device which improves
pharmacokinetics and bioavailability by delivering the substance at
a depth between 0.3 mm and 2 mm.
[0673] A method for delivering genes, enzymes and biological agents
to tissue cells has described by Desai and is taught for example in
US Patent Publication 20030073908, the contents of which are
incorporated herein by reference in their entirety. According to
Desai, multiple needles are incorporated into a device which is
inserted into a body and delivers a medication fluid through said
needles.
[0674] A method for treating cardiac arrhythmias with fibroblast
cells has been described by Lee et al and is taught for example in
US Patent Publication 20040005295, the contents of which are
incorporated herein by reference in their entirety. According to
Lee, multiple needles are incorporated into the device which
delivers fibroblast cells into the local region of the tissue.
[0675] A method using a magnetically controlled pump for treating a
brain tumor has been described by Shachar et al. and is taught for
example in U.S. Pat. No. 7,799,012 (method) and 7799016 (device),
the contents of which are incorporated herein by reference in their
entirety. According Shachar, multiple needles were incorporated
into the pump which pushes a medicating agent through the needles
at a controlled rate.
[0676] Methods of treating functional disorders of the bladder in
mammalian females have been described by Versi et al. and are
taught for example in U.S. Pat. No. 8,029,496, the contents of
which are incorporated herein by reference in their entirety.
According to Versi, an array of micro-needles is incorporated into
a device which delivers a therapeutic agent through the needles
directly into the trigone of the bladder.
[0677] A micro-needle transdermal transport device has been
described by Angel et al and is taught for example in U.S. Pat. No.
7,364,568, the contents of which are incorporated herein by
reference in their entirety. According to Angel, multiple needles
are incorporated into the device which transports a substance into
a body surface through the needles which are inserted into the
surface from different directions. The micro-needle transdermal
transport device may be a solid micro-needle system or a hollow
micro-needle system. As a non-limiting example, the solid
micro-needle system may have up to a 0.5 mg capacity, with 300-1500
solid micro-needles per cm.sup.2 about 150-700 .mu.m tall coated
with a drug. The micro-needles penetrate the stratum corneum and
remain in the skin for short duration (e.g., 20 seconds to 15
minutes). In another example, the hollow micro-needle system has up
to a 3 mL capacity to deliver liquid formulations using 15-20
microneedles per cm2 being approximately 950 .mu.m tall. The
micro-needles penetrate the skin to allow the liquid formulations
to flow from the device into the skin. The hollow micro-needle
system may be worn from 1 to 30 minutes depending on the
formulation volume and viscocity.
[0678] A device for subcutaneous infusion has been described by
Dalton et al and is taught for example in U.S. Pat. No. 7,150,726,
the contents of which are incorporated herein by reference in their
entirety. According to Dalton, multiple needles are incorporated
into the device which delivers fluid through the needles into a
subcutaneous tissue.
[0679] A device and a method for intradermal delivery of vaccines
and gene therapeutic agents through microcannula have been
described by Mikszta et al. and are taught for example in U.S. Pat.
No. 7,473,247, the contents of which are incorporated herein by
reference in their entirety. According to Mitszta, at least one
hollow micro-needle is incorporated into the device which delivers
the vaccines to the subject's skin to a depth of between 0.025 mm
and 2 mm.
[0680] A method of delivering insulin has been described by Pettis
et al and is taught for example in U.S. Pat. No. 7,722,595, the
contents of which are incorporated herein by reference in their
entirety. According to Pettis, two needles are incorporated into a
device wherein both needles insert essentially simultaneously into
the skin with the first at a depth of less than 2.5 mm to deliver
insulin to intradermal compartment and the second at a depth of
greater than 2.5 mm and less than 5.0 mm to deliver insulin to
subcutaneous compartment.
[0681] Cutaneous injection delivery under suction has been
described by Kochamba et al. and is taught for example in U.S. Pat.
No. 6,896,666, the contents of which are incorporated herein by
reference in their entirety. According to Kochamba, multiple
needles in relative adjacency with each other are incorporated into
a device which injects a fluid below the cutaneous layer.
[0682] A device for withdrawing or delivering a substance through
the skin has been described by Down et al and is taught for example
in U.S. Pat. No. 6,607,513, the contents of which are incorporated
herein by reference in their entirety. According to Down, multiple
skin penetrating members which are incorporated into the device
have lengths of about 100 microns to about 2000 microns and are
about 30 to 50 gauge.
[0683] A device for delivering a substance to the skin has been
described by Palmer et al and is taught for example in U.S. Pat.
No. 6,537,242, the contents of which are incorporated herein by
reference in their entirety. According to Palmer, an array of
micro-needles is incorporated into the device which uses a
stretching assembly to enhance the contact of the needles with the
skin and provides a more uniform delivery of the substance.
[0684] A perfusion device for localized drug delivery has been
described by Zamoyski and is taught for example in U.S. Pat. No.
6,468,247, the contents of which are incorporated herein by
reference in their entirety. According to Zamoyski, multiple
hypodermic needles are incorporated into the device which injects
the contents of the hypodermics into a tissue as said hypodermics
are being retracted.
[0685] A method for enhanced transport of drugs and biological
molecules across tissue by improving the interaction between
micro-needles and human skin has been described by Prausnitz et al.
and is taught for example in U.S. Pat. No. 6,743,211, the contents
of which are incorporated herein by reference in their entirety.
According to Prausnitz, multiple micro-needles are incorporated
into a device which is able to present a more rigid and less
deformable surface to which the micro-needles are applied.
[0686] A device for intraorgan administration of medicinal agents
has been described by Ting et al and is taught for example in U.S.
Pat. No. 6,077,251, the contents of which are incorporated herein
by reference in their entirety. According to Ting, multiple needles
having side openings for enhanced administration are incorporated
into a device which by extending and retracting said needles from
and into the needle chamber forces a medicinal agent from a
reservoir into said needles and injects said medicinal agent into a
target organ.
[0687] A multiple needle holder and a subcutaneous multiple channel
infusion port has been described by Brown and is taught for example
in U.S. Pat. No. 4,695,273, the contents of which are incorporated
herein by reference in their entirety. According to Brown, multiple
needles on the needle holder are inserted through the septum of the
infusion port and communicate with isolated chambers in said
infusion port.
[0688] A dual hypodermic syringe has been described by Horn and is
taught for example in U.S. Pat. No. 3,552,394, the contents of
which are incorporated herein by reference in their entirety.
According to Horn, two needles incorporated into the device are
spaced apart less than 68 mm and may be of different styles and
lengths, thus enabling injections to be made to different
depths.
[0689] A syringe with multiple needles and multiple fluid
compartments has been described by Hershberg and is taught for
example in U.S. Pat. No. 3,572,336, the contents of which are
incorporated herein by reference in their entirety. According to
Hershberg, multiple needles are incorporated into the syringe which
has multiple fluid compartments and is capable of simultaneously
administering incompatible drugs which are not able to be mixed for
one injection.
[0690] A surgical instrument for intradermal injection of fluids
has been described by Eliscu et al. and is taught for example in
U.S. Pat. No. 2,588,623, the contents of which are incorporated
herein by reference in their entirety. According to Eliscu,
multiple needles are incorporated into the instrument which injects
fluids intradermally with a wider disperse.
[0691] An apparatus for simultaneous delivery of a substance to
multiple breast milk ducts has been described by Hung and is taught
for example in EP 1818017, the contents of which are incorporated
herein by reference in their entirety. According to Hung, multiple
lumens are incorporated into the device which inserts though the
orifices of the ductal networks and delivers a fluid to the ductal
networks.
[0692] A catheter for introduction of medications to the tissue of
a heart or other organs has been described by Tkebuchava and is
taught for example in WO2006138109, the contents of which are
incorporated herein by reference in their entirety. According to
Tkebuchava, two curved needles are incorporated which enter the
organ wall in a flattened trajectory.
[0693] Devices for delivering medical agents have been described by
Mckay et al. and are taught for example in WO2006118804, the
content of which are incorporated herein by reference in their
entirety. According to Mckay, multiple needles with multiple
orifices on each needle are incorporated into the devices to
facilitate regional delivery to a tissue, such as the interior disc
space of a spinal disc.
[0694] A method for directly delivering an immunomodulatory
substance into an intradermal space within a mammalian skin has
been described by Pettis and is taught for example in WO2004020014,
the contents of which are incorporated herein by reference in their
entirety. According to Pettis, multiple needles are incorporated
into a device which delivers the substance through the needles to a
depth between 0.3 mm and 2 mm.
[0695] Methods and devices for administration of substances into at
least two compartments in skin for systemic absorption and improved
pharmacokinetics have been described by Pettis et al. and are
taught for example in WO2003094995, the contents of which are
incorporated herein by reference in their entirety. According to
Pettis, multiple needles having lengths between about 300 .mu.m and
about 5 mm are incorporated into a device which delivers to
intradermal and subcutaneous tissue compartments
simultaneously.
[0696] A drug delivery device with needles and a roller has been
described by Zimmerman et al. and is taught for example in
WO2012006259, the contents of which are incorporated herein by
reference in their entirety. According to Zimmerman, multiple
hollow needles positioned in a roller are incorporated into the
device which delivers the content in a reservoir through the
needles as the roller rotates.
Methods and Devices Utilizing Catheters and/or Lumens
[0697] Methods and devices using catheters and lumens may be
employed to administer the mmRNA of the present invention on a
single, multi- or split dosing schedule. Such methods and devices
are described below.
[0698] A catheter-based delivery of skeletal myoblasts to the
myocardium of damaged hearts has been described by Jacoby et al and
is taught for example in US Patent Publication 20060263338, the
contents of which are incorporated herein by reference in their
entirety. According to Jacoby, multiple needles are incorporated
into the device at least part of which is inserted into a blood
vessel and delivers the cell composition through the needles into
the localized region of the subject's heart.
[0699] An apparatus for treating asthma using neurotoxin has been
described by Deem et al and is taught for example in US Patent
Publication 20060225742, the contents of which are incorporated
herein by reference in their entirety. According to Deem, multiple
needles are incorporated into the device which delivers neurotoxin
through the needles into the bronchial tissue.
[0700] A method for administering multiple-component therapies has
been described by Nayak and is taught for example in U.S. Pat. No.
7,699,803, the contents of which are incorporated herein by
reference in their entirety. According to Nayak, multiple injection
cannulas may be incorporated into a device wherein depth slots may
be included for controlling the depth at which the therapeutic
substance is delivered within the tissue.
[0701] A surgical device for ablating a channel and delivering at
least one therapeutic agent into a desired region of the tissue has
been described by McIntyre et al and is taught for example in U.S.
Pat. No. 8,012,096, the contents of which are incorporated herein
by reference in their entirety. According to McIntyre, multiple
needles are incorporated into the device which dispenses a
therapeutic agent into a region of tissue surrounding the channel
and is particularly well suited for transmyocardial
revascularization operations.
[0702] Methods of treating functional disorders of the bladder in
mammalian females have been described by Versi et al and are taught
for example in U.S. Pat. No. 8,029,496, the contents of which are
incorporated herein by reference in their entirety. According to
Versi, an array of micro-needles is incorporated into a device
which delivers a therapeutic agent through the needles directly
into the trigone of the bladder.
[0703] A device and a method for delivering fluid into a flexible
biological barrier have been described by Yeshurun et al. and are
taught for example in U.S. Pat. No. 7,998,119 (device) and U.S.
Pat. No. 8,007,466 (method), the contents of which are incorporated
herein by reference in their entirety. According to Yeshurun, the
micro-needles on the device penetrate and extend into the flexible
biological barrier and fluid is injected through the bore of the
hollow micro-needles.
[0704] A method for epicardially injecting a substance into an area
of tissue of a heart having an epicardial surface and disposed
within a torso has been described by Bonner et al and is taught for
example in U.S. Pat. No. 7,628,780, the contents of which are
incorporated herein by reference in their entirety. According to
Bonner, the devices have elongate shafts and distal injection heads
for driving needles into tissue and injecting medical agents into
the tissue through the needles.
[0705] A device for sealing a puncture has been described by
Nielsen et al and is taught for example in U.S. Pat. No. 7,972,358,
the contents of which are incorporated herein by reference in their
entirety. According to Nielsen, multiple needles are incorporated
into the device which delivers a closure agent into the tissue
surrounding the puncture tract.
[0706] A method for myogenesis and angiogenesis has been described
by Chiu et al. and is taught for example in U.S. Pat. No.
6,551,338, the contents of which are incorporated herein by
reference in their entirety. According to Chiu, 5 to 15 needles
having a maximum diameter of at least 1.25 mm and a length
effective to provide a puncture depth of 6 to 20 mm are
incorporated into a device which inserts into proximity with a
myocardium and supplies an exogeneous angiogenic or myogenic factor
to said myocardium through the conduits which are in at least some
of said needles.
[0707] A method for the treatment of prostate tissue has been
described by Bolmsj et al. and is taught for example in U.S. Pat.
No. 6,524,270, the contents of which are incorporated herein by
reference in their entirety. According to Bolmsj, a device
comprising a catheter which is inserted through the urethra has at
least one hollow tip extendible into the surrounding prostate
tissue. An astringent and analgesic medicine is administered
through said tip into said prostate tissue.
[0708] A method for infusing fluids to an intraosseous site has
been described by Findlay et al. and is taught for example in U.S.
Pat. No. 6,761,726, the contents of which are incorporated herein
by reference in their entirety. According to Findlay, multiple
needles are incorporated into a device which is capable of
penetrating a hard shell of material covered by a layer of soft
material and delivers a fluid at a predetermined distance below
said hard shell of material.
[0709] A device for injecting medications into a vessel wall has
been described by Vigil et al. and is taught for example in U.S.
Pat. No. 5,713,863, the contents of which are incorporated herein
by reference in their entirety. According to Vigil, multiple
injectors are mounted on each of the flexible tubes in the device
which introduces a medication fluid through a multi-lumen catheter,
into said flexible tubes and out of said injectors for infusion
into the vessel wall.
[0710] A catheter for delivering therapeutic and/or diagnostic
agents to the tissue surrounding a bodily passageway has been
described by Faxon et al. and is taught for example in U.S. Pat.
No. 5,464,395, the contents of which are incorporated herein by
reference in their entirety. According to Faxon, at least one
needle cannula is incorporated into the catheter which delivers the
desired agents to the tissue through said needles which project
outboard of the catheter.
[0711] Balloon catheters for delivering therapeutic agents have
been described by Orr and are taught for example in WO2010024871,
the contents of which are incorporated herein by reference in their
entirety. According to Orr, multiple needles are incorporated into
the devices which deliver the therapeutic agents to different
depths within the tissue.
Methods and Devices Utilizing Electrical Current
[0712] Methods and devices utilizing electric current may be
employed to deliver the mmRNA of the present invention according to
the single, multi- or split dosing regimens taught herein. Such
methods and devices are described below.
[0713] An electro collagen induction therapy device has been
described by Marquez and is taught for example in US Patent
Publication 20090137945, the contents of which are incorporated
herein by reference in their entirety. According to Marquez,
multiple needles are incorporated into the device which repeatedly
pierce the skin and draw in the skin a portion of the substance
which is applied to the skin first.
[0714] An electrokinetic system has been described by Etheredge et
al. and is taught for example in US Patent Publication 20070185432,
the contents of which are incorporated herein by reference in their
entirety. According to Etheredge, micro-needles are incorporated
into a device which drives by an electrical current the medication
through the needles into the targeted treatment site.
[0715] An iontophoresis device has been described by Matsumura et
al. and is taught for example in U.S. Pat. No. 7,437,189, the
contents of which are incorporated herein by reference in their
entirety. According to Matsumura, multiple needles are incorporated
into the device which is capable of delivering ionizable drug into
a living body at higher speed or with higher efficiency.
[0716] Intradermal delivery of biologically active agents by
needle-free injection and electroporation has been described by
Hoffmann et al and is taught for example in U.S. Pat. No.
7,171,264, the contents of which are incorporated herein by
reference in their entirety. According to Hoffmann, one or more
needle-free injectors are incorporated into an electroporation
device and the combination of needle-free injection and
electroporation is sufficient to introduce the agent into cells in
skin, muscle or mucosa.
[0717] A method for electropermeabilization-mediated intracellular
delivery has been described by Lundkvist et al. and is taught for
example in U.S. Pat. No. 6,625,486, the contents of which are
incorporated herein by reference in their entirety. According to
Lundkvist, a pair of needle electrodes is incorporated into a
catheter. Said catheter is positioned into a body lumen followed by
extending said needle electrodes to penetrate into the tissue
surrounding said lumen. Then the device introduces an agent through
at least one of said needle electrodes and applies electric field
by said pair of needle electrodes to allow said agent pass through
the cell membranes into the cells at the treatment site.
[0718] A delivery system for transdermal immunization has been
described by Levin et al. and is taught for example in
WO2006003659, the contents of which are incorporated herein by
reference in their entirety. According to Levin, multiple
electrodes are incorporated into the device which applies
electrical energy between the electrodes to generate micro channels
in the skin to facilitate transdermal delivery.
[0719] A method for delivering RF energy into skin has been
described by Schomacker and is taught for example in WO2011163264,
the contents of which are incorporated herein by reference in their
entirety. According to Schomacker, multiple needles are
incorporated into a device which applies vacuum to draw skin into
contact with a plate so that needles insert into skin through the
holes on the plate and deliver RF energy.
VII. Definitions
[0720] At various places in the present specification, substituents
of compounds of the present disclosure are disclosed in groups or
in ranges. It is specifically intended that the present disclosure
include each and every individual subcombination of the members of
such groups and ranges. For example, the term "C.sub.1-6 alkyl" is
specifically intended to individually disclose methyl, ethyl,
C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl, and C.sub.6 alkyl.
[0721] About: As used herein, the term "about" means+/-10% of the
recited value.
[0722] Administered in combination: As used herein, the term
"administered in combination" or "combined administration" means
that two or more agents are administered to a subject at the same
time or within an interval such that there may be an overlap of an
effect of each agent on the patient. In some embodiments, they are
administered within about 60, 30, 15, 10, 5, or 1 minute of one
another. In some embodiments, the administrations of the agents are
spaced sufficiently closely together such that a combinatorial
(e.g., a synergistic) effect is achieved.
[0723] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans at any stage of development. In some embodiments,
"animal" refers to non-human animals at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, or a pig). In some embodiments, animals include,
but are not limited to, mammals, birds, reptiles, amphibians, fish,
and worms. In some embodiments, the animal is a transgenic animal,
genetically-engineered animal, or a clone.
[0724] Antigens of interest or desired antigens: As used herein,
the terms "antigens of interest" or "desired antigens" include
those proteins and other biomolecules provided herein that are
immunospecifically bound by the antibodies and fragments, mutants,
variants, and alterations thereof described herein. Examples of
antigens of interest include, but are not limited to, insulin,
insulin-like growth factor, hGH, tPA, cytokines, such as
interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or
IFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF
beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.
[0725] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest, refers to a
value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range
of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in
either direction (greater than or less than) of the stated
reference value unless otherwise stated or otherwise evident from
the context (except where such number would exceed 100% of a
possible value).
[0726] Associated with: As used herein, the terms "associated
with," "conjugated," "linked," "attached," and "tethered," when
used with respect to two or more moieties, means that the moieties
are physically associated or connected with one another, either
directly or via one or more additional moieties that serves as a
linking agent, to form a structure that is sufficiently stable so
that the moieties remain physically associated under the conditions
in which the structure is used, e.g., physiological conditions. An
"association" need not be strictly through direct covalent chemical
bonding. It may also suggest ionic or hydrogen bonding or a
hybridization based connectivity sufficiently stable such that the
"associated" entities remain physically associated.
[0727] Bifunctional: As used herein, the term "bifunctional" refers
to any substance, molecule or moiety which is capable of or
maintains at least two functions. The functions may effect the same
outcome or a different outcome. The structure that produces the
function may be the same or different. For example, bifunctional
modified RNAs of the present invention may encode a cytotoxic
peptide (a first function) while those nucleosides which comprise
the encoding RNA are, in and of themselves, cytotoxic (second
function). In this example, delivery of the bifunctional modified
RNA to a cancer cell would produce not only a peptide or protein
molecule which may ameliorate or treat the cancer but would also
deliver a cytotoxic payload of nucleosides to the cell should
degradation, instead of translation of the modified RNA, occur.
[0728] Biocompatible: As used herein, the term "biocompatible"
means compatible with living cells, tissues, organs or systems
posing little to no risk of injury, toxicity or rejection by the
immune system.
[0729] Biodegradable: As used herein, the term "biodegradable"
means capable of being broken down into innocuous products by the
action of living things.
[0730] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any substance
that has activity in a biological system and/or organism. For
instance, a substance that, when administered to an organism, has a
biological effect on that organism, is considered to be
biologically active. In particular embodiments, signal-sensor
polynucleotide, primary construct or mmRNA of the present invention
may be considered biologically active if even a portion of the
signal-sensor polynucleotide, primary construct or mmRNA is
biologically active or mimics an activity considered biologically
relevant.
[0731] Cancer: As used herein, the term "cancer" in a subject
refers to the presence of cells possessing characteristics, such as
uncontrolled proliferation, immortality, metastatic potential,
rapid growth and proliferation rate, and certain characteristic
morphological features. Often, cancer cells will be in the form of
a tumor, but such cells may exist alone within a subject, or may
circulate in the blood stream as independent cells, such as
leukemic cells.
[0732] Cell growth: As used herein, the term "cell growth" is
principally associated with growth in cell numbers, which occurs by
means of cell reproduction (i.e. proliferation) when the rate of
the latter is greater than the rate of cell death (e.g. by
apoptosis or necrosis).
[0733] Chemical terms: The following provides the definition of
various chemical terms from "acyl" to "thiol."
[0734] The term "acyl," as used herein, represents a hydrogen or an
alkyl group (e.g., a haloalkyl group), as defined herein, that is
attached to the parent molecular group through a carbonyl group, as
defined herein, and is exemplified by formyl (i.e., a
carboxyaldehyde group), acetyl, propionyl, butanoyl and the like.
Exemplary unsubstituted acyl groups include from 1 to 7, from 1 to
11, or from 1 to 21 carbons. In some embodiments, the alkyl group
is further substituted with 1, 2, 3, or 4 substituents as described
herein.
[0735] The term "acylamino," as used herein, represents an acyl
group, as defined herein, attached to the parent molecular group
though an amino group, as defined herein (i.e.,
--N(R.sup.N1)--C(O)--R, where R is H or an optionally substituted
C.sub.1-6, C.sub.1-10, or C.sub.1-20 alkyl group and R.sup.N1 is as
defined herein). Exemplary unsubstituted acylamino groups include
from 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to
21, from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41
carbons). In some embodiments, the alkyl group is further
substituted with 1, 2, 3, or 4 substituents as described herein,
and/or the amino group is --NH.sub.2 or --NHR.sup.N1, wherein
R.sup.N1 is, independently, OH, NO.sub.2, NH.sub.2,
NR.sup.N2.sub.2, SO.sub.2OR.sup.N2, SO.sub.2R.sup.N2, SOR.sup.N2,
alkyl, or aryl, and each R.sup.N2 can be H, alkyl, or aryl.
[0736] The term "acyloxy," as used herein, represents an acyl
group, as defined herein, attached to the parent molecular group
though an oxygen atom (i.e., --O--C(O)--R, where R is H or an
optionally substituted C.sub.1-6, C.sub.1-10, or C.sub.1-20 alkyl
group). Exemplary unsubstituted acyloxy groups include from 1 to 21
carbons (e.g., from 1 to 7 or from 1 to 11 carbons). In some
embodiments, the alkyl group is further substituted with 1, 2, 3,
or 4 substituents as described herein, and/or the amino group is
--NH.sub.2 or --NHR.sup.N1, wherein R.sup.N1 is, independently, OH,
NO.sub.2, NH.sub.2, NR.sup.N2.sub.2, SO.sub.2OR.sup.N2,
SO.sub.2R.sup.N2, SOR.sup.N2, alkyl, or aryl, and each R.sup.N2 can
be H, alkyl, or aryl.
[0737] The term "alkaryl," as used herein, represents an aryl
group, as defined herein, attached to the parent molecular group
through an alkylene group, as defined herein. Exemplary
unsubstituted alkaryl groups are from 7 to 30 carbons (e.g., from 7
to 16 or from 7 to 20 carbons, such as C.sub.1-6 alk-C.sub.6-10
aryl, C.sub.1-10 alk-C.sub.6-10 aryl, or C.sub.1-20 alk-C.sub.6-10
aryl). In some embodiments, the alkylene and the aryl each can be
further substituted with 1, 2, 3, or 4 substituent groups as
defined herein for the respective groups. Other groups preceded by
the prefix "alk-" are defined in the same manner, where "alk"
refers to a C.sub.1-6 alkylene, unless otherwise noted, and the
attached chemical structure is as defined herein.
[0738] The term "alkcycloalkyl" represents a cycloalkyl group, as
defined herein, attached to the parent molecular group through an
alkylene group, as defined herein (e.g., an alkylene group of from
1 to 4, from 1 to 6, from 1 to 10, or form 1 to 20 carbons). In
some embodiments, the alkylene and the cycloalkyl each can be
further substituted with 1, 2, 3, or 4 substituent groups as
defined herein for the respective group.
[0739] The term "alkenyl," as used herein, represents monovalent
straight or branched chain groups of, unless otherwise specified,
from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons)
containing one or more carbon-carbon double bonds and is
exemplified by ethenyl, 1-propenyl, 2-propenyl,
2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyls
include both cis and trans isomers. Alkenyl groups may be
optionally substituted with 1, 2, 3, or 4 substituent groups that
are selected, independently, from amino, aryl, cycloalkyl, or
heterocyclyl (e.g., heteroaryl), as defined herein, or any of the
exemplary alkyl substituent groups described herein.
[0740] The term "alkenyloxy" represents a chemical substituent of
formula --OR, where R is a C.sub.2-20 alkenyl group (e.g.,
C.sub.2-6 or C.sub.2-10 alkenyl), unless otherwise specified.
Exemplary alkenyloxy groups include ethenyloxy, propenyloxy, and
the like. In some embodiments, the alkenyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
(e.g., a hydroxy group).
[0741] The term "alkheteroaryl" refers to a heteroaryl group, as
defined herein, attached to the parent molecular group through an
alkylene group, as defined herein. Exemplary unsubstituted
alkheteroaryl groups are from 2 to 32 carbons (e.g., from 2 to 22,
from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2 to
14, from 2 to 13, or from 2 to 12 carbons, such as C.sub.1-6
alk-C.sub.1-12 heteroaryl, C.sub.1-10 alk-C.sub.1-12 heteroaryl, or
C.sub.1-20 alk-C.sub.1-12 heteroaryl). In some embodiments, the
alkylene and the heteroaryl each can be further substituted with 1,
2, 3, or 4 substituent groups as defined herein for the respective
group. Alkheteroaryl groups are a subset of alkheterocyclyl
groups.
[0742] The term "alkheterocyclyl" represents a heterocyclyl group,
as defined herein, attached to the parent molecular group through
an alkylene group, as defined herein. Exemplary unsubstituted
alkheterocyclyl groups are from 2 to 32 carbons (e.g., from 2 to
22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2
to 14, from 2 to 13, or from 2 to 12 carbons, such as C.sub.1-6
alk-C.sub.1-12 heterocyclyl, C.sub.1-10 alk-C.sub.1-12
heterocyclyl, or C.sub.1-20 alk-C.sub.1-12 heterocyclyl). In some
embodiments, the alkylene and the heterocyclyl each can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
for the respective group.
[0743] The term "alkoxy" represents a chemical substituent of
formula --OR, where R is a C.sub.1-20 alkyl group (e.g., C.sub.1-6
or C.sub.1-10 alkyl), unless otherwise specified. Exemplary alkoxy
groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and
isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl
group can be further substituted with 1, 2, 3, or 4 substituent
groups as defined herein (e.g., hydroxy or alkoxy).
[0744] The term "alkoxyalkoxy" represents an alkoxy group that is
substituted with an alkoxy group. Exemplary unsubstituted
alkoxyalkoxy groups include between 2 to 40 carbons (e.g., from 2
to 12 or from 2 to 20 carbons, such as C.sub.1-6 alkoxy-C.sub.1-6
alkoxy, C.sub.1-10 alkoxy-C.sub.1-10 alkoxy, or C.sub.1-20
alkoxy-C.sub.1-20 alkoxy). In some embodiments, the each alkoxy
group can be further substituted with 1, 2, 3, or 4 substituent
groups as defined herein.
[0745] The term "alkoxyalkyl" represents an alkyl group that is
substituted with an alkoxy group. Exemplary unsubstituted
alkoxyalkyl groups include between 2 to 40 carbons (e.g., from 2 to
12 or from 2 to 20 carbons, such as C.sub.1-6 alkoxy-C.sub.1-6
alkyl, C.sub.1-10 alkoxy-C.sub.1-10 alkyl, or C.sub.1-20
alkoxy-C.sub.1-20 alkyl). In some embodiments, the alkyl and the
alkoxy each can be further substituted with 1, 2, 3, or 4
substituent groups as defined herein for the respective group.
[0746] The term "alkoxycarbonyl," as used herein, represents an
alkoxy, as defined herein, attached to the parent molecular group
through a carbonyl atom (e.g., --C(O)--OR, where R is H or an
optionally substituted C.sub.1-6, C.sub.1-10, or C.sub.1-20 alkyl
group). Exemplary unsubstituted alkoxycarbonyl include from 1 to 21
carbons (e.g., from 1 to 11 or from 1 to 7 carbons). In some
embodiments, the alkoxy group is further substituted with 1, 2, 3,
or 4 substituents as described herein.
[0747] The term "alkoxycarbonylalkoxy," as used herein, represents
an alkoxy group, as defined herein, that is substituted with an
alkoxycarbonyl group, as defined herein (e.g., --O-alkyl-C(O)--OR,
where R is an optionally substituted C.sub.1-6, C.sub.1-10, or
C.sub.1-20 alkyl group). Exemplary unsubstituted
alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g., from 3 to
10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31
carbons, such as C.sub.1-6 alkoxycarbonyl-C.sub.1-6 alkoxy,
C.sub.1-10 alkoxycarbonyl-C.sub.1-10 alkoxy, or C.sub.1-20
alkoxycarbonyl-C.sub.1-20 alkoxy). In some embodiments, each alkoxy
group is further independently substituted with 1, 2, 3, or 4
substituents, as described herein (e.g., a hydroxy group).
[0748] The term "alkoxycarbonylalkyl," as used herein, represents
an alkyl group, as defined herein, that is substituted with an
alkoxycarbonyl group, as defined herein (e.g., -alkyl-C(O)--OR,
where R is an optionally substituted C.sub.1-20, C.sub.1-10, or
C.sub.1-6 alkyl group). Exemplary unsubstituted alkoxycarbonylalkyl
include from 3 to 41 carbons (e.g., from 3 to 10, from 3 to 13,
from 3 to 17, from 3 to 21, or from 3 to 31 carbons, such as
C.sub.1-6 alkoxycarbonyl-C.sub.1-6 alkyl, C.sub.1-10
alkoxycarbonyl-C.sub.1-10 alkyl, or C.sub.1-20
alkoxycarbonyl-C.sub.1-20 alkyl). In some embodiments, each alkyl
and alkoxy group is further independently substituted with 1, 2, 3,
or 4 substituents as described herein (e.g., a hydroxy group).
[0749] The term "alkyl," as used herein, is inclusive of both
straight chain and branched chain saturated groups from 1 to 20
carbons (e.g., from 1 to 10 or from 1 to 6), unless otherwise
specified. Alkyl groups are exemplified by methyl, ethyl, n- and
iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, and the like,
and may be optionally substituted with one, two, three, or, in the
case of alkyl groups of two carbons or more, four substituents
independently selected from the group consisting of: (1) C.sub.1-6
alkoxy; (2) C.sub.1-6 alkylsulfinyl; (3) amino, as defined herein
(e.g., unsubstituted amino (i.e., --NH.sub.2) or a substituted
amino (i.e., --N(R.sup.N1).sub.2, where R.sup.N1 is as defined for
amino); (4) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (5) azido; (6) halo;
(7) (C.sub.2-9 heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo
(e.g., carboxyaldehyde or acyl); (11) C.sub.1-7 spirocyclyl; (12)
thioalkoxy; (13) thiol; (14) --CO.sub.2R.sup.A', where R.sup.A' is
selected from the group consisting of (a) C.sub.1-20 alkyl (e.g.,
C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6 alkenyl),
(c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6 alk-C.sub.6-10
aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (15)
--C(O)NR.sup.B'R.sup.C', where each of R.sup.B' and R.sup.C' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (16) --SO.sub.2R.sup.D', where R.sup.D' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) C.sub.1-6 alk-C.sub.6-10 aryl, and (d)
hydroxy; (17) --SO.sub.2NR.sup.E'R.sup.F', where each of R.sup.E'
and R.sup.F' is, independently, selected from the group consisting
of (a) hydrogen, (b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18) --C(O)R.sup.G', where R.sup.G'
is selected from the group consisting of (a) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6
alkenyl), (c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6
alk-C.sub.6-10 aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (19)
--NR.sup.H'C(O)R.sup.I', wherein R.sup.H' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.I' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (20)
--NR.sup.J'C(O)OR.sup.K', wherein R.sup.J' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.K' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; and (21)
amidine. In some embodiments, each of these groups can be further
substituted as described herein. For example, the alkylene group of
a C.sub.1-alkaryl can be further substituted with an oxo group to
afford the respective aryloyl substituent.
[0750] The term "alkylene" and the prefix "alk-," as used herein,
represent a saturated divalent hydrocarbon group derived from a
straight or branched chain saturated hydrocarbon by the removal of
two hydrogen atoms, and is exemplified by methylene, ethylene,
isopropylene, and the like. The term "C.sub.x-y alkylene" and the
prefix "C.sub.x-y alk-" represent alkylene groups having between x
and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, and
exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, or 20 (e.g., C.sub.1-6, C.sub.1-10, C.sub.2-20, C.sub.2-6,
C.sub.2-10, or C.sub.2-20 alkylene). In some embodiments, the
alkylene can be further substituted with 1, 2, 3, or 4 substituent
groups as defined herein for an alkyl group.
[0751] The term "alkylsulfinyl," as used herein, represents an
alkyl group attached to the parent molecular group through an
--S(O)-- group. Exemplary unsubstituted alkylsulfinyl groups are
from 1 to 6, from 1 to 10, or from 1 to 20 carbons. In some
embodiments, the alkyl group can be further substituted with 1, 2,
3, or 4 substituent groups as defined herein.
[0752] The term "alkylsulfinylalkyl," as used herein, represents an
alkyl group, as defined herein, substituted by an alkylsulfinyl
group. Exemplary unsubstituted alkylsulfinylalkyl groups are from 2
to 12, from 2 to 20, or from 2 to 40 carbons. In some embodiments,
each alkyl group can be further substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0753] The term "alkynyl," as used herein, represents monovalent
straight or branched chain groups from 2 to 20 carbon atoms (e.g.,
from 2 to 4, from 2 to 6, or from 2 to 10 carbons) containing a
carbon-carbon triple bond and is exemplified by ethynyl,
1-propynyl, and the like. Alkynyl groups may be optionally
substituted with 1, 2, 3, or 4 substituent groups that are
selected, independently, from aryl, cycloalkyl, or heterocyclyl
(e.g., heteroaryl), as defined herein, or any of the exemplary
alkyl substituent groups described herein.
[0754] The term "alkynyloxy" represents a chemical substituent of
formula --OR, where R is a C.sub.2-20 alkynyl group (e.g.,
C.sub.2-6 or C.sub.2-10 alkynyl), unless otherwise specified.
Exemplary alkynyloxy groups include ethynyloxy, propynyloxy, and
the like. In some embodiments, the alkynyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
(e.g., a hydroxy group).
[0755] The term "amidine," as used herein, represents a
--C(.dbd.NH)NH.sub.2 group.
[0756] The term "amino," as used herein, represents
--N(R.sup.N1).sub.2, wherein each R.sup.N1 is, independently, H,
OH, NO.sub.2, N(R.sup.N2).sub.2, SO.sub.2OR.sup.N2,
SO.sub.2R.sup.N2, SOR.sup.N2, an N-protecting group, alkyl,
alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl,
carboxyalkyl, sulfoalkyl, heterocyclyl (e.g., heteroaryl), or
alkheterocyclyl (e.g., alkheteroaryl), wherein each of these
recited R.sup.N1 groups can be optionally substituted, as defined
herein for each group; or two R.sup.N1 combine to form a
heterocyclyl or an N-protecting group, and wherein each R.sup.N2
is, independently, H, alkyl, or aryl. The amino groups of the
invention can be an unsubstituted amino (i.e., --NH.sub.2) or a
substituted amino (i.e., --N(R.sup.N1).sub.2). In a preferred
embodiment, amino is --NH.sub.2 or --NHR.sup.N1, wherein R.sup.N1
is, independently, OH, NO.sub.2, NH.sub.2, NR.sup.N2.sub.2,
SO.sub.2OR.sup.N2, SO.sub.2R.sup.N2, SOR.sup.N2, alkyl,
carboxyalkyl, sulfoalkyl, or aryl, and each R.sup.N2 can be H,
C.sub.1-20 alkyl (e.g., C.sub.1-6 alkyl), or C.sub.6-10 aryl.
[0757] The term "amino acid," as described herein, refers to a
molecule having a side chain, an amino group, and an acid group
(e.g., a carboxy group of --CO.sub.2H or a sulfo group of
--SO.sub.3H), wherein the amino acid is attached to the parent
molecular group by the side chain, amino group, or acid group
(e.g., the side chain). In some embodiments, the amino acid is
attached to the parent molecular group by a carbonyl group, where
the side chain or amino group is attached to the carbonyl group.
Exemplary side chains include an optionally substituted alkyl,
aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl,
carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include
alanine, arginine, asparagine, aspartic acid, cysteine, glutamic
acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine,
leucine, lysine, methionine, norvaline, ornithine, phenylalanine,
proline, pyrrolysine, selenocysteine, serine, taurine, threonine,
tryptophan, tyrosine, and valine. Amino acid groups may be
optionally substituted with one, two, three, or, in the case of
amino acid groups of two carbons or more, four substituents
independently selected from the group consisting of: (1) C.sub.1-6
alkoxy; (2) C.sub.1-6 alkylsulfinyl; (3) amino, as defined herein
(e.g., unsubstituted amino (i.e., --NH.sub.2) or a substituted
amino (i.e., --N(R.sup.N1).sub.2, where R.sup.N1 is as defined for
amino); (4) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (5) azido; (6) halo;
(7) (C.sub.2-9 heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo
(e.g., carboxyaldehyde or acyl); (11) C.sub.1-7 spirocyclyl; (12)
thioalkoxy; (13) thiol; (14) --CO.sub.2R.sup.A', where R.sup.A' is
selected from the group consisting of (a) C.sub.1-20 alkyl (e.g.,
C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6 alkenyl),
(c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6 alk-C.sub.6-10
aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (15)
--C(O)NR.sup.B'R.sup.C', where each of R.sup.B' and R.sup.C' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (16) --SO.sub.2R.sup.D', where R.sup.D' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) C.sub.1-6 alk-C.sub.6-10 aryl, and (d)
hydroxy; (17) --SO.sub.2NR.sup.E'R.sup.F', where each of R.sup.E'
and R.sup.F' is, independently, selected from the group consisting
of (a) hydrogen, (b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18) --C(O)R.sup.G', where R.sup.G'
is selected from the group consisting of (a) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl), (b) C.sub.2-20 alkenyl (e.g., C.sub.2-6
alkenyl), (c) C.sub.6-10 aryl, (d) hydrogen, (e) C.sub.1-6
alk-C.sub.6-10 aryl, (f) amino-C.sub.1-20 alkyl, (g) polyethylene
glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (19)
--NR.sup.H'C(O)R.sup.I', wherein R.sup.H' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.I' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; (20)
--NR.sup.J'C(O)OR.sup.K', wherein R.sup.J' is selected from the
group consisting of (a1) hydrogen and (b1) C.sub.1-6 alkyl, and
R.sup.K' is selected from the group consisting of (a2) C.sub.1-20
alkyl (e.g., C.sub.1-6 alkyl), (b2) C.sub.2-20 alkenyl (e.g.,
C.sub.2-6 alkenyl), (c2) C.sub.6-10 aryl, (d2) hydrogen, (e2)
C.sub.1-6 alk-C.sub.6-10 aryl, (f2) amino-C.sub.1-20 alkyl, (g2)
polyethylene glycol of
--(CH.sub.2).sub.s2(OCH.sub.2CH.sub.2).sub.s1(CH.sub.2).sub.s3OR',
wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1
to 4), each of s2 and s3, independently, is an integer from 0 to 10
(e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from
1 to 10), and R' is H or C.sub.1-20 alkyl, and (h2)
amino-polyethylene glycol of
--NR.sup.N1(CH.sub.2).sub.s2(CH.sub.2CH.sub.2O).sub.s1(CH.sub.2).sub.s3NR-
.sup.N1, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6
or from 1 to 4), each of s2 and s3, independently, is an integer
from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1
to 6, or from 1 to 10), and each R.sup.N1 is, independently,
hydrogen or optionally substituted C.sub.1-6 alkyl; and (21)
amidine. In some embodiments, each of these groups can be further
substituted as described herein.
[0758] The term "aminoalkoxy," as used herein, represents an alkoxy
group, as defined herein, substituted by an amino group, as defined
herein. The alkyl and amino each can be further substituted with 1,
2, 3, or 4 substituent groups as described herein for the
respective group (e.g., CO.sub.2R.sup.A', where R.sup.A' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) hydrogen, and (d) C.sub.1-6 alk-C.sub.6-10
aryl, e.g., carboxy).
[0759] The term "aminoalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by an amino group, as defined
herein. The alkyl and amino each can be further substituted with 1,
2, 3, or 4 substituent groups as described herein for the
respective group (e.g., CO.sub.2R.sup.A', where R.sup.A' is
selected from the group consisting of (a) C.sub.1-6 alkyl, (b)
C.sub.6-10 aryl, (c) hydrogen, and (d) C.sub.1-6 alk-C.sub.6-10
aryl, e.g., carboxy).
[0760] The term "aryl," as used herein, represents a mono-,
bicyclic, or multicyclic carbocyclic ring system having one or two
aromatic rings and is exemplified by phenyl, naphthyl,
1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl,
phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and may
be optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from the group consisting of: (1) C.sub.1-7
acyl (e.g., carboxyaldehyde); (2) C.sub.1-20 alkyl (e.g., C.sub.1-6
alkyl, C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6
alkylsulfinyl-C.sub.1-6 alkyl, amino-C.sub.1-6 alkyl,
azido-C.sub.1-6 alkyl, (carboxyaldehyde)-C.sub.1-6 alkyl,
halo-C.sub.1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C.sub.1-6
alkyl, nitro-C.sub.1-6 alkyl, or C.sub.1-6 thioalkoxy-C.sub.1-6
alkyl); (3) C.sub.1-20 alkoxy (e.g., C.sub.1-6 alkoxy, such as
perfluoroalkoxy); (4) C.sub.1-6 alkylsulfinyl; (5) C.sub.6-10 aryl;
(6) amino; (7) C.sub.1-6 alk-C.sub.6-10 aryl; (8) azido; (9)
C.sub.3-8 cycloalkyl; (10) C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11)
halo; (12) C.sub.1-12 heterocyclyl (e.g., C.sub.1-12 heteroaryl);
(13) (C.sub.1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16)
C.sub.1-20 thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A', where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.1-6
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) alkyl, (b) C.sub.6-10 aryl, and (c)
alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (25)
C.sub.1-6 alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6
alk-C.sub.1-12 heteroaryl); (26) C.sub.2-20 alkenyl; and (27)
C.sub.2-20 alkynyl. In some embodiments, each of these groups can
be further substituted as described herein. For example, the
alkylene group of a C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl
can be further substituted with an oxo group to afford the
respective aryloyl and (heterocyclyl)oyl substituent group.
[0761] The term "arylalkoxy," as used herein, represents an alkaryl
group, as defined herein, attached to the parent molecular group
through an oxygen atom. Exemplary unsubstituted alkoxyalkyl groups
include from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20
carbons, such as C.sub.6-10 aryl-C.sub.1-6 alkoxy, C.sub.6-10
aryl-C.sub.1-10 alkoxy, or C.sub.6-10 aryl-C.sub.1-20 alkoxy). In
some embodiments, the arylalkoxy group can be substituted with 1,
2, 3, or 4 substituents as defined herein
[0762] The term "aryloxy" represents a chemical substituent of
formula --OR', where R' is an aryl group of 6 to 18 carbons, unless
otherwise specified. In some embodiments, the aryl group can be
substituted with 1, 2, 3, or 4 substituents as defined herein.
[0763] The term "aryloyl," as used herein, represents an aryl
group, as defined herein, that is attached to the parent molecular
group through a carbonyl group. Exemplary unsubstituted aryloyl
groups are of 7 to 11 carbons. In some embodiments, the aryl group
can be substituted with 1, 2, 3, or 4 substituents as defined
herein.
[0764] The term "azido" represents an --N.sub.3 group, which can
also be represented as --N.dbd.N.dbd.N.
[0765] The term "bicyclic," as used herein, refer to a structure
having two rings, which may be aromatic or non-aromatic. Bicyclic
structures include spirocyclyl groups, as defined herein, and two
rings that share one or more bridges, where such bridges can
include one atom or a chain including two, three, or more atoms.
Exemplary bicyclic groups include a bicyclic carbocyclyl group,
where the first and second rings are carbocyclyl groups, as defined
herein; a bicyclic aryl groups, where the first and second rings
are aryl groups, as defined herein; bicyclic heterocyclyl groups,
where the first ring is a heterocyclyl group and the second ring is
a carbocyclyl (e.g., aryl) or heterocyclyl (e.g., heteroaryl)
group; and bicyclic heteroaryl groups, where the first ring is a
heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)
or heterocyclyl (e.g., heteroaryl) group. In some embodiments, the
bicyclic group can be substituted with 1, 2, 3, or 4 substituents
as defined herein for cycloalkyl, heterocyclyl, and aryl
groups.
[0766] The terms "carbocyclic" and "carbocyclyl," as used herein,
refer to an optionally substituted C.sub.3-12 monocyclic, bicyclic,
or tricyclic structure in which the rings, which may be aromatic or
non-aromatic, are formed by carbon atoms. Carbocyclic structures
include cycloalkyl, cycloalkenyl, and aryl groups.
[0767] The term "carbamoyl," as used herein, represents
--C(O)--N(R.sup.N1).sub.2, where the meaning of each R.sup.N1 is
found in the definition of "amino" provided herein.
[0768] The term "carbamoylalkyl," as used herein, represents an
alkyl group, as defined herein, substituted by a carbamoyl group,
as defined herein. The alkyl group can be further substituted with
1, 2, 3, or 4 substituent groups as described herein.
[0769] The term "carbamyl," as used herein, refers to a carbamate
group having the structure --NR.sup.N1C(.dbd.O)OR or
--OC(.dbd.O)N(R.sup.N1).sub.2, where the meaning of each R.sup.N1
is found in the definition of "amino" provided herein, and R is
alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl
(e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as
defined herein.
[0770] The term "carbonyl," as used herein, represents a C(O)
group, which can also be represented as C.dbd.O.
[0771] The term "carboxyaldehyde" represents an acyl group having
the structure --CHO.
[0772] The term "carboxy," as used herein, means --CO.sub.2H.
[0773] The term "carboxyalkoxy," as used herein, represents an
alkoxy group, as defined herein, substituted by a carboxy group, as
defined herein. The alkoxy group can be further substituted with 1,
2, 3, or 4 substituent groups as described herein for the alkyl
group.
[0774] The term "carboxyalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by a carboxy group, as
defined herein. The alkyl group can be further substituted with 1,
2, 3, or 4 substituent groups as described herein.
[0775] The term "cyano," as used herein, represents an --CN
group.
[0776] The term "cycloalkoxy" represents a chemical substituent of
formula --OR, where R is a C.sub.3-8 cycloalkyl group, as defined
herein, unless otherwise specified. The cycloalkyl group can be
further substituted with 1, 2, 3, or 4 substituent groups as
described herein. Exemplary unsubstituted cycloalkoxy groups are
from 3 to 8 carbons. In some embodiment, the cycloalkyl group can
be further substituted with 1, 2, 3, or 4 substituent groups as
described herein.
[0777] The term "cycloalkyl," as used herein represents a
monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon
group from three to eight carbons, unless otherwise specified, and
is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, bicyclo[2.2.1]heptyl, and the like. When the
cycloalkyl group includes one carbon-carbon double bond, the
cycloalkyl group can be referred to as a "cycloalkenyl" group.
Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl,
and the like. The cycloalkyl groups of this invention can be
optionally substituted with: (1) C.sub.1-7 acyl (e.g.,
carboxyaldehyde); (2) C.sub.1-20 alkyl (e.g., C.sub.1-6 alkyl,
C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6 alkylsulfinyl-C.sub.1-6
alkyl, amino-C.sub.1-6 alkyl, azido-C.sub.1-6 alkyl,
(carboxyaldehyde)-C.sub.1-6 alkyl, halo-C.sub.1-6 alkyl (e.g.,
perfluoroalkyl), hydroxy-C.sub.1-6 alkyl, nitro-C.sub.1-6 alkyl, or
C.sub.1-6 thioalkoxy-C.sub.1-6 alkyl); (3) C.sub.1-20 alkoxy (e.g.,
C.sub.1-6 alkoxy, such as perfluoroalkoxy); (4) C.sub.1-6
alkylsulfinyl; (5) C.sub.6-10 aryl; (6) amino; (7) C.sub.1-6
alk-C.sub.6-10 aryl; (8) azido; (9) C.sub.3-8 cycloalkyl; (10)
C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11) halo; (12) C.sub.1-12
heterocyclyl (e.g., C.sub.1-12 heteroaryl); (13) (C.sub.1-12
heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C.sub.1-20
thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A', where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.A'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.6-10
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) C.sub.6-10 alkyl, (b) C.sub.6-10 aryl, and (c)
C.sub.1-6 alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.6-10 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) C.sub.6-10 aryl-C.sub.1-6 alkoxy; (25)
C.sub.1-6 alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6
alk-C.sub.1-12 heteroaryl); (26) oxo; (27) C.sub.2-20 alkenyl; and
(28) C.sub.2-20 alkynyl. In some embodiments, each of these groups
can be further substituted as described herein. For example, the
alkylene group of a C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl
can be further substituted with an oxo group to afford the
respective aryloyl and (heterocyclyl)oyl substituent group.
[0778] The term "diastereomer," as used herein means stereoisomers
that are not mirror images of one another and are
non-superimposable on one another.
[0779] The term "effective amount" of an agent, as used herein, is
that amount sufficient to effect beneficial or desired results, for
example, clinical results, and, as such, an "effective amount"
depends upon the context in which it is being applied. For example,
in the context of administering an agent that treats cancer, an
effective amount of an agent is, for example, an amount sufficient
to achieve treatment, as defined herein, of cancer, as compared to
the response obtained without administration of the agent.
[0780] The term "enantiomer," as used herein, means each individual
optically active form of a compound of the invention, having an
optical purity or enantiomeric excess (as determined by methods
standard in the art) of at least 80% (i.e., at least 90% of one
enantiomer and at most 10% of the other enantiomer), preferably at
least 90% and more preferably at least 98%.
[0781] The term "halo," as used herein, represents a halogen
selected from bromine, chlorine, iodine, or fluorine.
[0782] The term "haloalkoxy," as used herein, represents an alkoxy
group, as defined herein, substituted by a halogen group (i.e., F,
Cl, Br, or I). A haloalkoxy may be substituted with one, two,
three, or, in the case of alkyl groups of two carbons or more, four
halogens. Haloalkoxy groups include perfluoroalkoxys (e.g.,
--OCF.sub.3), --OCHF.sub.2, --OCH.sub.2F, --OCCl.sub.3,
--OCH.sub.2CH.sub.2Br, --OCH.sub.2CH(CH.sub.2CH.sub.2Br)CH.sub.3,
and --OCHICH.sub.3. In some embodiments, the haloalkoxy group can
be further substituted with 1, 2, 3, or 4 substituent groups as
described herein for alkyl groups.
[0783] The term "haloalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by a halogen group (i.e., F,
Cl, Br, or I). A haloalkyl may be substituted with one, two, three,
or, in the case of alkyl groups of two carbons or more, four
halogens. Haloalkyl groups include perfluoroalkyls (e.g.,
--CF.sub.3), --CHF.sub.2, --CH.sub.2F, --CCl.sub.3,
--CH.sub.2CH.sub.2Br, --CH.sub.2CH(CH.sub.2CH.sub.2Br)CH.sub.3, and
--CHICH.sub.3. In some embodiments, the haloalkyl group can be
further substituted with 1, 2, 3, or 4 substituent groups as
described herein for alkyl groups.
[0784] The term "heteroalkylene," as used herein, refers to an
alkylene group, as defined herein, in which one or two of the
constituent carbon atoms have each been replaced by nitrogen,
oxygen, or sulfur. In some embodiments, the heteroalkylene group
can be further substituted with 1, 2, 3, or 4 substituent groups as
described herein for alkylene groups.
[0785] The term "heteroaryl," as used herein, represents that
subset of heterocyclyls, as defined herein, which are aromatic:
i.e., they contain 4n+2 pi electrons within the mono- or
multicyclic ring system. Exemplary unsubstituted heteroaryl groups
are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2
to 10, or 2 to 9) carbons. In some embodiment, the heteroaryl is
substituted with 1, 2, 3, or 4 substituents groups as defined for a
heterocyclyl group.
[0786] The term "heterocyclyl," as used herein represents a 5-, 6-
or 7-membered ring, unless otherwise specified, containing one,
two, three, or four heteroatoms independently selected from the
group consisting of nitrogen, oxygen, and sulfur. The 5-membered
ring has zero to two double bonds, and the 6- and 7-membered rings
have zero to three double bonds. Exemplary unsubstituted
heterocyclyl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9,
2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term
"heterocyclyl" also represents a heterocyclic compound having a
bridged multicyclic structure in which one or more carbons and/or
heteroatoms bridges two non-adjacent members of a monocyclic ring,
e.g., a quinuclidinyl group. The term "heterocyclyl" includes
bicyclic, tricyclic, and tetracyclic groups in which any of the
above heterocyclic rings is fused to one, two, or three carbocyclic
rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring,
a cyclopentane ring, a cyclopentene ring, or another monocyclic
heterocyclic ring, such as indolyl, quinolyl, isoquinolyl,
tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples
of fused heterocyclyls include tropanes and
1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl,
pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,
imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl,
homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,
oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl,
thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,
isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,
quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,
phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,
benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,
triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl),
purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl),
tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
dihydrothienyl, dihydroindolyl, dihydroquinolyl,
tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl,
pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,
isobenzofuranyl, benzothienyl, and the like, including dihydro and
tetrahydro forms thereof, where one or more double bonds are
reduced and replaced with hydrogens. Still other exemplary
heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl;
2,3-dihydro-2-oxo-1H-imidazolyl;
2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,
2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);
2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,
2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);
2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,
2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);
4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino
5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,
1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);
2,6-dioxo-piperidinyl (e.g.,
2,6-dioxo-3-ethyl-3-phenylpiperidinyl);
1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,
2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);
1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,
1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);
1,6-dihydro-6-oxo-pyridazinyl (e.g.,
1,6-dihydro-6-oxo-3-ethylpyridazinyl);
1,6-dihydro-6-oxo-1,2,4-triazinyl (e.g.,
1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);
2,3-dihydro-2-oxo-1H-indolyl (e.g.,
3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and
2,3-dihydro-2-oxo-3,3'-spiropropane-1H-indol-1-yl);
1,3-dihydro-1-oxo-2H-iso-indolyl;
1,3-dihydro-1,3-dioxo-2H-iso-indolyl; 1H-benzopyrazolyl (e.g.,
1-(ethoxycarbonyl)-1H-benzopyrazolyl);
2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,
3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);
2,3-dihydro-2-oxo-benzoxazolyl (e.g.,
5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);
2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;
1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,
4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,
2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);
1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,
1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);
1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,
1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H -purinyl);
1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,
1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);
2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl;
and 1,8-naphthylenedicarboxamido. Additional heterocyclics include
3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and
2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or
diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl,
benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and
thiocanyl. Heterocyclic groups also include groups of the
formula
##STR00001##
where
[0787] E' is selected from the group consisting of --N-- and
--CH--; F' is selected from the group consisting of --N.dbd.CH--,
--NH--CH.sub.2--, --NH--C(O)--, --NH--, --CH.dbd.N--,
--CH.sub.2--NH--, --C(O)--NH--, --CH.dbd.CH--, --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2O--, --OCH.sub.2--, --O--, and
--S--; and G' is selected from the group consisting of --CH-- and
--N--. Any of the heterocyclyl groups mentioned herein may be
optionally substituted with one, two, three, four or five
substituents independently selected from the group consisting of:
(1) C.sub.1-7 acyl (e.g., carboxyaldehyde); (2) C.sub.1-20 alkyl
(e.g., C.sub.1-6 alkyl, C.sub.1-6 alkoxy-C.sub.1-6 alkyl, C.sub.1-6
alkylsulfinyl-C.sub.1-6 alkyl, amino-C.sub.1-6 alkyl,
azido-C.sub.1-6 alkyl, (carboxyaldehyde)-C.sub.1-6 alkyl,
halo-C.sub.1-6 alkyl (e.g., perfluoroalkyl), hydroxy-C.sub.1-6
alkyl, nitro-C.sub.1-6 alkyl, or C.sub.1-6 thioalkoxy-C.sub.1-6
alkyl); (3) C.sub.1-20 alkoxy (e.g., C.sub.1-6 alkoxy, such as
perfluoroalkoxy); (4) C.sub.1-6 alkylsulfinyl; (5) C.sub.6-10 aryl;
(6) amino; (7) C.sub.1-6 alk-C.sub.6-10 aryl; (8) azido; (9)
C.sub.3-8 cycloalkyl; (10) C.sub.1-6 alk-C.sub.3-8 cycloalkyl; (11)
halo; (12) C.sub.1-12 heterocyclyl (e.g., C.sub.2-12 heteroaryl);
(13) (C.sub.1-12 heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16)
C.sub.1-20 thioalkoxy (e.g., C.sub.1-6 thioalkoxy); (17)
--(CH.sub.2).sub.qCO.sub.2R.sup.A', where q is an integer from zero
to four, and R.sup.A' is selected from the group consisting of (a)
C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, (c) hydrogen, and (d)
C.sub.1-6 alk-C.sub.6-10 aryl; (18)
--(CH.sub.2).sub.qCONR.sup.B'R.sup.C', where q is an integer from
zero to four and where R.sup.B' and R.sup.C' are independently
selected from the group consisting of (a) hydrogen, (b) C.sub.1-6
alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6 alk-C.sub.6-10 aryl;
(19) --(CH.sub.2).sub.qSO.sub.2R.sup.D', where q is an integer from
zero to four and where R.sup.D' is selected from the group
consisting of (a) C.sub.1-6 alkyl, (b) C.sub.6-10 aryl, and (c)
C.sub.1-6 alk-C.sub.6-10 aryl; (20)
--(CH.sub.2).sub.qSO.sub.2NR.sup.E'R.sup.F', where q is an integer
from zero to four and where each of R.sup.E' and R.sup.F' is,
independently, selected from the group consisting of (a) hydrogen,
(b) C.sub.1-6 alkyl, (c) C.sub.6-10 aryl, and (d) C.sub.1-6
alk-C.sub.6-10 aryl; (21) thiol; (22) C.sub.6-10 aryloxy; (23)
C.sub.3-8 cycloalkoxy; (24) arylalkoxy; (25) C.sub.1-6
alk-C.sub.1-12 heterocyclyl (e.g., C.sub.1-6 alk-C.sub.1-12
heteroaryl); (26) oxo; (27) (C.sub.1-12 heterocyclyl)imino; (28)
C.sub.2-20 alkenyl; and (29) C.sub.2-20 alkynyl. In some
embodiments, each of these groups can be further substituted as
described herein. For example, the alkylene group of a
C.sub.1-alkaryl or a C.sub.1-alkheterocyclyl can be further
substituted with an oxo group to afford the respective aryloyl and
(heterocyclyl)oyl substituent group.
[0788] The term "(heterocyclyl)imino," as used herein, represents a
heterocyclyl group, as defined herein, attached to the parent
molecular group through an imino group. In some embodiments, the
heterocyclyl group can be substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0789] The term "(heterocyclyl)oxy," as used herein, represents a
heterocyclyl group, as defined herein, attached to the parent
molecular group through an oxygen atom. In some embodiments, the
heterocyclyl group can be substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0790] The term "(heterocyclyl)oyl," as used herein, represents a
heterocyclyl group, as defined herein, attached to the parent
molecular group through a carbonyl group. In some embodiments, the
heterocyclyl group can be substituted with 1, 2, 3, or 4
substituent groups as defined herein.
[0791] The term "hydrocarbon," as used herein, represents a group
consisting only of carbon and hydrogen atoms.
[0792] The term "hydroxy," as used herein, represents an --OH
group.
[0793] The term "hydroxyalkenyl," as used herein, represents an
alkenyl group, as defined herein, substituted by one to three
hydroxy groups, with the proviso that no more than one hydroxy
group may be attached to a single carbon atom of the alkyl group,
and is exemplified by dihydroxypropenyl, hydroxyisopentenyl, and
the like.
[0794] The term "hydroxyalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by one to three hydroxy
groups, with the proviso that no more than one hydroxy group may be
attached to a single carbon atom of the alkyl group, and is
exemplified by hydroxymethyl, dihydroxypropyl, and the like.
[0795] The term "isomer," as used herein, means any tautomer,
stereoisomer, enantiomer, or diastereomer of any compound of the
invention. It is recognized that the compounds of the invention can
have one or more chiral centers and/or double bonds and, therefore,
exist as stereoisomers, such as double-bond isomers (i.e.,
geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e.,
(+) or (-)) or cis/trans isomers). According to the invention, the
chemical structures depicted herein, and therefore the compounds of
the invention, encompass all of the corresponding stereoisomers,
that is, both the stereomerically pure form (e.g., geometrically
pure, enantiomerically pure, or diastereomerically pure) and
enantiomeric and stereoisomeric mixtures, e.g., racemates.
Enantiomeric and stereoisomeric mixtures of compounds of the
invention can typically be resolved into their component
enantiomers or stereoisomers by well-known methods, such as
chiral-phase gas chromatography, chiral-phase high performance
liquid chromatography, crystallizing the compound as a chiral salt
complex, or crystallizing the compound in a chiral solvent.
Enantiomers and stereoisomers can also be obtained from
stereomerically or enantiomerically pure intermediates, reagents,
and catalysts by well-known asymmetric synthetic methods.
[0796] The term "N-protected amino," as used herein, refers to an
amino group, as defined herein, to which is attached one or two
N-protecting groups, as defined herein.
[0797] The term "N-protecting group," as used herein, represents
those groups intended to protect an amino group against undesirable
reactions during synthetic procedures. Commonly used N-protecting
groups are disclosed in Greene, "Protective Groups in Organic
Synthesis," 3.sup.rd Edition (John Wiley & Sons, New York,
1999), which is incorporated herein by reference. N-protecting
groups include acyl, aryloyl, or carbamyl groups such as formyl,
acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,
2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl,
o-nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl,
4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral
auxiliaries such as protected or unprotected D, L or D, L-amino
acids such as alanine, leucine, phenylalanine, and the like;
sulfonyl-containing groups such as benzenesulfonyl,
p-toluenesulfonyl, and the like; carbamate forming groups such as
benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,
2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxy carbonyl, t-butyloxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxy carbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl,
and the like, alkaryl groups such as benzyl, triphenylmethyl,
benzyloxymethyl, and the like and silyl groups, such as
trimethylsilyl, and the like. Preferred N-protecting groups are
formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl,
phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and
benzyloxycarbonyl (Cbz).
[0798] The term "nitro," as used herein, represents an --NO.sub.2
group.
[0799] The term "oxo" as used herein, represents .dbd.O.
[0800] The term "perfluoroalkyl," as used herein, represents an
alkyl group, as defined herein, where each hydrogen radical bound
to the alkyl group has been replaced by a fluoride radical.
Perfluoroalkyl groups are exemplified by trifluoromethyl,
pentafluoroethyl, and the like.
[0801] The term "perfluoroalkoxy," as used herein, represents an
alkoxy group, as defined herein, where each hydrogen radical bound
to the alkoxy group has been replaced by a fluoride radical.
Perfluoroalkoxy groups are exemplified by trifluoromethoxy,
pentafluoroethoxy, and the like.
[0802] The term "spirocyclyl," as used herein, represents a
C.sub.2-7 alkylene diradical, both ends of which are bonded to the
same carbon atom of the parent group to form a spirocyclic group,
and also a C.sub.1-6 heteroalkylene diradical, both ends of which
are bonded to the same atom. The heteroalkylene radical forming the
spirocyclyl group can containing one, two, three, or four
heteroatoms independently selected from the group consisting of
nitrogen, oxygen, and sulfur. In some embodiments, the spirocyclyl
group includes one to seven carbons, excluding the carbon atom to
which the diradical is attached. The spirocyclyl groups of the
invention may be optionally substituted with 1, 2, 3, or 4
substituents provided herein as optional substituents for
cycloalkyl and/or heterocyclyl groups.
[0803] The term "stereoisomer," as used herein, refers to all
possible different isomeric as well as conformational forms which a
compound may possess (e.g., a compound of any formula described
herein), in particular all possible stereochemically and
conformationally isomeric forms, all diastereomers, enantiomers
and/or conformers of the basic molecular structure. Some compounds
of the present invention may exist in different tautomeric forms,
all of the latter being included within the scope of the present
invention.
[0804] The term "sulfoalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by a sulfo group of
--SO.sub.3H. In some embodiments, the alkyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein.
[0805] The term "sulfonyl," as used herein, represents an
--S(O).sub.2-- group.
[0806] The term "thioalkaryl," as used herein, represents a
chemical substituent of formula --SR, where R is an alkaryl group.
In some embodiments, the alkaryl group can be further substituted
with 1, 2, 3, or 4 substituent groups as described herein.
[0807] The term "thioalkheterocyclyl," as used herein, represents a
chemical substituent of formula --SR, where R is an alkheterocyclyl
group. In some embodiments, the alkheterocyclyl group can be
further substituted with 1, 2, 3, or 4 substituent groups as
described herein.
[0808] The term "thioalkoxy," as used herein, represents a chemical
substituent of formula --SR, where R is an alkyl group, as defined
herein. In some embodiments, the alkyl group can be further
substituted with 1, 2, 3, or 4 substituent groups as described
herein.
[0809] The term "thiol" represents an --SH group.
[0810] Compound: As used herein, the term "compound," is meant to
include all stereoisomers, geometric isomers, tautomers, and
isotopes of the structures depicted.
[0811] The compounds described herein can be asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereomers, are intended unless otherwise
indicated. Compounds of the present disclosure that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present disclosure. Cis and trans geometric
isomers of the compounds of the present disclosure are described
and may be isolated as a mixture of isomers or as separated
isomeric forms.
[0812] Compounds of the present disclosure also include tautomeric
forms. Tautomeric forms result from the swapping of a single bond
with an adjacent double bond and the concomitant migration of a
proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and
total charge. Examples prototropic tautomers include ketone-enol
pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic
acid pairs, enamine-imine pairs, and annular forms where a proton
can occupy two or more positions of a heterocyclic system, such as,
1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and
2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in
equilibrium or sterically locked into one form by appropriate
substitution.
[0813] Compounds of the present disclosure also include all of the
isotopes of the atoms occurring in the intermediate or final
compounds. "Isotopes" refers to atoms having the same atomic number
but different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium.
[0814] The compounds and salts of the present disclosure can be
prepared in combination with solvent or water molecules to form
solvates and hydrates by routine methods.
[0815] Condition: As used herein, the term "condition" refers to a
disorder that presents with observable symptoms.
[0816] Conserved: As used herein, the term "conserved" refers to
nucleotides or amino acid residues of a polynucleotide sequence or
polypeptide sequence, respectively, that are those that occur
unaltered in the same position of two or more sequences being
compared. Nucleotides or amino acids that are relatively conserved
are those that are conserved amongst more related sequences than
nucleotides or amino acids appearing elsewhere in the
sequences.
[0817] In some embodiments, two or more sequences are said to be
"completely conserved" if they are 100% identical to one another.
In some embodiments, two or more sequences are said to be "highly
conserved" if they are at least 70% identical, at least 80%
identical, at least 90% identical, or at least 95% identical to one
another. In some embodiments, two or more sequences are said to be
"highly conserved" if they are about 70% identical, about 80%
identical, about 90% identical, about 95%, about 98%, or about 99%
identical to one another. In some embodiments, two or more
sequences are said to be "conserved" if they are at least 30%
identical, at least 40% identical, at least 50% identical, at least
60% identical, at least 70% identical, at least 80% identical, at
least 90% identical, or at least 95% identical to one another. In
some embodiments, two or more sequences are said to be "conserved"
if they are about 30% identical, about 40% identical, about 50%
identical, about 60% identical, about 70% identical, about 80%
identical, about 90% identical, about 95% identical, about 98%
identical, or about 99% identical to one another. Conservation of
sequence may apply to the entire length of an oligonucleotide or
polypeptide or may apply to a portion, region or feature
thereof.
[0818] Cyclic or Cyclized: As used herein, the term "cyclic" refers
to the presence of a continuous loop. Cyclic molecules need not be
circular, only joined to form an unbroken chain of subunits. Cyclic
molecules such as the engineered RNA or mRNA of the present
invention may be single units or multimers or comprise one or more
components of a complex or higher order structure.
[0819] Cytostatic: As used herein, "cytostatic" refers to
inhibiting, reducing, suppressing the growth, division, or
multiplication of a cell (e.g., a mammalian cell (e.g., a human
cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a
combination thereof
[0820] Cytotoxic: As used herein, "cytotoxic" refers to killing or
causing injurious, toxic, or deadly effect on a cell (e.g., a
mammalian cell (e.g., a human cell)), bacterium, virus, fungus,
protozoan, parasite, prion, or a combination thereof.
[0821] Delivery: As used herein, "delivery" refers to the act or
manner of delivering a compound, substance, entity, moiety, cargo
or payload.
[0822] Delivery Agent: As used herein, "delivery agent" refers to
any substance which facilitates, at least in part, the in vivo
delivery of signal-sensor polynucleotide, primary construct or
mmRNA to targeted cells.
[0823] Destabilized: As used herein, the term "destable,"
"destabilize," or "destabilizing region" means a region or molecule
that is less stable than a starting, wild-type or native form of
the same region or molecule.
[0824] Detectable label: As used herein, "detectable label" refers
to one or more markers, signals, or moieties which are attached,
incorporated or associated with another entity that is readily
detected by methods known in the art including radiography,
fluorescence, chemiluminescence, enzymatic activity, absorbance and
the like. Detectable labels include radioisotopes, fluorophores,
chromophores, enzymes, dyes, metal ions, ligands such as biotin,
avidin, streptavidin and haptens, quantum dots, and the like.
Detectable labels may be located at any position in the peptides or
proteins disclosed herein. They may be within the amino acids, the
peptides, or proteins, or located at the N- or C-termini.
[0825] Disease: As used herein, the term "disease" refers to an
abnormal condition affecting the body of an organism often showing
specific bodily symptoms.
[0826] Disorder: As used herein, the term "disorder," refers to a
disruption of or an interference with normal functions or
established systems of the body.
[0827] Digest: As used herein, the term "digest" means to break
apart into smaller pieces or components. When referring to
polypeptides or proteins, digestion results in the production of
peptides.
[0828] Distal: As used herein, the term "distal" means situated
away from the center or away from a point or region of
interest.
[0829] Dose splitting factor (DSF)-ratio of PUD of dose split
treatment divided by PUD of total daily dose or single unit dose.
The value is derived from comparison of dosing regimens groups.
[0830] Encoded protein cleavage signal: As used herein, "encoded
protein cleavage signal" refers to the nucleotide sequence which
encodes a protein cleavage signal.
[0831] Engineered: As used herein, embodiments of the invention are
"engineered" when they are designed to have a feature or property,
whether structural or chemical, that varies from a starting point,
wild type or native molecule.
[0832] Exosome: As used herein, "exosome" is a vesicle secreted by
mammalian cells or a complex involved in RNA degradation.
[0833] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end processing); (3)
translation of an RNA into a polypeptide or protein; and (4)
post-translational modification of a polypeptide or protein.
[0834] Feature: As used herein, a "feature" refers to a
characteristic, a property, or a distinctive element.
[0835] Formulation: As used herein, a "formulation" includes at
least a signal-sensor polynucleotide, primary construct or mmRNA
and a delivery agent.
[0836] Fragment: A "fragment," as used herein, refers to a portion.
For example, fragments of proteins may comprise polypeptides
obtained by digesting full-length protein isolated from cultured
cells.
[0837] Functional: As used herein, a "functional" biological
molecule is a biological molecule in a form in which it exhibits a
property and/or activity by which it is characterized.
[0838] Genotype: As used herein, "genotype" refers to the change in
the genotype, or genetic makeup, of a subject, cell, tissue, organ
and/or organism.
[0839] Homology: As used herein, the term "homology" refers to the
overall relatedness between polymeric molecules, e.g. between
nucleic acid molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. In some embodiments,
polymeric molecules are considered to be "homologous" to one
another if their sequences are at least 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical
or similar. The term "homologous" necessarily refers to a
comparison between at least two sequences (polynucleotide or
polypeptide sequences). In accordance with the invention, two
polynucleotide sequences are considered to be homologous if the
polypeptides they encode are at least about 50%, 60%, 70%, 80%,
90%, 95%, or even 99% for at least one stretch of at least about 20
amino acids. In some embodiments, homologous polynucleotide
sequences are characterized by the ability to encode a stretch of
at least 4-5 uniquely specified amino acids. For polynucleotide
sequences less than 60 nucleotides in length, homology is
determined by the ability to encode a stretch of at least 4-5
uniquely specified amino acids. In accordance with the invention,
two protein sequences are considered to be homologous if the
proteins are at least about 50%, 60%, 70%, 80%, or 90% identical
for at least one stretch of at least about 20 amino acids.
[0840] Identity: As used herein, the term "identity" refers to the
overall relatedness between polymeric molecules, e.g., between
oligonucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of the percent
identity of two polynucleotide sequences, for example, can be
performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second nucleic acid sequences for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a sequence aligned
for comparison purposes is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or 100% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which needs
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. For example, the percent identity between two nucleotide
sequences can be determined using methods such as those described
in Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,
M Stockton Press, New York, 1991; each of which is incorporated
herein by reference. For example, the percent identity between two
nucleotide sequences can be determined using the algorithm of
Meyers and Miller (CABIOS, 1989, 4:11-17), which has been
incorporated into the ALIGN program (version 2.0) using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. The percent identity between two nucleotide sequences can,
alternatively, be determined using the GAP program in the GCG
software package using an NWSgapdna.CMP matrix. Methods commonly
employed to determine percent identity between sequences include,
but are not limited to those disclosed in Carillo, H., and Lipman,
D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by
reference. Techniques for determining identity are codified in
publicly available computer programs. Exemplary computer software
to determine homology between two sequences include, but are not
limited to, GCG program package, Devereux, J., et al., Nucleic
Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA
Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
[0841] Inhibit expression of a gene: As used herein, the phrase
"inhibit expression of a gene" means to cause a reduction in the
amount of an expression product of the gene. The expression product
can be an RNA transcribed from the gene (e.g., an mRNA) or a
polypeptide translated from an mRNA transcribed from the gene.
Typically a reduction in the level of an mRNA results in a
reduction in the level of a polypeptide translated therefrom. The
level of expression may be determined using standard techniques for
measuring mRNA or protein.
[0842] In vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, in a Petri dish, etc.,
rather than within an organism (e.g., animal, plant, or
microbe).
[0843] In vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant, or microbe or
cell or tissue thereof).
[0844] Isolated: As used herein, the term "isolated" refers to a
substance or entity that has been separated from at least some of
the components with which it was associated (whether in nature or
in an experimental setting). Isolated substances may have varying
levels of purity in reference to the substances from which they
have been associated. Isolated substances and/or entities may be
separated from at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, or more of
the other components with which they were initially associated. In
some embodiments, isolated agents are more than about 80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, or more than about
99% pure. As used herein, a substance is "pure" if it is
substantially free of other components. Substantially isolated: By
"substantially isolated" is meant that the compound is
substantially separated from the environment in which it was formed
or detected. Partial separation can include, for example, a
composition enriched in the compound of the present disclosure.
Substantial separation can include compositions containing at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 95%, at least about 97%, or
at least about 99% by weight of the compound of the present
disclosure, or salt thereof. Methods for isolating compounds and
their salts are routine in the art.
[0845] Linker: As used herein, a linker refers to a group of atoms,
e.g., 10-1,000 atoms, and can be comprised of the atoms or groups
such as, but not limited to, carbon, amino, alkylamino, oxygen,
sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be
attached to a modified nucleoside or nucleotide on the nucleobase
or sugar moiety at a first end, and to a payload, e.g., a
detectable or therapeutic agent, at a second end. The linker may be
of sufficient length as to not interfere with incorporation into a
nucleic acid sequence. The linker can be used for any useful
purpose, such as to form mmRNA multimers (e.g., through linkage of
two or more signal-sensor polynucleotides, primary constructs, or
mmRNA molecules) or mmRNA conjugates, as well as to administer a
payload, as described herein. Examples of chemical groups that can
be incorporated into the linker include, but are not limited to,
alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester,
alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can
be optionally substituted, as described herein. Examples of linkers
include, but are not limited to, unsaturated alkanes, polyethylene
glycols (e.g., ethylene or propylene glycol monomeric units, e.g.,
diethylene glycol, dipropylene glycol, triethylene glycol,
tripropylene glycol, tetraethylene glycol, or tetraethylene
glycol), and dextran polymers, Other examples include, but are not
limited to, cleavable moieties within the linker, such as, for
example, a disulfide bond (--S--S--) or an azo bond (--N.dbd.N--),
which can be cleaved using a reducing agent or photolysis.
Non-limiting examples of a selectively cleavable bond include an
amido bond can be cleaved for example by the use of
tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,
and/or photolysis, as well as an ester bond can be cleaved for
example by acidic or basic hydrolysis.
[0846] Metastasis: As used herein, the term "metastasis" means the
process by which cancer spreads from the place at which it first
arose as a primary tumor to distant locations in the body.
[0847] Method of Treating: The phrase "a method of treating" or its
equivalent, when applied to, for example, cancer refers to a
procedure or course of action that is designed to reduce or
eliminate the number of cancer cells, prevent the increase in the
number of cancer cells, or to alleviate the symptoms of a cancer in
a subject. A method of treating cancer or another oncology-related
disorder does not necessarily mean that the cancer cells or other
disorder will, in fact, be completely eliminated, that the number
of cells or disorder will, in fact, be reduced, or that the
symptoms of a cancer or other disorder will, in fact, be
alleviated. Often, a method of treating cancer will be performed
even with a low likelihood of success, but which, given the medical
history and estimated survival expectancy of a subject, is
nevertheless deemed an overall beneficial course of action.
[0848] MicroRNA (miRNA) binding site: As used herein, a microRNA
(miRNA) binding site represents a nucleotide location or region of
a nucleic acid transcript to which at least the "seed" region of a
miRNA binds.
[0849] Modified: As used herein "modified" refers to a changed
state or structure of a molecule of the invention. Molecules may be
modified in many ways including chemically, structurally, and
functionally. In one embodiment, the mRNA molecules of the present
invention are modified by the introduction of non-natural
nucleosides and/or nucleotides, e.g., as it relates to the natural
ribonucleotides A, U, G, and C. Noncanonical nucleotides such as
the cap structures are not considered "modified" although they
differ from the chemical structure of the A, C, G, U
ribonucleotides.
[0850] Mucus: As used herein, "mucus" refers to the natural
substance that is viscous and comprises mucin glycoproteins.
[0851] Naturally occurring: As used herein, "naturally occurring"
means existing in nature without artificial aid.
[0852] Non-human vertebrate: As used herein, a "non human
vertebrate" includes all vertebrates except Homo sapiens, including
wild and domesticated species. Examples of non-human vertebrates
include, but are not limited to, mammals, such as alpaca, banteng,
bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea
pig, horse, llama, mule, pig, rabbit, reindeer, sheep water
buffalo, and yak.
[0853] Off-target: As used herein, "off target" refers to any
unintended effect on any one or more target, gene, or cellular
transcript.
[0854] Oncology-related: As used herein, the term
"oncology-related" refers to any disease, disorder, condition,
treatment, process, substance or compound related to any aspect of
one or more hyperproliferative diseases, disorders and/or
conditions including, but not limited to, cancer.
[0855] Open reading frame: As used herein, "open reading frame" or
"ORF" refers to a sequence which does not contain a stop codon in a
given reading frame.
[0856] Operably linked: As used herein, the phrase "operably
linked" refers to a functional connection between two or more
molecules, constructs, transcripts, entities, moieties or the
like.
[0857] Paratope: As used herein, a "paratope" refers to the
antigen-binding site of an antibody.
[0858] Patient: As used herein, "patient" refers to a subject who
may seek or be in need of treatment, requires treatment, is
receiving treatment, will receive treatment, or a subject who is
under care by a trained professional for a particular disease or
condition.
[0859] Optionally substituted: Herein a phrase of the form
"optionally substituted X" (e.g., optionally substituted alkyl) is
intended to be equivalent to "X, wherein X is optionally
substituted" (e.g., "alkyl, wherein said alkyl is optionally
substituted"). It is not intended to mean that the feature "X"
(e.g. alkyl) per se is optional.
[0860] Peptide: As used herein, "peptide" is less than or equal to
50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45,
or 50 amino acids long.
[0861] Pharmaceutical composition: The phrase "pharmaceutical
composition" refers to a composition that alters the etiology of a
disease, disorder and/or condition.
[0862] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0863] Pharmaceutically acceptable excipients: The phrase
"pharmaceutically acceptable excipient," as used herein, refers any
ingredient other than the compounds described herein (for example,
a vehicle capable of suspending or dissolving the active compound)
and having the properties of being substantially nontoxic and
non-inflammatory in a patient. Excipients may include, for example:
antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes (colors), emollients, emulsifiers, fillers
(diluents), film formers or coatings, flavors, fragrances, glidants
(flow enhancers), lubricants, preservatives, printing inks,
sorbents, suspensing or dispersing agents, sweeteners, and waters
of hydration. Exemplary excipients include, but are not limited to:
butylated hydroxytoluene (BHT), calcium carbonate, calcium
phosphate (dibasic), calcium stearate, croscarmellose, crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,
ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, lactose, magnesium stearate, maltitol, mannitol,
methionine, methylcellulose, methyl paraben, microcrystalline
cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch glycolate, sorbitol, starch (corn), stearic acid,
sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
and xylitol.
[0864] Pharmaceutically acceptable salts: The present disclosure
also includes pharmaceutically acceptable salts of the compounds
described herein. As used herein, "pharmaceutically acceptable
salts" refers to derivatives of the disclosed compounds wherein the
parent compound is modified by converting an existing acid or base
moiety to its salt form (e.g., by reacting the free base group with
a suitable organic acid). Examples of pharmaceutically acceptable
salts include, but are not limited to, mineral or organic acid
salts of basic residues such as amines; alkali or organic salts of
acidic residues such as carboxylic acids; and the like.
Representative acid addition salts include acetate, adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,
hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like, as well as
nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine, and the like. The pharmaceutically
acceptable salts of the present disclosure include the conventional
non-toxic salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. The pharmaceutically
acceptable salts of the present disclosure can be synthesized from
the parent compound which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts can be
prepared by reacting the free acid or base forms of these compounds
with a stoichiometric amount of the appropriate base or acid in
water or in an organic solvent, or in a mixture of the two;
generally, nonaqueous media like ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile are preferred. Lists of suitable salts
are found in Remington's Pharmaceutical Sciences, 17.sup.th ed.,
Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical
Salts: Properties, Selection, and Use, P. H. Stahl and C. G.
Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of
Pharmaceutical Science, 66, 1-19 (1977), each of which is
incorporated herein by reference in its entirety.
[0865] Pharmaceutically acceptable solvate: The term
"pharmaceutically acceptable solvate," as used herein, means a
compound of the invention wherein molecules of a suitable solvent
are incorporated in the crystal lattice. A suitable solvent is
physiologically tolerable at the dosage administered. For example,
solvates may be prepared by crystallization, recrystallization, or
precipitation from a solution that includes organic solvents,
water, or a mixture thereof. Examples of suitable solvents are
ethanol, water (for example, mono-, di-, and tri-hydrates),
N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),
N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC),
1,3-dimethyl-2-imidazolidinone (DMEU),
1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU),
acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water
is the solvent, the solvate is referred to as a "hydrate."
[0866] Pharmacokinetic: As used herein, "pharmacokinetic" refers to
any one or more properties of a molecule or compound as it relates
to the determination of the fate of substances administered to a
living organism. Pharmacokinetics is divided into several areas
including the extent and rate of absorption, distribution,
metabolism and excretion. This is commonly referred to as ADME
where: (A) Absorption is the process of a substance entering the
blood circulation; (D) Distribution is the dispersion or
dissemination of substances throughout the fluids and tissues of
the body; (M) Metabolism (or Biotransformation) is the irreversible
transformation of parent compounds into daughter metabolites; and
(E) Excretion (or Elimination) refers to the elimination of the
substances from the body. In rare cases, some drugs irreversibly
accumulate in body tissue.
[0867] Phenotype: As used herein, "phenotype" refers to the set of
observable characteristics of a subject, cell, tissue, organ and/or
organism.
[0868] Physicochemical: As used herein, "physicochemical" means of
or relating to a physical and/or chemical property.
[0869] Preventing: As used herein, the term "preventing" refers to
partially or completely delaying onset of an infection, disease,
disorder and/or condition; partially or completely delaying onset
of one or more symptoms, features, or clinical manifestations of a
particular infection, disease, disorder, and/or condition;
partially or completely delaying onset of one or more symptoms,
features, or manifestations of a particular infection, disease,
disorder, and/or condition; partially or completely delaying
progression from an infection, a particular disease, disorder
and/or condition; and/or decreasing the risk of developing
pathology associated with the infection, the disease, disorder,
and/or condition.
[0870] Prodrug: The present disclosure also includes prodrugs of
the compounds described herein. As used herein, "prodrugs" refer to
any substance, molecule or entity which is in a form predicate for
that substance, molecule or entity to act as a therapeutic upon
chemical or physical alteration. Prodrugs may by covalently bonded
or sequestered in some way and which release or are converted into
the active drug moiety prior to, upon or after administered to a
mammalian subject. Prodrugs can be prepared by modifying functional
groups present in the compounds in such a way that the
modifications are cleaved, either in routine manipulation or in
vivo, to the parent compounds. Prodrugs include compounds wherein
hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any
group that, when administered to a mammalian subject, cleaves to
form a free hydroxyl, amino, sulfhydryl, or carboxyl group
respectively. Preparation and use of prodrugs is discussed in T.
Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol.
14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in
Drug Design, ed. Edward B. Roche, American Pharmaceutical
Association and Pergamon Press, 1987, both of which are hereby
incorporated by reference in their entirety.
[0871] Proliferate: As used herein, the term "proliferate" means to
grow, expand or increase or cause to grow, expand or increase
rapidly. "Proliferative" means having the ability to proliferate.
"Anti-proliferative" means having properties counter to or
inapposite to proliferative properties.
[0872] Protein cleavage site: As used herein, "protein cleavage
site" refers to a site where controlled cleavage of the amino acid
chain can be accomplished by chemical, enzymatic or photochemical
means.
[0873] Protein cleavage signal: As used herein "protein cleavage
signal" refers to at least one amino acid that flags or marks a
polypeptide for cleavage.
[0874] Progression: As used herein, the term "progression" (e.g.,
cancer progression) means the advancement or worsening of or toward
a disease or condition.
[0875] Protein of interest: As used herein, the terms "proteins of
interest" or "desired proteins" include those provided herein and
fragments, mutants, variants, and alterations thereof
[0876] Proximal: As used herein, the term "proximal" means situated
nearer to the center or to a point or region of interest.
[0877] Pseudouridine: As used herein, pseudouridine refers to the
C-glycoside isomer of the nucleoside uridine. A "pseudouridine
analog" is any modification, variant, isoform or derivative of
pseudouridine. For example, pseudouridine analogs include but are
not limited to 1-carboxymethyl-pseudouridine,
1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine,
1-taurinomethyl-4-thio-pseudouridine, 1-methyl-pseudouridine
(m.sup.1.psi.), 1-methyl-4-thio-pseudouridine
(m.sup.1s.sup.4.psi.), 4-thio-1-methyl-pseudouridine,
3-methyl-pseudouridine (m.sup.3.psi.),
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,
4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,
1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp.sup.3
.psi.), and 2'-O-methyl-pseudouridine (.psi.m).
[0878] Purified: As used herein, "purify," "purified,"
"purification" means to make substantially pure or clear from
unwanted components, material defilement, admixture or
imperfection.
[0879] Regression: As used herein, the term "regression" or "degree
of regression" refers to the reversal, either phenotypically or
genotypically, of a cancer progression. Slowing or stopping cancer
progression may be considered regression.
[0880] Reducing the effect: As used herein, the phrase "reducing
the effect" when referring to symptoms, means reducing, eliminating
or alleviating the symptom in the subject. It does not necessarily
mean that the symptom will, in fact, be completely eliminated,
reduced or alleviated.
[0881] Sample: As used herein, the term "sample" or "biological
sample" refers to a subset of its tissues, cells or component parts
(e.g. body fluids, including but not limited to blood, mucus,
lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva,
amniotic fluid, amniotic cord blood, urine, vaginal fluid and
semen). A sample further may include a homogenate, lysate or
extract prepared from a whole organism or a subset of its tissues,
cells or component parts, or a fraction or portion thereof,
including but not limited to, for example, plasma, serum, spinal
fluid, lymph fluid, the external sections of the skin, respiratory,
intestinal, and genitourinary tracts, tears, saliva, milk, blood
cells, tumors, organs. A sample further refers to a medium, such as
a nutrient broth or gel, which may contain cellular components,
such as proteins or nucleic acid molecule.
[0882] Side effect: As used herein, the phrase "side effect" refers
to a secondary effect of treatment.
[0883] Signal Peptide Sequences: As used herein, the phrase "signal
peptide sequences" refers to a sequence which can direct the
transport or localization of a protein.
[0884] Signal-sensor polynucleotide: As used herein, "signal-sensor
polynucleotides" are nucleic acid transcripts which encode one or
more oncology-related polypeptides of interest that, when
translated, delivers a "signal" to the cell (cancer or
noncancerous) which results in the therapeutic benefit to the
organism of either being detrimental to the cancer cell or
beneficial to normal cells or both detrimental to cancer cells and
advantageous to normal cells. The signal-sensor polynucleotides may
optionally further comprise a sequence (translatable or not) which
"senses" the microenvironment of the polynucleotide and alters (a)
the function or phenotypic outcome associated with the peptide or
protein which is translated, (b) the expression level of the
signal-sensor polynucleotide, and/or both.
[0885] Single unit dose: As used herein, a "single unit dose" is a
dose of any therapeutic administed in one dose/at one time/single
route/single point of contact, i.e., single administration
event.
[0886] Similarity: As used herein, the term "similarity" refers to
the overall relatedness between polymeric molecules, e.g. between
polynucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of percent
similarity of polymeric molecules to one another can be performed
in the same manner as a calculation of percent identity, except
that calculation of percent similarity takes into account
conservative substitutions as is understood in the art.
[0887] Skin: The term "skin" is the thin layer of tissue forming
the natural outer covering of the body of a subject and includes
the epidermis and the dermis. The dermis is the thick layer of
living tissue below the epidermis which is the surface epithelium
of the skin.
[0888] Split dose: As used herein, a "split dose" is the division
of single unit dose or total daily dose into two or more doses.
[0889] Stable: As used herein "stable" refers to a compound that is
sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and preferably capable of
formulation into an efficacious therapeutic agent.
[0890] Stabilized: As used herein, the term "stabilize",
"stabilized," "stabilized region" means to make or become
stable.
[0891] Subject: As used herein, the term "subject" or "patient"
refers to any organism to which a composition in accordance with
the invention may be administered, e.g., for experimental,
diagnostic, prophylactic, and/or therapeutic purposes. Typical
subjects include animals (e.g., mammals such as mice, rats,
rabbits, non-human primates, and humans) and/or plants.
[0892] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0893] Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
[0894] Substantially simultaneously: As used herein and as it
relates to plurality of doses, the term means within 2 seconds.
[0895] Suffering from: An individual who is "suffering from" a
disease, disorder, and/or condition has been diagnosed with or
displays one or more symptoms of a disease, disorder, and/or
condition.
[0896] Susceptible to: An individual who is "susceptible to" a
disease, disorder, and/or condition has not been diagnosed with
and/or may not exhibit symptoms of the disease, disorder, and/or
condition but harbors a propensity to develop a disease or its
symptoms. In some embodiments, an individual who is susceptible to
a disease, disorder, and/or condition (for example, cancer) may be
characterized by one or more of the following: (1) a genetic
mutation associated with development of the disease, disorder,
and/or condition; (2) a genetic polymorphism associated with
development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
and/or nucleic acid associated with the disease, disorder, and/or
condition; (4) habits and/or lifestyles associated with development
of the disease, disorder, and/or condition; (5) a family history of
the disease, disorder, and/or condition; and (6) exposure to and/or
infection with a microbe associated with development of the
disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[0897] Symptom: As used herein, the term "symptom" is a signal of a
disease, disorder and/or condition. For example, symptoms may be
felt or noticed by the subject who has them but may not be easily
accessed by looking at a subject's outward appearance or behaviors.
Examples of symptoms include, but are not limited to, weakness,
aches and pains, fever, fatigue, weight loss, blood clots,
increased blood calcium levels, low white blood cell count, short
of breath, dizziness, headaches, hyperpigmentation, jaundice,
erthema, pruritis, excessive hair growth, change in bowel habits,
change in bladder function, long-lasting sores, white patches
inside the mouth, white spots on the tongue, unusual bleeding or
discharge, thickening or lump on parts of the body, indigestion,
trouble swallowing, changes in warts or moles, change in new skin
and nagging cough or hoarseness.
[0898] Synthetic: The term "synthetic" means produced, prepared,
and/or manufactured by the hand of man. Synthesis of
polynucleotides or polypeptides or other molecules of the present
invention may be chemical or enzymatic.
[0899] Targeted Cells: As used herein, "targeted cells" refers to
any one or more cells of interest. The cells may be found in vitro,
in vivo, in situ or in the tissue or organ of an organism. The
organism may be an animal, preferably a mammal, more preferably a
human and most preferably a patient.
[0900] Therapeutic Agent: The term "therapeutic agent" refers to
any agent that, when administered to a subject, has a therapeutic,
diagnostic, and/or prophylactic effect and/or elicits a desired
biological and/or pharmacological effect.
[0901] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of an agent to
be delivered (e.g., nucleic acid, drug, therapeutic agent,
diagnostic agent, prophylactic agent, etc.) that is sufficient,
when administered to a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition.
[0902] Therapeutically effective outcome: As used herein, the term
"therapeutically effective outcome" means an outcome that is
sufficient in a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition.
[0903] Total daily dose: As used herein, a "total daily dose" is an
amount given or prescribed in 24 hr period. It may be administered
as a single unit dose.
[0904] Transcription factor: As used herein, the term
"transcription factor" refers to a DNA-binding protein that
regulates transcription of DNA into RNA, for example, by activation
or repression of transcription. Some transcription factors effect
regulation of transcription alone, while others act in concert with
other proteins. Some transcription factor can both activate and
repress transcription under certain conditions. In general,
transcription factors bind a specific target sequence or sequences
highly similar to a specific consensus sequence in a regulatory
region of a target gene. Transcription factors may regulate
transcription of a target gene alone or in a complex with other
molecules.
[0905] Treating: As used herein, the term "treating" refers to
partially or completely alleviating, ameliorating, improving,
relieving, delaying onset of, inhibiting progression of, reducing
severity of, and/or reducing incidence of one or more symptoms or
features of a particular infection, disease, disorder, and/or
condition. For example, "treating" cancer may refer to inhibiting
survival, growth, and/or spread of a tumor. Treatment may be
administered to a subject who does not exhibit signs of a disease,
disorder, and/or condition and/or to a subject who exhibits only
early signs of a disease, disorder, and/or condition for the
purpose of decreasing the risk of developing pathology associated
with the disease, disorder, and/or condition.
[0906] Tumor: As used herein, a "tumor" is an abnormal growth of
tissue, whether benign or malignant.
[0907] Tumor growth: As used herein, the term "tumor growth" or
"tumor metastases" means an increased mass or volume of the tumor
or expansion of the tumor distribution.
[0908] Unmodified: As used herein, "unmodified" refers to any
substance, compound or molecule prior to being changed in any way.
Unmodified may, but does not always, refer to the wild type or
native form of a biomolecule. Molecules may undergo a series of
modifications whereby each modified molecule may serve as the
"unmodified" starting molecule for a subsequent modification.
EQUIVALENTS AND SCOPE
[0909] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0910] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0911] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0912] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0913] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any nucleic acid or protein
encoded thereby; any method of production; any method of use; etc.)
can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[0914] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
[0915] Section and table headings are not intended to be
limiting.
EXAMPLES
Example 1
Signal-Sensor Polynucleotide Production
[0916] Modified signal-sensor mRNAs (mmRNA) according to the
invention may be made using standard laboratory methods and
materials. The open reading frame (ORF) of the gene of interest may
be flanked by a 5' untranslated region (UTR) which may contain a
strong Kozak translational initiation signal and/or an alpha-globin
3' UTR which may include an oligo(dT) sequence for templated
addition of a poly-A tail. The modified mRNAs may be modified to
reduce the cellular innate immune response. The modifications to
reduce the cellular response may include pseudouridine (.psi.) and
5-methyl-cytidine (5meC, 5mc or m.sup.5C). (See, Kariko K et al.
Immunity 23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40
(2008), Anderson B R et al. NAR (2010); herein incorporated by
reference).
[0917] The ORF may also include various upstream or downstream
additions (such as, but not limited to, .beta.-globin, tags, etc.)
may be ordered from an optimization service such as, but limited
to, DNA2.0 (Menlo Park, Calif.) and may contain multiple cloning
sites which may have XbaI recognition. Upon receipt of the
construct, it may be reconstituted and transformed into chemically
competent E. coli.
[0918] For the present invention, NEB DH5-alpha Competent E. coli
may be used. Transformations are performed according to NEB
instructions using 100 ng of plasmid. The protocol is as
follows:
[0919] Thaw a tube of NEB 5-alpha Competent E. coli cells on ice
for 10 minutes.
[0920] Add 1-5 .mu.l containing 1 pg-100 ng of plasmid DNA to the
cell mixture. Carefully flick the tube 4-5 times to mix cells and
DNA. Do not vortex.
[0921] Place the mixture on ice for 30 minutes. Do not mix.
[0922] Heat shock at 42.degree. C. for exactly 30 seconds. Do not
mix.
[0923] Place on ice for 5 minutes. Do not mix.
[0924] Pipette 950 .mu.l of room temperature SOC into the
mixture.
[0925] Place at 37.degree. C. for 60 minutes. Shake vigorously (250
rpm) or rotate.
[0926] Warm selection plates to 37.degree. C.
[0927] Mix the cells thoroughly by flicking the tube and
inverting.
[0928] Spread 50-100 .mu.l of each dilution onto a selection plate
and incubate overnight at 37.degree. C. Alternatively, incubate at
30.degree. C. for 24-36 hours or 25.degree. C. for 48 hours.
[0929] A single colony is then used to inoculate 5 ml of LB growth
media using the appropriate antibiotic and then allowed to grow
(250 RPM, 37.degree. C.) for 5 hours. This is then used to
inoculate a 200 ml culture medium and allowed to grow overnight
under the same conditions.
[0930] To isolate the plasmid (up to 850 .mu.g), a maxi prep is
performed using the Invitrogen PURELINK.TM. HiPure Maxiprep Kit
(Carlsbad, Calif.), following the manufacturer's instructions.
[0931] In order to generate cDNA for In Vitro Transcription (IVT),
the plasmid is first linearized using a restriction enzyme such as
XbaI. A typical restriction digest with XbaI will comprise the
following: Plasmid 1.0 .mu.g; 10.times. Buffer 1.0 .mu.l; XbaI 1.5
.mu.l; dH.sub.2O up to 10 .mu.l; incubated at 37.degree. C. for 1
hr. If performing at lab scale (<5 .mu.g), the reaction is
cleaned up using Invitrogen's PURELINK.TM. PCR Micro Kit (Carlsbad,
Calif.) per manufacturer's instructions. Larger scale purifications
may need to be done with a product that has a larger load capacity
such as Invitrogen's standard PURELINK.TM. PCR Kit (Carlsbad,
Calif.). Following the cleanup, the linearized vector is quantified
using the NanoDrop and analyzed to confirm linearization using
agarose gel electrophoresis.
[0932] As a non-limiting example, G-CSF may represent the
polypeptide of interest. Sequences used in the steps outlined in
Examples 1-5 are shown in Table 12. It should be noted that the
start codon (ATG) has been underlined in each sequence of Table
12.
TABLE-US-00012 TABLE 12 G-CSF Sequences SEQ ID NO Description 6592
cDNAsequence: ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCT
GCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGG
GCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAA
GTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTG
CCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCT
CTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCA
GCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGG
GGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTG
GACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCA
GATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCC
ATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGT
TGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCC
ACCTTGCCCAGCCCTGA 6593 cDNA having T7 polymerase site, Afel and Xba
restriction site: TAATACGACTCACTATA
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC
ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCT
GCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGG
GCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAA
GTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTG
CCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCT
CTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCA
GCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGG
GGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTG
GACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCA
GATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCC
ATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGT
TGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCC
ACCTTGCCCAGCCCTGA
AGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCC
TTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGC
CGCTCGAGCATGCATCTAGA 6594 Optimized sequence; containing T7
polymerase site, AfeI and Xba restriction site TAATACGACTCACTATA
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC
ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTT
GCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCG
GACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAG
GTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCG
CGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGC
TTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAG
TTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGA
CTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGA
CACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGA
TGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAAT
GCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAG
CGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACAT CTTGCGCAGCCGTGA
AGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCC
TTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGC
CGCTCGAGCATGCATCTAGA 6595 mRNA sequence (transcribed)
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC
AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAG
UUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCU
CUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUG
GAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAG
CUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUC
GGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCG
CAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUG
UUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAA
UUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCA
ACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUG
CAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGC
AGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAA
GUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGA
AGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUC
CCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG
Example 2
PCR for cDNA Production
[0933] PCR procedures for the preparation of cDNA are performed
using 2.times. KAPA HIF1.TM. HotStart ReadyMix by Kapa Biosystems
(Woburn, Mass.). This system includes 2.times. KAPA ReadyMix 12.5
.mu.l; Forward Primer (10 uM) 0.75 .mu.l; Reverse Primer (10 uM)
0.75 .mu.l; Template cDNA 100 ng; and dH.sub.2O diluted to 25.0
.mu.l. The reaction conditions are at 95.degree. C. for 5 min. and
25 cycles of 98.degree. C. for 20 sec, then 58.degree. C. for 15
sec, then 72.degree. C. for 45 sec, then 72.degree. C. for 5 min.
then 4.degree. C. to termination.
[0934] The reverse primer of the instant invention incorporates a
poly-T.sub.120 for a poly-A.sub.120 in the mRNA. Other reverse
primers with longer or shorter poly(T) tracts can be used to adjust
the length of the poly(A) tail in the mRNA.
[0935] The reaction is cleaned up using Invitrogen's PURELINK.TM.
PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions
(up to 5 .mu.g). Larger reactions will require a cleanup using a
product with a larger capacity. Following the cleanup, the cDNA is
quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the cDNA is the expected size. The cDNA
is then submitted for sequencing analysis before proceeding to the
in vitro transcription reaction.
Example 3
In Vitro Transcription (IVT)
[0936] The in vitro transcription reaction generates mRNA
containing modified nucleotides or modified RNA. The input
nucleotide triphosphate (NTP) mix is made in-house using natural
and un-natural NTPs.
[0937] A typical in vitro transcription reaction includes the
following:
TABLE-US-00013 Template cDNA 1.0 .mu.g 10x transcription buffer
(400 mM Tris- 2.0 .mu.l HCl pH 8.0, 190 mM MgCl.sub.2, 50 mM DTT,
10 mM Spermidine) Custom NTPs (25 mM each) 7.2 .mu.l RNase
Inhibitor 20 U T7 RNA polymerase 3000 U dH.sub.20 Up to 20.0 .mu.l.
and Incubation at 37.degree. C. for 3 hr-5 hrs.
[0938] The crude IVT mix may be stored at 4.degree. C. overnight
for cleanup the next day. 1 U of RNase-free DNase is then used to
digest the original template. After 15 minutes of incubation at
37.degree. C., the mRNA is purified using Ambion's MEGACLEAR.TM.
Kit (Austin, Tex.) following the manufacturer's instructions. This
kit can purify up to 500 .mu.g of RNA. Following the cleanup, the
RNA is quantified using the NanoDrop and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred.
Example 4
Enzymatic Capping of mRNA
[0939] Capping of the mRNA is performed as follows where the
mixture includes: IVT RNA 60 .mu.g-180 .mu.g and dH.sub.2O up to 72
.mu.l. The mixture is incubated at 65.degree. C. for 5 minutes to
denature RNA, and then is transferred immediately to ice.
[0940] The protocol then involves the mixing of 10.times. Capping
Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl.sub.2)
(10.0 .mu.l); 20 mM GTP (5.0 .mu.l); 20 mM S-Adenosyl Methionine
(2.5 .mu.l); RNase Inhibitor (100 U); 2'-O-Methyltransferase (400
U); Vaccinia capping enzyme (Guanylyl transferase) (40 U);
dH.sub.2O (Up to 28 .mu.l); and incubation at 37.degree. C. for 30
minutes for 60 .mu.g RNA or up to 2 hours for 180 .mu.g of RNA.
[0941] The mRNA is then purified using Ambion's MEGACLEAR.TM. Kit
(Austin, Tex.) following the manufacturer's instructions. Following
the cleanup, the RNA is quantified using the NANODROP.TM.
(ThermoFisher, Waltham, Mass.) and analyzed by agarose gel
electrophoresis to confirm the RNA is the proper size and that no
degradation of the RNA has occurred. The RNA product may also be
sequenced by running a reverse-transcription-PCR to generate the
cDNA for sequencing.
Example 5
PolyA Tailing Reaction
[0942] Without a poly-T in the cDNA, a poly-A tailing reaction must
be performed before cleaning the final product. This is done by
mixing Capped IVT RNA (100 .mu.l); RNase Inhibitor (20 U);
10.times. Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100
mM MgCl.sub.2)(12.0 .mu.l); 20 mM ATP (6.0 .mu.l); Poly-A
Polymerase (20 U); dH.sub.2O up to 123.5 pi and incubation at
37.degree. C. for 30 min. If the poly-A tail is already in the
transcript, then the tailing reaction may be skipped and proceed
directly to cleanup with Ambion's MEGACLEAR.TM. kit (Austin, Tex.)
(up to 500 .mu.g). Poly-A Polymerase is preferably a recombinant
enzyme expressed in yeast.
[0943] For studies performed and described herein, the poly-A tail
is encoded in the IVT template to comprise 160 nucleotides in
length. However, it should be understood that the processivity or
integrity of the polyA tailing reaction may not always result in
exactly 160 nucleotides. Hence polyA tails of approximately 160
nucleotides, e.g, about 150-165, 155, 156, 157, 158, 159, 160, 161,
162, 163, 164 or 165 are within the scope of the invention.
Example 6
Natural 5' Caps and 5' Cap Analogues
[0944] 5'-capping of modified RNA may be completed concomitantly
during the in vitro-transcription reaction using the following
chemical RNA cap analogs to generate the 5'-guanosine cap structure
according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the
ARCA cap]; G(5)ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A;
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). 5'-capping
of modified RNA may be completed post-transcriptionally using a
Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure:
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1
structure may be generated using both Vaccinia Virus Capping Enzyme
and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from
the Cap 1 structure followed by the 2'-O-methylation of the
5'-antepenultimate nucleotide using a 2'-O methyl-transferase. Cap
3 structure may be generated from the Cap 2 structure followed by
the 2'-O-methylation of the 5'-preantepenultimate nucleotide using
a 2'-O methyl-transferase. Enzymes are preferably derived from a
recombinant source.
[0945] When transfected into mammalian cells, the modified mRNAs
have a stability of between 12-18 hours or more than 18 hours,
e.g., 24, 36, 48, 60, 72 or greater than 72 hours.
Example 7
Capping
A. Protein Expression Assay
[0946] Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID
NO: 6567; mRNA sequence fully modified with 5-methylcytidine at
each cytidine and pseudouridine replacement at each uridine site
shown in SEQ ID NO: 6570 with a polyA tail approximately 160
nucleotides in length not shown in sequence) containing the ARCA
(3' O-Me-m7G(5')ppp(5')G) cap analog or the Cap1 structure can be
transfected into human primary keratinocytes at equal
concentrations. 6, 12, 24 and 36 hours post-transfection the amount
of G-CSF secreted into the culture medium can be assayed by ELISA.
Synthetic mRNAs that secrete higher levels of G-CSF into the medium
would correspond to a synthetic mRNA with a higher
translationally-competent Cap structure.
B. Purity Analysis Synthesis
[0947] Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID
NO: 6567; mRNA sequence fully modified with 5-methylcytidine at
each cytidine and pseudouridine replacement at each uridine site
shown in SEQ ID NO: 6570 with a polyA tail approximately 160
nucleotides in length not shown in sequence) containing the ARCA
cap analog or the Cap1 structure crude synthesis products can be
compared for purity using denaturing Agarose-Urea gel
electrophoresis or HPLC analysis. Synthetic mRNAs with a single,
consolidated band by electrophoresis correspond to the higher
purity product compared to a synthetic mRNA with multiple bands or
streaking bands. Synthetic mRNAs with a single HPLC peak would also
correspond to a higher purity product. The capping reaction with a
higher efficiency would provide a more pure mRNA population.
C. Cytokine Analysis
[0948] Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID
NO: 6567; mRNA sequence fully modified with 5-methylcytidine at
each cytidine and pseudouridine replacement at each uridine site
shown in SEQ ID NO: 6570 with a polyA tail approximately 160
nucleotides in length not shown in sequence) containing the ARCA
cap analog or the Cap1 structure can be transfected into human
primary keratinocytes at multiple concentrations. 6, 12, 24 and 36
hours post-transfection the amount of pro-inflammatory cytokines
such as TNF-alpha and IFN-beta secreted into the culture medium can
be assayed by ELISA. Synthetic mRNAs that secrete higher levels of
pro-inflammatory cytokines into the medium would correspond to a
synthetic mRNA containing an immune-activating cap structure.
D. Capping Reaction Efficiency
[0949] Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID
NO: 6567; mRNA sequence fully modified with 5-methylcytidine at
each cytidine and pseudouridine replacement at each uridine site
shown in SEQ ID NO: 6570 with a polyA tail approximately 160
nucleotides in length not shown in sequence) containing the ARCA
cap analog or the Cap1 structure can be analyzed for capping
reaction efficiency by LC-MS after capped mRNA nuclease treatment.
Nuclease treatment of capped mRNAs would yield a mixture of free
nucleotides and the capped 5'-5-triphosphate cap structure
detectable by LC-MS. The amount of capped product on the LC-MS
spectra can be expressed as a percent of total mRNA from the
reaction and would correspond to capping reaction efficiency. The
cap structure with higher capping reaction efficiency would have a
higher amount of capped product by LC-MS.
Example 8
Agarose Gel Electrophoresis of Modified RNA or RT PCR Products
[0950] Individual modified RNAs (200-400 ng in a 20 .mu.l volume)
or reverse transcribed PCR products (200-400 ng) are loaded into a
well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad,
Calif.) and run for 12-15 minutes according to the manufacturer
protocol.
Example 9
Nanodrop Modified RNA Quantification and UV Spectral Data
[0951] Modified RNAs in TE buffer (1 .mu.l) are used for Nanodrop
UV absorbance readings to quantitate the yield of each modified RNA
from an in vitro transcription reaction.
Example 10
Formulation of Signal-Sensor Polynucleotides
[0952] Signal-sensor polynucleotides may be formulated for in vitro
and in vivo experiments according to the methods taught in
International Application PCT/US12/069610 filed Dec. 14, 2012, the
contents of which are incorporated herein by reference in their
entirety.
Example 11
Assays and Methods of Detection or Analysis of Signal-Sensor
Polynucleotides
[0953] Signal-sensor polynucleotides may be investigated using the
methods described in co-pending International Patent application
No. PCT/US2013/030070 filed Mar. 9, 2013 and U.S. Patent
Application No. U.S. 61/681,742 filed Aug. 10, 2012 (MNC2), the
contents of which are incorporated herein by reference in their
entirety.
Example 12
Cell Lines for the Study of Signal-Sensor Polynucleotides
[0954] Signal-sensor polynucleotides may be investigated in any
number of cancer or normal cell lines. Cell lines useful in the
present invention include those from ATCC (Manassas, Va.) and are
listed in Table 13.
TABLE-US-00014 TABLE 13 Cell lines ATCC Number Hybridoma or Cell
line Description Name CCL-171 Homo sapiens (human) Source: Organ:
lung MRC-5 Disease: normal Cell Type: fibroblast CCL-185 Homo
sapiens (human) Source: Organ: lung A549 Disease: carcinoma CCL-248
Homo sapiens (human) Source: Organ: colon T84 Disease: colorectal
carcinoma Derived from metastatic site: lung CCL-256 Homo sapiens
(human) Source: Organ: lung NCI-H2126 Disease: adenocarcinoma;
non-small cell lung cancer [H2126] Derived from metastatic site:
pleural effusion CCL-257 Homo sapiens (human) Source: Organ: lung
NCI-H1688 Disease: carcinoma; classic small cell lung cancer
[H1688] CCL-75 Homo sapiens (human) Source: Organ: lung WI-38
Disease: normal Cell Type: fibroblast CCL-75.1 Homo sapiens (human)
Source: Organ: lung WI-38 VA-13 Cell Type: fibroblastSV40
transformed subline 2RA CCL-95.1 Homo sapiens (human) Source:
Organ: lung WI-26 VA4 Cell Type: SV40 transformed CRL-10741 Homo
sapiens (human) Source: Organ: liver C3A [HepG2/C3A, Disease:
hepatocellular carcinoma derivative of Hep G2 (ATCC HB- 8065)]
CRL-11233 Homo sapiens (human) Source: Organ: liver THLE-3 Tissue:
left lobe Cell Type: epithelialimmortalized with SV40 large T
antigen CRL-11351 Homo sapiens (human) Source: Organ: lung H69AR
Disease: carcinoma; small cell lung cancer; multidrug resistant
Cell Type: epithelial CRL-1848 Homo sapiens (human) Source: Organ:
lung NCI-H292 [H292] Disease: mucoepidermoid pulmonary carcinoma
CRL-1918 Homo sapiens (human) Source: Organ: pancreas CFPAC-1
Disease: ductal adenocarcinoma; cystic fibrosis Derived from
metastatic site: liver metastasis CRL-1973 Homo sapiens (human)
Source: Organ: testis NTERA-2 cl.D1 Disease: malignant pluripotent
embryonal carcinoma [NT2/D1] Derived from metastatic site: lung
CRL-2049 Homo sapiens (human) Source: Organ: lung DMS 79 Disease:
carcinoma; small cell lung cancer CRL-2062 Homo sapiens (human)
Source: Organ: lung DMS 53 Disease: carcinoma; small cell lung
cancer CRL-2064 Homo sapiens (human) Source: Organ: lung DMS 153
Disease: carcinoma; small cell lung cancer Derived from metastatic
site: liver CRL-2066 Homo sapiens (human) Source: Organ: lung DMS
114 Disease: carcinoma; small cell lung cancer CRL-2081 Homo
sapiens (human) Source: Disease: biphasic MSTO-211H mesothelioma
Derived from metastatic site: lung CRL-2170 Homo sapiens (human)
Source: Organ: lung SW 1573 [SW- Disease: alveolar cell carcinoma
1573, SW1573] CRL-2177 Homo sapiens (human) Source: Organ: lung SW
1271 [SW- Disease: carcinoma; small cell lung cancer 1271, SW1271]
CRL-2195 Homo sapiens (human) Source: Organ: lung SHP-77 Disease:
carcinoma; small cell lung cancer Cell Type: large cell, variant;
CRL-2233 Homo sapiens (human) Source: Organ: liver SNU-398 Disease:
hepatocellular carcinoma CRL-2234 Homo sapiens (human) Source:
Organ: liver SNU-449 Tumor Stage: grade II-III/IV Disease:
hepatocellular carcinoma CRL-2235 Homo sapiens (human) Source:
Organ: liver SNU-182 Tumor Stage: grade III/IV Disease:
hepatocellular carcinoma CRL-2236 Homo sapiens (human) Source:
Organ: liver SNU-475 Tumor Stage: grade II-IV/V Disease:
hepatocellular carcinoma CRL-2237 Homo sapiens (human) Source:
Organ: liver SNU-387 Tumor Stage: grade IV/V Disease: pleomorphic
hepatocellular carcinoma CRL-2238 Homo sapiens (human) Source:
Organ: liver SNU-423 Tumor Stage: grade III/IV Disease: pleomorphic
hepatocellular carcinoma CRL-2503 Homo sapiens (human) Source:
Organ: lung NL20 Tissue: bronchus Disease: normal CRL-2504 Homo
sapiens (human) Source: Organ: lung NL20-TA [NL20T- Tissue:
bronchus A] Disease: normal CRL-2706 Homo sapiens (human) Source:
Organ: liver THLE-2 Tissue: left lobe Cell Type: epithelialSV40
transformed CRL-2741 Homo sapiens (human) Source: Organ: lung
HBE135-E6E7 Tissue: bronchus Cell Type: epithelialHPV-16 E6/E7
transformed CRL-2868 Homo sapiens (human) Source: Organ: lung
HCC827 Disease: adenocarcinoma Cell Type: epithelial CRL-2871 Homo
sapiens (human) Source: Organ: lung HCC4006 Disease: adenocarcinoma
Derived from metastatic site: pleural effusion Cell Type:
epithelial CRL-5800 Homo sapiens (human) Source: Organ: lung
NCI-H23 [H23] Disease: adenocarcinoma; non-small cell lung cancer
CRL-5803 Homo sapiens (human) Source: Organ: lung NCI-H1299
Disease: carcinoma; non-small cell lung cancer Derived from
metastatic site: lymph node CRL-5804 Homo sapiens (human) Source:
Organ: lung NCI-H187 [H187] Disease: carcinoma; classic small cell
lung cancer Derived from metastatic site: pleural effusion CRL-5807
Homo sapiens (human) Source: Organ: lung NCI-H358 [H-358, Tissue:
bronchiole; alveolus H358] Disease: bronchioalveolar carcinoma;
non-small cell lung cancer CRL-5808 Homo sapiens (human) Source:
Organ: lung NCI-H378 [H378] Tumor Stage: stage E Disease:
carcinoma; classic small cell lung cancer Derived from metastatic
site: pleural effusion CRL-5810 Homo sapiens (human) Source: Organ:
lung NCI-H522 [H522] Tumor Stage: stage 2 Disease: adenocarcinoma;
non-small cell lung cancer CRL-5811 Homo sapiens (human) Source:
Organ: lung NCI-H526 [H526] Tumor Stage: stage E Disease:
carcinoma; variant small cell lung cancer Derived from metastatic
site: bone marrow CRL-5815 Homo sapiens (human) Source: Organ: lung
NCI-H727 [H727] Tissue: bronchus Disease: carcinoid CRL-5816 Homo
sapiens (human) Source: Organ: lung NCI-H810 [H810] Tumor Stage:
stage 2 Disease: carcinoma; non-small cell lung cancer CRL-5817
Homo sapiens (human) Source: Organ: lung NCI-H889 [H889] Tumor
Stage: stage E Disease: carcinoma; classic small cell lung cancer
Derived from metastatic site: lymph node CRL-5818 Homo sapiens
(human) Source: Organ: lung NCI-H1155 Disease: carcinoma; non-small
cell lung cancer [H1155] Derived from metastatic site: lymph node
CRL-5819 Homo sapiens (human) Source: Organ: lung NCI-H1404
Disease: papillary adenocarcinoma [H1404] Derived from metastatic
site: lymph node CRL-5822 Homo sapiens (human) Source: Organ:
stomach NCI-N87 [N87] Disease: gastric carcinoma Derived from
metastatic site: liver CRL-5823 Homo sapiens (human) Source: Organ:
lung NCI-H196 [H196] Tumor Stage: stage E Disease: carcinoma;
variant small cell lung cancer Derived from metastatic site:
pleural effusion CRL-5824 Homo sapiens (human) Source: Organ: lung
NCI-H211 [H211] Tumor Stage: stage E Disease: carcinoma; small cell
lung cancer Derived from metastatic site: bone marrow CRL-5825 Homo
sapiens (human) Source: Organ: lung NCI-H220 [H220] Tumor Stage:
stage E Disease: carcinoma; classic small cell lung cancer Derived
from metastatic site: pleural effusion CRL-5828 Homo sapiens
(human) Source: Organ: lung NCI-H250 [H250] Tumor Stage: stage E
Disease: carcinoma; classic small cell lung cancer Derived from
metastatic site: brain CRL-5831 Homo sapiens (human) Source: Organ:
lung NCI-H524 [H524] Tumor Stage: stage L Disease: carcinoma;
variant small cell lung cancer Derived from metastatic site: lymph
node CRL-5834 Homo sapiens (human) Source: Organ: lung NCI-H647
[H647] Tumor Stage: stage 3A Disease: adenosquamous carcinoma;
non-small cell lung cancer Derived from metastatic site: pleural
effusion CRL-5835 Homo sapiens (human) Source: Organ: lung NCI-H650
[H650] Disease: bronchioalveolar carcinoma; non-small cell lung
cancer Derived from metastatic site: lymph node CRL-5836 Homo
sapiens (human) Source: Organ: lung NCI-H711 [H711] Tumor Stage:
stage E Disease: carcinoma; classic small cell lung cancer Derived
from metastatic site: bone marrow CRL-5837 Homo sapiens (human)
Source: Organ: lung NCI-H719 [H719] Tumor Stage: stage E Disease:
carcinoma; classic small cell lung cancer Derived from metastatic
site: bone marrow CRL-5840 Homo sapiens (human) Source: Organ: lung
NCI-H740 [H740] Tumor Stage: stage E Disease: carcinoma; classic
small cell lung cancer Derived from metastatic site: lymph node
CRL-5841 Homo sapiens (human) Source: Organ: lung NCI-H748 [H748]
Tumor Stage: stage E Disease: carcinoma; classic small cell lung
cancer Derived from metastatic site: lymph node CRL-5842 Homo
sapiens (human) Source: Organ: lung NCI-H774 [H774] Tumor Stage:
stage E Disease: carcinoma; classic small cell lung cancer Derived
from metastatic site: soft tissue CRL-5844 Homo sapiens (human)
Source: Organ: lung NCI-H838 [H838] Tumor stage: 3B Disease:
adenocarcinoma; non-small cell lung cancer Derived from metastatic
site: lymph node CRL-5845 Homo sapiens (human) Source: Organ: lung
NCI-H841 [H841] Tumor Stage: stage L Disease: carcinoma; variant
small cell lung cancer Derived from metastatic site: lymph node
CRL-5846 Homo sapiens (human) Source: Organ: lung NCI-H847 [H847]
Tumor Stage: stage L Disease: carcinoma; classic small cell lung
cancer Derived from metastatic site: pleural effusion CRL-5849 Homo
sapiens (human) Source: Organ: lung NCI-H865 [H865] Tumor Stage:
stage L Disease: carcinoma; classic small cell lung cancer Derived
from metastatic site: pleural effusion CRL-5850 Homo sapiens
(human) Source: Organ: lung NCI-H920 [H920] Tumor Stage: stage 4
Disease: adenocarcinoma; non-small cell lung cancer Derived from
metastatic site: lymph node CRL-5853 Homo sapiens (human) Source:
Organ: lung NCI-H1048 Disease: carcinoma; small cell lung cancer
[H1048] Derived from metastatic site: pleural effusion CRL-5855
Homo sapiens (human) Source: Organ: lung NCI-H1092 Tumor Stage:
stage E [H1092] Disease: carcinoma; classic small cell lung cancer
Derived from metastatic site: bone marrow CRL-5856 Homo sapiens
(human) Source: Organ: lung NCI-H1105 Tumor Stage: stage E [H1105]
Disease: carcinoma; classic small cell lung cancer Derived from
metastatic site: lymph node CRL-5858 Homo sapiens (human) Source:
Organ: lung NCI-H1184 Tumor Stage: stage L [H1184] Disease:
carcinoma; small cell lung cancer Derived from metastatic site:
lymph node CRL-5859 Homo sapiens (human) Source: Organ: lung
NCI-H1238 Tumor Stage: stage E [H1238] Disease: carcinoma; small
cell lung cancer Derived from metastatic site: bone marrow CRL-5864
Homo sapiens (human) Source: Organ: lung NCI-H1341 Disease:
carcinoma; small cell lung cancer [H1341] Derived from metastatic
site: cervix CRL-5867 Homo sapiens (human) Source: Organ: lung
NCI-H1385 Tumor Stage: stage 3A [H1385] Disease: carcinoma;
non-small cell lung cancer Derived from metastatic site: lymph node
CRL-5869 Homo sapiens (human) Source: Organ: lung NCI-H1417 Tumor
Stage: stage E [H1417] Disease: carcinoma; classic small cell lung
cancer CRL-5870 Homo sapiens (human) Source: Organ: lung NCI-H1435
Disease: adenocarcinoma; non-small cell lung cancer [H1435]
CRL-5871 Homo sapiens (human) Source: Organ: lung NCI-H1436 Tumor
Stage: stage E [H1436] Disease: carcinoma; classic small cell lung
cancer Derived from metastatic site: lymph node CRL-5872 Homo
sapiens (human) Source: Organ: lung NCI-H1437 Tumor Stage: stage 1
[H1437] Disease: adenocarcinoma; non-small cell lung cancer
Derived from metastatic site: pleural effusion CRL-5874 Homo
sapiens (human) Source: Organ: lung NCI-H1522 Tumor Stage: stage E
[H1522] Disease: carcinoma; small cell lung cancer Derived from
metastatic site: pleural effusion CRL-5875 Homo sapiens (human)
Source: Organ: lung NCI-H1563 Disease: adenocarcinoma; non-small
cell lung cancer [H1563] CRL-5876 Homo sapiens (human) Source:
Organ: lung NCI-H1568 Disease: adenocarcinoma; non-small cell lung
cancer [H1568] Derived from metastatic site: lymph node CRL-5877
Homo sapiens (human) Source: Organ: lung NCI-H1573 Tumor Stage:
stage 4 [H1573] Disease: adenocarcinoma Derived from metastatic
site: soft tissue CRL-5878 Homo sapiens (human) Source: Organ: lung
NCI-H1581 Tumor Stage: stage 4 [H1581] Disease: non-small cell lung
cancer Cell Type: large cell; CRL-5879 Homo sapiens (human) Source:
Tumor Stage: stage E NCI-H1618 Disease: carcinoma; small cell lung
cancer [H1618] Derived from metastatic site: bone marrow CRL-5881
Homo sapiens (human) Source: Organ: lung NCI-H1623 Tumor Stage:
stage 3B [H1623] Disease: adenocarcinoma; non-small cell lung
cancer Derived from metastatic site: lymph node CRL-5883 Homo
sapiens (human) Source: Organ: lung NCI-H1650 [H- Tumor Stage:
stage 3B 1650, H1650] Disease: adenocarcinoma; bronchoalveolar
carcinoma Derived from metastatic site: pleural effusion CRL-5884
Homo sapiens (human) Source: Organ: lung NCI-H1651 Disease:
adenocarcinoma; non-small cell lung cancer [H1651] CRL-5885 Homo
sapiens (human) Source: Organ: lung NCI-H1666 [H- Disease:
adenocarcinoma; bronchoalveolar carcinoma 1666, H1666] Derived from
metastatic site: pleural effusion CRL-5886 Homo sapiens (human)
Source: Organ: lung NCI-H1672 Tumor Stage: stage L [H1672] Disease:
carcinoma; classic small cell lung cancer CRL-5887 Homo sapiens
(human) Source: Organ: lung NCI-H1693 Tumor Stage: stage 3B [H1693]
Disease: adenocarcinoma; non-small cell lung cancer Derived from
metastatic site: lymph node CRL-5888 Homo sapiens (human) Source:
Organ: lung NCI-H1694 Tumor Stage: stage E [H1694] Disease:
carcinoma; classic small cell lung cancer Derived from metastatic
site: ascites CRL-5889 Homo sapiens (human) Source: Organ: lung
NCI-H1703 Tumor Stage: stage 1 [H1703] Disease: non-small cell lung
cancer Cell Type: squamous cell; CRL-5891 Homo sapiens (human)
Source: Organ: lung NCI-H1734 [H- Disease: adenocarcinoma;
non-small cell lung cancer 1734, H1734] CRL-5892 Homo sapiens
(human) Source: Organ: lung NCI-H1755 Tumor Stage: stage 4 [H1755]
Disease: adenocarcinoma; non-small cell lung cancer Derived from
metastatic site: liver CRL-5892 Homo sapiens (human) Source: Organ:
lung NCI-H1755 Tumor Stage: stage 4 [H1755] Disease:
adenocarcinoma; non-small cell lung cancer Derived from metastatic
site: liver CRL-5893 Homo sapiens (human) Source: Organ: lung
NCI-H1770 Tumor Stage: stage 4 [H1770] Disease: carcinoma;
non-small cell lung cancer Derived from metastatic site: lymph node
Cell Type: neuroendocrine; CRL-5896 Homo sapiens (human) Source:
Organ: lung NCI-H1793 Disease: adenocarcinoma; non-small cell lung
cancer [H1793] CRL-5898 Homo sapiens (human) Source: Organ: lung
NCI-H1836 Tumor Stage: stage L [H1836] Disease: carcinoma; classic
small cell lung cancer CRL-5899 Homo sapiens (human) Source: Organ:
lung NCI-H1838 Disease: adenocarcinoma; non-small cell lung cancer
[H1838] CRL-5900 Homo sapiens (human) Source: Organ: lung NCI-H1869
Tumor Stage: stage 4 [H1869] Disease: non-small cell lung cancer
Derived from metastatic site: pleural effusion Cell Type: squamous
cell; CRL-5902 Homo sapiens (human) Source: Organ: lung NCI-H1876
Tumor Stage: stage E [H1876] Disease: carcinoma; classic small cell
lung cancer Derived from metastatic site: lymph node CRL-5903 Homo
sapiens (human) Source: Organ: lung NCI-H1882 Tumor Stage: stage E
[H1882] Disease: carcinoma; small cell lung cancer Derived from
metastatic site: bone marrow CRL-5904 Homo sapiens (human) Source:
Organ: lung NCI-H1915 Tumor Stage: stage 4 [H1915] Disease: poorly
differentiated carcinoma; non-small cell lung cancer Derived from
metastatic site: brain Cell Type: large cell; CRL-5906 Homo sapiens
(human) Source: Organ: lung NCI-H1930 Tumor Stage: stage L [H1930]
Disease: carcinoma; classic small cell lung cancer Derived from
metastatic site: lymph node CRL-5907 Homo sapiens (human) Source:
Organ: lung NCI-H1944 Tumor Stage: stage 3B [H1944] Disease:
adenocarcinoma; non-small cell lung cancer Derived from metastatic
site: soft tissue CRL-5908 Homo sapiens (human) Source: Organ: lung
NCI-H1975 [H- Disease: adenocarcinoma; non-small cell lung cancer
1975, H1975] CRL-5909 Homo sapiens (human) Source: Organ: lung
NCI-H1993 Tumor Stage: stage 3A [H1993] Disease: adenocarcinoma;
non-small cell lung cancer Derived from metastatic site: lymph node
CRL-5912 Homo sapiens (human) Source: Organ: lung NCI-H2023 Tumor
Stage: stage 3A [H2023] Disease: adenocarcinoma; non-small cell
lung cancer Derived from metastatic site: lymph node CRL-5913 Homo
sapiens (human) Source: Organ: lung NCI-H2029 Tumor Stage: stage E
[H2029] Disease: carcinoma; small cell lung cancer Derived from
metastatic site: lymph node CRL-5914 Homo sapiens (human) Source:
Organ: lung NCI-H2030 Disease: adenocarcinoma; non-small cell lung
cancer [H2030] Derived from metastatic site: lymph node CRL-5917
Homo sapiens (human) Source: Organ: lung NCI-H2066 Tumor Stage:
stage 1 [H2066] Disease: mixed; small cell lung cancer;
adenocarcinoma; squamous cell carcinoma CRL-5918 Homo sapiens
(human) Source: Organ: lung NCI-H2073 Tumor Stage: stage 3A [H2073]
Disease: adenocarcinoma; non-small cell lung cancer CRL-5920 Homo
sapiens (human) Source: Organ: lung NCI-H2081 Tumor Stage: stage E
[H2081] Disease: carcinoma; classic small cell lung cancer Derived
from metastatic site: pleural effusion CRL-5921 Homo sapiens
(human) Source: Organ: lung NCI-H2085 Disease: adenocarcinoma;
non-small cell lung cancer [H2085] CRL-5922 Homo sapiens (human)
Source: Organ: lung NCI-H2087 Tumor Stage: stage 1 [H2087] Disease:
adenocarcinoma; non-small cell lung cancer Derived from metastatic
site: lymph node CRL-5923 Homo sapiens (human) Source: Organ: lung
NCI-H2106 Tissue: neuroendocrine [H2106] Tumor Stage: stage 4
Disease: non-small cell lung cancer Derived from metastatic site:
lymph node CRL-5924 Homo sapiens (human) Source: Organ: lung
NCI-H2110 Disease: non-small cell lung cancer [H2110] Derived from
metastatic site: pleural effusion CRL-5926 Homo sapiens (human)
Source: Organ: lung NCI-H2135 Disease: non-small cell lung cancer
[H2135] CRL-5927 Homo sapiens (human) Source: Organ: lung NCI-H2141
Tumor Stage: stage E [H2141] Disease: carcinoma; small cell lung
cancer Derived from metastatic site: lymph node CRL-5929 Homo
sapiens (human) Source: Organ: lung NCI-H2171 Tumor Stage: stage E
[H2171] Disease: carcinoma; small cell lung cancer Derived from
metastatic site: pleural effusion CRL-5930 Homo sapiens (human)
Source: Organ: lung NCI-H2172 Disease: non-small cell lung cancer
[H2172] CRL-5931 Homo sapiens (human) Source: Organ: lung NCI-H2195
Tumor Stage: stage E [H2195] Disease: carcinoma; small cell lung
cancer Derived from metastatic site: bone marrow CRL-5932 Homo
sapiens (human) Source: Organ: lung NCI-H2196 Tumor Stage: stage E
[H2196] Disease: carcinoma; small cell lung cancer Derived from
metastatic site: bone marrow CRL-5933 Homo sapiens (human) Source:
Organ: lung NCI-H2198 Tumor Stage: stage E [H2198] Disease:
carcinoma; small cell lung cancer Derived from metastatic site:
lymph node CRL-5934 Homo sapiens (human) Source: Organ: lung
NCI-H2227 Tumor Stage: stage E [H2227] Disease: carcinoma; small
cell lung cancer CRL-5935 Homo sapiens (human) Source: Organ: lung
NCI-H2228 Disease: adenocarcinoma; non-small cell lung cancer
[H2228] CRL-5938 Homo sapiens (human) Source: Organ: lung NCI-H2286
Tumor Stage: stage 1 [H2286] Disease: mixed; small cell lung
cancer; adenocarcinoma; squamous cell carcinoma CRL-5939 Homo
sapiens (human) Source: Organ: lung NCI-H2291 Disease:
adenocarcinoma; non-small cell lung cancer [H2291] Derived from
metastatic site: lymph node CRL-5940 Homo sapiens (human) Source:
Organ: lung NCI-H2330 Tumor Stage: stage L [H2330] Disease:
carcinoma; small cell lung cancer Derived from metastatic site:
lymph node CRL-5941 Homo sapiens (human) Source: Organ: lung
NCI-H2342 Tumor Stage: stage 3A [H2342] Disease: adenocarcinoma;
non-small cell lung cancer CRL-5942 Homo sapiens (human) Source:
Organ: lung NCI-H2347 Tumor Stage: stage 1 [H2347] Disease:
adenocarcinoma; non-small cell lung cancer CRL-5944 Homo sapiens
(human) Source: Organ: lung NCI-H2405 Tumor Stage: stage 4 [H2405]
Disease: adenocarcinoma; non-small cell lung cancer Derived from
metastatic site: ascites CRL-5945 Homo sapiens (human) Source:
Organ: lung NCI-H2444 Disease: non-small cell lung cancer [H2444]
CRL-5975 Homo sapiens (human) Source: Organ: lung UMC-11 Disease:
carcinoid CRL-5976 Homo sapiens (human) Source: Organ: lung NCI-H64
[H64] Tumor Stage: stage E Disease: carcinoma; small cell lung
cancer Derived from metastatic site: lymph node CRL-5978 Homo
sapiens (human) Source: Organ: lung NCI-H735 [H735] Tumor Stage:
stage E Disease: carcinoma; small cell lung cancer Derived from
metastatic site: liver CRL-5978 Homo sapiens (human) Source: Organ:
lung NCI-H735 [H735] Tumor Stage: stage E Disease: carcinoma; small
cell lung cancer Derived from metastatic site: liver CRL-5982 Homo
sapiens (human) Source: Organ: lung NCI-H1963 Tumor Stage: stage L
[H1963] Disease: carcinoma; small cell lung cancer CRL-5983 Homo
sapiens (human) Source: Organ: lung NCI-H2107 Tumor Stage: stage E
[H2107] Disease: carcinoma; small cell lung cancer Derived from
metastatic site: bone marrow CRL-5984 Homo sapiens (human) Source:
Organ: lung NCI-H2108 Tumor Stage: stage E [H2108] Disease:
carcinoma; small cell lung cancer Derived from metastatic site:
bone marrow CRL-5985 Homo sapiens (human) Source: Organ: lung
NCI-H2122 Tumor Stage: stage 4 [H2122] Disease: adenocarcinoma;
non-small cell lung cancer Derived from metastatic site: pleural
effusion CRL-7343 Homo sapiens (human) Source: Organ: lung Hs 573.T
Disease: cancer CRL-7344 Homo sapiens (human) Source: Organ: lung
Hs 573.Lu CRL-8024 Homo sapiens (human) Source: Organ: liver
PLC/PRF/5 Disease: hepatoma Cell Type: Alexander cells; CRL-9609
Homo sapiens (human) Source: Organ: lung BEAS-2B Tissue: bronchus
Disease: normal Cell Type: epithelialvirus transformed HB-8065 Homo
sapiens (human) Source: Organ: liver Hep G2 Disease: hepatocellular
carcinoma HTB-105 Homo sapiens (human) Source: Organ: testes Tera-1
Disease: embryonal carcinoma, malignant Derived from metastatic
site: lung HTB-106 Homo sapiens (human) Source: Disease: malignant
Tera-2 embryonal carcinoma Derived from metastatic site: lung
HTB-119 Homo sapiens (human) Source: Organ: lung NCI-H69 [H69]
Disease: carcinoma; small cell lung cancer HTB-120 Homo sapiens
(human) Source: Organ: lung NCI-H128 [H128] Disease: carcinoma;
small cell lung cancer Derived from metastatic site: pleural
effusion HTB-168 Homo sapiens (human) Source: Organ: lung ChaGo-K-1
Tissue: bronchus Disease: bronchogenic carcinoma HTB-171 Homo
sapiens (human) Source: Organ: lung NCI-H446 [H446] Disease:
carcinoma; small cell lung cancer Derived from metastatic site:
pleural effusion HTB-172 Homo sapiens (human) Source: Organ: lung
NCI-H209 [H209]
Disease: carcinoma; small cell lung cancer Derived from metastatic
site: bone marrow HTB-173 Homo sapiens (human) Source: Organ: lung
NCI-H146 [H146] Disease: carcinoma; small cell lung cancer Derived
from metastatic site: bone marrow HTB-174 Homo sapiens (human)
Source: Organ: lung NCI-H441 [H441] Disease: papillary
adenocarcinoma HTB-175 Homo sapiens (human) Source: Organ: lung
NCI-H82 [H82] Disease: carcinoma; small cell lung cancer Derived
from metastatic site: pleural effusion HTB-177 Homo sapiens (human)
Source: Organ: lung NCI-H460 [H460] Disease: carcinoma; large cell
lung cancer Derived from metastatic site: pleural effusion HTB-178
Homo sapiens (human) Source: Organ: lung NCI-H596 [H596] Disease:
adenosquamous carcinoma HTB-179 Homo sapiens (human) Source: Organ:
lung NCI-H676B Disease: adenocarcinoma [H676B] Derived from
metastatic site: pleural effusion HTB-180 Homo sapiens (human)
Source: Organ: lung NCI-H345 [H345] Disease: carcinoma; small cell
lung cancer Derived from metastatic site: bone marrow HTB-181 Homo
sapiens (human) Source: Organ: lung NCI-H820 [H820] Disease:
papillary adenocarcinoma Derived from metastatic site: lymph node
HTB-182 Homo sapiens (human) Source: Organ: lung NCI-H520 [H520]
Disease: squamous cell carcinoma HTB-183 Homo sapiens (human)
Source: Organ: lung NCI-H661 [H661] Disease: carcinoma; large cell
lung cancer Derived from metastatic site: lymph node HTB-184 Homo
sapiens (human) Source: Organ: lung NCI-H510A Disease: carcinoma;
small cell lung cancer; [H510A, NCI- extrapulmonary origin H510]
Derived from metastatic site: adrenal gland HTB-52 Homo sapiens
(human) Source: Organ: liver SK-HEP-1 Tissue: ascites Disease:
adenocarcinoma HTB-53 Homo sapiens (human) Source: Organ: lung
A-427 Disease: carcinoma HTB-54 Homo sapiens (human) Source: Organ:
lung Calu-1 Tumor Stage: grade III Disease: epidermoid carcinoma
Derived from metastatic site: pleura HTB-55 Homo sapiens (human)
Source: Organ: lung Calu-3 Disease: adenocarcinoma Derived from
metastatic site: pleural effusion HTB-56 Homo sapiens (human)
Source: Organ: unknown, Calu-6 probably lung Disease: anaplastic
carcinoma HTB-57 Homo sapiens (human) Source: Organ: lung SK-LU-1
Disease: adenocarcinoma HTB-58 Homo sapiens (human) Source: Organ:
lung SK-MES-1 Disease: squamous cell carcinoma Derived from
metastatic site: pleural effusion HTB-59 Homo sapiens (human)
Source: Organ: lung SW 900 [SW-900, Tumor Stage: grade IV SW900]
Disease: squamous cell carcinoma HTB-64 Homo sapiens (human)
Source: Disease: malignant Malme-3M melanoma Derived from
metastatic site: lung HTB-79 Homo sapiens (human) Source: Organ:
pancreas Capan-1 Disease: adenocarcinoma Derived from metastatic
site: liver
Example 13
Animal Models for the Study of Signal-Sensor Polynucleotides
[0955] Various animal models are available for the study of the
signal-sensor polynucleotides of the present invention. These
include, among others, models for lung and liver cancers.
[0956] The lung cancer model of Fukazawa et al (Anticancer
Research, 2010; 30: 4193-4200) is employed in studies of
signal-sensor polynucleotides. Briefly, a congenic mouse is created
by crossing a ubiquitously expressing dominant negative Myc
(Omomyc) mouse with a KRAS mutation-positive lung cancer model
mouse. In the presence of Omomyc, lung tumors caused by the
expression of mutated KRAS regresses in the congenic mouse,
indicating that Omomyc caused tumor cell death of KRAS
mutation-positive lung cancer.
[0957] Human lung cancer xenografts are also prepared by the method
of Fukazawa. Briefly, human lung cancer xenografts are established
in 4-week-old female BALB/C nude mice (Charles River Laboratories
Japan, Kanagawa, Japan) by subcutaneous inoculation of 4.times.106
A549 cells into the dorsal flank. The mice are randomly assigned
into six groups (n=6/group). After the tumors reach a diameter of
about 0.5 cm (approximately 6 days after tumor inoculations), each
group of mice are injected with 100 .mu.l solution containing PBS,
5.times.1010 vp of control or signal-sensor polynucleotide into the
dorsalflank tumor for the selected dosing regimen. Animals are then
observed closely and survival studiesor other analyses are
performed.
[0958] The LSL-KRAS.sup.G12D: TRE Omomyc:CMV rtTA triple transgenic
model involves the use of an adenovirus expressing Cre recombinase
which is administered via inhalation to induce oncogene expression
via excision of the floxed STOP codon, and ubiquitous Omomyc
expression is controlled via doxycycline. The model is reported in
Soucek et al. (Nature, 1-5 (2008)). The mice of Soucek may be
crossed with the LSLKRAS.sup.G12D single transgenic mice (Jackson
Laboratories) and may be used for inhalation delivered or otherwise
lung-delivered studies of signal-sensor polynucleotides expressing
MYC inhibitor D or other oncology related polypeptide.
[0959] Mouse-in-mouse models may also be employed. Such models are
akin to the p53-/-:c-Myc overexpressing HCC model of Zender (Cell.
2006 Jun. 30; 125(7): 1253-1267).
[0960] Design of such models involve starting with the WT or tumor
suppressor deleted (such as p53-/-) 129 Sv/Ev Mm ES cell clone;
introduction of liver activated protein (LAP) promoter directed
tetracycline transactivator (tTA) and tetO-luciferase for liver
specific imaging; freezing the resulting LAP-tTA: tetO-luciferase
clones to be used for c-Myc as well as other liver relevant
programs oncogene; adding tetO driven oncogene, e.g. tetOcMyc;
Freeze resulting LAP-tTA: tetO-luciferase: tetO-MYC clones;
injecting resulting ES clones into C.sub.57B1/6 blastocytes and
implant in pseudo pregnant mothers whereby the resulting chimeric
animals are the tumor model upon removal of doxycycline (i.e.
Tet--Off). The model will ideally evince inducible nodules of
c-Myc-driven, luciferase-expressing HCC surrounded by normal
hepatocytes.
[0961] Orthotopic HCC models using the HEP3B cell lines in mice may
also be used (Crown Bio).
[0962] Nongermline genetically engineered mouse model (NGEMM)
platform is a platform that could also be utilized for exploring
signal-sensor polynucleotides.
Example 14
Inhibition of HIF1-Alpha: SHARP1 and CITED4
[0963] Hypoxia-inducible factors (HIFs) control cellular adaptation
to oxygen deprivation. Cancer cells engage HIFs to sustain their
growth in adverse conditions, thus promoting a cellular
reprograming that includes metabolism, proliferation, survival and
mobility. HIFs overexpression in human cancer biopsies correlates
with high metastasis and mortality.
[0964] Hifs regulate genes related to metabolism such as GLUT1,
GLUT3, ALDOA, ENO.sub.1, GAPDH, HK1, HK2, PFKL, PGK1, PKM2, LDHA,
proliferation such as IGF-2, TGFA, VEGFA, survival such as TERT,
NANOG, OCT4 and cell migration-invasion such as ZEB1, ZEB2, SNAI2,
MMP14, MMP9, AMF, MET, PTHrP. (Keith, et al Nat Rev Cancer 2012;
12:9-22).
[0965] To investigate the destabilization of cancer, one or more
signal-sensor polynucleotides may be administered to the cancer
cell. The selection of the sequence, dose or administrative route
is optionally informed by diagnostic evaluation of the cell, tumor,
tissue or organism including, but not limited to, expression
profiling of the cancer, metabolic evaluation (hypoxic, acidotic),
apoptotic vs. survival profiling, cell cycle vs. senescent
profiling, immune sensitivities, and/or evaluation of stromal
factors.
[0966] In one arm of the study signal-sensor polynucleotides
encoding either or both oncology related polypeptides, CITED4 and
SHARP1 are administered where administration of either or both
results in the inhibition of the transcriptome of HIF-1alpha in
cancer cells. Suppression of HIF1-alpha gene regulated expression
occurs upon administration with higher suppression when both
polynucleotides are administered together. Reporter constructs such
as luciferase under HIF1-alpha are used in the manner similar to
the methods disclosed in van de Sluis et al, (J Clin Invest. 2010;
120(6):2119-2130). It is known that both CITED4 and SHARP1
expression results in decreased HIF1-alpha and concomitant
reduction in HIF1-alpha regulated gene expression. Evaluation of
cell death and/or proliferation isalso performed.
[0967] Additional experiments involve the use a cancer cell line
where CITED4 and SHARP1 are themselves down regulated either under
hypoxic conditions. Therefore a positive result demonstrates that
specifically targeting the metabolic profile (in this case
hypoxic-adaptations of CITED4 and SHAPR1) with replacement of
native proteins via signal-sensor polynucleotides can directly
impact the transcriptome and survival advantage of cancer cells
with this profile. Further, the data would show that the relative
impact of signal-sensor polynucleotide vs. vehicle under hypoxic
conditions was more significant for cancer cells than for normal
cells. (i.e., the cancer cells have a disproportionate survival
advantage based on their CITED4+SHARP1 down regulation) that makes
them more sensitive to the replacement of this protein then a
normal cell is to overproduction of it. It is understood that a
cancer cell will likely be experiencing hypoxic conditions and that
a normal cell under normoxic conditions might be able to tolerate
CITED4 and SHARP1 over expression because the normal cell is not
dependent on HIF1alpha transctiptome for survival advantage.
[0968] In vivo experiments are performed according to the design of
the in vitro experiments where the animal model is one evincing
metastasis in the cancer setting because HIF-1 alpha appears to
confer the largest portion of its advantage in metastasis. Animals
are administered the signal-sensor polynucleotide compared to no
treatment or a control polynucleotide. Animal cells, tissues and/or
organs are then evaluated for alterations in gene expression
profiles or transcriptome levels.
Example 15
Alteration of Signal-Sensor Polynucleotide Trafficking: NLS and
NES
[0969] Two nuclear export signals (NES) which may be incorporated
into the Signal-sensor polynucleotides of the present invention
include those reported by Muller, et al (Traffic, 2009, 10:
514-527) and are associated with signaling via the gene COMMD1.
These are NEST, PVAIIELEL (SEQ ID NO 6596) and NES2, VNQILKTLSE
(SEQ ID NO 6597).
[0970] Nuclear localization signals may also be used. One such
sequence is PKKKRKV (SEQ ID NO: 6598).
[0971] Cell lines or mice are administered one or more
signal-sensor polynucleotides having a NLS or NES encoded therein.
Upon administration the polynucleotide is trafficked to an
alternate location, e.g., into the nucleus using the NLS. The
oncology related polypeptide having the NLS would be trafficked to
the nucleus where it would deliver either a survival or death
signal to the nuclear microenvironment. Polypeptides which may be
localized to the nucleus include those with altered binding
properties for DNA which will function to alter the expression
profile of the cell in a therapeutically benefical manner for the
cell, tissue or organism.
[0972] In one experiment, the signal-sensor polynucleotide encodes
a COMMD1 mut1/mut 2+NLS (e.g., both NES signals disrupted plus a
NLS added) following the methods of Muller et al, (Traffic 2009;
10: 514-527) and van de Sluis et al, (J Clin Invest. 2010; 120
(6):2119-2130). The signal sequence may encode an oncology related
polypeptide or a scrambled sequence which is not translatable. The
signal sequence encoded would interact with HIF1-alpha to alter the
transcritome of the cancer cells.
[0973] The experiment is repeated under normal and hypoxic
conditions.
[0974] Once identified the HIF1-alpha dependent signal-sensor
polynucleotide is tested in cancer cell lines clonal survival or a
marker of apoptosis is measured and compared to control or mock
treated cells.
Example 16
Signal-Sensor Polynucleotides in the Treatment of Hepatocellular
Carcinoma (HCC): Disruption of Cancer Cell Transcriptome
[0975] Using the animal models outlined in Example 13, animals are
treated with signal-sensor polynucleotide for MYC inhibitor D vs.
negative control (untranslatable mRNA for MYC inhibitor D) vs.
vehicle. For the KRas model addition of the existing transduced
OmoMyc model may also be utilized. Animals are then evaluated for
gene expression, tumor status or for any of the hallmarks
associated with cancer phenotypes or genotypes.
Example 17
Cytoprotective Signal-Sensor Polynucleotides
[0976] Deliver one or multiple mRNA therapeutics resulting in a
protein (native or non-native, intracellular or extracellular) that
confers a cytoprotective advantage to normal cells in the setting
of cancer therapeutics (both mRNA and non-mRNA)
Example 18
miRNA Binding Sites (BS) Useful as Sensor Sequences in
Signal-Sensor Polynucleotides
[0977] miRNA-binding sites are used in the 3'UTR of mRNA
therapeutics to direct cytotoxic or cytoprotective mRNA
therapeutics to specific cells (normal and/or cancerous).
[0978] A strong apoptotic signal (i.e., AIFsh--Apoptosis Inducing
Factor short isoform, constitutively active (C.A.) caspase 6 (also
known as Rev-caspase-6)--is a a component of HSV1-tk--herpes
simplex virus 1-thymidine kinase) is encoded as the
oncology-related polypeptide or "signal" and is encoded along with
a series of 3'UTR miR binding sites, such as that for mir-122a,
that would make the signal-sensor polynucleotide relatively much
more stable in cancerous cells than in normal cells.
[0979] Experiments comparing cancer vs. normal heaptic cell lines
where the cancer cell lines have a specific miR signature are
performed in vitro. SNU449 or HEP3B (human derived HCC cell lines)
are used because both have been shown to have "undetectable
miR-122a", whereas normal hepatocytes should have very high
miR-122a levels.
A. AIFsh Encoded Polypeptide Study
[0980] First a cancer cell is selected which is sensitive to AIFsh
signal-sensor polynucleotide (i.e., it results in apoptosis).
[0981] Three miR-122a binding sites are encoded into the 3'UTR of
an mRNA sequence for AIFsh and the study arms include 2 cell lines
(normal hepatocyte, SNU449 or HEP3B).times.5 treatments (vehicle
alone, signal-sensor polynucleotide untranslatabe, signal-sensor
polynucleotide AIFsh (no miR BS in 3'UTR), 3'UTR[miR122a
BS.times.3]-signal-sensor polynucleotide untranslatable,
3'UTR[miR122a BS.times.3]--signal-sensor polynucleotide AIFsh).
[0982] The expected result would be significant apoptosis in the
face of signal-sensor polynucleotide AIFsh in both normal and
cancer (HEP3B or SNU449) cell lines in the absence of any
3'UTR-miR122a BS. However, a significant difference in the relative
apoptosis of normal vs. cancer cell lines in the face of 3'UTR
[miR122a BS.times.3]--signal-sensor polynucleotide AIFsh.
[0983] Reversibility of the effect is shown with the
co-administration of miR122a to the cancer cell line (e.g., through
some transduction of the miR122a activity back into the cancer cell
line).
B. C.a. Caspase 6 Encoded Polypeptide Study
[0984] First a cancer cell is selected which is sensitive to C.A.
caspase 6 signal-sensor polynucleotide (i.e., it results in
apoptosis).
[0985] Three miR-122a binding sites are encoded into the 3'UTR of
an mRNA sequence for C.A. caspase 6 and the study arms include 2
cell lines (normal hepatocyte, SNU449 or HEP3B).times.5 treatments
(vehicle alone, signal-sensor polynucleotide untranslatabe,
signal-sensor polynucleotide C.A. caspase 6 (no miR BS in 3'UTR),
3'UTR[miR122a BS.times.3]--signal-sensor polynucleotide
untranslatable, 3'UTR[miR122a BS.times.3]--signal-sensor
polynucleotide C.A. caspase 6).
[0986] The expected result would be significant apoptosis in the
face of signal-sensor polynucleotide C.A. caspase 6 in both normal
and cancer (HEP3B or SNU449) cell lines in the absence of any
3'UTR-miR122a BS. However, a significant difference in the relative
apoptosis of normal vs. cancer cell lines in the face of 3'UTR
[miR122a BS.times.3]-signal-sensor polynucleotide C.A. caspase
6.
C. HSV1-tk Encoded Polypeptide Study
[0987] First a cancer cell is selected which is sensitive to
HSV1-tk signal-sensor polynucleotide (i.e., it results in
apoptosis).
[0988] Three miR-122a binding sites are encoded into the 3'UTR of
an mRNA sequence for HSV1-tk and the study arms include 2 cell
lines (normal hepatocyte, SNU449 or HEP3B).times.5 treatments
(vehicle alone, signal-sensor polynucleotide untranslatabe,
signal-sensor polynucleotide HSV1-tk (no miR BS in 3'UTR),
3'UTR[miR122a BS.times.3]--signal-sensor polynucleotide
untranslatable, 3'UTR[miR122a BS.times.3]--signal-sensor
polynucleotide HSV1-tk).
[0989] The expected result would be significant apoptosis in the
face of signal-sensor polynucleotide HSV1-tk in both normal and
cancer (HEP3B or SNU449) cell lines in the absence of any
3'UTR-miR122a BS. However, a significant difference in the relative
apoptosis of normal vs. cancer cell lines in the face of 3'UTR
[miR122a BS.times.3]--signal-sensor polynucleotide HSV1-tk.
[0990] Reversibility of the effect is shown with the
co-administration of miR122a to the cancer cell line (e.g., through
some transduction of the miR122a activity back into the cancer cell
line).
D. In Vivo Study of Signal-Sensor Polynucleotides
[0991] In vivo animal studies are performed for AIFsh, C.A. caspase
6 and HSV1-tk using any of the models disclosed herein or a
commercially available orthotopic HCC model.
Example 19
Expression of Modified Nucleic Acid with microRNA Binding Site
[0992] Human embryonic kidney epithelial cells (HEK293A) and
primary human hepatocytes (Hepatocytes) were seeded at a density of
200,000 per well in 500 ul cell culture medium (InVitro GRO medium
from Celsis, Chicago, Ill.). G-CSF mRNA having an alpha-globin
3'UTR (G-CSF alpha) (mRNA sequence is shown in SEQ ID NO: 6599;
polyA tail of approximately 160 nucleotides not shown in sequence;
5'Cap, Cap1; fully modified with 5-methylcytidine and
pseudouridine) G-CSF mRNA having an alpha-globin 3'UTR and a
miR-122 binding site (G-CSF miR-122) (mRNA sequence is shown in SEQ
ID NO: 6600; polyA tail of approximately 160 nucleotides not shown
in sequence; 5'Cap, Cap1; fully modified with 5-methylcytidine and
pseudouridine) or G-CSF mRNA having an alpha-globin 3'UTR with four
miR-122 binding sites with the seed deleted (G-CSF no seed) (mRNA
sequence is shown in SEQ ID NO: 6601; polyA tail of approximately
160 nucleotides not shown in sequence; 5'Cap, Cap1; fully modified
with 5-methylcytidine and pseudouridine) was tested at a
concentration of 250 ng per well in 24 well plates. The expression
of G-CSF was measured by ELISA and the results are shown in Table
14.
TABLE-US-00015 TABLE 14 miR-122 Binding Sites HEK293A Hepatocytes
Protein Protein Expression Expression (ng/mL) (ng/mL) G-CSF alpha
99.85 8.18 G-CSF miR-122 87.67 0 G-CSF no seed 200.2 8.05
[0993] Since HEK293 cells do not express miR-122 there was no
down-regulation of G-CSF protein from the sequence containing
miR-122. Whereas, the human hepatocytes express high levels of
miR-122 and there was a drastic down-regulation of G-CSF protein
observed when the G-CSF sequence contained the miR-122 target
sequence. Consequently, the mRNA functioned as an auxotrophic
mRNA.
Example 20
MYC Inhibitor D Study in Cell Lines
[0994] Cell lines of liver and lung cancer, such as those described
herein, are transfected with MYC inhibitor D modified mRNA in
saline or formulated as described herein or in International
Application No PCT/US2012/69610, herein incorporated by reference
in its entirety. To evaluate the selectivity and/or the effects of
therapy with MYC inhibitor D modified mRNA, normal hepatocytes are
also transfected with the MYC inhibitor D modified mRNA.
Example 21
Formulation of Signal-Sensor Polynucleotides
[0995] Signal-sensor polynucleotides are formulated in lipid
nanoparticles as described herein, known in the art, and/or as
described in International Application No PCT/US2012/69610, herein
incorporated by reference in its entirety. For tumor delivery, the
lipid nanoparticle formulations are adapted for efficient delivery
prior to in vitro or in vivo administration. For targeted delivery
and/or to reduce toxicity the signal-senor polynucleotides include
at least one miR binding site.
[0996] The lipid nanoparticle formulations are administered by
methods known in the art or described herein (e.g., intraveneous,
intramuscular and/or intranasal) to liver and lung cancer models
(e.g., those described herein and subcutaneous human xenografts in
mice, orthotopic human xenografts in mice and
transgenic/genetically engineered mouse models).
Example 22
Delivery of Signal-Sensor Polynucleotides to Mammals
[0997] Signal-sensor polynucleotides are formulated for in vivo
delivery in a lung and/or liver cancer model (e.g., those described
herein). The signal-sensor polynucleotides are formulated in lipid
nanoparticles as described herein, known in the art and/or
described in International Application No PCT/US2012/69610, herein
incorporated by reference in its entirety.
[0998] The lung and/or liver cancer models are analyzed for protein
expression, apoptosis, toxicity, efficacy through tumor volume,
liver enzyme levels and effect on tumor tissue to evaluate the
effect of administration of the formulated signal-sensor
polynucleotides on the lung and/or liver cancer models. Assays are
used to evaluate protein expression of the signal-sensor
polynucleotides. Apoptosis, toxicity, efficacy through tumor
volume, liver enzyme levels and tumor tissue are evaluate using
common methods known in the art.
Example 23
Dose Response
[0999] Nanoparticle formulations of 98N12-5 (NPA-005) and
DLin-KC2-DMA (NPA-003) were tested at varying concentrations to
determine the MFI of FL4 or mCherry (mRNA sequence shown in SEQ ID
NO: 6602; polyA tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytidine and
pseudouridine) over a range of doses. The formulations tested are
outlined in Table 15. To determine the optimal concentration of
nanoparticle formulations of 98N12-5, varying concentrations of
formulated modified RNA (100 ng, 10 ng, 1.0 ng, 0.1 ng and 0.01 ng
per well) were tested in a 24-well plate of HEK293.
[1000] Human embryonic kidney epithelial (HEK293) (LGC standards
GmbH, Wesel, Germany) were seeded on 96-well plates (Greiner
Bio-one GmbH, Frickenhausen, Germany) and plates were precoated
with collagen typel. HEK293 were seeded at a density of 30,000
cells per well in 100 .mu.l cell culture medium. The cell culture
medium was DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mM
Sodiumpyruvate and 1.times. non-essential amino acids (Biochrom AG,
Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich,
Munich, Germany). Formulations containing mCherry mRNA were added
in quadruplicates directly after seeding the cells and
incubated.
[1001] Cells were harvested by transferring the culture media
supernatants to a 96-well Pro-Bind U-bottom plate (Beckton
Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with
1/2 volume Trypsin/EDTA (Biochrom AG, Berlin, Germany), pooled with
respective supernatants and fixed by adding one volume PBS/2% FCS
(both Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck,
Darmstadt, Germany). Samples then were submitted to a flow
cytometer measurement with a 532 nm excitation laser and the 610/20
filter for PE-Texas Red in a LSRII cytometer (Beckton Dickinson
GmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI)
of all events were analyzed and the results of the FL4 MFI of each
dose are shown in Table 16. Likewise, to determine the optimal
concentration of nanoparticle formulations of DLin-KC2-DMA, varying
concentrations of formulated modified RNA (250 ng 100 ng, 10 ng,
1.0 ng, 0.1 ng and 0.01 ng per well) were tested in a 24-well plate
of HEK293, and the results of the FL4 MFI of each dose are shown in
Table 17. Nanoparticle formulations of DLin-KC2-DMA were also
tested at varying concentrations of formulated modified RNA (250
ng, 100 ng and 30 ng per well) in a 24 well plate of HEK293, and
the results of the FL4 MFI of each dose are shown in Table 18. A
dose of 1 ng/well for 98N12-5 and a dose of 10 ng/well for
DLin-KC2-DMA were found to resemble the FL4 MFI of the
background.
[1002] To determine how close the concentrations resembled the
background, we utilized a flow cytometer with optimized filter sets
for detection of mCherry expression, and were able to obtain
results with increased sensitivity relative to background levels.
Doses of 25 ng/well, 0.25 ng/well, 0.025 ng/well and 0.0025 ng/well
were analyzed for 98N12-5 (NPA-005) and DLin-KC2-DMA (NPA-003) to
determine the MFI of mCherry. As shown in Table 19, the
concentration of 0.025 ng/well and lesser concentrations are
similar to the background MFI level of mCherry which is about
386.125.
TABLE-US-00016 TABLE 15 Formulations Formulation # NPA-003 NPA-005
Lipid DLin-KC2-DMA 98N12-5 Lipid/RNA 20 15 wt/wt Mean size 114 nm
106 nm PDI: 0.08 PDI: 0.12
TABLE-US-00017 TABLE 16 HEK293, NPA-005, 24-well, n = 4 Formulation
FL4 MFI Untreated control 0.246 NPA-005 100 ng 2.2175 NPA-005 10 ng
0.651 NPA-005 1.0 ng 0.28425 NPA-005 0.1 ng 0.27675 NPA-005 0.01 ng
0.2865
TABLE-US-00018 TABLE 17 HEK293, NPA-003, 24-well, n = 4 Formulation
FL4 MFI Untreated control 0.3225 NPA-003 250 ng 2.9575 NPA-003 100
ng 1.255 NPA-003 10 ng 0.40025 NPA-003 1 ng 0.33025 NPA-003 0.1 ng
0.34625 NPA-003 0.01 ng 0.3475
TABLE-US-00019 TABLE 18 HEK293, NPA-003, 24-well, n = 4 Formulation
FL4 MFI Untreated control 0.27425 NPA-003 250 ng 5.6075 NPA-003 100
ng 3.7825 NPA-003 30 ng 1.5525
TABLE-US-00020 TABLE 19 Concentration and MFI MFI mCherry
Formulation NPA-003 NPA-005 25 ng/well 11963.25 12256.75 0.25
ng/well 1349.75 2572.75 0.025 ng/well 459.50 534.75 0.0025 ng/well
310.75 471.75
Example 24
LNP Formulations
[1003] Formulations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5,
C.sub.12-200 and DLin-MC.sub.3-DMA were incubated at a
concentration of 60 ng/well or 62.5 ng/well in a plate of HEK293
and 62.5 ng/well in a plate of HepG2 cells for 24 hours to
determine the MFI of mCherry (mRNA sequence shown in SEQ ID NO:
6602; polyA tail of approximately 160 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytidine and
pseudouridine) for each formulation.
[1004] Human embryonic kidney epithelial (HEK293) and
hepatocellular carcinoma epithelial (HepG2) cells (LGC standards
GmbH, Wesel, Germany) were seeded on 96-well plates (Greiner
Bio-one GmbH, Frickenhausen, Germany) and plates for HEK293 cells
were precoated with collagen typel. HEK293 were seeded at a density
of 30,000 and HepG2 were seeded at a density of 35,000 cells per
well in 100 .mu.l cell culture medium. For HEK293 the cell culture
medium was DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mM
Sodiumpyruvate and 1.times. non-essential amino acids (Biochrom AG,
Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich,
Munich, Germany) and for HepG2 the culture medium was MEM (Gibco
Life Technologies, Darmstadt, Germany), 10% FCS adding 2 mM
L-Glutamine, 1 mM Sodiumpyruvate and 1.times. non-essential amino
acids (Biochrom AG, Berlin, Germany. Formulations containing
mCherry mRNA (mRNA sequence shown in SEQ ID NO: 6602; polyA tail of
approximately 160 nucleotides not shown in sequence; 5' cap, Cap1);
were added in quadruplicates directly after seeding the cells and
incubated. The mCherry cDNA with the T7 promoter, 5' untranslated
region (UTR) and 3' UTR used in in vitro transcription (IVT) is
given in SEQ ID NO: 6603. The mCherry mRNA was modified with 5meC
at each cytidine and pseudouridine replacement at each uridine
site.
[1005] Cells were harvested by transferring the culture media
supernatants to a 96-well Pro-Bind U-bottom plate (Beckton
Dickinson GmbH, Heidelberg, Germany). Cells were trypsinized with
1/2 volume Trypsin/EDTA (Biochrom AG, Berlin, Germany), pooled with
respective supernatants and fixed by adding one volume PBS/2% FCS
(both Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck,
Darmstadt, Germany). Samples then were submitted to a flow
cytometer measurement with a 532 nm excitation laser and the 610/20
filter for PE-Texas Red in a LSRII cytometer (Beckton Dickinson
GmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI)
of all events was determined.
[1006] The formulations tested are outlined in Table 20 below. As
shown in Table 21 for the 60 ng/well and Tables 22, 23, 24 and 25
for the 62.5 ng/well, the formulation of NPA-003 and NPA-018 have
the highest mCherry MFI and the formulations of NPA-008, NPA-010
and NPA-013 are most the similar to the background sample mCherry
MFI value.
TABLE-US-00021 TABLE 20 Formulations Formulation Lipid/RNA # Lipid
wt/wt Mean size (nm) NPA-001 DLin-KC2-DMA 10 155 nm PDI: 0.08
NPA-002 DLin-KC2-DMA 15 140 nm PDI: 0.11 NPA-002-2 DLin-KC2-DMA 15
105 nm PDI: 0.04 NPA-003 DLin-KC2-DMA 20 114 nm PDI: 0.08 NPA-003-2
DLin-KC2-DMA 20 95 nm PDI: 0.02 NPA-005 98N12-5 15 127 nm PDI: 0.12
NPA-006 98N12-5 20 126 nm PDI: 0.08 NPA-007 DLin-DMA 15 148 nm PDI:
0.09 NPA-008 DLin-K-DMA 15 121 nm PDI: 0.08 NPA-009 C12-200 15 138
nm PDI: 0.15 NPA-010 DLin-MC3-DMA 15 126 nm PDI: 0.09 NPA-012
DLin-DMA 20 86 nm PDI: 0.08 NPA-013 DLin-K-DMA 20 104 nm PDI: 0.03
NPA-014 C12-200 20 101 nm PDI: 0.06 NPA-015 DLin-MC3-DMA 20 109 nm
PDI: 0.07
TABLE-US-00022 TABLE 21 HEK293, 96-well, 60 ng Modified RNA/well
Formulation MFI mCherry Untreated 871.81 NPA-001 6407.25 NPA-002
14995 NPA-003 29499.5 NPA-005 3762 NPA-006 2676 NPA-007 9905.5
NPA-008 1648.75 NPA-009 2348.25 NPA-010 4426.75 NPA-012 11466
NPA-013 2098.25 NPA-014 3194.25 NPA-015 14524
TABLE-US-00023 TABLE 22 HEK293, 62.5 ng/well Formulation MFI
mCherry Untreated 871.81 NPA-001 6407.25 NPA-002 14995 NPA-003
29499.5 NPA-005 3762 NPA-006 2676 NPA-007 9905.5 NPA-008 1648.75
NPA-009 2348.25 NPA-010 4426.75 NPA-012 11466 NPA-013 2098.25
NPA-014 3194.25 NPA-015 14524
TABLE-US-00024 TABLE 23 HEK293, 62.5 ng/well Formulation MFI
mCherry Untreated 295 NPA-007 3504 NPA-012 8286 NPA-017 6128
NPA-003-2 17528 NPA-018 34142 NPA-010 1095 NPA-015 5859 NPA-019
3229
TABLE-US-00025 TABLE 24 HepG2, 62.5 ng/well Formulation MFI mCherry
Untreated 649.94 NPA-001 6006.25 NPA-002 8705 NPA-002-2 15860.25
NPA-003 15059.25 NPA-003-2 28881 NPA-005 1676 NPA-006 1473 NPA-007
15678 NPA-008 2976.25 NPA-009 961.75 NPA-010 3301.75 NPA-012
18333.25 NPA-013 5853 NPA-014 2257 NPA-015 16225.75
TABLE-US-00026 TABLE 25 HepG2, 62.5 ng/well Formulation MFI mCherry
Untreated control 656 NPA-007 16798 NPA-012 21993 NPA-017 20377
NPA-003-2 35651 NPA-018 40154 NPA-010 2496 NPA-015 19741 NPA-019
16373
Example 25
LNP In Vivo Studies
[1007] mCherry mRNA (SEQ ID NO: 6604; polyA tail of approximately
160 nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytidine and pseudouridine) was formulated as a lipid
nanoparticle (LNP) using the syringe pump method. The LNP was
formulated at a 20:1 weight ratio of total lipid to modified mRNA
with a final lipid molar ratio of 50:10:38.5:1.5
(DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). The mCherry
formulation, listed in Table 26, was characterized by particle
size, zeta potential, and encapsulation.
TABLE-US-00027 TABLE 26 mCherry Formulation Formulation # NPA-003-5
Modified mRNA mCherry Mean size 105 nm PDI: 0.09 Zeta at pH 7.4 1.8
mV Encaps. (RiboGr) 100%
[1008] The LNP formulation was administered to mice (n=5)
intravenously at a modified mRNA dose of 100 ug. Mice were
sacrificed at 24 hrs after dosing. The liver and spleen from the
mice administered with mCherry modified mRNA formulations were
analyzed by immunohistochemistry (IHC), western blot, or
fluorescence-activated cell sorting (FACS).
[1009] Histology of the liver showed uniform mCherry expression
throughout the section, while untreated animals did not express
mCherry. Western blots were also used to confirm mCherry expression
in the treated animals, whereas mCherry was not detected in the
untreated animals. Tubulin was used as a control marker and was
detected in both treated and untreated mice, indicating that normal
protein expression in hepatocytes was unaffected.
[1010] FACS and IHC were also performed on the spleens of mCherry
and untreated mice. All leukocyte cell populations were negative
for mCherry expression by FACS analysis. By IHC, there were also no
observable differences in the spleen in the spleen between mCherry
treated and untreated mice.
Example 26
Titration of the Binding Affinity Between Two Cofactors
[1011] Experiments are conducted in order to titrate the binding
affinity between two cofactors. As used herein, the term "titrate"
refers to a method whereby one or more factors are introduced
systematically (such as at increasing levels or wherein the one or
more factors are systematically modified) to a solution, scenario
or series thereof in order to assess a property of interest. In
this embodiment, the property of interest is the binding affinity
between two cofactors. In one embodiment, constructs encoding the
two cofactors are obtained and/or synthesized and a series of
mutant constructs are prepared and/or synthesized. Mutant
constructs encode cofactor mutants that may include truncated
mutants (mutant proteins lacking one or more amino acids from
either the N- or C-terminal domains), mutants with regional
deletions [proteins wherein internal regions (comprising one or
more amino acids) of the protein are absent], mutants with single
amino acid substitutions (wherein a normally expressed amino acid
is replaced with an alternative amino acid), mutants with one or
more additional amino acids added internally or at either terminus,
mutants with regional substitutions [proteins wherein internal
regions (comprising one or more amino acids) of the protein are
substituted with alternative regions (comprising one or more amino
acids) and/or combinations of any of these. Mutant constructs are
mutated randomly or subjected to targeted mutation based on
existing knowledge of the molecular interactions necessary for
binding between the two cofactors being investigated.
[1012] In some embodiments, a series of mutant proteins are
designed such that the mutations follow a progressive pattern along
the polypeptide chain. Such series may allow for a better
understanding of a particular aspect or feature of the interaction
between cofactors. A mutant series may include, for example, the
production of a series of mutants, each with a single amino acid
substitution, wherein each mutant has a different amino acid along
it's polypeptide sequence mutated (e.g. alanine is substituted,
thereby eliminating the influence of an amino acid side chain at
each position). In another example, a series of mutants are
designed such that the mutants in the series comprise truncations
of increasing size. In another example, amino acids capable of
being post-translationally modified (e.g. phosphorylated,
acetylated, ubiquitinated, glycosylated, etc.) in a similar manner
may be mutated along the polypeptide sequence in a series of
mutants.
[1013] For titration experiments with mutant cofactors, a baseline
affinity between the two cofactors is established by combining both
cofactors under conditions favorable for binding and the binding
affinity between the cofactors is assayed. Binding affinity may be
assessed using any of a variety of methods known in the art. Such
methods may include, but are not limited to Western blot analysis,
immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),
fluorescence resonance energy transfer (FRET), fluorescence
recovery after photobleaching (FRAP), fluorescence polarization
technologies and/or surface plasmon resonance (SPR) based
technologies. For titration, according to one method, a mutant
series of one or both cofactors are combined with the two unmutated
cofactors (to allow for binding competition between the wild type
and mutated proteins). Changes in affinity between the two
cofactors in the presence of increasing concentrations of different
mutants are assessed and compared and/or plotted against the
specific mutations present in the series of mutants that are
competing for binding. Alternatively, mutant cofactors in a series
are individually combined with a corresponding unmutated binding
partner and assessed for binding affinity. Increasing
concentrations of the wild type cofactor (corresponding to the
mutant cofactor) are introduced and changes in binding between the
mutant cofactors and the corresponding unmutated binding partner
are assessed. Comparisons are made between the resulting binding
curves and the binding curves of other mutants tested.
[1014] In some embodiments, titration of the binding affinity
between two cofactors is assessed in the presence or absence of
increasing concentrations of a third factor. Such a third factor
may be an inhibitor or activator of binding between the two
cofactors. A series of mutants, as described above, may be
generated for a third factor and such a series may be used in
titration experiments to assess the effect of mutations on binding
between the two cofactors.
[1015] Information obtained from titration experiments may be used
to design modified mRNA molecules to encode factors that modulate
the interaction between cofactors.
[1016] In some embodiments, titration experiments are carried out
wherein the binding affinity between HIF1 subunits (HIF1-alpha,
HIF2-alpha and ARNT) and/or mutated HIF1 subunits and/or other
proteins that interact with HIF1 is assessed. Titration experiments
may utilize mutant series generated using constructs for one or
more of HIF1-alpha, HIF2-alpha, ARNT and/or a third interacting
factor. In some embodiments, a mutant series is generated for
HIF1-alpha. HIF1-alpha and HIF2-alpha are hyrdroxylated by HIF
hydroxylase enzymes under normal levels of oxygen in the cell,
facilitating degredation and/or blocking transcriptional activity.
Hyrdorxylation decreases as oxygen levels drop, allowing HIF1-alpha
and/or HIF2-alpha to associate with their cofactor, ARNT leading to
elevated expression of genes comprising HIF-response elements
(HREs) (Keith, B. et al., HIF1.alpha. and HIF2.alpha.: sibling
rivalry in hypoxic tumour growth and progression. Nat Rev Cancer.
2011 Dec. 15; 12(1):9-22). In one embodiment, HIF1-alpha mutant
series are generated wherein mutations in the series progressively
eliminate one or more hydroxylation sites along the polypeptide
chain (including, but not limited to proline 402, proline 564
and/or asparagine 803), thereby modulating stability and/or
transcriptional activity in mutant versions of HIF1-alpha. In
another embodiment, an alternative cofactor, HIF2-alpha is used to
generate a mutant series. Such a mutant series may progressively
eliminate one or more hydroxylation sites along the polypeptide
chain (including, but not limited to proline 405, proline 531
and/or asparagine 847), thereby modulating stability and/or
transcriptional activity in mutant versions of HIF2-alpha. In
another embodiment, HIF1-alpha and/or HIF2-alpha mutant series are
generated that progressively mutate regions necessary for
interaction with ARNT, thereby creating mutants with altered
abilities to bind ARNT and modulate HIF-dependent gene expression.
In another embodiment, ARNT mutant series are generated that
progressively mutate regions necessary for interactions with other
HIF subunits, thereby creating mutants with altered abilities to
bind HIF subunits and modulate HIF-dependent gene expression.
[1017] In some embodiments, mutant series are generated for Von
Hippel-Landau tumor suppressor protein (pVHL). This protein binds
hydroxylated HIF1-alpha and HIF2-alpha, facilitating their
ubiquitination and degradation. In one embodiment, mutant series
are generated that progressively mutate regions necessary for
interaction with HIF1 subunits, thereby creating mutants with
altered abilities to bind HIF1 subunits and modulate HIF-dependent
gene expression.
[1018] Shown in Table 27 and 28 are the transcript sequences and
polypeptide sequences (respectively) for protein targets for use in
titration experiments. The name and description of the gene
encoding the polypeptide of interest are accompanied by the ENSEMBL
Transcript ID (ENST) and transcript sequence (Table 27) or the
ENSEMBL Protein ID (ENSP) and peptide sequence (Table 28). In some
embodiments of the present invention, modified mRNAs may be
designed to encode factors that modulate the affinity between HIF
subunits and/or HIF-dependent gene expression. Such modified mRNAs
may be designed using knowledge gained from titration
experiments.
TABLE-US-00028 TABLE 27 Transcript sequences for additional targets
for titration experiments SEQ Target ENST ID Target Description ID
Transcript Sequence NO HIF2-alpha hypoxia 263734
GCTTTACACTCGCGAGCGGACCGCCACACGG 6605 inducible
GTCCGGTGCCCGCTGCGCTTCCGCCCCAGCGC factor 2,
TCCTGAGGCGGCCGTACAATCCTCGGCAGTGT alpha
CCTGAGACTGTATGGTCAGCTCAGCCCGGCCT subunit;
CCGACTCCTTCCGACTCCCAGCATTCGAGCCA endothelial
CTTTTTTTTTTCTTTGAAAACTCAGAAAAGTG PAS
ACTCCTTTTCCAGGGAAAAAGGAACTTGGGTT domain
CCCTTCTCTCCGTCCTCTTTTCGGGTCTGACAG protein 1
CCTCCACCCACTCCTTCCCCGGACCCCGCCTC CGCGCGCAGGTTCCTCCCAGTCACCTTTCTCC
ACCCCCGCCCCCGCACCTAGCCCGCCGCGCG CCACCTTCCACCTGACTGCGCGGGGCGCTCGG
GACCTGCGCGCACCTCGGACCTTCACCACCCG CCCGGGCCGCGGGGAGCGGACGAGGGCCACA
GCCCCCCACCCGCCAGGGAGCCCAGGTGCTC GGCGTCTGAACGTCTCAAAGGGCCACAGCGA
CAATGACAGCTGACAAGGAGAAGAAAAGGA GTAGCTCGGAGAGGAGGAAGGAGAAGTCCCG
GGATGCTGCGCGGTGCCGGCGGAGCAAGGAG ACGGAGGTGTTCTATGAGCTGGCCCATGAGC
TGCCTCTGCCCCACAGTGTGAGCTCCCATCTG GACAAGGCCTCCATCATGCGACTGGCAATCA
GCTTCCTGCGAACACACAAGCTCCTCTCCTCA GTTTGCTCTGAAAACGAGTCCGAAGCCGAAG
CTGACCAGCAGATGGACAACTTGTACCTGAA AGCCTTGGAGGGTTTCATTGCCGTGGTGACCC
AAGATGGCGACATGATCTTTCTGTCAGAAAA CATCAGCAAGTTCATGGGACTTACACAGGTG
GAGCTAACAGGACATAGTATCTTTGACTTCAC TCATCCCTGCGACCATGAGGAGATTCGTGAG
AACCTGAGTCTCAAAAATGGCTCTGGTTTTGG GAAAAAAAGCAAAGACATGTCCACAGAGCG
GGACTTCTTCATGAGGATGAAGTGCACGGTC ACCAACAGAGGCCGTACTGTCAACCTCAAGT
CAGCCACCTGGAAGGTCTTGCACTGCACGGG CCAGGTGAAAGTCTACAACAACTGCCCTCCTC
ACAATAGTCTGTGTGGCTACAAGGAGCCCCT GCTGTCCTGCCTCATCATCATGTGTGAACCAA
TCCAGCACCCATCCCACATGGACATCCCCCTG GATAGCAAGACCTTCCTGAGCCGCCACAGCA
TGGACATGAAGTTCACCTACTGTGATGACAG AATCACAGAACTGATTGGTTACCACCCTGAG
GAGCTGCTTGGCCGCTCAGCCTATGAATTCTA CCATGCGCTAGACTCCGAGAACATGACCAAG
AGTCACCAGAACTTGTGCACCAAGGGTCAGG TAGTAAGTGGCCAGTACCGGATGCTCGCAAA
GCATGGGGGCTACGTGTGGCTGGAGACCCAG GGGACGGTCATCTACAACCCTCGCAACCTGC
AGCCCCAGTGCATCATGTGTGTCAACTACGTC CTGAGTGAGATTGAGAAGAATGACGTGGTGT
TCTCCATGGACCAGACTGAATCCCTGTTCAAG CCCCACCTGATGGCCATGAACAGCATCTTTGA
TAGCAGTGGCAAGGGGGCTGTGTCTGAGAAG AGTAACTTCCTATTCACCAAGCTAAAGGAGG
AGCCCGAGGAGCTGGCCCAGCTGGCTCCCAC CCCAGGAGACGCCATCATCTCTCTGGATTTCG
GGAATCAGAACTTCGAGGAGTCCTCAGCCTA TGGCAAGGCCATCCTGCCCCCGAGCCAGCCA
TGGGCCACGGAGTTGAGGAGCCACAGCACCC AGAGCGAGGCTGGGAGCCTGCCTGCCTTCAC
CGTGCCCCAGGCAGCTGCCCCGGGCAGCACC ACCCCCAGTGCCACCAGCAGCAGCAGCAGCT
GCTCCACGCCCAATAGCCCTGAAGACTATTAC ACATCTTTGGATAACGACCTGAAGATTGAAG
TGATTGAGAAGCTCTTCGCCATGGACACAGA GGCCAAGGACCAATGCAGTACCCAGACGGAT
TTCAATGAGCTGGACTTGGAGACACTGGCAC CCTATATCCCCATGGACGGGGAAGACTTCCA
GCTAAGCCCCATCTGCCCCGAGGAGCGGCTC TTGGCGGAGAACCCACAGTCCACCCCCCAGC
ACTGCTTCAGTGCCATGACAAACATCTTCCAG CCACTGGCCCCTGTAGCCCCGCACAGTCCCTT
CCTCCTGGACAAGTTTCAGCAGCAGCTGGAG AGCAAGAAGACAGAGCCCGAGCACCGGCCCA
TGTCCTCCATCTTCTTTGATGCCGGAAGCAAA GCATCCCTGCCACCGTGCTGTGGCCAGGCCA
GCACCCCTCTCTCTTCCATGGGGGGCAGATCC AATACCCAGTGGCCCCCAGATCCACCATTAC
ATTTTGGGCCCACAAAGTGGGCCGTCGGGGA TCAGCGCACAGAGTTCTTGGGAGCAGCGCCG
TTGGGGCCCCCTGTCTCTCCACCCCATGTCTC CACCTTCAAGACAAGGTCTGCAAAGGGTTTT
GGGGCTCGAGGCCCAGACGTGCTGAGTCCGG CCATGGTAGCCCTCTCCAACAAGCTGAAGCT
GAAGCGACAGCTGGAGTATGAAGAGCAAGCC TTCCAGGACCTGAGCGGGGGGGACCCACCTG
GTGGCAGCACCTCACATTTGATGTGGAAACG GATGAAGAACCTCAGGGGTGGGAGCTGCCCT
TTGATGCCGGACAAGCCACTGAGCGCAAATG TACCCAATGATAAGTTCACCCAAAACCCCAT
GAGGGGCCTGGGCCATCCCCTGAGACATCTG CCGCTGCCACAGCCTCCATCTGCCATCAGTCC
CGGGGAGAACAGCAAGAGCAGGTTCCCCCCA CAGTGCTACGCCACCCAGTACCAGGACTACA
GCCTGTCGTCAGCCCACAAGGTGTCAGGCAT GGCAAGCCGGCTGCTCGGGCCCTCATTTGAGT
CCTACCTGCTGCCCGAACTGACCAGATATGAC TGTGAGGTGAACGTGCCCGTGCTGGGAAGCT
CCACGCTCCTGCAAGGAGGGGACCTCCTCAG AGCCCTGGACCAGGCCACCTGAGCCAGGCCT
TCTACCTGGGCAGCACCTCTGCCGACGCCGTC CCACCAGCTTCACTCTCTCCGTCTGTTTTTGCA
ACTAGGTATTTCTAACGCCAGCACACTATTTA CAAGATGGACTTACCTGGCAGACTTGCCCAG
GTCACCAAGCAGTGGCCTTTTTCTGAGATGCT CACTTTATTATCCCTATTTTTAAAGTACACAA
TTGTTTTACCTGTTCTGAAATGTTCTTAAATTT TGTAGGATTTTTTTCCTCCCCACCTTCAATGA
CTTCTAATTTATATTATCCATAGGTTTCTCTCC CTCCTTCTCCTTCTCACACACAACTGTCCATA
CTAACAAGTTTGGTGCATGTCTGTTCTTCTGT AGGGAGAAGCTTTAGCTTCATTTTACTAAAAA
GATTCCTCGTTATTGTTGTTGCCAAAGAGAAA CAAAAATGATTTTGCTTTCCAAGCTTGGTTTG
TGGCGTCTCCCTCGCAGAGCCCTTCTCGTTTC TTTTTTAAACTAATCACCATATTGTAAATTTC
AGGGTTTTTTTTTTTTTGTTTAAGCTGACTCTT TGCTCTAATTTTGGAAAAAAAGAAATGTGAA
GGGTCAACTCCAACGTATGTGGTTATCTGTGA AAGTTGCACAGCGTGGCTTTTCCTAAACTGGT
GTTTTTCCCCCGCATTTGGTGGATTTTTTATTA TTATTCAAAAACATAACTGAGTTTTTTAAAAG
AGGAGAAAATTTATATCTGGGTTAAGTGTTTA TCATATATATGGGTACTTTGTAATATCTAAAA
ACTTAGAAACGGAAATGGAATCCTGCTCACA AAATCACTTTAAGATCTTTTCGAAGCTGTTAA
TTTTTCTTAGTGTTGTGGACACTGCAGACTTG TCCAGTGCTCCCACGGCCTGTACGGACACTGT
GGAAGGCCTCCCTCTGTCGGCTTTTTGCCATC TGTGATATGCCATAGGTGTGACAATCCGAGC
AGTGGAGTCATTCAGCGGGAGCACTGCGCGC TATCCCCTCACATTCTCTATGTACTATGTATGT
ATGTATTATTATTATTGCTGCCAAGAGGGTCT GATGGCACGTTGTGGGGTCGGGGGGTGGGGC
GGGGAAGTGCTCTAACTTTTCTTAAGGTTTTG TTGCTAGCCCTTCAAGTGCACTGAGCTATGTG
ACTCGGATGGTCTTTCACACGGCACATTTGGA CATTTCCAGAACTACCATGAGATGGTTTAGAC
GGGAATTCATGCAAATGAGGGGTCAAAAATG GTATAGTGACCCCGTCCACGTCCTCCAAGCTC
ACGACCTTGGAGCCCCGTGGAGCTGGACTGA GGAGGAGGCTGCACAGCGGGAGAGCAGCTG
GTCCAGACCAGCCCTGCAGCCCCCACTCAGC CGGCAGCCAGATGGCCCCGCAAGGCCTCCAG
GGATGGCCCCTAGCCACAGGCCCTGGCTGAG GTCTCTGGGTCGGTCAGTGACATGTAGGTAG
GAAGCACTGAAAATAGTGTTCCCAGAGCACT TTGCAACTCCCTGGGTAAGAGGGACGACACC
TCTGGTTTTTCAATACCAATTACATGGAACTT TTCTGTAATGGGTACAATGAAGAAGTTTCTAA
AAACACACACAAAGCACATTGGGCCAACTAT TTAGTAAGCCCGGATAGACTTATTGCCAAAA
ACAAAAAATAGCTTTCAAAAGAAATTTAAGT TCTATGAGAAATTCCTTAGTCATGGTGTTGCG
TAAATCATATTTTAGCTGCACGGCATTACCCC ACACAGGGTGGCAGAACTTGAAGGGTTACTG
ACGTGTAAATGCTGGTATTTGATTTCCTGTGT GTGTTGCCCTGGCATTAAGGGCATTTTACCCT
TGCAGTTTTACTAAAACACTGAAAAATATTCC AAGCTTCATATTAACCCTACCTGTCAACGTAA
CGATTTCATGAACGTTATTATATTGTCGAATT CCTACTGACAACATTATAACTGTATGGGAGCT
TAACTTTATAAGGAAATGTATTTTGACACTGG TATCTTATTAAAGTATTCTGATCCTA pVHL
von Hippel- 256474 TGAGTGTTTATGTTTGTAGTTTTAATTGCTCTG 6606 Lindau
AAGTAAATATCTGATTTTCCAATTTCCACCAG tumor
AGTGCTCTGCACATAGTAGGTCTAATTATTTT suppressor
TCCCTCTTTACTAATCACCCATGCCTTGTAAG AATTCAGTTAGTTGACTTTTTGTACTTTATAA
GCGTGATGATTGGGTGTTCCCGTGTGAGATGC GCCACCCTCGAACCTTGTTACGACGTCGGCAC
ATTGCGCGTCTGACATGAAGAAAAAAAAAAT TCAGTTAGTCCACCAGGCACAGTGGCTAAGG
CCTGTAATCCCTGCACTTTGAGAGGCCAAGGC AGGAGGATCACTTGAACCCAGGAGTTCGAGA
CCAGCCTAGGCAACATAGCGAGACTCCGTTT CAAACAACAAATAAAAATAATTAGTCGGGCA
TGGTGGTGCGCGCCTACAGTACCAACTACTCG GGAGGCTGAGGCGAGACGATCGCTTGAGCCA
GGGAGGTCAAGGCTGCAGTGAGCCAAGCTCG CGCCACTGCACTCCAGCCCGGGCGACAGAGT
GAGACCCTGTCTCAAAAAAAAAAAAAACACC AAACCTTAGAGGGGCGAAAAAAAATTTTATA
GTGGAAATACAGTAACGAGTTGGCCTAGCCT CGCCTCCGTTACAACGGCCTACGGTGCTGGA
GGATCCTTCTGCGCACGCGCACAGCCTCCGGC CGGCTATTTCCGCGAGCGCGTTCCATCCTCTA
CCGAGCGCGCGCGAAGACTACGGAGGTCGAC TCGGGAGCGCGCACGCAGCTCCGCCCCGCGT
CCGACCCGCGGATCCCGCGGCGTCCGGCCCG GGTGGTCTGGATCGCGGAGGGAATGCCCCGG
AGGGCGGAGAACTGGGACGAGGCCGAGGTA GGCGCGGAGGAGGCAGGCGTCGAAGAGTAC
GGCCCTGAAGAAGACGGCGGGGAGGAGTCG GGCGCCGAGGAGTCCGGCCCGGAAGAGTCCG
GCCCGGAGGAACTGGGCGCCGAGGAGGAGAT GGAGGCCGGGCGGCCGCGGCCCGTGCTGCGC
TCGGTGAACTCGCGCGAGCCCTCCCAGGTCAT CTTCTGCAATCGCAGTCCGCGCGTCGTGCTGC
CCGTATGGCTCAACTTCGACGGCGAGCCGCA GCCCTACCCAACGCTGCCGCCTGGCACGGGC
CGCCGCATCCACAGCTACCGAGGTCACCTTTG GCTCTTCAGAGATGCAGGGACACACGATGGG
CTTCTGGTTAACCAAACTGAATTATTTGTGCC ATCTCTCAATGTTGACGGACAGCCTATTTTTG
CCAATATCACACTGCCAGTGTATACTCTGAAA GAGCGATGCCTCCAGGTTGTCCGGAGCCTAG
TCAAGCCTGAGAATTACAGGAGACTGGACAT CGTCAGGTCGCTCTACGAAGATCTGGAAGAC
CACCCAAATGTGCAGAAAGACCTGGAGCGGC TGACACAGGAGCGCATTGCACATCAACGGAT
GGGAGATTGAAGATTTCTGTTGAAACTTACAC TGTTTCATCTCAGCTTTTGATGGTACTGATGA
GTCTTGATCTAGATACAGGACTGGTTCCTTCC TTAGTTTCAAAGTGTCTCATTCTCAGAGTAAA
ATAGGCACCATTGCTTAAAAGAAAGTTAACT GACTTCACTAGGCATTGTGATGTTTAGGGGCA
AACATCACAAAATGTAATTTAATGCCTGCCCA TTAGAGAAGTATTTATCAGGAGAAGGTGGTG
GCATTTTTGCTTCCTAGTAAGTCAGGACAGCT TGTATGTAAGGAGGTTTGTATAAGTAATTCAG
TGGGAATTGCAGCATATCGTTTAATTTTAAGA AGGCATTGGCATCTGCTTTTAATGGATGTATA
ATACATCCATTCTACATCCGTAGCGGTTGGTG ACTTGTCTGCCTCCTGCTTTGGGAAGACTGAG
GCATCCGTGAGGCAGGGACAAGTCTTTCTCCT CTTTGAGACCCCAGTGCCTGCACATCATGAGC
CTTCAGTCAGGGTTTGTCAGAGGAACAAACC AGGGGACACTTTGTTAGAAAGTGCTTAGAGG
TTCTGCCTCTATTTTTGTTGGGGGGTGGGAGA GGGGACCTTAAAATGTGTACAGTGAACAAAT
GTCTTAAAGGGAATCATTTTTGTAGGAAGCAT TTTTTATAATTTTCTAAGTCGTGCACTTTCTCG
GTCCACTCTTGTTGAAGTGCTGTTTTATTACT GTTTCTAAACTAGGATTGACATTCTACAGTTG
TGATAATAGCATTTTTGTAACTTGCCATCCGC ACAGAAAATACGAGAAAATCTGCATGTTTGA
TTATAGTATTAATGGACAAATAAGTTTTTGCT AAATGTGAGTATTTCTGTTCCTTTTTGTAAAT
ATGTGACATTCCTGATTGATTTGGGTTTTTTTG
TTGTTGTTGTTTTGTTTTGTTTTGTTTTTTTGAG
ATGGAGTCTCACTCTTGTCACCCAGGCTGGAG TGCAGTGGCGCCATCTCGGCTCACTGCAACCT
CTGCCTCCTGGGTTCACGTAATCCTCCTGAGT AGCTGGGATTACAGGCGCCTGCCACCACGCT
GGCCAATTTTTGTACTTTTAGTAGAGACAGTG TTTCGTCATGTTGGCCAGGCTGGTTTCAAACT
CCTGACCTCAGGTGATCCGCCCACCTCAGCCT CCCAAAATGGTGGGATTACAGGTGTGTGGGC
CACCGTGCCTGGCTGATTCAGCATTTTTTATC AGGCAGGACCAGGTGGCACTTCCACCTCCAG
CCTCTGGTCCTACCAATGGATTCATGGAGTAG CCTGGACTGTTTCATAGTTTTCTAAATGTACA
AATTCTTATAGGCTAGACTTAGATTCATTAAC TCAAATTCAATGCTTCTATCAGACTCAGTTTT
TTGTAACTAATAGATTTTTTTTTCCACTTTTGT TCTACTCCTTCCCTAATAGCTTTTTAAAAAAA
TCTCCCCAGTAGAGAAACATTTGGAAAAGAC AGAAAACTAAAAAGGAAGAAAAAAGATCCC
TATTAGATACACTTCTTAAATACAATCACATT AACATTTTGAGCTATTTCCTTCCAGCCTTTTTA
GGGCAGATTTTGGTTGGTTTTTACATAGTTGA GATTGTACTGTTCATACAGTTTTATACCCTTTT
TCATTTAACTTTATAACTTAAATATTGCTCTAT GTTAGTATAAGCTTTTCACAAACATTAGTATA
GTCTCCCTTTTATAATTAATGTTTGTGGGTATT TCTTGGCATGCATCTTTAATTCCTTATCCTAGC
CTTTGGGCACAATTCCTGTGCTCAAAAATGAG AGTGACGGCTGGCATGGTGGCTCCCGCCTGT
AATCCCAGTACTTTGGAAAGCCAAGGTAAGA GGATTGCTTGAGCCCAGAACTTCAAGATGAG
CCTGGGCTCATAGTGAGAACCCATCTATACA AAAAATTTTTAAAAATTAGCATGGCGGCACA
CATCTGTAATCCTAGCTACTTGGCAGGCTGAG GTGAGAAGATCATTGGAGTTTAGGAATTGGA
GGCTGCAGTGAGCCATGAGTATGCCACTGCA CTCCAGCCTGGGGGACAGAGCAAGACCCTGC
CTCAAAAAAAAAAAAAAAAAAAAAATCAGG CCGGGCATGGTGGCTCACGCCTGTAATCCCA
GCACTTTGGGAGGTCGAGGTGGGCAGATCAC CTGAGGTCAGGAGTTCGAGACCAGCCTGGCC
AACATGGTAAAACCCCATTTCTACTAAAAAA TACAAGAAT pVHL von Hippel- 345392
CCCGCGTCCGACCCGCGGATCCCGCGGCGTC 6607 Lindau
CGGCCCGGGTGGTCTGGATCGCGGAGGGAAT tumor
GCCCCGGAGGGCGGAGAACTGGGACGAGGCC suppressor
GAGGTAGGCGCGGAGGAGGCAGGCGTCGAA GAGTACGGCCCTGAAGAAGACGGCGGGGAG
GAGTCGGGCGCCGAGGAGTCCGGCCCGGAAG AGTCCGGCCCGGAGGAACTGGGCGCCGAGGA
GGAGATGGAGGCCGGGCGGCCGCGGCCCGTG CTGCGCTCGGTGAACTCGCGCGAGCCCTCCCA
GGTCATCTTCTGCAATCGCAGTCCGCGCGTCG TGCTGCCCGTATGGCTCAACTTCGACGGCGAG
CCGCAGCCCTACCCAACGCTGCCGCCTGGCA CGGGCCGCCGCATCCACAGCTACCGAGTGTA
TACTCTGAAAGAGCGATGCCTCCAGGTTGTCC GGAGCCTAGTCAAGCCTGAGAATTACAGGAG
ACTGGACATCGTCAGGTCGCTCTACGAAGAT CTGGAAGACCACCCAAATGTGCAGAAAGACC
TGGAGCGGCTGACACAGGAGCGCATTGCACA TCAACGGATGGGAGATTGAAGATTTCTGTTG
AAACTTACACTGTTTCATCTCAGCTTTTGATG GTACTGATGAGTCTTGATCTAGATACAGGACT
GGTTCCTTCCTTAGTTTCAAAGTGTCTCATTCT CAGAGTAAAATAGGCACCATTGCTTAAAAGA
AAGTTAACTGACTTCACTAGGCATTGTGATGT TTAGGGGCAAACATCACAAAATGTAATTTAA
TGCCTGCCCATTAGAGAAGTATTTATCAGGAG AAGGTGGTGGCATTTTTGCTTCCTAGTAAGTC
AGGACAGCTTGTATGTAAGGAGGTTTGTATA AGTAATTCAGTGGGAATTGCAGCATATCGTTT
AATTTTAAGAAGGCATTGGCATCTGCTTTTAA TGGATGTATAATACATCCATTCTACATCCGTA
GCGGTTGGTGACTTGTCTGCCTCCTGCTTTGG GAAGACTGAGGCATCCGTGAGGCAGGGACAA
GTCTTTCTCCTCTTTGAGACCCCAGTGCCTGC ACATCATGAGCCTTCAGTCAGGGTTTGTCAGA
GGAACAAACCAGGGGACACTTTGTTAGAAAG TGCTTAGAGGTTCTGCCTCTATTTTTGTTGGG
GGGTGGGAGAGGGGACCTTAAAATGTGTACA GTGAACAAATGTCTTAAAGGGAATCATTTTTG
TAGGAAGCATTTTTTATAATTTTCTAAGTCGT GCACTTTCTCGGTCCACTCTTGTTGAAGTGCT
GTTTTATTACTGTTTCTAAACTAGGATTGACA TTCTACAGTTGTGATAATAGCATTTTTGTAAC
TTGCCATCCGCACAGAAAATACGAGAAAATC TGCATGTTTGATTATAGTATTAATGGACAAAT
AAGTTTTTGCTAAATGTGAGTATTTCTGTTCC TTTTTGTAAATATGTGACATTCCTGATTGATTT
GGGTTTTTTTGTTGTTGTTGTTTTGTTTTGTTTT GTTTTTTTGAGATGGAGTCTCACTCTTGTCAC
CCAGGCTGGAGTGCAGTGGCGCCATCTCGGC TCACTGCAACCTCTGCCTCCTGGGTTCACGTA
ATCCTCCTGAGTAGCTGGGATTACAGGCGCCT GCCACCACGCTGGCCAATTTTTGTACTTTTAG
TAGAGACAGTGTTTCGTCATGTTGGCCAGGCT GGTTTCAAACTCCTGACCTCAGGTGATCCGCC
CACCTCAGCCTCCCAAAATGGTGGGATTACA GGTGTGTGGGCCACCGTGCCTGGCTGATTCAG
CATTTTTTATCAGGCAGGACCAGGTGGCACTT CCACCTCCAGCCTCTGGTCCTACCAATGGATT
CATGGAGTAGCCTGGACTGTTTCATAGTTTTC TAAATGTACAAATTCTTATAGGCTAGACTTAG
ATTCATTAACTCAAATTCAATGCTTCTATCAG ACTCAGTTTTTTGTAACTAATAGATTTTTTTTT
CCACTTTTGTTCTACTCCTTCCCTAATAGCTTT TTAAAAAAATCTCCCCAGTAGAGAAACATTT
GGAAAAGACAGAAAACTAAAAAGGAAGAAA AAAGATCCCTATTAGATACACTTCTTAAATAC
AATCACATTAACATTTTGAGCTATTTCCTTCC AGCCTTTTTAGGGCAGATTTTGGTTGGTTTTT
ACATAGTTGAGATTGTACTGTTCATACAGTTT TATACCCTTTTTCATTTAACTTTATAACTTAAA
TATTGCTCTATGTTAGTATAAGCTTTTCACAA ACATTAGTATAGTCTCCCTTTTATAATTAATG
TTTGTGGGTATTTCTTGGCATGCATCTTTAATT CCTTATCCTAGCCTTTGGGCACAATTCCTGTG
CTCAAAAATGAGAGTGACGGCTGGCATGGTG GCTCCCGCCTGTAATCCCAGTACTTTGGAAAG
CCAAGGTAAGAGGATTGCTTGAGCCCAGAAC TTCAAGATGAGCCTGGGCTCATAGTGAGAAC
CCATCTATACAAAAAATTTTTAAAAATTAGCA TGGCGGCACACATCTGTAATCCTAGCTACTTG
GCAGGCTGAGGTGAGAAGATCATTGGAGTTT AGGAATTGGAGGCTGCAGTGAGCCATGAGTA
TGCCACTGCACTCCAGCCTGGGGGACAGAGC AAGACCCTGCCTCAAAAAAAAAAAAAAAAA
AAAAA pVHL von Hippel- 450183 GGATCCCGCGGCGTCCGGCCCGGGTGGTCTG 6608
Lindau GATCGCGGAGGGAATGCCCCGGAGGGCGGAG tumor
AACTGGGACGAGGCCGAGGTAGGCGCGGAG suppressor
GAGGCAGGCGTCGAAGAGTACGGCCCTGAAG AAGACAGCTACCGAGGTCACCTTTGGCTCTTC
AGAGATGCAGGGACACACGATGGGCTTCTGG TTAACCAAACTGAATTATTTGTGCCATCTCTC
AATGTTGACGGACAGCCTATTTTTGCCAATAT CACACTGCCAGTGTATACTCTGAAAGAGCGA
TGCCTCCAGGTTGTCCGGAGCCTAGTCAAGCC TGAGAATTACAGGAGACTGGACATCGTCAGG
TCGCTCTACGAAGATCTGGAAGACCACCCAA ATGTGCAGAAAGACCTGGAGCGGCTGACACA
GGAGCGCATTGCACATCAACGGATGGGAGAT TGAAGATTTCTGTTGAAACTTACACTGTTTCA
TCTCAGCTTTTGATGGTACTGATGAGTCTTGA TCTAGATACAGGACTGGTTCCTTCCTTAGTTT
CAAAGTGTCTCATTCTCAGAGTAAAATAGGC ACCATTGCTTAAAAGAAAGTTAACTGACTTCA
CTAGGCATTGTGATGTTTAGGGGCAAACATC ACAAAATGTAATTTAATGCCTGCCCATTAGAG
AAGTATTTATCAGGAGAAGGTGGTGGCATTTT TGCTTCCTAGTAAGTCAGGACAGCTTGTATGT
AAGGAGGTTTGTATAAGTAATTCAGTGGGAA TTGCAGCATATCGTTTAATTTTAAGAAGGCAT
TGGCATCTGCTTTTAATGGATGTATAATACAT CCATTCTACATCCGTAGCGGTTGGTGACTTGT
CTGCCTCCTGCTTTGGGAAGACTGAGGCATCC GTGAGGCAGGGACAAGTCTTTCTCCTCTTTGA
GACCCCAGTGCCTGCACATCATGAGCCTTCAG TCAGGGTTTGTCAGAGGAACAAACCAGGGGA
CACTTTGTTAGAAAGTGCTTAGAGGTTCTGCC TCTATTTTTGTTGGGGGGTGGGAGAGGGGAC
CTTAAAATGTGTACAGTGAACAAATGTCTTAA AGGGAATCATTTTTGTAGGAAGCATTTTTTAT
AATTTTCTAAGTCGTGCACTTTCTCGGTCCAC TCTTGTT HIF1-alpha hypoxia 557538
ATTTGAAAACTTGGCAACCTTGGATTGGATGG 6609 inducible
ATTCATATTTCTTAGTATAGAAGTTCTTGATA factor 1,
TAACTGAAAAATTAAGTTAAACACTTAATAA alpha
GTGGTGGTTACTCAGCACTTTTAGATGCTGTT subunit
TATAATAGATGACCTTTTCTAACTAATTTACA (basic helix-
GTTTTTTGAAAGATAACTGAGAGGTTGAGGG loop-helix
ACGGAGATTTTCTTCAAGCAATTTTTTTTTTCA transcription
TTTTAAATGAGCTCCCAATGTCGGAGTTTGGA factor)
AAACAAATTTGTCTTTTTAAAAGAAGGTCTAG GAAACTCAAAACCTGAAGAATTGGAAGAAAT
CAGAATAGAAAATGGTAGGATAAGTTCTGAA CGTCGAAAAGAAAAGTCTCGAGATGCAGCCA
GATCTCGGCGAAGTAAAGAATCTGAAGTTTTT TATGAGCTTGCTCATCAGTTGCCACTTCCACA
TAATGTGAGTTCGCATCTTGATAAGGCCTCTG TGATGAGGCTTACCATCAGCTATTTGCGTGTG
AGGAAACTTCTGGATGCTGGTGATTTGGATAT TGAAGATGACATGAAAGCACAGATGAATTGC
TTTTATTTGAAAGCCTTGGATGGTTTTGTTAT GGTTCTCACAGATGATGGTGACATGATTTACA
TTTCTGATAATGTGAACAAATACATGGGATTA ACTCAGTTTGAACTAACTGGACACAGTGTGTT
TGATTTTACTCATCCATGTGACCATGAGGAAA TGAGAGAAATGCTTACACACAGAAATGGCCT
TGTGAAAAAGGGTAAAGAACAAAACACACA GCGAAGCTTTTTTCTCAGAATGAAGTGTACCC
TAACTAGCCGAGGAAGAACTATGAACATAAA GTCTGCAACATGGAAGGTATTGCACTGCACA
GGCCACATTCACGTATATGATACCAACAGTA ACCAACCTCAGTGTGGGTATAAGAAACCACC
TATGACCTGCTTGGTGCTGATTTGTGAACCCA TTCCTCACCCATCAAATATTGAAATTCCTTTA
GATAGCAAGACTTTCCTCAGTCGACACAGCCT GGATATGAAATTTTCTTATTGTGATGAAAGAA
TTACCGAATTGATGGGATATGAGCCAGAAGA ACTTTTAGGCCGCTCAATTTATGAATATTATC
ATGCTTTGGACTCTGATCATCTGACCAAAACT CATCATGATATGTTTACTAAAGGACAAGTCAC
CACAGGACAGTACAGGATGCTTGCCAAAAGA GGTGGATATGTCTGGGTTGAAACTCAAGCAA
CTGTCATATATAACACCAAGAATTCTCAACCA CAGTGCATTGTATGTGTGAATTACGTTGTGAG
TGGTATTATTCAGCACGACTTGATTTTCTCCC TTCAACAAACAGAATGTGTCCTTAAACCGGTT
GAATCTTCAGATATGAAAATGACTCAGCTATT CACCAAAGTTGAATCAGAAGATACAAGTAGC
CTCTTTGACAAACTTAAGAAGGAACCTGATG CTTTAACTTTGCTGGCCCCAGCCGCTGGAGAC
ACAATCATATCTTTAGATTTTGGCAGCAACGA CACAGAAACTGATGACCAGCAACTTGAGGAA
GTACCATTATATAATGATGTAATGCTCCCCTC ACCCAACGAAAAATTACAGAATATAAATTTG
GCAATGTCTCCATTACCCACCGCTGAAACGCC AAAGCCACTTCGAAGTAGTGCTGACCCTGCA
CTCAATCAAGAAGTTGCATTAAAATTAGAAC CAAATCCAGAGTCACTGGAACTTTCTTTTACC
ATGCCCCAGATTCAGGATCAGACACCTAGTC CTTCCGATGGAAGCACTAGACAAAGTTCACC
TGAGCCTAATAGTCCCAGTGAATATTGTTTTT ATGTGGATAGTGATATGGTCAATGAATTCAA
GTTGGAATTGGTAGAAAAACTTTTTGCTGAAG ACACAGAAGCAAAGAACCCATTTTCTACTCA
GGACACAGATTTAGACTTGGAGATGTTAGCT CCCTATATCCCAATGGATGATGACTTCCAGTT
ACGTTCCTTCGATCAGTTGTCACCATTAGAAA GCAGTTCCGCAAGCCCTGAAAGCGCAAGTCC
TCAAAGCACAGTTACAGTATTCCAGCAGACT CAAATACAAGAACCTACTGCTAATGCCACCA
CTACCACTGCCACCACTGATGAATTAAAAAC AGTGACAAAAGACCGTATGGAAGACATTAAA
ATATTGATTGCATCTCCATCTCCTACCCACAT ACATAAAGAAACTACTAGTGCCACATCATCA
CCATATAGAGATACTCAAAGTCGGACAGCCT CACCAAACAGAGCAGGAAAAGGAGTCATAG
AACAGACAGAAAAATCTCATCCAAGAAGCCC TAACGTGTTATCTGTCGCTTTGAGTCAAAGAA
CTACAGTTCCTGAGGAAGAACTAAATCCAAA GATACTAGCTTTGCAGAATGCTCAGAGAAAG
CGAAAAATGGAACATGATGGTTCACTTTTTCA AGCAGTAGGAATTGGAACATTATTACAGCAG
CCAGACGATCATGCAGCTACTACATCACTTTC TTGGAAACGTGTAAAAGGATGCAAATCTAGT
GAACAGAATGGAATGGAGCAAAAGACAATTA
TTTTAATACCCTCTGATTTAGCATGTAGACTG CTGGGGCAATCAATGGATGAAAGTGGATTAC
CACAGCTGACCAGTTATGATTGTGAAGTTAAT GCTCCTATACAAGGCAGCAGAAACCTACTGC
AGGGTGAAGAATTACTCAGAGCTTTGGATCA AGTTAACTGAGCTTTTTCTTAATTTCATTCCTT
TTTTTGGACACTGGTGGCTCATTACCTAAAGC AGTCTATTTATATTTTCTACATCTAATTTTAGA
AGCCTGGCTACAATACTGCACAAACTTGGTTA GTTCAATTTTGATCCCCTTTCTACTTAATTTAC
ATTAATGCTCTTTTTTAGTATGTTCTTTAATGC TGGATCACAGACAGCTCATTTTCTCAGTTTTT
TGGTATTTAAACCATTGCATTGCAGTAGCATC ATTTTAAAAAATGCACCTTTTTATTTATTTATT
TTTGGCTAGGGAGTTTATCCCTTTTTCGAATT ATTTTTAAGAAGATGCCAATATAATTTTTGTA
AGAAGGCAGTAACCTTTCATCATGATCATAG GCAGTTGAAAAATTTTTACACCTTTTTTTTCA
CATTTTACATAAATAATAATGCTTTGCCAGCA GTACGTGGTAGCCACAATTGCACAATATATTT
TCTTAAAAAATACCAGCAGTTACTCATGGAAT ATATTCTGCGTTTATAAAACTAGTTTTTAAGA
AGAAATTTTTTTTGGCCTATGAAATTGTTAAA CCTGGAACATGACATTGTTAATCATATAATAA
TGATTCTTAAATGCTGTATGGTTTATTATTTA AATGGGTAAAGCCATTTACATAATATAGAAA
GATATGCATATATCTAGAAGG HIF1-alpha hypoxia 394997
GACAGGAGGATCACCCTCTTCGTCGCTTCGGC 6610 inducible
CAGTGTGTCGGGCTGGGCCCTGACAAGCCAC factor 1,
CTGAGGAGAGGCTCGGAGCCGGGCCCGGACC alpha
CCGGCGATTGCCGCCCGCTTCTCTCTAGTCTC subunit
ACGAGGGGTTTCCCGCCTCGCACCCCCACCTC (basic helix-
TGGACTTGCCTTTCCTTCTCTTCTCCGCGTGTG loop-helix
GAGGGAGCCAGCGCTTAGGCCGGAGCGAGCC transcription
TGGGGGCCGCCCGCCGTGAAGACATCGCGGG factor)
GACCGATTCACCATGGAGGGCGCCGGCGGCG CGAACGACAAGAAAAATAGGATAAGTTCTGA
ACGTCGAAAAGAAAAGTCTCGAGATGCAGCC AGATCTCGGCGAAGTAAAGAATCTGAAGTTT
TTTATGAGCTTGCTCATCAGTTGCCACTTCCA CATAATGTGAGTTCGCATCTTGATAAGGCCTC
TGTGATGAGGCTTACCATCAGCTATTTGCGTG TGAGGAAACTTCTGGATGCTGGTGATTTGGAT
ATTGAAGATGACATGAAAGCACAGATGAATT GCTTTTATTTGAAAGCCTTGGATGGTTTTGTT
ATGGTTCTCACAGATGATGGTGACATGATTTA CATTTCTGATAATGTGAACAAATACATGGGAT
TAACTCAGTTTGAACTAACTGGACACAGTGTG TTTGATTTTACTCATCCATGTGACCATGAGGA
AATGAGAGAAATGCTTACACACAGAAATGGC CTTGTGAAAAAGGGTAAAGAACAAAACACAC
AGCGAAGCTTTTTTCTCAGAATGAAGTGTACC CTAACTAGCCGAGGAAGAACTATGAACATAA
AGTCTGCAACATGGAAGGTATTGCACTGCAC AGGCCACATTCACGTATATGATACCAACAGT
AACCAACCTCAGTGTGGGTATAAGAAACCAC CTATGACCTGCTTGGTGCTGATTTGTGAACCC
ATTCCTCACCCATCAAATATTGAAATTCCTTT AGATAGCAAGACTTTCCTCAGTCGACACAGC
CTGGATATGAAATTTTCTTATTGTGATGAAAG AATTACCGAATTGATGGGATATGAGCCAGAA
GAACTTTTAGGCCGCTCAATTTATGAATATTA TCATGCTTTGGACTCTGATCATCTGACCAAAA
CTCATCATGATATGTTTACTAAAGGACAAGTC ACCACAGGACAGTACAGGATGCTTGCCAAAA
GAGGTGGATATGTCTGGGTTGAAACTCAAGC AACTGTCATATATAACACCAAGAATTCTCAAC
CACAGTGCATTGTATGTGTGAATTACGTTGTG AGTGGTATTATTCAGCACGACTTGATTTTCTC
CCTTCAACAAACAGAATGTGTCCTTAAACCG GTTGAATCTTCAGATATGAAAATGACTCAGCT
ATTCACCAAAGTTGAATCAGAAGATACAAGT AGCCTCTTTGACAAACTTAAGAAGGAACCTG
ATGCTTTAACTTTGCTGGCCCCAGCCGCTGGA GACACAATCATATCTTTAGATTTTGGCAGCAA
CGACACAGAAACTGATGACCAGCAACTTGAG GAAGTACCATTATATAATGATGTAATGCTCCC
CTCACCCAACGAAAAATTACAGAATATAAAT TTGGCAATGTCTCCATTACCCACCGCTGAAAC
GCCAAAGCCACTTCGAAGTAGTGCTGACCCT GCACTCAATCAAGAAGTTGCATTAAAATTAG
AACCAAATCCAGAGTCACTGGAACTTTCTTTT ACCATGCCCCAGATTCAGGATCAGACACCTA
GTCCTTCCGATGGAAGCACTAGACAAAGTTC ACCTGAGCCTAATAGTCCCAGTGAATATTGTT
TTTATGTGGATAGTGATATGGTCAATGAATTC AAGTTGGAATTGGTAGAAAAACTTTTTGCTGA
AGACACAGAAGCAAAGAACCCATTTTCTACT CAGGACACAGATTTAGACTTGGAGATGTTAG
CTCCCTATATCCCAATGGATGATGACTTCCAG TTACGTTCCTTCGATCAGTTGTCACCATTAGA
AAGCAGTTCCGCAAGCCCTGAAAGCGCAAGT CCTCAAAGCACAGTTACAGTATTCCAGCAGA
CTCAAATACAAGAACCTACTGCTAATGCCAC CACTACCACTGCCACCACTGATGAATTAAAA
ACAGTGACAAAAGACCGTATGGAAGACATTA AAATATTGATTGCATCTCCATCTCCTACCCAC
ATACATAAAGAAACTACTAGTGCCACATCAT CACCATATAGAGATACTCAAAGTCGGACAGC
CTCACCAAACAGAGCAGGAAAAGGAGTCATA GAACAGACAGAAAAATCTCATCCAAGAAGCC
CTAACGTGTTATCTGTCGCTTTGAGTCAAAGA ACTACAGTTCCTGAGGAAGAACTAAATCCAA
AGATACTAGCTTTGCAGAATGCTCAGAGAAA GCGAAAAATGGAACATGATGGTTCACTTTTTC
AAGCAGTAGGAATTGGAACATTATTACAGCA GCCAGACGATCATGCAGCTACTACATCACTTT
CTTGGAAACGTGTAAAAGGATGCAAATCTAG TGAACAGAATGGAATGGAGCAAAAGACAATT
ATTTTAATACCCTCTGATTTAGCATGTAGACT GCTGGGGCAATCAATGGATGAAAGTGGATTA
CCACAGCTGACCAGTTATGATTGTGAAGTTAA TGCTCCTATACAAGGCAGCAGAAACCTACTG
CAGGGTGAAGAATTACTCAGAGCTTTGGATC AAGTTAACTGAGCTTTTTCTTAATTTCATTCCT
TTTTTTGGACACTGGTGGCTCATTACCTAAAG CAGTCTATTTATATTTTCTACATCTAATTTTAG
AAGCCTGGCTACAATACTGCACAAACTTGGTT AGTTCAATTTTGATCCCCTTTCTACTTAATTTA
CATTAATGCTCTTTTTTAGTATGTTCTTTAATG CTGGATCACAGACAGCTCATTTTCTCAGTTTT
TTGGTATTTAAACCATTGCATTGCAGTAGCAT CATTTTAAAAAATGCACCTTTTTATTTATTTAT
TTTTGGCTAGGGAGTTTATCCCTTTTTCGAATT ATTTTTAAGAAGATGCCAATATAATTTTTGTA
AGAAGGCAGTAACCTTTCATCATGATCATAG GCAGTTGAAAAATTTTTACACCTTTTTTTTCA
CATTTTACATAAATAATAATGCTTTGCCAGCA GTACGTGGTAGCCACAATTGCACAATATATTT
TCTTAAAAAATACCAGCAGTTACTCATGGAAT ATATTCTGCGTTTATAAAACTAGTTTTTAAGA
AGAAATTTTTTTTGGCCTATGAAATTGTTAAA CCTGGAACATGACATTGTTAATCATATAATAA
TGATTCTTAAATGCTGTATGGTTTATTATTTA AATGGGTAAAGCCATTTACATAATATAGAAA
GATATGCATATATCTAGAAGGTATGTGGCATT TATTTGGATAAAATTCTCAATTCAGAGAAATC
ATCTGATGTTTCTATAGTCACTTTGCCAGCTC AAAAGAAAACAATACCCTATGTAGTTGTGGA
AGTTTATGCTAATATTGTGTAACTGATATTAA ACCTAAATGTTCTGCCTACCCTGTTGGTATAA
AGATATTTTGAGCAGACTGTAAACAAGAAAA AAAAAATCATGCATTCTTAGCAAAATTGCCTA
GTATGTTAATTTGCTCAAAATACAATGTTTGA TTTTATGCACTTTGTCGCTATTAACATCCTTTT
TTTCATGTAGATTTCAATAATTGAGTAATTTT AGAAGCATTATTTTAGGAATATATAGTTGTCA
CAGTAAATATCTTGTTTTTTCTATGTACATTGT ACAAATTTTTCATTCCTTTTGCTCTTTGTGGTT
GGATCTAACACTAACTGTATTGTTTTGTTACA TCAAATAAACATCTTCTGTGGACCAGG
TABLE-US-00029 TABLE 28 Peptide sequences for additional targets
for titration experiments SEQ Target ENSP ID Target Description ID
Protein Sequence NO HIF2- hypoxia 263734
MTADKEKKRSSSERRKEKSRDAARCRRSKETE 6611 alpha inducible
VFYELAHELPLPHSVSSHLDKASIMRLAISFLRT factor 2,
HKLLSSVCSENESEAEADQQMDNLYLKALEGFI alpha
AVVTQDGDMIFLSENISKFMGLTQVELTGHSIF subunit;
DFTHPCDHEEIRENLSLKNGSGFGKKSKDMSTE endothelial
RDFFMRMKCTVTNRGRTVNLKSATWKVLHCT PAS
GQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQ domain
HPSHMDIPLDSKTFLSRHSMDMKFTYCDDRITE protein 1
LIGYHPEELLGRSAYEFYHALDSENMTKSHQNL CTKGQVVSGQYRMLAKHGGYVWLETQGTVIY
NPRNLQPQCIMCVNYVLSEIEKNDVVFSMDQTE SLFKPHLMAMNSIFDSSGKGAVSEKSNFLFTKL
KEEPEELAQLAPTPGDAIISLDFGNQNFEESSAY
GKAILPPSQPWATELRSHSTQSEAGSLPAFTVPQ
AAAPGSTTPSATSSSSSCSTPNSPEDYYTSLDND
LKIEVIEKLFAMDTEAKDQCSTQTDFNELDLET
LAPYIPMDGEDFQLSPICPEERLLAENPQSTPQH
CFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKK
TEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSS MGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFL
GAAPLGPPVSPPHVSTFKTRSAKGFGARGPDVL SPAMVALSNKLKLKRQLEYEEQAFQDLSGGDP
PGGSTSHLMWKRMKNLRGGSCPLMPDKPLSAN VPNDKFTQNPMRGLGHPLRHLPLPQPPSAISPGE
NSKSRFPPQCYATQYQDYSLSSAHKVSGMASR LLGPSFESYLLPELTRYDCEVNVPVLGSSTLLQG
GDLLRALDQAT pVHL von Hippel- 256474 MPRRAENWDEAEVGAEEAGVEEYGPEEDGGE
6612 Lindau ESGAEESGPEESGPEELGAEEEMEAGRPRPVLRS tumor
VNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPY suppressor
PTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVN
QTELFVPSLNVDGQPIFANITLPVYTLKERCLQV
VRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDL ERLTQERIAHQRMGD pVHL von Hippel-
344757 MPRRAENWDEAEVGAEEAGVEEYGPEEDGGE 6613 Lindau
ESGAEESGPEESGPEELGAEEEMEAGRPRPVLRS tumor
VNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPY suppressor
PTLPPGTGRRIHSYRVYTLKERCLQVVRSLVKP ENYRRLDIVRSLYEDLEDHPNVQKDLERLTQER
IAHQRMGD pVHL von Hippel- 395399 MPRRAENWDEAEVGAEEAGVEEYGPEEDSYR
6614 Lindau GHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQ tumor
PIFANITLPVYTLKERCLQVVRSLVKPENYRRLD suppressor
IVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD HIF1- hypoxia 451696
MRLTISYLRVRKLLDAGDLDIEDDMKAQMNCF 6615 alpha inducible
YLKALDGFVMVLTDDGDMIYISDNVNKYMGL factor 1,
TQFELTGHSVFDFTHPCDHEEMREMLTHRNGL alpha
VKKGKEQNTQRSFFLRMKCTLTSRGRTMNIKS subunit
ATWKVLHCTGHIHVYDTNSNQPQCGYKKPPMT (basic helix-
CLVLICEPIPHPSNIEIPLDSKTFLSRHSLDMKFSY loop-helix
CDERITELMGYEPEELLGRSIYEYYHALDSDHL transcription
TKTHHDMFTKGQVTTGQYRMLAKRGGYVWV factor)
ETQATVIYNTKNSQPQCIVCVNYVVSGIIQHDLI
FSLQQTECVLKPVESSDMKMTQLFTKVESEDTS
SLFDKLKKEPDALTLLAPAAGDTIISLDFGSNDT ETDDQQLEEVPLYNDVMLPSPNEKLQNINLAM
SPLPTAETPKPLRSSADPALNQEVALKLEPNPES
LELSFTMPQIQDQTPSPSDGSTRQSSPEPNSPSEY
CFYVDSDMVNEFKLELVEKLFAEDTEAKNPFST QDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLES
SSASPESASPQSTVTVFQQTQIQEPTANATTTTA
TTDELKTVTKDRMEDIKILIASPSPTHIHKETTSA
TSSPYRDTQSRTASPNRAGKGVIEQTEKSHPRSP
NVLSVALSQRTTVPEEELNPKILALQNAQRKRK MEHDGSLFQAVGIGTLLQQPDDHAATTSLSWK
RVKGCKSSEQNGMEQKTIILIPSDLACRLLGQS MDESGLPQLTSYDCEVNAPIQGSRNLLQGEELL
RALDQVN HIF1- hypoxia 378446 MEGAGGANDKKNRISSERRKEKSRDAARSRRS 6616
alpha inducible KESEVFYELAHQLPLPHNVSSHLDKASVMRLTI factor 1,
SYLRVRKLLDAGDLDIEDDMKAQMNCFYLKAL alpha
DGFVMVLTDDGDMIYISDNVNKYMGLTQFELT subunit
GHSVFDFTHPCDHEEMREMLTHRNGLVKKGKE (basic helix-
QNTQRSFFLRMKCTLTSRGRTMNIKSATWKVL loop-helix
HCTGHIHVYDTNSNQPQCGYKKPPMTCLVLICE transcription
PIPHPSNIEIPLDSKTFLSRHSLDMKFSYCDERITE factor)
LMGYEPEELLGRSIYEYYHALDSDHLTKTHHD MFTKGQVTTGQYRMLAKRGGYVWVETQATVI
YNTKNSQPQCIVCVNYVVSGIIQHDLIFSLQQTE
CVLKPVESSDMKMTQLFTKVESEDTSSLFDKLK
KEPDALTLLAPAAGDTIISLDFGSNDTETDDQQL
EEVPLYNDVMLPSPNEKLQNINLAMSPLPTAET
PKPLRSSADPALNQEVALKLEPNPESLELSFTMP
QIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSD
MVNEFKLELVEKLFAEDTEAKNPFSTQDTDLDL
EMLAPYIPMDDDFQLRSFDQLSPLESSSASPESA
SPQSTVTVFQQTQIQEPTANATTTTATTDELKTV
TKDRMEDIKILIASPSPTHIHKETTSATSSPYRDT
QSRTASPNRAGKGVIEQTEKSHPRSPNVLSVAL SQRTTVPEEELNPKILALQNAQRKRKMEHDGSL
FQAVGIGTLLQQPDDHAATTSLSWKRVKGCKS SEQNGMEQKTIILIPSDLACRLLGQSMDESGLPQ
LTSYDCEVNAPIQGSRNLLQGEELLRALDQVN
Materials for Examples 27-33
[1019] Table 29 describes the modified mRNA sequences described in
Examples 27-33.
TABLE-US-00030 TABLE 29 SEQ ID Target mRNA Sequence (polyA tail and
5'cap not shown in sequence) NO Apoptosis-
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC 6617 inducing
ACCAUGGAAAAAGUCAGACGAGAGGGGGUUAAGGUGAUGCCCAA factor short
UGCUAUUGUGCAAUCCGUUGGAGUCAGCAGUGGCAAGUUACUUA (AIFsh)
UCAAGCUGAAAGACGGCAGGAAGGUAGAAACUGACCACAUAGUG
GCAGCUGUGGGCCUGGAGCCCAAUGUUGAGUUGGCCAAGACUGG
UGGCCUGGAAAUAGACUCAGAUUUUGGUGGCUUCCGGGUAAAUG
CAGAGCUACAAGCACGCUCUAACAUCUGGGUGGCAGGAGAUGCU
GCAUGCUUCUACGAUAUAAAGUUGGGAAGGAGGCGGGUAGAGCA
CCAUGAUCACGCUGUUGUGAGUGGAAGAUUGGCUGGAGAAAAUA
UGACUGGAGCUGCUAAGCCGUACUGGCAUCAGUCAAUGUUCUGG
AGUGAUUUGGGCCCCGAUGUUGGCUAUGAAGCUAUUGGUCUUGU
GGACAGUAGUUUGCCCACAGUUGGUGUUUUUGCAAAAGCAACUG
CACAAGACAACCCCAAAUCUGCCACAGAGCAGUCAGGAACUGGUA
UCCGAUCAGAGAGUGAGACAGAGUCCGAGGCCUCAGAAAUUACU
AUUCCUCCCAGCACCCCGGCAGUUCCACAGGCUCCCGUCCAGGGG
GAGGACUACGGCAAAGGUGUCAUCUUCUACCUCAGGGACAAAGU
GGUCGUGGGGAUUGUGCUAUGGAACAUCUUUAACCGAAUGCCAA
UAGCAAGGAAGAUCAUUAAGGACGGUGAGCAGCAUGAAGAUCUC
AAUGAAGUAGCCAAACUAUUCAACAUUCAUGAAGACUGAUAAUA
GGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCC
CCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUG AAUAAAGUCUGAGUGGGCGGC
Siah E3 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC 6618 ubiquitin
ACCAUGAGCCGUCAGACUGCUACAGCAUUACCUACCGGUACCUCG protein
AAGUGUCCACCAUCCCAGAGGGUGCCUGCCCUGACUGGCACAACU ligase 1
GCAUCCAACAAUGACUUGGCGAGUCUUUUUGAGUGUCCAGUCUG (SIAH1)
CUUUGACUAUGUGUUACCGCCCAUUCUUCAAUGUCAGAGUGGCC
AUCUUGUUUGUAGCAACUGUCGCCCAAAGCUCACAUGUUGUCCA
ACUUGCCGGGGCCCUUUGGGAUCCAUUCGCAACUUGGCUAUGGA
GAAAGUGGCUAAUUCAGUACUUUUCCCCUGUAAAUAUGCGUCUU
CUGGAUGUGAAAUAACUCUGCCACACACAGAAAAAGCAGACCAU
GAAGAGCUCUGUGAGUUUAGGCCUUAUUCCUGUCCGUGCCCUGG
UGCUUCCUGUAAAUGGCAAGGCUCUCUGGAUGCUGUAAUGCCCC
AUCUGAUGCAUCAGCAUAAGUCCAUUACAACCCUACAGGGAGAG
GAUAUAGUUUUUCUUGCUACAGACAUUAAUCUUCCUGGUGCUGU
UGACUGGGUGAUGAUGCAGUCCUGUUUUGGCUUUCACUUCAUGU
UAGUCUUAGAGAAACAGGAAAAAUACGAUGGUCACCAGCAGUUC
UUCGCAAUCGUACAGCUGAUAGGAACACGCAAGCAAGCUGAAAA
UUUUGCUUACCGACUUGAGCUAAAUGGUCAUAGGCGACGAUUGA
CUUGGGAAGCGACUCCUCGAUCUAUUCAUGAAGGAAUUGCAACA
GCCAUUAUGAAUAGCGACUGUCUAGUCUUUGACACCAGCAUUGC
ACAGCUUUUUGCAGAAAAUGGCAAUUUAGGCAUCAAUGUAACUA
UUUCCAUGUGUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUU
CUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC
CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC Constitively
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC 6619 active
ACCAUGAUUGAGACAGACAGUGGUGUUGAUGAUGACAUGGCGUG (C.A.
UCAUAAAAUACCAGUGGAGGCCGACUUCUUGUAUGCAUACUCCA caspase 3
CAGCACCUGGUUAUUAUUCUUGGCGAAAUUCAAAGGAUGGCUCC (also
UGGUUCAUCCAGUCGCUUUGUGCCAUGCUGAAACAGUAUGCCGA known as
CAAGCUUGAAUUUAUGCACAUUCUUACCCGGGUUAACCGAAAGG reverse
UGGCAACAGAAUUUGAGUCCUUUUCCUUUGACGCUACUUUUCAU caspase 3
GCAAAGAAACAGAUUCCAUGUAUUGUUUCCAUGCUCACAAAAGA (Rev-
ACUCUAUUUUUAUCACGAUGAAGUUGAUGGGGGAUCCCCCAUGG Caspase 3))
AGAACACUGAAAACUCAGUGGAUUCAAAAUCCAUUAAAAAUUUG
GAACCAAAGAUCAUACAUGGAAGCGAAUCAAUGGACUCUGGAAU
AUCCCUGGACAACAGUUAUAAAAUGGAUUAUCCUGAGAUGGGUU
UAUGUAUAAUAAUUAAUAAUAAGAAUUUUCAUAAGAGCACUGGA
AUGACAUCUCGGUCUGGUACAGAUGUCGAUGCAGCAAACCUCAG
GGAAACAUUCAGAAACUUGAAAUAUGAAGUCAGGAAUAAAAAUG
AUCUUACACGUGAAGAAAUUGUGGAAUUGAUGCGUGAUGUUUCU
AAAGAAGAUCACAGCAAAAGGAGCAGUUUUGUUUGUGUGCUUCU
GAGCCAUGGUGAAGAAGGAAUAAUUUUUGGAACAAAUGGACCUG
UUGACCUGAAAAAAAUAACAAACUUUUUCAGAGGGGAUCGUUGU
AGAAGUCUAACUGGAAAACCCAAACUUUUCAUUAUUCAGGCCUG
CCGUGGUACAGAACUGGACUGUGGCAUUGAGACAGACUGAUAAU
AGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUU
GAAUAAAGUCUGAGUGGGCGGC Granulysin
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC 6620
ACCAUGGCAACUUGGGCCCUGCUGCUUCUUGCAGCCAUGUUGCUC
GGAAAUCCUGGUCUGGUGUUUUCGCGCCUUUCACCGGAGUACUA
CGAUCUCGCUCGCGCACAUCUGCGCGACGAGGAGAAGUCGUGCCC
AUGUCUCGCACAAGAAGGGCCACAGGGUGACCUUUUGACCAAGA
CGCAAGAACUUGGCAGGGACUACCGAACCUGUCUGACCAUCGUGC
AAAAGCUGAAGAAAAUGGUCGAUAAACCUACCCAAAGAAGCGUG
UCCAACGCAGCGACUCGGGUGUGCCGGACUGGCAGAUCCAGAUG
GCGGGAUGUGUGUAGAAACUUCAUGAGAAGGUACCAGAGCCGUG
UUACUCAGGGACUGGUCGCGGGAGAAACUGCCCAACAGAUUUGC
GAAGAUCUGCGACUCUGUAUUCCUUCAACCGGACCCCUUUGAUA
AUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUC
CCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUU
UGAAUAAAGUCUGAGUGGGCGGC MYC
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC 6621 inhibitor D
ACCAUGACCGAAGAAAACGUCAAGAGAAGAACCCAUAAUGUCCU
CGAGCGCCAGCGGCGCAAUGAGCUCAAGCGCAGCUUCUUUGCACU
CAGGGACCAAAUUCCAGAGUUGGAGAACAACGAAAAGGCCCCGA
AGGUGGUGAUCCUUAAGAAGGCGACUGCCUACAUCCUGUCGGUG
CAGGCUGAGACUCAAAAGCUGAUCUCCGAAAUCGAUCUGCUCCG
GAAACAGAACGAACAACUGAAACACAAACUGGAACAGCUGCGGA
AUUCAUGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUU
GCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGU
ACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC
Example 27
Detection of Apoptosis-Inducing Factor Short Protein: Western
Blot
[1020] CD1 mice (Harlan Laboratories, South Easton, Mass.) were
administered intravenously lipolexed apoptosis-inducing factor
short (AIFsh) modified mRNA (mRNA sequence shown in Table 29; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1) fully modified with 5-methylcytidine and pseudouridine
(5 mC/pU), fully modified with 5-methylcytidine and
1-methylpseudouridine (5 mC/1 mpU), 25% of uridine modified with
2-thiouridine and 25% of cytidine modified with 5-methylcytidine
(s2U and 5 mC), fully modified with pseudouridine (pU) or fully
modified with 1-methylpseudouridine (1 mpU). The mice were
administered a dose of 2 ug of mRNA complexed with 2 ul
Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) in 100 ul
sterile basal DMEM medium (w/o additives, LifeTechnologies, Grand
Island, N.Y.).
[1021] After 6 hours, the animals were sacrificed and serum &
spleen are taken. Spleens were transferred to 6-well plates and
kept on ice in presence of 1 ml PBS. One spleen was cut with a
scalpel several times and with a rubber cell scraper splenocytes
were squeezed out until the PBS turns turbid due to cell
release.
[1022] Leaving fibrous components behind, the cells were
transferred to a 100 um cell strainer (BD Biosciences, San Jose,
Calif.) sitting on a 12-well cell culture plate. By gravity the
cells passed through the cell strainer and were collected beneath
in the 12-well culture dish. 1 ml of PBS was transferred with the
free-floating splenocytes to an Eppendorf tube and spun for 5 min
at 2000 rpm. The PBS was discarded and the cell pellet combined
with 500 ul fresh PBS. The spenocytes were resuspended by brief
vortexing for 5 mins at 2000 rpm. The PBS was discarded and 1 ml BD
Pharmlyse was added to the cell pellet. The splenocytes were
resuspended by brief vortexing. The cells were incubated at room
temperature for 3 minutes and then spun at 200 rpm for 5 minutes.
The cells were washed twice with 500 ul PBS and spun as described
above. The cells were resuspended with 500 ul of PBS and spun as
described.
[1023] 250 ul of splenocytes were combined with 1.times. Pharmlyse
buffer and vortexed briefly or resuspended with a pipet and then
spun for 2 minutes at 2000 rpm.
[1024] In one tube, resuspend cell pellet in 500 ul RIPA buffer
with protease inhibitor cocktail for mammalian cells
(BostonBioproducts, Ashland, Mass.) and freeze lysate or continue
with BCA assay immediately. In a second tube, add 250 ul FACS
staining kit fixation solution (4% formaldehyde; R and D Systems,
Minneapolis, Minn.) and then incubate for 10 minutes at room
temperature. The cells were washed twice with 500 ul PBS and spun
as described above. The cell pellet was resuspended in 500 PBS and
stored at 4.degree. C.
[1025] Protein lysates were loaded on NuPage SDS-PAGE system
(chambers and power supply) with 1.5 mm ready-to-use Bis-Tris gels
and 4-12% acrylamide gradient with MOPS-buffer as running aid (all
Life Technologies, Grand Island, N.Y.). Each lysate sample was
prepared to 40 ul final volume. This sample contained 25 ug protein
lysate in variable volume, RIPA buffer to make up volume to 26 ul,
4 ul of 10.times. reducing agent and 10 ul 4.times.SDS loading
buffer (both from Life Technologies, Grand Island, N.Y.). Samples
were heated at 95.degree. C. for 5 min and loaded on the gel.
Standard settings were chosen by the manufacturer, 200V, 120 mA and
max. 25 W. Run time was 60 min, but no longer than running dye
reaching the lower end of the gel.
[1026] After the run was terminated, the plastic case was cracked
and the encased gel transferred to a ready-to-use nitrocellulose
membrane kit and power supply (iBLOT; LifeTechnologies, Grand
Island, N.Y.). Using default settings, the protein lysate was
transferred by high Ampere electricity from the gel to the
membrane.
[1027] After the transfer, the membranes were incubated in 5% BSA
in 1.times.TBS for 15 minutes then in 5% BSA in 1.times.TBS+0.1%
Tween for another 15 minutes. Primary antibodies (AIFsh rabbit
polyclonal antibody; Abcam, Cambridge, Mass.) against AIFsh
proteins were applied in 3m1 of 5% BSA in 1.times.TBS solution at a
1:500 to 1:2000 dilution for 3 hours at room temperature and gentle
agitation on an orbital shaker. Membranes are washed 3 times with
1.times.TBS/0.1% Tween, 5 minutes each time with gentle agitation.
The secondary antibody (Goat anti-rabbit HRP conjugate; Abcam,
Cambridge, Mass.) was conjugated to horse radish peroxidase and
binds to the primary antibody antibodies. The secondary antibody
was diluted of 1:1000 to 1:5000 in 5% BSA in 1.times.TBS and
incubated for 3 hrs at RT.
[1028] At the end of incubation time, the membranes were washed 3
times with 1.times.TBS/0.1% Tween, 5 minutes each time with gentle
agitation. The membranes were developed in 5m1 Pierce WestPico
Chemiluminescent Subtrate (Thermo Fisher, Rockford, Ill.) as
directed.
[1029] As shown in FIGS. 3A and 3B the Western Blot detected
protein around the expected size of 60 kd for each of the 2 samples
evaluated for each chemistry.
Example 28
Detection of Siah E3 Ubiquitin Protein Ligase 1 Protein: Western
Blot
[1030] CD1 mice (Harlan Laboratories, South Easton, Mass.) were
administered intravenously lipolexed siah E3 ubiquitin protein
ligase 1 (SIAH1) modified mRNA (mRNA sequence shown in SEQ ID NO.
6618 (Table 29); polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1) fully modified with
5-methylcytidine and pseudouridine (5 mC/pU), fully modified with
5-methylcytidine and 1-methylpseudouridine (5 mC/1 mpU), 25% of
uridine modified with 2-thiouridine and 25% of cytidine modified
with 5-methylcytidine (s2U and 5 mC), fully modified with
pseudouridine (pU) or fully modified with 1-methylpseudouridine (1
mpU). The mice were administered a dose of 2 ug of mRNA complexed
with 2 ul Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)
in 100 ul sterile basal DMEM medium (w/o additives,
LifeTechnologies, Grand Island, N.Y.).
[1031] After 6 hours, the animals were sacrificed and serum &
spleen are taken. Spleens were transferred to 6-well plates and
kept on ice in presence of 1 ml PBS. One spleen was cut with a
scalpel several times and with a rubber cell scraper splenocytes
were squeezed out until the PBS turns turbid due to cell
release.
[1032] Leaving fibrous components behind, the cells were
transferred to a 100 um cell strainer (BD Biosciences, San Jose,
Calif.) sitting on a 12-well cell culture plate. By gravity the
cells passed through the cell strainer and were collected beneath
in the 12-well culture dish. 1 ml of PBS was transferred with the
free-floating splenocytes to an Eppendorf tube and spun for 5 min
at 2000 rpm. The PBS was discarded and the cell pellet combined
with 500 ul fresh PBS. The spenocytes were resuspended by brief
vortexing for 5 mins at 2000 rpm. The PBS was discarded and 1 ml BD
Pharmlyse was added to the cell pellet. The splenocytes were
resuspended by brief vortexing. The cells were incubated at room
temperature for 3 minutes and then spun at 200 rpm for 5 minutes.
The cells were washed twice with 500 ul PBS and spun as described
above. The cells were resuspended with 500 ul of PBS and spun as
described.
[1033] 250 ul of splenocytes were combined with 1.times. Pharmlyse
buffer and vortexed briefly or resuspended with a pipet and then
spun for 2 minutes at 2000 rpm.
[1034] In one tube, resuspend cell pellet in 500 ul RIPA buffer
with protease inhibitor cocktail for mammalian cells
(BostonBioproducts, Ashland, Mass.) and freeze lysate or continue
with BCA assay immediately. In a second tube, add 250 ul FACS
staining kit fixation solution (4% formaldehyde; R and D Systems,
Minneapolis, Minn.) and then incubate for 10 minutes at room
temperature. The cells were washed twice with 500 ul PBS and spun
as described above. The cell pellet was resuspended in 500 PBS and
stored at 4.degree. C.
[1035] Protein lysates were loaded on NuPage SDS-PAGE system
(chambers and power supply) with 1.5 mm ready-to-use Bis-Tris gels
and 4-12% acrylamide gradient with MOPS-buffer as running aid (all
Life Technologies, Grand Island, N.Y.). Each lysate sample was
prepared to 40 ul final volume. This sample contained 25 ug protein
lysate in variable volume, RIPA buffer to make up volume to 26 ul,
4 ul of 10.times. reducing agent and 10 ul 4.times.SDS loading
buffer (both from Life Technologies, Grand Island, N.Y.). Samples
were heated at 95.degree. C. for 5 min and loaded on the gel.
Standard settings were chosen by the manufacturer, 200V, 120 mA and
max. 25 W. Run time was 60 min, but no longer than running dye
reaching the lower end of the gel.
[1036] After the run was terminated, the plastic case was cracked
and the encased gel transferred to a ready-to-use nitrocellulose
membrane kit and power supply (iBLOT; LifeTechnologies, Grand
Island, N.Y.). Using default settings, the protein lysate was
transferred by high Ampere electricity from the gel to the
membrane.
[1037] After the transfer, the membranes were incubated in 5% BSA
in 1.times.TBS for 15 minutes then in 5% BSA in 1.times.TBS+0.1%
Tween for another 15 minutes. Primary antibodies (SIAH1 rabbit
polyclonal antibody; Abcam, Cambridge, Mass.) against SIAH1
proteins were applied in 3m1 of 5% BSA in 1.times.TBS solution at a
1:500 to 1:2000 dilution for 3 hours at room temperature and gentle
agitation on an orbital shaker. Membranes are washed 3 times with
1.times.TBS/0.1% Tween, 5 minutes each time with gentle agitation.
The secondary antibody (Goat anti-rabbit HRP conjugate; Abcam,
Cambridge, Mass.) was conjugated to horse radish peroxidase and
binds to the primary antibody antibodies. The secondary antibody
was diluted of 1:1000 to 1:5000 in 5% BSA in 1.times.TBS and
incubated for 3 hrs at RT.
[1038] At the end of incubation time, the membranes were washed 3
times with 1.times. TBS/0.1% Tween, 5 minutes each time with gentle
agitation. The membranes were developed in 5m1 Pierce WestPico
Chemiluminescent Subtrate (Thermo Fisher, Rockford, Ill.) as
directed.
[1039] As shown in FIGS. 4A and 4B the Western Blot detected
protein around the expected size of 31 kd for each of the 2 samples
evaluated for each chemistry.
Example 29
Detection of Reverse Caspase 3 Protein: Western Blot
[1040] CD1 mice (Harlan Laboratories, South Easton, Mass.) were
administered intravenously lipolexed constitutively active (C.A.)
caspase 3 (also known as Reverse-Caspase 3 or Rev-Caspase 3)
modified mRNA (mRNA sequence shown in SEQ ID NO. 6619 (Table 29);
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1) fully modified with 5-methylcytidine and
pseudouridine (5 mC/pU), fully modified with 5-methylcytidine and
1-methylpseudouridine (5 mC/1 mpU), 25% of uridine modified with
2-thiouridine and 25% of cytidine modified with 5-methylcytidine
(s2U and 5 mC), fully modified with pseudouridine (pU) or fully
modified with 1-methylpseudouridine (1 mpU). The mice were
administered a dose of 2 ug of mRNA complexed with 2 ul
Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) in 100 ul
sterile basal DMEM medium (w/o additives, LifeTechnologies, Grand
Island, N.Y.).
[1041] After 6 hours, the animals were sacrificed and serum &
spleen are taken. Spleens were transferred to 6-well plates and
kept on ice in presence of 1 ml PBS. One spleen was cut with a
scalpel several times and with a rubber cell scraper splenocytes
were squeezed out until the PBS turns turbid due to cell
release.
[1042] Leaving fibrous components behind, the cells were
transferred to a 100 um cell strainer (BD Biosciences, San Jose,
Calif.) sitting on a 12-well cell culture plate. By gravity the
cells passed through the cell strainer and were collected beneath
in the 12-well culture dish. 1 ml of PBS was transferred with the
free-floating splenocytes to an Eppendorf tube and spun for 5 min
at 2000 rpm. The PBS was discarded and the cell pellet combined
with 500 ul fresh PBS. The spenocytes were resuspended by brief
vortexing for 5 mins at 2000 rpm. The PBS was discarded and 1 ml BD
Pharmlyse was added to the cell pellet. The splenocytes were
resuspended by brief vortexing. The cells were incubated at room
temperature for 3 minutes and then spun at 200 rpm for 5 minutes.
The cells were washed twice with 500 ul PBS and spun as described
above. The cells were resuspended with 500 ul of PBS and spun as
described.
[1043] 250 ul of splenocytes were combined with 1.times. Pharmlyse
buffer and vortexed briefly or resuspended with a pipet and then
spun for 2 minutes at 2000 rpm.
[1044] In one tube, resuspend cell pellet in 500 ul RIPA buffer
with protease inhibitor cocktail for mammalian cells
(BostonBioproducts, Ashland, Mass.) and freeze lysate or continue
with BCA assay immediately. In a second tube, add 250 ul FACS
staining kit fixation solution (4% formaldehyde; R and D Systems,
Minneapolis, Minn.) and then incubate for 10 minutes at room
temperature. The cells were washed twice with 500 ul PBS and spun
as described above. The cell pellet was resuspended in 500 PBS and
stored at 4.degree. C.
[1045] Protein lysates were loaded on NuPage SDS-PAGE system
(chambers and power supply) with 1.5 mm ready-to-use Bis-Tris gels
and 4-12% acrylamide gradient with MOPS-buffer as running aid (all
Life Technologies, Grand Island, N.Y.). Each lysate sample was
prepared to 40 ul final volume. This sample contained 25 ug protein
lysate in variable volume, RIPA buffer to make up volume to 26 ul,
4 ul of 10.times. reducing agent and 10 ul 4.times.SDS loading
buffer (both from Life Technologies, Grand Island, N.Y.). Samples
were heated at 95.degree. C. for 5 min and loaded on the gel.
Standard settings were chosen by the manufacturer, 200V, 120 mA and
max. 25 W. Run time was 60 min, but no longer than running dye
reaching the lower end of the gel.
[1046] After the run was terminated, the plastic case was cracked
and the encased gel transferred to a ready-to-use nitrocellulose
membrane kit and power supply (iBLOT; LifeTechnologies, Grand
Island, N.Y.). Using default settings, the protein lysate was
transferred by high Ampere electricity from the gel to the
membrane.
[1047] After the transfer, the membranes were incubated in 5% BSA
in 1.times.TBS for 15 minutes then in 5% BSA in 1.times.TBS+0.1%
Tween for another 15 minutes. Primary antibodies (Caspase 3 rabbit
polyclonal antibody; Abcam, Cambridge, Mass.) against target
proteins were applied in 3m1 of 5% BSA in 1.times.TBS solution at a
1:500 to 1:2000 dilution for 3 hours at room temperature and gentle
agitation on an orbital shaker. Membranes are washed 3 times with
1.times.TBS/0.1% Tween, 5 minutes each time with gentle agitation.
The secondary antibody (Goat anti-rabbit HRP conjugate; Abcam,
Cambridge, Mass.) was conjugated to horse radish peroxidase and
binds to the primary antibody antibodies. The secondary antibody
was diluted of 1:1000 to 1:5000 in 5% BSA in 1.times.TBS and
incubated for 3 hrs at RT.
[1048] At the end of incubation time, the membranes were washed 3
times with 1.times.TBS/0.1% Tween, 5 minutes each time with gentle
agitation. The membranes were developed in 5m1 Pierce WestPico
Chemiluminescent Subtrate (Thermo Fisher, Rockford, Ill.) as
directed.
[1049] As shown in FIGS. 5A and 5B the Western Blot detected
protein around the expected size of 32 kd for each of the 2 samples
evaluated for each chemistry.
Example 30
Detection of Granulysin Protein: Western Blot
[1050] CD1 mice (Harlan Laboratories, South Easton, Mass.) were
administered intravenously lipolexed granulysin mRNA (mRNA sequence
shown in SEQ ID NO. 6620 (Table 29); polyA tail of approximately
140 nucleotides not shown in sequence; 5' cap, Cap1) fully modified
with 5-methylcytidine and pseudouridine (5 mC/pU), fully modified
with 5-methylcytidine and 1-methylpseudouridine (5 mC/1 mpU), 25%
of uridine modified with 2-thiouridine and 25% of cytidine modified
with 5-methylcytidine (s2U and 5 mC), fully modified with
pseudouridine (pU) or fully modified with 1-methylpseudouridine (1
mpU). The mice were administered a dose of 2 ug of mRNA complexed
with 2 ul Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)
in 100 ul sterile basal DMEM medium (w/o additives,
LifeTechnologies, Grand Island, N.Y.).
[1051] After 6 hours, the animals were sacrificed and serum &
spleen are taken. Spleens were transferred to 6-well plates and
kept on ice in presence of 1 ml PBS. One spleen was cut with a
scalpel several times and with a rubber cell scraper splenocytes
were squeezed out until the PBS turns turbid due to cell
release.
[1052] Leaving fibrous components behind, the cells were
transferred to a 100 um cell strainer (BD Biosciences, San Jose,
Calif.) sitting on a 12-well cell culture plate. By gravity the
cells passed through the cell strainer and were collected beneath
in the 12-well culture dish. 1 ml of PBS was transferred with the
free-floating splenocytes to an Eppendorf tube and spun for 5 min
at 2000 rpm. The PBS was discarded and the cell pellet combined
with 500 ul fresh PBS. The spenocytes were resuspended by brief
vortexing for 5 mins at 2000 rpm. The PBS was discarded and 1 ml BD
Pharmlyse was added to the cell pellet. The splenocytes were
resuspended by brief vortexing. The cells were incubated at room
temperature for 3 minutes and then spun at 200 rpm for 5 minutes.
The cells were washed twice with 500 ul PBS and spun as described
above. The cells were resuspended with 500 ul of PBS and spun as
described.
[1053] 250 ul of splenocytes were combined with 1.times. Pharmlyse
buffer and vortexed briefly or resuspended with a pipet and then
spun for 2 minutes at 2000 rpm.
[1054] In one tube, resuspend cell pellet in 500 ul RIPA buffer
with protease inhibitor cocktail for mammalian cells
(BostonBioproducts, Ashland, Mass.) and freeze lysate or continue
with BCA assay immediately. In a second tube, add 250 ul FACS
staining kit fixation solution (4% formaldehyde; R and D Systems,
Minneapolis, Minn.) and then incubate for 10 minutes at room
temperature. The cells were washed twice with 500 ul PBS and spun
as described above. The cell pellet was resuspended in 500 PBS and
stored at 4.degree. C.
[1055] Protein lysates were loaded on NuPage SDS-PAGE system
(chambers and power supply) with 1.5 mm ready-to-use Bis-Tris gels
and 4-12% acrylamide gradient with MOPS-buffer as running aid (all
Life Technologies, Grand Island, N.Y.). Each lysate sample was
prepared to 40 ul final volume. This sample contained 25 ug protein
lysate in variable volume, RIPA buffer to make up volume to 26 ul,
4 ul of 10.times. reducing agent and 10 ul 4.times.SDS loading
buffer (both from Life Technologies, Grand Island, N.Y.). Samples
were heated at 95.degree. C. for 5 min and loaded on the gel.
Standard settings were chosen by the manufacturer, 200V, 120 mA and
max. 25 W. Run time was 60 min, but no longer than running dye
reaching the lower end of the gel.
[1056] After the run was terminated, the plastic case was cracked
and the encased gel transferred to a ready-to-use nitrocellulose
membrane kit and power supply (iBLOT; LifeTechnologies, Grand
Island, N.Y.). Using default settings, the protein lysate was
transferred by high Ampere electricity from the gel to the
membrane.
[1057] After the transfer, the membranes were incubated in 5% BSA
in 1.times.TBS for 15 minutes then in 5% BSA in 1.times.TBS+0.1%
Tween for another 15 minutes. Primary antibodies (Granulysin mouse
monoclonal antibody; Abcam, Cambridge, Mass.) against granulysin
proteins were applied in 3m1 of 5% BSA in 1.times.TBS solution at a
1:500 to 1:2000 dilution for 3 hours at room temperature and gentle
agitation on an orbital shaker. Membranes are washed 3 times with
1.times.TBS/0.1% Tween, 5 minutes each time with gentle agitation.
The secondary antibody (Donkey anti-mouse HRP conjugate; Abcam,
Cambridge, Mass.) was conjugated to horse radish peroxidase and
binds to the primary antibody antibodies. The secondary antibody
was diluted of 1:1000 to 1:5000 in 5% BSA in 1.times.TBS and
incubated for 3 hrs at RT.
[1058] At the end of incubation time, the membranes were washed 3
times with 1.times.TBS/0.1% Tween, 5 minutes each time with gentle
agitation. The membranes were developed in 5m1 Pierce WestPico
Chemiluminescent Subtrate (Thermo Fisher, Rockford, Ill.) as
directed.
[1059] As shown in FIGS. 6A and 6B the Western Blot detected
protein around the expected size of 16 kd for each of the 2 samples
evaluated for each chemistry.
Example 31
Confirmation of Peptide Identity
[1060] Proteins can be evaluated using liquid chromatography-mass
spectrometry in tandem with mass spectrometry (LC-MS/MS) with
quantitative LC-multiple reaction monitoring (MRM) in order to
confirm the identity of the peptide. The identity of any protein
target described herein can be evaluated using the liquid
chromatography-mass spectrometry in tandem with mass spectrometry
(LC-MS/MS) with quantitative LC-multiple reaction monitoring (MRM)
Assay (Biognosys AG, Schlieren Switzerland). HeLa cell lysates
containing protein expressed from modified mRNA are evaluated using
LC-MS/MS with quantitative LC-MRM Assay (Biognosys, Schlieren
Switzerland) in order to confirm the identity of the peptides in
the cell lysates. The identified peptide fragments are compared
against known proteins including isoforms using methods known
and/or described in the art.
A. Sample Preparation
[1061] Protein in each sample in lysis buffer is reduced by
incubation for 1 hour at 37.degree. C. with 5 mM
tris(2-carboxyethyl)phosphine (TCEP). Alkylation is carried out
using 10 mM iodoacetamide for 30 minutes in the dark at room
temperature. Proteins are digested to peptides using trypsin
(sequence grade, PromegaCorporation, Madison, Wis.) at a
protease:protein ratio of 1:50. Digestion is carried out overnight
at 37.degree. C. (total digestion time is 12 hours). Peptides are
cleaned up for mass spectrometric analysis using C.sub.18 spin
columns (The Nest Group, Southborough, Mass.) according to the
manufacturer's instructions. Peptides are dried down to complete
dryness and resuspended in LC solvent A (1% acetonitrile, 0.1%
formic acid (FA)). All solvents are HPLC-grade from
SIGMA-ALDRICH.RTM. (St. Louis, Mo.) and all chemicals, where not
stated otherwise, are obtained from SIGMA-ALDRICH.RTM. (St. Louis,
Mo.).
B. LC-MS/MS and LC-MRM
[1062] Peptides are injected to a packed C.sub.18 column (Magic AQ,
3 um particle size, 200 .ANG. pore size, Michrom Bioresources, Inc
(Auburn, Calif.); 11 cm column length, 75 um inner diameter, New
Objective (Woburn, Mass.)) on a Proxeon Easy nLC nano-liquid
chromatography system for all mass spectrometric analysis. LC
solvents are A: 1% acetonitrile in water with 0.1% FA; B: 3% water
in acetonitrile with 0.1% FA. The LC gradient for shotgun analysis
is 5-35% solvent B in 120 minutes followed by 35-100% solvent B in
2 minutes and 100% solvent B for 8 minutes (total gradient length
is 130 minutes). LC-MS/MS shotgun runs for peptide discovery are
carried out on a Thermo Scientific (Thermo Fisher Scientific)
(Billerica, Mass.) Q Exactive mass spectrometer equipped with a
standard nano-electrospray source. The LC gradient for LC-MRM is
5-35% solvent B in 30 minutes followed by 35-100% solvent B in 2
minutes and 100% solvent B for 8 minutes (total gradient length is
40 minutes). The Thermo Scientific (Thermo Fisher Scientific)
(Billerica, Mass.) TSQ Vantage triple quadrupole mass spectrometer
is equipped with a standard nano-electrospray source. In
unscheduled MRM mode for recalibration it is operated at a dwell
time of 20 ms per transition. For relative quantification of the
peptides across samples, the TSQ Vantage is operated in scheduled
MRM mode with an acquisition window length of 4 minutes. The LC
eluent is electrosprayed at 1.9 kV and MRM analysis is performed
using a Q1 peak width of 0.7 Da. Collision energies are calculated
for the TSQ Vantage by a linear regression according to the
vendor's specifications.
C. Assay Design, Data Processing and Analysis
[1063] For the generation of LC-MRM assays, the 12 most intense
fragment ions from LC-MS/MS analysis are measured in scheduled
LC-MRM mode and data were processed using MQUEST.RTM. (Cluetec,
Karlsruhe, Germany), the scoring part of mProphet (Reiter et al,
mProphet: Automated data processing and statistical validation for
large-scale SRM experiments, Nature Methods, 2011 (8), 430-435; the
contents of which are herein incorporated by reference). Assays
were validated manually, exact fragment intensities are determined
and iRTs (indexed retention times) are assigned relative to
Biognosys's iRT-peptides (Escher et al. Using iRT, a normalized
retention time for more targeted measurement of peptides,
Proteomics, 2012 (12), 1111-1121; the contents of which are herein
incorporated by reference).
[1064] For the relative quantification of the peptides across the
sample series the 8 most intense transitions of each assay are
measured across the sample series. Data analysis is carried out
using SpectroDive.TM. (Biognosys, Schlieren Switzerland). Total
peak areas are compared for the selected peptides and a false
discover rate of 0.05 is applied. Peptides with a Qvalue below 0.05
are excluded and considered not detected in the respective
sample.
Example 32
Confirmation and of Peptide Identity from Chemically Modified
mRNA
[1065] Cell lysates containing protein produced from siah E3
ubiquitin protein ligase 1 (SIAH1) modified mRNA (mRNA sequence
shown in SEQ ID NO. 6618 (Table 29); polyA tail of approximately
140 nucleotides not shown in sequence; 5' cap, Cap1), MYC inhibitor
D (a unique dominant-negative 90 amino acid protein comprised of
the human c-Myc) modified mRNA (mRNA sequence shown in SEQ ID NO.
6621 (Table 29); polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1), fully modified with
5-methylcytidine and pseudouridine (5 mC and pU), fully modified
with 5-methylcytidine and 1-methylpsudouridine (5 mC and 1 mpU),
modified where 25% of uridine modified with 2-thiouridine and 25%
of cytidine modified with 5-methylcytidine (s2U and 5 mC), fully
modified with pseudouridine (pU), or fully modified with
1-methylpseudouridine (1 mpU) were evaluated using the LC-MS/MS
with quantitative LC-MRM as described in Example 31. Peptide
fragments identified for the evaluated proteins are shown in Table
30.
TABLE-US-00031 TABLE 30 Proteins and Peptide Fragment Sequences
Peptide 5mC 5mC s2U Fragment and and and SEQ ID NO pU 1mpU 5mC pU
1mpU SIAH1 GPLGSIR 6622 YES -- YES -- YES MYC INHIBITOR D
ATAYILSVQAET 6623 YES YES YES YES YES QK KATAYILSVQAE 6624 YES YES
YES YES YES TQK LISEIDLLRK 6625 YES YES YES YES YES
Example 33
Confirmation and of Peptide Identity from 1-Methylpseudouridine
Modified mRNA
[1066] Cell lysates containing protein produced from granulysin
mRNA (mRNA sequence shown in Table 29; polyA tail of approximately
140 nucleotides not shown in sequence; 5' cap, Cap1) fully modified
with 1-methylpseudouridine (1 mpU) were evaluated using the
LC-MS/MS with quantitative LC-MRM as described in Example 31.
Peptide fragments identified for the evaluated proteins are shown
in Table 31. In Table 31, "Uniprot ID" refers to the protein
identifier from the UniProt database when the peptide fragment
sequences were blasted against all review proteins in the
database.
TABLE-US-00032 TABLE 31 Proteins and Peptide Fragment Sequences
Peptide Fragment SEQ ID NO Uniprot ID GRANULYSIN SCPCLAQEGPQGDLLTK
6626 P22749
Example 34
Signal-Sensor Polynucleotides in the Treatment of Cancer (HCC):
Disruption of Cancer Cell Transcriptome Using Dominant Negative
STAT3 and Akt mRNA
[1067] Using the animal models outlined in Example 13, animals are
treated with signal-sensor polynucleotide encoding for a dominant
negative STAT3 molecule or a dominant negative Akt molecule whose
expression has been shown to interfere with PI-3 kinase induced
oncogenic transformation, including in glioblastoma cells (Vogt and
Hart, Cancer Discov, 2011 1:481-486; herein included by reference
in its entirety). Animals are injected with mRNA encoding dominant
negative STAT3 mRNA vs dominant negative Akt mRNA vs negative
control mRNA (non-translated version of the same mRNA containing
multiple stop codons) vs vehicle using an appropriate route of
delivery and formulation. Animals are then evaluated for gene
expression, tumor status or for any of the hallmarks associated
with cancer phenotypes or genotypes. Other examples of dominant
negative approaches for cancer are outlined and could similarly be
used with modified mRNA (Moss and Lemoine Chapter 15 RNA
Interference and Dominant Negative Approaches in Viral Therapy of
Cancer Harrington et al., eds. Wiley & Sons; herein
incorporated by reference in its entirety).
Example 35
Signal-Sensor Polynucleotides in the Treatment of Cancer (HCC):
Disruption of Cancer Cell Transcriptome Using Dominant Negative
hTERT mRNA
[1068] Using the animal models outlined in Example 13, animals are
treated with signal-sensor polynucleotide encoding for a dominant
negative hTERT whose expression has been shown to interfere with
telomerase activity and lead to apoptosis of cancer cells (Agrawal
et al. 2012 Recent Pat Anricancer Drug Discov 7:102-117, Samy et
al. 2012 Mol Cancer Ther 11:2384-2393, Nguyen et al. 2009 Cell
Cycle. 8:3227-3233; all herein included by reference in their
entirety). Telomerase, a specialised RNA-directed DNA polymerase
extends and stabilises the telomeres at the ends of the eukaryotic
chromosomes. The progressive loss of telomeres results in limited
number of cell divisions and has been linked to the mechanism of
human cellular ageing. Tumour cells marked by indefinite
proliferation have stable telomere length maintained by telomerase.
The differential expression of the telomerase enzyme in normal and
cancer cells have led to the evolution of tumour specific
anti-telomerase approaches which inhibit the telomerase enzyme
activity so as to destabilise and shorten the telomeres leading to
senescence in cancer cells. One such approach is to use modified
mRNA to express a dominant negative hTERT. As such animals are
injected with mRNA encoding dominant negative hTERT mRNA vs
negative control mRNA (non-translated version of the same mRNA
containing multiple stop codons) vs vehicle using an appropriate
route of delivery and formulation. Animals are then evaluated for
gene expression, tumor status or for any of the hallmarks
associated with cancer phenotypes or genotypes. Other examples of
dominant negative approaches for cancer are outlined and could
similarly be used with modified mRNA (Moss and Lemoine Chapter 15
RNA Interference and Dominant Negative Approaches in Viral Therapy
of Cancer Harrington et al., eds. Wiley & Sons; herein
incorporated by reference in its entirety).
Example 36
Signal-Sensor Polynucleotides in the Treatment of Cancer (HCC):
Disruption of Cancer Cell Transcriptome Using Dominant Negative
Survivin mRNA
[1069] Using the animal models outlined in Example 13, animals are
treated with signal-sensor polynucleotide encoding for a dominant
negative survivin (C84A and others) whose expression has been shown
to lead to apoptosis of cancer cells (Cheung et al. 2010 Cancer
Cell Int. 10:36; herein included by reference in its entirety).
Survivin is a member of the inhibitor-of-apoptosis (IAP) family
which is widely expressed by many different cancers. Overexpression
of survivin is associated with drug resistance in cancer cells, and
reduced patient survival after chemotherapy and radiotherapy.
Agents that antagonize the function of survivin hold promise for
treating many forms of cancer. One such approach is to use modified
mRNA to express a dominant negative survivin (C84A mutation is one
described example). As such animals are injected with mRNA encoding
dominant negative survivin mRNA vs negative control mRNA
(non-translated version of the same mRNA containing multiple stop
codons) vs vehicle using an appropriate route of delivery and
formulation. Animals are then evaluated for gene expression, tumor
status or for any of the hallmarks associated with cancer
phenotypes or genotypes. Other examples of dominant negative
approaches for cancer are outlined and could similarly be used with
modified mRNA (Moss and Lemoine Chapter 15_RNA Interference and
Dominant Negative Approaches in Viral Therapy of Cancer Harrington
et al., eds. Wiley & Sons; herein incorporated by reference in
its entirety).
Example 37
Expression of Modified Nucleic Acid with microRNA Binding Site
[1070] Human embryonic kidney epithelial cells (HEK293A), or
antigen presenting cells or cell lines with highly expressed
mir-142/146, such as monocyte-derived dendritic cells (MDDC) or
PBMC, are seeded at a density of 200,000 per well in 500 ul cell
culture medium (InVitro GRO medium from Celsis, Chicago, Ill.).
G-CSF mRNA (mRNA sequence is shown in SEQ ID NO: 6595; polyA tail
of at least 140 nucleotides not shown in sequence; 5'Cap, Cap1)
G-CSF mRNA having a miR-142-5p binding site (G-CSF miR-142-5p)
(cDNA sequence is shown in SEQ ID NO:6627; mRNA sequence is shown
in SEQ ID NO: 6628, polyA tail of at least 140 nucleotides not
shown in sequence; 5'Cap, Cap1), G-CSF mRNA having a seed sequence
from miR-142-5p binding site (G-CSF miR-142-5p-seed) (cDNA sequence
is shown in SEQ ID NO. 6629; mRNA sequence is shown in SEQ ID NO:
6630; polyA tail of at least 140 nucleotides not shown in sequence;
5'Cap, Cap1) G-CSF mRNA having a miR-142-5p binding site without
the seed sequence (G-CSF miR-142-5p-seedless) (cDNA sequence is
shown in SEQ ID NO: 6631, mRNA sequence is shown in SEQ ID NO:
6632; polyA tail of at least 140 nucleotides not shown in sequence;
5'Cap, Cap1) G-CSF mRNA having a miR-142-3p binding site (G-CSF
miR-142-3p) (cDNA sequence is shown in SEQ ID NO: 6633, mRNA
sequence is shown in SEQ ID NO: 6634; polyA tail of at least 140
nucleotides not shown in sequence; 5'Cap, Cap1; G-CSF mRNA having a
seed sequence from miR-142-3p binding site (G-CSF miR-142-3p-seed)
(cDNA sequence is shown in SEQ ID NO: 6635, mRNA sequence is shown
in SEQ ID NO: 6636; polyA tail of at least 140 nucleotides not
shown in sequence; 5'Cap, Cap1) G-CSF mRNA having a miR-142-3p
binding site without the seed sequence (G-CSF miR-142-3p-seedless)
(cDNA sequence is shown in SEQ ID NO: 6637; mRNA sequence is shown
in SEQ ID NO: 6638; polyA tail of at least 140 nucleotides not
shown in sequence; 5'Cap, Cap1) G-CSF mRNA having a miR-146a
binding site (G-CSF miR-146a) (cDNA sequence is shown in SEQ ID NO.
6639, mRNA sequence is shown in SEQ ID NO: 6640; polyA tail of at
least 140 nucleotides not shown in sequence; 5'Cap, Cap1) G-CSF
mRNA having a seed sequence from miR-146a binding site (G-CSF
miR-146a-seed) (cDNA sequence is shown in SEQ ID NO.6641, mRNA
sequence is shown in SEQ ID NO:6642; polyA tail at least 140
nucleotides not shown in sequence; 5'Cap, Cap1) or G-CSF mRNA
having a miR-146a binding site without the seed sequence (G-CSF
miR-146a-seedless) (cDNA sequence is shown in SEQ ID NO.6643, mRNA
sequence is shown in SEQ ID NO: 6644; polyA tail at least
nucleotides not shown in sequence; 5'Cap, Cap1) are tested at a
concentration of 250 ng per well in 24 well plates. The mRNA
sequences are evaluated with various chemical modifications
described herein and/or known in the art including, fully modified
with 5-methylcytidine and pseudouridine, fully modified with
5-methylcytidine and 1-methylpseudouridine, fully modified with
pseudouridine, fully modified with 1-methylpseudouridine and where
25% of the uridine residues are modified with 2-thiouridine and 25%
of the cytidine residues are modified with 5-methylcytidine. The
expression of G-CSF in each sample is measured by ELISA.
[1071] Shown in Table 32 are the DNA and mRNA G-CSF sequences with
the miR binding sites described above. In the table, the start
codon of each sequence is underlined.
TABLE-US-00033 TABLE 32 G-CSF constructs with miR binding sites SEQ
ID NO. Description SEQ 6627 DNA TAATACGACTCACTATA sequence
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF miR-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 142-5p
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTAGTAGTGCTTTCTACTTTATGTGGTCTTTGAATAAAGCCTGA
GTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 6628 mRNA
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence:
CACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGG G-CSF miR-
CCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAA 142-5p
GAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUU
CCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAU
GGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUG
CCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUC
CCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUG
GCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCA
GGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCC
CGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACA
ACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCU
GCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUC
AGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCA
UUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCC
GUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGC
CCUUCUUCUCUCCCUUGCACCUGUACCUCUAGUAGUGCUUUCUA
CUUUAUGUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 6629 DNA TAATACGACTCACTATA
sequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF miR-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 142-5p-seed
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTACTTTATTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGC CGCTCGAGCATGCATCTAGA
6630 mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence:
CACC G-CSF miR- AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCU
142-5p-seed GCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAG
CGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUU
UUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAG
CCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAU
CCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUG
GGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAG
GGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGA
CUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGAC
GCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCA
UCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAG
CCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCG
CAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUU
UGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG
UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCC
CUUCUUCUCUCCCUUGCACCUGUACCUCUACUUUAUUGGUCUUU GAAUAAAGCCUGAGUAGGAAG
6631 DNA TAATACGACTCACTATA sequence
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF miR-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 142-5p-
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC seedless
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTAGTAGTGCTTTCTGTGGTCTTTGAATAAAGCCTGAGTAGGAA
GGCGGCCGCTCGAGCATGCATCTAGA 6632 mRNA
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence: CACC G-CSF
miR- AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCU 142-5p-
GCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAG seedless
CGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUU
UUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAG
CCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAU
CCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUG
GGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAG
GGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGA
CUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGAC
GCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCA
UCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAG
CCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCG
CAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUU
UGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG
UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCC
CUUCUUCUCUCCCUUGCACCUGUACCUCUAGUAGUGCUUUCUGU
GGUCUUUGAAUAAAGCCUGAGUAGGAAG 6633 DNA TAATACGACTCACTATA sequence
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF miR-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 142-3p
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTTCCATAAAGTAGGAAACACTACATGGTCTTTGAATAAAGCCT
GAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 6634 mRNA
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence: CACC G-CSF
miR- AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCU 142-3p
GCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAG
CGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUU
UUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAG
CCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAU
CCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUG
GGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAG
GGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGA
CUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGAC
GCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCA
UCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAG
CCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCG
CAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUU
UGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG
UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCC
CUUCUUCUCUCCCUUGCACCUGUACCUCUUCCAUAAAGUAGGAA
ACACUACAUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 6635 DNA TAATACGACTCACTATA
sequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF miR-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 142-3p-seed
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTACACTACTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGC CGCTCGAGCATGCATCTAGA
6636 mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence:
CACC G-CSF miR- AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCU
142-3p-seed GCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAG
CGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUU
UUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAG
CCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAU
CCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUG
GGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAG
GGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGA
CUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGAC
GCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCA
UCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAG
CCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCG
CAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUU
UGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG
UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCC
CUUCUUCUCUCCCUUGCACCUGUACCUCUACACUACUGGUCUUU GAAUAAAGCCUGAGUAGGAAG
6637 DNA TAATACGACTCACTATA sequence
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF miR-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 142-3p-
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC seedless
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTTCCATAAAGTAGGAAATGGTCTTTGAATAAAGCCTGAGTAG
GAAGGCGGCCGCTCGAGCATGCATCTAGA 6638 mRNA
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence:
CACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGG G-CSF miR-
CCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAA 142-3p-
GAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUU seedless
CCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAU
GGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUG
CCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUC
CCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUG
GCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCA
GGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCC
CGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACA
ACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCU
GCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUC
AGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCA
UUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCC
GUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGC
CCUUCUUCUCUCCCUUGCACCUGUACCUCUUCCAUAAAGUAGGA
AAUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 6639 DNA TAATACGACTCACTATA sequence
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG
site and AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF miR-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 146a
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTAACCCATGGAATTCAGTTCTCATGGTCTTTGAATAAAGCCTG
AGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 6640 mRNA
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence:
CACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGG G-CSF miR-
CCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAA 146a
GAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUU
CCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAU
GGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUG
CCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUC
CCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUG
GCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCA
GGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCC
CGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACA
ACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCU
GCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUC
AGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCA
UUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCC
GUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGC
CCUUCUUCUCUCCCUUGCACCUGUACCUCUAACCCAUGGAAUUC
AGUUCUCAUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 6641 DNA TAATACGACTCACTATA
sequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 146a-seed
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTAGTTCTCTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGC CGCTCGAGCATGCATCTAGA
6642 mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence:
CACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGG G-CSF-
CCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAA 146a-seed
GAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUU
CCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAU
GGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUG
CCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUC
CCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUG
GCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCA
GGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCC
CGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACA
ACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCU
GCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUC
AGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCA
UUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCC
GUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGC
CCUUCUUCUCUCCCUUGCACCUGUACCUCUAGUUCUCUGGUCUU UGAAUAAAGCCUGAGUAGGAAG
6643 DNA TAATACGACTCACTATA sequence
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCC having the
ACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCC T7
CTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAG polymerase
CGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTG site and
AAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCA restriction
CTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAG sites:
GAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTC G-CSF-
TCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTC 146a-
CCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCC seedless
CTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTG
CAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATG
GAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGC
AATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGT
CCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGG
GTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTAC
CTCTAACCCATGGAATTCATGGTCTTTGAATAAAGCCTGAGTAGGA
AGGCGGCCGCTCGAGCATGCATCTAGA 6644 mRNA
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC sequence:
CACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGG G-CSF-
CCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAA 146a-
GAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUU seedless
CCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAU
GGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUG
CCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUC
CCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUG
GCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCA
GGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCC
CGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACA
ACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCU
GCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUC
AGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCA
UUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCC
GUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGC
CCUUCUUCUCUCCCUUGCACCUGUACCUCUAACCCAUGGAAUUC
AUGGUCUUUGAAUAAAGCCUGAGUAGGAAG
[1072] It is likely that the binding site "seed" sequence is
sufficient to induce mircoRNA binding, the expression of G-CSF
should be down-regulated in cells transfected with miR-142-3p,
miR-142-3p-seed, miR-142-5p, miR-142-5p-seed, miR-146a or
miR-146a-seed. Whereas, the miR-142-3p-seedless,
miR-142-5p-seedless, miR-146a-seedless should not change the
expression of G-CSF, as compared with cells transfected with G-CSF
mRNA without microRNA binding sites.
Example 38
APCs Specific microRNA Binding Sites to Suppress Modified Nucleic
Acid Mediated Immune Stimulation
[1073] The binding sites for microRNAs are used in the 3'UTR of
mRNA therapeutics to selectively degrade mRNA therapeutics in the
immune cells to subdue unwanted immunogenic reactions caused by
mRNA therapeutics delivery.
[1074] A signal-sensor polynucleotide comprising a series of 3'UTR
miR binding sites which make the signal sensor polynucleotide more
unstable in antigen presenting cells (APCs), such as, but not
limited to mir-142-5p, mir-142-3p, mir-146a-5p and mir-146a-3p,
encodes an oncology-related polypeptide of the present invention.
The addition of miR binding sites in the 3'UTR making a signal
sensnor polynucleotide unstable would subdue modified mRNA mediated
immune stimulation.
[1075] Experiments comparing the cytokine expression (e.g.
TNF-alpha) induced by the signal-sensor polypeptide with APCs
specific microRNA signature vs. without such signature is performed
in vitro by methods described herein and/or known in the art.
Example 39. In Vitro Expression of mRNAs with miR binding sites
[1076] Human embryonic kidney epithelial cells (HEK293A),
antigen-presenting cells or cell lines with highly expressed
mir-142/146, such as monocyte-derived dendritic cells (MDDC) or
PBMC, are seeded at a density of 200,000 per well in 500 ul cell
culture medium (InVitro GRO medium from Celsis, Chicago, Ill.).
Cultured cells are transfected with G-CSF mRNAs with or without
microRNA signature, as described in Example 37. The cells are
transfected for five consecutive days. The transfection complexes
are removed four hours after each round of transfection.
[1077] The culture supernatant is assayed for secreted G-CSF
(R&D Systems, catalog #DCS50), tumor necrosis factor-alpha
(TNF-alpha) and interferon alpha (IFN-alpha by ELISA every day
after transfection following manufacturer's protocols. The cells
are analyzed for viability using CELL TITER GLO.RTM. (Promega,
catalog #G7570) 6 hrs and 18 hrs after the first round of
transfection and every alternate day following that. At the same
time from the harvested cells, total RNA is isolated and treated
with DNASE.RTM. using the RNAEASY micro kit (catalog #74004)
following the manufacturer's protocol. 100 ng of total RNA is used
for cDNA synthesis using the High Capacity cDNA Reverse
Transcription kit (Applied Biosystems, cat #4368814) following the
manufacturer's protocol. The cDNA is then analyzed for the
expression of innate immune response genes by quantitative real
time PCR using SybrGreen in a Biorad CFX 384 instrument following
the manufacturer's protocol.
Example 40
In Vivo Detection of Innate Immune Response Study
[1078] To test the signal sensor protein expression and in vivo
immune response, female BALB/C mice (n=5) are injected
intramuscularly with G-CSF mRNA with or without microRNA signatures
as described in Example 37. Blood is collected at 8 hours after
dosing. The protein levels of G-CSF, TNF-alpha and IFN-alpha is
determined by ELISA.
[1079] The difference of cytokine production is seen as measured by
mouse TNF-alpha and IFN-alpha level in serum. Injection with G-CSF
modified mRNA having miR-142 and miR-146a binding site or binding
site seed shows a lower level of cytokine response in vivo.
Example 41
Expression of miR-122 in Primary Hepatocytes
[1080] Hepatocyte specific miR-122 level in rat and human primary
hepatocytes was measured. Hela Cells and primary rat and human
hepatocytes were cultured and RNAs were extracted from cell
lysates. The miR-122 level in rat and human primary hepatocytes was
compared with that in Hela cells. The miR-122 level is about 6 fold
increased in primary human hepatocytes and about 12 fold increased
in primary rat hepatocytes, respectively, as compared with that in
Hela cells.
Example 42
Expression of Modified Nucleic Acid with Mir-122 Binding Site in
Hepatocytes
[1081] Primary rat and human hepatocytes and Hela cells were seeded
at a density of 200,000 per well in 500 ul cell culture medium
(InVitro GRO medium from Celsis, Chicago, Ill.). G-CSF mRNA having
a miR-122 binding site in the 3'UTR (G-CSF miR-122-1X) (mRNA
sequence is shown in SEQ ID NO: 6600; polyA tail of approximately
140 nucleotides not shown in sequence; 5'Cap, Cap1) fully modified
with 5-methylcytidine and pseudouridine (5 mC/pU), or fully
modified with pseudouridine (pU) or G-CSF mRNA with four miR-122
binding sites with the seed deleted (G-CSF no seed) (mRNA sequence
is shown in SEQ ID NO: 6601; polyA tail of approximately 140
nucleotides not shown in sequence; 5'Cap, Cap1) fully modified with
5-methylcytidine and pseudouridine (5 mC/pU) or fully modified with
pseudouridine (pU) was tested at a concentration of 250 ng per well
in 24 well plates. The 24 hours after transfection, the expression
of G-CSF was measured by ELISA, and the results are shown in Table
33.
TABLE-US-00034 TABLE 33 G-CSF mir122 expression Primary human
Primary rat Hela cells Hepatocytes Hepatocytes Protein Protein
Protein Expression Expression Expression (ng/mL) (ng/mL) (ng/mL)
G-CSF miR-122 1X 167.34 67.60 3.40 (5mC/pU) G-CSF miR-122 1X 292.18
116.18 25.63 (pU) G-CSF no seed 194.78 129.77 8.39 (5mC/pU) G-CSF
no seed (pU) 335.78 462.88 84.93
Example 43
Expression of Modified Nucleic Acids with Mir-122 Binding Sites in
Hepatocytes
[1082] MicroRNA control gene expression through the translational
suppression and/or degradation of target messenger RNA. Mir-122
binding site containing G-CSF mRNA was translationally regulated in
hepatocytes.
[1083] Primary rat and human hepatocytes and Hela cells were seeded
at a density of 200,000 per well in 500 ul cell culture medium
(InVitro GRO medium from Celsis, Chicago, Ill.). G-CSF mRNA (G-CSF
alpha) (mRNA sequence is shown in SEQ ID NO: 6599; polyA tail of
approximately 140 nucleotides not shown in sequence; 5'Cap, Cap1)
fully modified with 5-methylcytidine and pseudouridine (5 mC/pU),
G-CSF mRNA having a miR-122 binding site in the 3'UTR (G-CSF
miR-122-1X) (mRNA sequence is shown in SEQ ID NO: 6600; polyA tail
of approximately 140 nucleotides not shown in sequence; 5'Cap, Cap
1) fully modified with 5-methylcytidine and pseudouridine (5mc/pU)
or G-CSF mRNA with four miR-122 binding sites with the seed deleted
(G-CSF no seed) (mRNA sequence is shown in SEQ ID NO: 6601; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'Cap,
Cap1) fully modified with 5-methylcytidine and pseudouridine (5
mC/pU) was tested at a concentration of 250 ng per well in 24 well
plates. 24 hours after transfection, the expression of G-CSF was
measured by ELISA. The G-CSF drug (mRNA) levels and protein levels
are shown in Table 34.
TABLE-US-00035 TABLE 34 G-CSF drug and protein levels Human
Hepatocytes Rat Hepatocytes Drug (mRNA) Protein Drug (mRNA) Protein
level (unit expres- level (unit expres- normalized sion normalized
sion to HPRT) (ng/ml) to HPRT) (ng/ml) G-CSF alpha 43237.6 247.26
26615.88 784.6 (5mC/pU) G-CSF miR-122- 46340.9 74.07 20171.07
40.628 1X (5mC/pU) G-CSF no seed 70239.7 298.28 23170.47 894.06
(5mC/pU)
Example 44
Microphysiological Systems
[1084] The polynucleotides, primary constructs and/or mmRNA of the
present invention are formulated using one of the methods described
herein such as in buffer, lipid nanoparticles and PLGA. These
formulations are then administered to or contacted with
microphysiological systems created from organ chips as described in
International Publication Nos. WO2013086502, WO2013086486 and
WO2013086505, the contents of each of which are herein incorporated
by reference in its entirety.
Example 45
Translation Enhancing Elements (TEEs) in Untranslated Regions
[1085] The 5' and/or 3' untranslated regions (UTRs) in the
signal-sensor polynucleotides, primary constructs and/or mmRNA
described herein may include at least one translation enhancing
element (TEE). Such TEE which may be included in the 5'UTR and/or
3'UTR include, but are not limited to, those listed in Table 35,
including portion and/or fragments thereof. The TEE sequence may
include at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 99% or more than 99% of the TEE sequences
disclosed in Table 35 and/or the TEE sequence may include a 5-30
nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide
fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of
the TEE sequences disclosed in Table 35.
TABLE-US-00036 TABLE 35 TEE Sequences TEE Identifier Sequence SEQ
ID NO TEE-001 MSCSGCNGMWA 6645 TEE-002 RNSGAGMGRMR 6646 TEE-003
RNSGAGMGRMRRR 6647 TEE-004 RMSCSGCNGMWR 6648 TEE-005 GCGAGAGAA --
TEE-006 GGGAGCGAA -- TEE-007 GCGAGAGGA -- TEE-008 GCGAGCGGA --
TEE-009 CGGAGCGAA -- TEE-010 CGGAGCGGA -- TEE-011 ACGAGAGGA --
TEE-012 ACGAGCGGA -- TEE-013 GACGAGAGGA 6649 TEE-014 GACGAGAGAA
6650 TEE-015 AGCGAGCG -- TEE-016 AGGAGAGGA -- TEE-017 GCCGAGAGA --
TEE-018 CGAGAGGCA -- TEE-019 GAGAGGAGC -- TEE-020 CGCGGCGGA --
TEE-021 CGCCGCCGC -- TEE-022 GCGGCTGAA -- TEE-023 CCGGCTGAA --
TEE-024 CGCCGCTGAA 6651 TEE-025 CGCCGCGGAA 6652 TEE-026 CGCCGCCGAA
6653 TEE-027 CCCGCGGAA -- TEE-028 CCCGCCGAA -- TEE-029 CCCGCTGAA --
TEE-030 CCCGGCGGA -- TEE-031 CGCGGCTGA -- TEE-032 CGGCTGCTA --
TEE-033 CCCGGCGGA -- TEE-034 AGCCGCCGCA 6654 TEE-035 ACGCCGCCGA
6655 TEE-036 GGCATTCATCGT 6656 TEE-037 GCATTAGTATCT 6657 TEE-038
TCGGTTATTGTT 6658 TEE-039 TCCAATTGGGAA 6659 TEE-040 ATCTATTGGCCA
6660 TEE-041 TTACTGGGTGTT 6661 TEE-042 AGGGTGAAGGTC 6662 TEE-043
GGTGGGTGTGTC 6663 TEE-044 CGCTTCAATGCT 6664 TEE-045 TGCTTCAATGCC
6665 TEE-046 TGTGTCTTTGCA 6666 TEE-047 CACGGGGACAGC 6667 TEE-048
AAGCTGTACATG 6668 TEE-049 GATGGGGGCACA 6669 TEE-050 ATATGTGCCCTT
6670 TEE-051 TCCTTCTGGGTC 6671 TEE-052 GGTGGGTGTGTC 6672 TEE-053
GAATGGATGGGG 6673 TEE-054 CAXGTGATATTC 6674 TEE-055 AGGAGGGTTTGT
6675 TEE-056 TGGGCGAGTGGG 6676 TEE-057 CGGCTCACCAGT 6677 TEE-058
GGTTTCXATAAC 6678 TEE-059 GGTGGGTGTGTC 6679 TEE-060 TTACTGGGTGTT
6680 TEE-061 AAGTCTTTGGGT 6681 TEE-062 CCGGCGGGU -- TEE-063
CCGGCGGG -- TEE-064 CCGGCGG -- TEE-065 CCGGCG -- TEE-066 CCGGC --
TEE-067 CGGCGGGU -- TEE-068 GGGAGACGGCGGCGGTGGCGGCGCGGGCAGAGCAAG
6682 GACGCGGCGGATCCCACTCGCACAGCAGCGCACTCGG TGCCCCGCGCAGGGTCG
TEE-069 AAAGAAATGGAATCGAAGAGAATGGAAACAAATGGA 6683
ATGGAATTGAATGGAATGGAATTGA ATGGAATGGGAACG TEE-070
AAAGAAATGGAATCGAAGAGAATGGAAACAAATGGA 6684 ATGGAATTGAATGGAATGGAATTGA
ATGGAATGGGAACG TEE-071 AGACAGTCAGACAATCACAAAGAAACAAGAATGAAA 6685
ATGAATGAACAAAACCTTCAAGAAATATGGGATTATG AAGAGGCCAAATGT TEE-072
AAAAGGAAATACAAGACAACAAACACAGAAACACAA 6686
CCATCGGGCATCATGAAACCTCGTGAAGATAATCATCA GGGT TEE-073
AGACCCTAATATCACAGTTAAACGAACTAGAGAAGGA 6687
AGAGCAAACAAATTCAAAAGCTAGCGGAAAGCAAGA AATAACTAAGACCAG TEE-074
AAAGACTTAAACATAAGACCTAAAACCATAAAAACCA 6688
CAGAAGAAAACATAGGCAATGCCATTCAGGACATAGG CATGGGCAAAGACTTC TEE-075
AGCAATAACCAAACAACCTCATTAAAAAGTAGGCAAA 6689
GGACATAAACAGACACTTTTCAAAAGAAGACATACAC GTGGCCAACAAACATATG TEE-076
AGAAAGAATCAAGAGGAAATGCAAGAAATCCAAAAC 6690
ACTGTAACAGATATGATGAATAATGAGGTATGCACTC ATCAGCAGACTCGACAT TEE-077
GCACTAGTCAGATCAAGACAGAAAGTCAACGAACAAA 6691
GAACAGACTTAAACTACACTCTAGA ACAAATGGACCTA TEE-078
AGCAGCCAACAAGCATATGAAATAATGCTCCACAACA 6692
CTCATCATCAGAGAAATGCAAATCA AAACCAAAAT TEE-079
AATATACGCAAATCAATAAATGTAATCCAGCATATAA 6693
ACAGTACTAAAGACAAAAACCACAT GATTATCTCAATAGATGCAGAAAAGGCC TEE-080
ATGTACACAAATCAATAAATGCAGTCCAGCATATAAA 6694
CAGAACCAAACACAAAAACCACATG ATTATCTCAATAGATGCAGAAAAGGCCTTT TEE-081
TATACCACACAAATGCAAAAGATTATTAGCAACAATT 6695
ATCAACAGCAATATGTCAACAAGTT GACAAACCTAGAGGACATGGAT TEE-082
AAACACACAAAGCAACAAAAGAACGAAGCAACAAAA 6696 GCATAGATTTATTGAAATGAAAGTA
CATTCTACAGAGTGGGGGCAGGCT TEE-083
GAAATCATCATCAAACGGAATCGAATGGAATCATTGA 6697
ATGGAATGGAATGGAATCATCATGG AATGGAAACG TEE-084
AACAGAATGGAATCAAATCGAATGAAATGGAATGGAA 6698 TAGAAAGGAATGGAATGAAATGGA
ATGGAAAGGATTCGAATGGAATGCAATCG TEE-085
TACAAAGAACTCAAACAAATCAGCAAGAACAAAAACA 6699 ATCCCAACAAAATGTTGGACAAAG
ACATGAATAGACAATTCTCGAAAGAAGATGTACAAAT GGCT TEE-086
TGTTGAGAGAAATTAAACAAAGCACAGATAAATGGAA 6700
AAACGTGTTCATAGATTGAAAGACT TCATGTTGTATGGTGTC TEE-087
AAACGATTGGACAGGAATGGAATCACCATCGAATGGA 6701
AACGAATGGAATCTTCGAATGGAAT TGAATGAAATTATTGAACGGAATCAAATAGAATCATC
ATTGAACAGAATCAAATTGGATCAT TEE-088
AACAATAAACAAACTCCAACTAGACACAATAGTCAAA 6702
TTGCTGAAAATGAAATATAAAGGAA CAATCTCGATGGTAGCCCAAGGA TEE-089
AAATCAATAAATGTAATTCAGCATATAAACAGAACCA 6703
AAGACAAAAACCACATGATTATCTC AATAGATGCAGAAAAGGCCTTT TEE-090
GCTCAAGGAAATAAAATAGGACACAAAGAAATGGAA 6704 AAACATTCCATACTCATGGATAGAA
AGAATCAATATCATGAAATGGCC TEE-091
AACATACGCAAATCAATAAATGTAATCCAGCATATAA 6705
ACAGAACCAAAGACAAAAACCACAT GATTATCTCAATAGATGCAGAAAAGGCC TEE-092
AACAATCACTAGTCCTTAAGTAAGAGACAACACCTTTT 6706
GTCACACACAGTTTGTCCTAACTTT ATCTTGGTAATTGGGGAGACC TEE-093
AGAAAACACACAGACAACAAAAAACACAGAACGACA 6707 ATGACAAAATGGCCAAGC
TEE-094 ACACAACAACCAAGAAACAACCCCATTAAGAAGTGGG 6708
AAAAATACATGAATAAACACATCTC AAAAGAAGACAAACAAGTGGCTAAC TEE-095
ACAGCAGAAAACGAACATCAGAAAATCACTCTACATG 6709
ATGCTTAAATACAGAGGGCAAGCAA CCCAAGAGAAAACACCACTTCCTAAT TEE-096
GAATAGAACAGAATGGAATCAAATCGAATGAAATGGA 6710 ATGGAATAGAAAGGAATGGAATGA
AATGGAATGGAAAGGATTCGAATGGAATG TEE-097
TAAGCAGAGAAAATATCAACACGAAAATAATGCAAGG 6711 AGAAAAATACAGAACAATCCAAAA
TGTGGCC TEE-098 GAACAATCAATGGAAGCAGAAACAAATAAACCAAGGT 6712
GTGCATCAAGGAATACATTCACGC ATGATGGCTGTATGAGTAAAATG TEE-099
GATCAATAAATGTAATTCATCATATAAACAGAGAACT 6713
AAAGACAAAAACACATGATTATCGC AATACATGCAGAAAAGGCC TEE-100
GACAAGAGTTCAGAAAGGAAGACTACACAGAAATACG 6714
CATTTTAAAGTCACTGACATGGAGA TGACACTTAAAACCATGAACATGGATGGG TEE-101
AAGCAAAGAAAGAATGAAGCAGCAAAAGAACGAAAG 6715 CAGGAATTTATTGAAAACCAAAGTA
CACTCCACAGTATGGGAGCGGACCCGAGCA TEE-102
ACCAACATAAGACAAAGAAACATCCAGCAGCTGCCTA 6716
TGGCAAAAGATTACAATGTGTCAAA CAAGAGGGCAATG TEE-103
GGACAAATTGCTAGAAATAAACAAATTACCAAAAATG 6717 ATTCAAGTAGAGACAGAGAATCAA
AATAGAACTACACATAAGTGGGCCAAG TEE-104
AACATAATCCATCAAATAAACAGAACCAAAGACAAAA 6718
ACCACATGATTATCTCAATAGATGC AGAAAAGGCCTTC TEE-105
AAAATCAATATGAAAACAAACACAAGCAGACAAAGA 6719 AAATTGGGCAAAAGGTTTGAGCAGA
CACTTCACCAAAGAAGTACAAATGGCAAATCAGCA TEE-106
AACCAAATTAGACAAATTGGAAATCATTACACATAAC 6720
AAAAGTAATAAACTGTCAGCCTCAG TAGTATTCATTGTACATAAACTGGCC TEE-107
AAGGAATTTAAGCAAATCAACAAGCAAAACCAAAATA 6721 ATCCCATTAAAAAGTGGGTAAAGG
ACATGAATACACACTTGTCAATAGAGGACATTCAAGT GGCCAAC TEE-108
TAACCTGATTTGCCATAATCCACGATACGCTTACAACA 6722
GTGATATACAAGTTACATGAGAAAC ACAAACATTTTGCAAGGAAACTGTGGCCAGATG TEE-109
AACTAACACAAGAACAGAAAACCAAACATCACATGTT 6723
CTCACTCATAAGCGGGAGCTGAACA ATGAGAACACACGGACACAGGGAGAGGAACATG TEE-110
TAAACTGACACAAACACAGACACACAGATACACACAT 6724
ACATACAGAAATACACATTCACACA CAGACCTGGTCTTTGGAGCCAGAGATG TEE-111
ATCAACAGACAACAGAAACAAATCCACAAAGCACTTA 6725
GTTATTAGAACTGTCATACAGACTG TACAACAACCACATTTACCAT TEE-112
AAATAAGCCAACGGTCATAAATTGCAAAGCCTTTTACA 6726
ATCCAAACATGATGGAAACGATAT GCCATTTTGAAGGTGATTTGAAAAGCACATGGTTT
TEE-113 AAACAGTTCAAAAATTATTGCAACAAAATGAGAGAGA 6727
TGAGTTTATCTTGCAAACTAATGGA TGGTAGCAGTGACAGTGGCAAAACGTGGTTTGATTCT
TEE-114 TAAGCAACTTCAGCAAAGTCTCAGGATACAAAATCAA 6728
TGTACAAAAATCACAAGCATTCTTA TACACCAACAACAGACAAACAAGAGTGCCAAATCATG
TEE-115 AGCAAACAAACAAACAAACAAACAAACTATGACAGG 6729
AACAAAACGTCACATATCAACATTA ACAAAGAATGTAAACAGCCTAAATGCTTCACTTAAAA
GTTATAGACAGGGGCTGGGCATGGT GGCTCACGCC TEE-116
GGAAATAACAGAGAACACAAACAAATGGGAAAACATT 6730 CCATGTTCATGGATAGGAAGAATC
AATATTGTGAAAATGGCCATACT TEE-117
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 6731
TACAAAAATCACAAGCATTCTTATA CACCAATAACAGACAAACAGAGAGCC TEE-118
AGATAAGAATAAGGCAAACATAGTAATAGGGAGTTCA 6732 TGAATAACACACGGAAAGAGAACT
TACAGGGCTGTGATCAGGAAACG TEE-119
AGGAAATAAAAGAAGACACAAACAAATGGAAGAACA 6733 TTCCATGCTTATGGATAGGGAGAAT
CAGTATCGTGAAAATGGCCATACT TEE-120
AACATACGAAAATCAATAAACGTAATCCAGCATATAA 6734 ACAGAACCAAAGACAAAAACCACA
TGATTATCTCAATAGATGCAGAAAAGGCCTTT TEE-121
AATGGACTCGAATGAAATCATCATCAAACGGAATCGA 6735
ATGGAATCATTGAATGGAATGGAAT GGAATCATCATGGAATGGAAACG TEE-122
AAGATTTAAACATAAGACCTAAAACGACAAAAATCCT 6736
AGGAGAAAACCTAAGCAATACCATT CAGGACATAGGCATGGGCAAAGACTTCATG TEE-123
TAATGAGAAGACACAGACAACACAAAGAATCACAGAA 6737 ACATGACACAGGTGACAAGAACAG
GCAAGGACCTGCAGTGCACAGGAGCC TEE-124
TAAACGTTAGACCTAAAACCATAAAAACCCTAGAAGA 6738
AAACCTAGGCATTACCATTCAGGAC ATAGGCATGGGCAAGGAC TEE-125
GAATTGAATTGAATGGAATGGAATGCAATGGAATCTA 6739 ATGAAACGGAAAGGAAAGGAATGG
AATGGAATGGAATG TEE-126 GTAATGGAATGGAATGGAAAGGAATCGAAACGAAAG 6740
GAATGGAGACAGATGGAATGGAATG GAACAGAG TEE-127
AGAGAAATGCAAATCAAAACCACAATGGAATACCATC 6741
TCACGCCAGTCAGAATGGCAATTAT TAAAAAATCACAACAATTAATGATGGCAAGGCTGTGG
TEE-128 AACATACACAAATCAATAAACGTAATCCAGCTTATAA 6742
ACAGAACCAAAGACAAAAACCACAT GATTATCTCAATAGATGCGGAAAAGGCC TEE-129
TAAACAGAACCAAAGACAAAAATCACATGATTATCTC 6743 AATAGATGCAGAAAAGGCC
TEE-130 AATGGAATGCAATCGAATGGAATGGAATCGAACGGAA 6744
TGGAATAAAATGGAAGAAAACTGG CAAGAAATGGAATCG TEE-131
AGATAAAAAGAACAGCAGCCAAAATGACAAAAGCAA 6745 AAAGCAAAATCGTGTTAGAGCCAGG
TGTGGTGATGTGTGCT TEE-132 AGGAAAGTTTTCAATATGAGAAAGATACAAACCAACA 6746
GAATAAGCAAACTGGATAAACAGA AAATACAGAGAGAGCCAAGG TEE-133
GCAATCTCAGGATACAAAATCAATGTGCAAAAATCAC 6747
AAGCATTCTCATACACCAATAACAG ACAAACAGAGCCAAATCATG TEE-134
AGCATTCATATCTTGCAGTGTTGGGAAAGAGTGAGAG 6748
GTTGTGATGTCAAGAAGGATAGGTC AGAAGTGGAAGGTATGGGGGATTGTGCCTGCTGTCAT
GGCT TEE-135 AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 6749
TGCAAAAATCACAAGCATTCTTATA CACCAATAACAGACAAACAGAGAGCC TEE-136
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 6750
TGCAAAAATCACAAGCATTCTTATA CACCAACAACAGACAAACAGAGAGCC TEE-137
TAAGCCGATAAGCAACTTCAGCAAAGTCTCAGGAGAC 6751
AAAATCAATGTGCAAAAAATCACAA GCATTCTTATACACTAATAACAGACAAACAGAGAGCC
AAATCATG TEE-138 AACGTGACATACATACAAAAAGTTTTTAGAGCAAGTG 6752
AAATTTTAGCTGCTATATGTTAATTG GTGGTAATCCC TEE-139
TACGCAAATCGATAAATGTAATCCAGCATATAAACAG 6753
AACCAAAGACAAAAACCACATGATT ATCTCAATAGATGCAGAAAAGGCC TEE-140
GCAATCGAATGGAATGGAATCGAACGGAATGGAATAA 6754 AATGGAAGAAAACTGGCAAGAAAT
GGAATCG TEE-141 TTGAATCGAATGGAATCGAATGGATTGGAAAGGAATA 6755
GAATGGAATGGAATGGAATTGACTC AAATGGAATG TEE-142
TAAAGAAAAACAAACAAACAGAAATCAATGAAAATCC 6756 CATTCAAAGGTCAGCAACCTCAAA
GACTGAAGGTAGATAAGCCCACAAGGATG TEE-143
GTCATATTTGGGATTTATCATCTGTTTCTATTGTTGTTG 6757
TTTTAGTACACACAAAGCCACAATA AATATTCTAGGCT TEE-144
AAAAGTACAGAAGACAACAAAAAATGAGAGAGAGAA 6758 AGATAACAGACTATAGCAGCATTGG
TGATCAGAGCCACCAG TEE-145 AACCCACAAAGACAACAGAAGAAAAGACAACAGTAG 6759
ACAAGGATGTCAACCACATTTTGGA AGAGACAAGTAATCAAACACATGGCA TEE-146
AAAGACCGAAACAACAACAGAAACAGAAACAAACAA 6760 CAATAAGAAAAAATGTTAAGCAAAA
CAAATGATTGCACAACTTACATGATTACTGAGTGTTCT AATGGT TEE-147
AATCAGTAAACGTAATACAGCATATAAACAGAACCAA 6761
AGACAAAAACCACATGATTATCTCA ATAGATGCAGAAAAGGCC TEE-148
AAGCAACTTCAGCAAAGTCTCAGGACACAAAATCAAT 6762
ATGCGAAAATCACAAGCATTCCTAT ACACCAATAATAGACAAACAGAGAGCCAAATCATG
TEE-149 AGCAACTTCAGCAAAATCTCAGGATACAAAATCAATG 6763
TACAAAAATCACAAGCATTCTTATA CACCAACAACAGACAAACAGAGAGCC TEE-150
TAATGCAAACTAAAACGACAATGAGATATCAATACAT 6764
AACTACCAGAAAGGCTAACAAAAAA ACAGTCATAACACACCAAAGGCTGATGAGTGAGGATG
TGCAG TEE-151 AGCAACTTCAGCAAAGTCTCAGGATACAAAATCGATG 6765
TGCAAAAATCACAAGCATTCTTATA CACCAATAACAGGCAAACAGAGAGCC TEE-152
GATATATAAACAAGAAAACAACTAATCACAACTCAAT 6766
ATCAAAGTGCAATGATGGTGCAAAA TGCAAGTATGGTGGGGACAGAGAAAGGATGC TEE-153
AAGACAGAACACTGAAACTCAACAGAGAAGTAACAAG 6767 AACACCTAAGACAAGGAAGGAGAG
GGAAGGCAGGCAG TEE-154 TAAGACACATAGAAAACATAAAGCAAAATGGCAGATG 6768
TAAATGCAACCTATCAATCAAAACA TTACGAATGGCTT TEE-155
TGAAACAAATGATAATGAAAATACAACATACCAAACA 6769
TACGAGATACAGTAAAAGCAGTACT AAGATGCAAGTATATATTGCTACAAGTGCCTAC TEE-156
AATGTAATCCAGCATATAAACAGAGCCAAAGACAAAA 6770
ACCACATGATTATCTCAATAGATGC
AGAAAAAGCCTTTGACAAAATTCAACAACCCTTCATGC TAAAAACTCTCAATAAATTAGGTAT
TGATGGGACG TEE-157 ACAAAATTGATAGACCACTAGCAAGACTAATAAAGAA 6771
GAAAAGAGAGAAGAATCATTACCA TTCAGGACATAGGCATGGGCAAGGAC TEE-158
AAGGATTCGAATGGAATGCAATCGAATGGAATGGAAT 6772 CGAACGGAATGGAATAAAATGGAA
GAAAACTGGCAAGAAATGGAATCG TEE-159
GATCATCAGAGAAACAGAGAAATGCAAATTAAAACCA 6773
CAATGAGATACTATCTCCACACAAG TCAGAATGGCTAT TEE-160
ATCAAAAGAAAAGCAACCTAACAAATACGGGAAGAAT 6774
ATTTGAATAGACATTTCACAGGAAA AGATATATGAATGGCCAAAAAGCAAATGAAAAG TEE-161
AACAGCAATGACAATGATCAGTAACAACAAGACTTTT 6775 AACTTTGAAAAAATCAGGACC
TEE-162 AAGAGCCTGAATAGCTAAAGTGATCATAAGCAAAAAG 6776
AACAAAGTCGGAAGCATCACATTAC CTGACTTCAAACTATACTCAAAGGCTATG TEE-163
ACTCAGGAAAAATAACGAATCCAACTCACAGGAGAAA 6777 GAAGTACAAACCAGAAACCAATTT
CAAATTACAAGGACCAGAATACTCATGTTGGCTGGCC AGT TEE-164
TTGACCAGAACACATTACACAATGCTAATCAACTGCAA 6778
AGGAGAATATGAACAGAGAGGAGG ACATGGATATTTTGTG TEE-165
AACATATGGAAAAAAACTCAACATCACTGATCATTAG 6779
AGAAATGCAAATCAAAACCACAATG AGATACCATCTCACGCCAGTCAGAATGGCG TEE-166
AGCAACTTCAGCAAAGACTCAGGATACAAAATCAATG 6780
TGCAAAAATCACAAGCATTCTTATA CACCAATAACAGACAGAGAGCCAAAT TEE-167
TGGGATATGGGTGAAAGAACAAGTTTGCAGAAAAGAT 6781
ACAGTGAATTATGGACCATGAGTTC GGGAAAGAAGGGTAGGACTGCG TEE-168
AGCAGTGCAAGAACAACATAACATACAAGTAAACAAA 6782 CACATGGGGCCAGGTAATAAAAAG
TCAGGCTCAAGAGGTCAG TEE-169 AAGGAAAAGTAAAAGGAACTTAACACCTTCAAGAAAA
6783 GACAGACAAATAACAAAACAGCAG TTTGATAGAATGAGATATCAGGGGATGGCA
TEE-170 GCTAGTTCAACATATGCAAATCAATAAACGTAATCCAT 6784
CACATAAACAGAACCAATGACAAA AACCACGATTATCTCAATAGATGCAGAAAAGGCC TEE-171
AACATCACTGATCATTAGAAACACACAAATCAAAACC 6785
ACAATAAGATACCATCTAACACCAG TCACAATGGCTATT TEE-172
AGAGCATCCACAAGGCCCAATTCAAAGAATCTGAAAT 6786
AATGTATTGTTACTGCAACAGTTGTG AGTACCAGTGGCATCAG TEE-173
GGAATAACAACAACAACAACCAAAAGACATATAGAAA 6787 ACAAACAGCACGATGGCAGATGTA
AAGCCTACC TEE-174 AAACGCAGAAACAAATCAACGAAAGAACGAAGCAAT 6788
GAAAGACAAAGCAACAAAAGAATG GAGTAAGAAAGCACACTCCACAAAGTGGAAGCAGGCT
GGGACA TEE-175 AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 6789
TGCAAAAATCACAAGCATTCCTATA CACCAACAACAGACAAACAGAGAGCC TEE-176
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 6790
GGAAAAAATCACAAGCATTCCTATA CATCAATAACAGACAAACAGAGAGCC TEE-177
ACACATTTCAAGGAAGGAAACAAGAACAGACAGAAAC 6791
ACAACATACTTCATGAAACCACATT TTAGCATCCTGGCCGAGTATTCATCA TEE-178
AGCAACTTCAGCAAAGTCTCAGGACACAAAATCAATG 6792
TGCAAAAATCACAAGCATTCTTATA CACCAATAACAGACAAACAGAGAGCC TEE-179
TATTTTACCAGATTATTCAAGCAATATATAGACAGCTT 6793
AAAGCATACAAGAAGACATGTATAG ATTTACATGCAAACACTGCACCACTTTACATAAGGGAC
TTGAGCAC TEE-180 CCCAACTTCAAATTATACTACAAGGCTACAGTAATCAA 6794
AAAAGCATAGTACTATTACAAAAA CAGACACACAGGCCAATGGAATACAAT TEE-181
AGAAAGGATTCGAATGGAATGAAAAAGAATTGAATGG 6795 AATAGAACAGAATGGAATCAAATC
GAATGAAATGGAATGGAATAGAAAGGAATGGAATG TEE-182
GTTTACAGTCAAGTGTACAAACAGAATATAAGCAAAC 6796
AAAAGAGAACATATACTTACAAACT ATGCTAAGTGCCATGAAGGAAAAG TEE-183
AAGAGTATTGAAGTTGACATATCTAGACTGATCAAGA 6797
ACAAAGACAAAAGGTACAGATTATC AAGAAAATGAGCGGGCAAAGCAAGATGGCC TEE-184
AGTAGAATTGCAATTGCAAATTTCACACATATACTCAC 6798
ACACAAGTACACACATCCACTTTTA CAACTAAAAAAACTAGCACCCAGGACAGGTGCAGTGG CT
TEE-185 TGAATGCTATAGAGCAGTAAAAACAAATAAATGAACT 6799
ACATTACAGCTACTTACAACCATAT GAAAGAATATAACCATAACAATGATGAGTGGACAAAA
GCTAAGTGTGAAAGAATGCATAGT GCTACAGCAGCCAACATTTACAGC TEE-186
GAATGGAATCAAATAGAATGGAATCGAAACAAATGGA 6800 ATGGAATGGAATGGGAGCTGAGAT
TGTGTCACTGCAC TEE-187 TAAAAGTGTGCTCAACATCATTGATCATCAGAGAAATG 6801
CAAATCAAAACTACAATGAGATAT CATCTCATCCCAGTCAAAGTGGCT TEE-188
TCAGACCATAGCAGATAACATGCACATTAGCAATACG 6802 ATTGCCATGACAGAGTGGTTGGTG
TEE-189 ACAAACAATCCAATTCGAAAATGGGCAAGATATTTCA 6803
CCAAAGACATGAGCTGATATTTCAC TEE-190
AGGAAAAACAACAACAACAACAGGAAAACAACCTCA 6804
GTATGAAGACAAGTACATTGATTTAT TCAACATTTACTGATCACTTTTCAGGTGGTAGGCAG
TEE-191 AACAAAACAAAAACCCAACTCAATAACAAGAAGACAA 6805
ACAACCCAATTTAAAATGAGCAAA GAACTTGATAAACATGTCTCCAAAGAAGATACGGCCA
AAGAGCAC TEE-192 ATACAACTAAAGCAAATATAAGCAACTAAAGCAACAG 6806
TACAACTAAAGCAAAACAGAACAA GACTGCCAGGGCCTAGAAAAGCCAAGAAC TEE-193
AACAACAACAACAACAGGAAAACAACCTCAGTATGAA 6807
GACAAGTACATTGATTTATTCAACA TTTACTGATCACTTTTCAGGTGGTAGGCAGACC TEE-194
AGAGAGTATTCATCATGAGGAGTATTACTGGACAAAT 6808
AATTCACAAACGAACAAACCAAAGC GATCATCTTTGTACTGGCTGGCTA TEE-195
AGTAAATCACCATAAAGAAGGTAAGAGTTCATTCACA 6809
AAAACAACAAACTGAAGAATCAGGC CATAGTA TEE-196
AAAATAGAATGAAAGAGAATCAAATGGAATTGAATCG 6810 AATGGAATCGAATGGATTGGAAAG
GAATAGAATGGAATGGAATGGAATG TEE-197
AAAAGATGCAAAAGTAGCAAATGCAATGTTAAAACAA 6811 GCAAAGAAAGAATCAGGTGGACCA
CATAGTGCAGTGCTTCTC TEE-198 TTCACAGCAGCATTACGCACAATAGCCAGAAGGTGGG
6812 AACAGACAAAATGCCTTTTGATGGG TEE-199
CCATAACACAATTAAAAACAACCTAAATGTCTAATAG 6813
AAGAACACTGTTCAGACCGGGCATG GTGGCTTATACC TEE-200
TGGATTTCAGATATTTAACACAAAATAGTCAAAGCAG 6814
ATAAATACTAGCAACTTATTTTTAAT GGGTAACATCATATGTTCGTGCCTT TEE-201
ATCATTGAATGCAATCACATGGAATCATCACAGAATG 6815
GAATCGTACGGAATCATCATCGAAT GGAATTGAATGGAATCATCAATTGGACTCGAATGGAA
ACATCAAATGGAATCGATTGGAAGT GTCGAATGGACTCG TEE-202
AGAAACAGCCAGAAAACAATTATTACCTACAGCATTA 6816
AAACTATTCAAATGACAGCATATTTT TCAGCAGAAATCATGAAGGCCAGAAGGACGTGTCAT
TEE-203 AAAATGATCATGAGAAAATTCAGCAACAAAACCATGA 6817
AATTGCAAAGATATTACTTTTGGGA TGGAACAGAGCTGGAAGGCAAAGAG TEE-204
AACCACTGCTCAAGGAAATAAGAGAGAACACAAACAA 6818
ATGAAAAAACATTCCATGCTCATGG ATAGGAAGAATCAG TEE-205
TACTCTCAGAAGGGAAGCAGATATTCAGCATAAATCA 6819
TATTGTTTGTACAAAGAGTCTGGGCA TGGTGAATGACACT TEE-206
TATAGTTGAATGAACACACATACACACACACATGCCA 6820
CAAAACAAAAACAAAGTTATCCTCA CACACAGGATAGAAACCAAACCAAATCCCAACACATG
GCAAGATGAT TEE-207 GCTCAAAGAAATCAGAAATGACACAAGCAAATGGAAA 6821
AACATGCCATGTTCATGAATATGAA GAATCAATATTGTTAAAATGGCCATACTGCTCA TEE-208
GGATACAAAATCAATGTACAAAAATCACAAGCATTCT 6822
TATACACCAATAACAGACAAACAGA GAGCC TEE-209
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 6823
TACAAAAATCACAAGCATTCTTATA CACCAACAACAGACAAACAGAGAGCC TEE-210
AGGAGAATAGCAGTAGAATGACAAAATTAGATTTTCA 6824
CATGAAACTTGATGACAGTGTAGGA AATGGACTGAAAGGACAAGAC TEE-211
AGCAACTTCAGCAAAGTCTCGGGATACAAAATCAATG 6825
TGCAAAAATCACAAGCATTCCTATA CACCAATAACAGGCAAACAGAGAGCC TEE-212
AAGTTCAAACATCAGTATTAACCTTGAACATCAATGGC 6826
CTACATGCATCACTTAAAACATACA GACAGGCAAATTGGGTTAAGAAAACAAACAAGCAAAC
AAAACATGTTCCAAACATTTGTTGG CTAT TEE-213
AAGAAACAATCAAAAGGAAGTGCTAGAAATAAAACAC 6827 ACTGTAATAGAAAAGAAGAATGCC
TTATGGGCTTATCAATAGACTAGACATGGCCAGG TEE-214
AAAGAAAGACAGAGAACAAACGTAATTCAAGATGACT 6828
GATTACATATCCAAGAACATTAGAT GGTCAAAGACTTTAAGAAGGAATACATTCAAAGGCAA
AAAGTCACTTACTGATTTTGGTGGA GTTTGCCACATGGAC TEE-215
AGCAACTTCAGCAAAGTTTCAGGATACAAAATCAATG 6829
TGCAAAAATCACAAGCATTCTTATA CACCAACAACAGACAAACAGAGAGCC TEE-216
AGAATCAAATGGAATTGAATCGAATGGAATCGAATGG 6830
ATTGGAAAGGAATAGAATGGAATG GAATGGAATG TEE-217
AAACAGAACCACAGATATCTGTAAAGGATTACACTAT 6831
AGTATTCAACAGAGTATGGAACAGA GTATAGTATTCAACAGAGTATGCAAAGAAACTAAGGC
CAGAAAG TEE-218 AAAAAATGTTCAACATCACTAGTCAGCAGAGAAATGC 6832
AAATCAAAATCACAATGAGATAACT TCTCACACCAGACAGCATGGC TEE-219
GAATCCATGTTCATAGCACAACAACCAAACAGAAGAA 6833
ATCACTGTGAAATAAGAAACAAAGC AAAACACAGATGTCGACACATGGCA TEE-220
AGGATACAAAATCAAAGTGCAAAAATCACAAGCATTC 6834
TTATACACCAATAACAGACAAACAG AGAGCC TEE-221
AACAGATTTAAACAAACCAACAAGCAAAAAACGAACA 6835 ACTCCATTCAAACATGGACAAAAG
ACACGAACAGACACTTTTCAAAGAAGACATACATGTG GCC TEE-222
AAAGACAATATACAAATGGCCAATAAGCACATGAAAA 6836
GACGCTCAACATCCTTAGTCGTTAA GGCAATGCAAATCAAAACCACAATG TEE-223
TAAACAACGAGAACACATGAACACAAAGAGGGGAAC 6837 AACAGACACCAAGACCTTCTTGAGG
GTGGAGGATGGGAGGAGGGAG TEE-224
GGTTCAACTTACAATATTTTGACTTGACAACAGTGCAA 6838
AAGCAATACACGATTAGTAGAAAC ACACTTCCAATGCCCATAGGACCATTCTGC TEE-225
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 6839
AGCAAAAATCACAAGCATTCTTACA CACCAATAACAGACAAACAGAGAGCC TEE-226
AATCCAGCATATAAACAGAACCAAAGACAAAAACCAC 6840
ATGATTATCTCAATAGATGCAGAAA AGGCC TEE-227
TGAAAATACAAATGACCATGCAAGTAATTCCGCAGGG 6841 AGAGAGCGGATATGAACAAACAGA
AGAAATCAGATGGGATAGTGCTGGCGGGAAGTCA TEE-228
GCAAATGATTATAAGTGCTGTTATAGAAACATTCAAAG 6842
ACCAGAAAAGGACCACAATGGCTG ACCAC TEE-229
AGTCAATAACAAGAAGACAAACAACCCAATTACAAAA 6843
TGGGATATGAATTTAATAGATGTTA CTCCAAGGAAGATACACAAATGGCCAAC TEE-230
ATGGTTAAAACTCAACAATGAAAACACAAACAGCGCA 6844 ATTTAAAAATGGGCAAAATGACAG
GCCAGACCCAGTGGCTCATGCG TEE-231
TAACTACTCACAGAACTCAACAAAACACTATACATGC 6845
ATTTACCAGTTTATTATAAAGATACA AGTCAGGAACAGCCAAATGGAAGAAATGTAAATGGCA AG
TEE-232 AACAGACCATAAATAAACACAGAAGACACACGAGTGT 6846
AAAGTCAGTGCCCCGCTGCGAATTA AATCGGGGTGATGTGATGGCGAGTGAGTGGGTAGTT
TEE-233 GAATAGAATAGAATGGAATCATCGAATGGAATCGAAT 6847
GGAATCATCATGATATGGAATTGAG TGGAATC TEE-234
GGAATCTATAATACAGCTGTTTATAGCCAAGCACTAAA 6848
TCATATGATACAGAAAACAAATGC AGATGGTTTGAAGGGTGGG TEE-235
AAGATAGAGTTGAAACAGTGGACAATTAAAGAGTAAT 6849
TTGGAAGAATGGTGAAATTACAGCC ATGCTTTGAATCAGGCGGGTTCACTGGC TEE-236
TGAAAAGAAGAATGACCATAAGCAAGCAGATGAAAA 6850
ACAAAACAGAATTTTTACAGACGTCT TGGACTGATATCTTGGGC TEE-237
AGGAATCTATAATACAGCTGTTTATAGCCAAGCACTAA 6851
ATCATATGATACAGAAAACAAATG CAGATGGTTTGAAGGGTGGG TEE-238
AGGAAAAGAAAGAAATAGAAAATGCGAAATGGTAAG 6852 AAAAAACAGCATAATAAACATTTGT
ATGGTGTTGATGGACAATGCATT TEE-239
TAACAGTACCAAAAAACAGTCATAATCTTCAAGAGCTT 6853
AAATTTAGCATGAAAGGAAGACAT TCATCAAAGAATCACACAAAGGAATGTAAAATTAAAT
GGAGATTAGTGCCAGGAAAGAGC TEE-240
GCAAAACACAAACAACGCCATAAAAAACTGGGCAAAG 6854
GATATGAACAGACATTTTTCAAAAC AAAACATACTTATGGCCAAC TEE-241
AACAAAATTGAACAACATGCAAAGAAACATAAACGAA 6855 GCAATGAAAGTGTGCAGATCCACT
GAAATGAAAGTGCTGTCCAGAGTGGGAGCCAGCTCGA GA TEE-242
GAATGGAATCAACATCAAACGGAATCAAACGGAATTA 6856
TCGAATGGAATCGAAGAGAATCATC GAATGGCCACGAATGGAATCATCTAATGGAATGGAAT
GGAATAATCCATGG TEE-243 TACAAGAAAATCACAGTAACATTTATAAAACACAGAA 6857
GTGTGAACACACAGCTATTGACCTT GAAAACAGTGAAAGAGGGTCAGCTGTAGAACTAAGAC
ATAAGCAAAGTTTTTCAATCAAGAA TACATGGGTGGCC TEE-244
AAGAATTGGACAAAACACACAAACAAAGCAAGGAAG 6858 GAATGAAAGGATTTGTTGAAAATGA
AAGTACACTCCACAGTGTGGGAGCAG TEE-245
ACAGTTAACAAAAACCGAACAATCTAATTACGAAATG 6859
AACAAAAGATATGAACAGACATTTC ACCCGAGAGTATACAGGGGCCAGGCATGGT TEE-246
AAACGCACAAACAAAGCAAGGAAAGAATGAAGCAAC 6860 AAAAGCAGAGATTTATTGAAAATGA
AAAATACACTCCACAGGGTGGG TEE-247
CACCATGAGTCATTAGGTAAATGCAAATCAAAACCAC 6861
AATGAAATACTTCACACCCATGAAG ATGGCTATAATAAAAAAACAGACA TEE-248
AGCAACTTCAGCAAAGTCTCAGGAGACAAAATCAATG 6862
TACAAAAATCACAAGCATTCTTATA CACCAATAACAGACAAACAGAGAGCC TEE-249
TGACATGCAAGAAATAAGGAAGTGCAAAAACAAACAA 6863 ACAAACAACAACAACAACAACAAC
AACAACAACAAAAAACAGTCCCAAAAGGATGGGCAG TEE-250
AGACTTGAAAAGCACAGACAACGAAAGCAAAAATGG 6864 ACAAATGGAATCACATCAAGCTAAA
AGGTTTTGCATGGCAAAGG TEE-251 GCAAAAGAAACAATCAGTAGAGTAAACAGACAACTCA
6865 TAGAATGCAAGAAAATCATCGCAA TCTGTACATCCAACAAAGGGCT TEE-252
ACAAAATCAAACTAACCTCGATAAGAATGCAAGTGAA 6866
TCAAAATGAGTTTCAAGGGGTTGTG GCTAGTACACGCTTTCTACAGCTG TEE-253
ACAAACCACTGCTCAAGGAAATAAGGACACAAACAAA 6867
TGGAACAACATTCCGTGCTCATGGA TAGGAAGAATCAATATCGTGAAAATGGCCATACT
TEE-254 GAACGATTTATCACTGAAAATTAATACTCATGCAAGTA 6868
GTAAACGAATGTAATGACCATGAT AAGGAGACGGACGGTGGTGATAGT TEE-255
AGCAGAAGAAATAACTGAAATCAGAGTGAAACTGAAT 6869 CAAATTGAGATGCAAAAATACATA
CGAAATGGCCAG TEE-256 TGAATAGACACACAGACCAATGGAACAGAATAGAGAA 6870
CACAGAATAAATCTGCACACTTATA GCCAGCTGATTTTTGACAAATTTGCCAAG TEE-257
AGCAACTTCAGCAGTCTCAGTATACAAAAACAATGTG 6871
CAAAAATCACAAGCATTCCTATATG CCAATAACAGACAAACAGAGAGCC TEE-258
ACCAATCAAGAAAACAATGCAACCCACAGAGAATGGA 6872 CAAAAGCAAGGCAGGACAATGGCT
TEE-259 GCCACAATTTTGAAACAACCATAATAATGAGAATACA 6873
CAAGACAACTCCAATAATGTGGGAA GACAAACTTTGCAATTCACATCATGGC TEE-260
GAAAATGAACAATATGAACAAACAAACAAAATTACTA 6874
CCCTTACGAAAGTACGTGCATTCTA GTATGGTGACAAAAAGGAAA TEE-261
TATGCAAATCAATAAACATAATCCATCACATAAACAG 6875
AAACAAAGACAAAATGACATGATTA TCTCAATAGATGCAGAAAAGGCC TEE-262
CACCCATCTGTAGGACCAGGAAGCCTGATGTGGGAGA 6876
GAACAGCAGGCTAAATCCAGGGTTG GTCTCTACAGCAGAGGGAATCACAAGCCTGTTAGCAA
GTGAAGAACCAACACTGGCAAGAGT GTGAAGGCC TEE-263
AGGATACAAAATCAATGTACAAAAATCACAAACATTC 6877
TTATACACCAACAACAGACAAACAG AGAGCCAAATCATGGGTG TEE-264
AGGAAAATGCAAATCAGAACGACTATAACACACCATC 6878
TCAAACTCGTTAGGATGGCTATTAT CAAAAAGTCAAGAGATAACAAATGTGGGCAAGGG
TEE-265 GTAACAAAACAGACTCATAGACCAATAGAACAGAATA 6879
GAGAATTCAGAAATAAGACTGCACT TCTATGACCATGTGATCTTAGACAAACCT TEE-266
AAAGGAAAACTACAAAACACTGCTGAAAGAAATCATT 6880 GACAACACAAACAAATGGAAACAC
ATCCCAAGATCATGGGTGGGTGGAATCAAT TEE-267
ACACACATACCAACAGAACATGACAAAAGAACAAAAC 6881 CAGCCGCATGCATACTCGATGGAG
ACAAAGGTAACACTGCAGAATGGTGAAGGAAGAACAG TCATTTTAATGACAGTGTTGGCT
TEE-268 AACTAAGACAACAGATTGATTTACACTACTATTTTCAC 6882
ACAGCCAAAAATATCACTATGGCAA TCGTCAAAAGGTCAATTCAAAGATGGGACAGT TEE-269
GATCAGCTTAGAATACAATGGAACAGAACAGATTAGA 6883
ACAATGTGATTTTATTAGGGGCCAC AGCACTGTTGACTCAAGTACAAGTTCTGACTCATGTAG
AACTAACACTTTT TEE-270 GAATGGAATCAAATCGAATGAAATGGAATGGAATAGA 6884
AAGGAATGGAATGAAATGGAATGG AAAGGATTCGAAT TEE-271
AAATGAACAAAACTAGAGGAATGACATTACCTGACTT 6885
CAAATTATACTACAGAGCTATAGTA ACCAAAACAGCATGGTACAGGCAT TEE-272
GGACAACATACACAAATCAGTCAAGATACATCATTTC 6886
AACAGAATGAAAGACAAAAACCATT TGATCACTTCAATCGATGATGAAAAAGCA TEE-273
AACTTCAGCAAATTCTCAGGATACAAAATCAATGTGCA 6887
AAAACCACAAGCATTCCTATACAC CAATAATAGACAGTGAGCCAAAT TEE-274
TATGACTTTCACAAATTACAGAAAAAGACACCCATTTG 6888
ACAAGGGAACTGAAGGTGGTGAAG ACATACTGGCAGGCTAC TEE-275
AACAGCAATAGACACAAAGTCAGCACTTACAGTACAA 6889 AAACTAATGGCAAAAGCACATGAA
GTGGGACAT TEE-276 TGTAACACTGCAAACCATAAAAACCGTAGAAGAAAAC 6890
CTAGACAATACTATTCAGGACATAG GCATGGGCAAAGAC TEE-277
GAAGAAGAAAAAACATGGATATACAATGTCAACAGAA 6891 ATCAAGGAGAAACGGAATTTCACC
AATCAATTTAGTGATCTGGGTT TEE-278
AAAACACACAAACATACATGTGGATGCACATATAAAC 6892
ATGCACATACACACACACATAAATG CACAAACACACTTAACACAAGCACACATGCAAACAAA
CACATGG TEE-279 TAGAAGGAATTTGATACATGCTCAGAAATACAGGCAA 6893
AGGAAGTAGGTGCCTGCCAGTGAAC ACAGGGGAACTATGGCTCCTA TEE-280
TGACTAAACAGAGTTGAACAAGAACAAAAAGCAAATT 6894
TGCAGAAATGAAATACATACTAATT GAAAGTCCATGGACAGGCTCAACAGATGATATAGATA
CAGCTAAAGAGATAATTAGTGAAAT GGATCAG TEE-281
AAGTAATAAGACTGAATTAGTAATACAAAGTGTCTCA 6895
ACAAAGAAAATTGCGGGACTGTTCA TGCTCATGGACAGGAAGAATCAATATCATGAAAATGG CC
TEE-282 ACAGACAGAGATTTAAAACAATAAACAAGCAGTAAGC 6896
AAACACAGATAACAAAATGACATG ATCCAACAAATACTCAGAAGGAGACTTAGAAATGAAT
TGAGGGTC TEE-283 AGAAAAAAACAAACAGCCCATTAAAAGGTAGACAAA 6897
GGACATGAACACTTTTCAAAAGAAG ACATACATGTGGCCAAACAGCATG TEE-284
AAAAATGACCAGAGCAATAGAATGCATTGACCAGATA 6898
AAGACCTTCACGTATGTTGAACTAA AATGTGTGGTGCAGGTG TEE-285
AATCAGTCTAGATCTTAAAGGAACACCAGAGGGAGTA 6899
TTTAAATGTGCCCAATAAGCAAGAA TTATGGTGATGTGGAAGTA TEE-286
GAATGGAATGGAAAGGAATCGAAACGAAAGGAATGG 6900 AGACAGATGGAATGGAATGGAACAG
AGAGCAATGG TEE-287 GGAATGGAATGAACACGAATGTAATGCAACCCAATAG 6901
AATGGAATCGAATGGCATGGAATAT AAAGAAATGGAATCGAAGAGAATGGAAACAAATGGA
ATGGAATTG TEE-288 AGGACATGAATAGACAATTCTCAAAAGAAGATACACA 6902
AGTGGCAAACAAACACATGAAAAA AGACTCAACATTAGTAATGACCATGGAAATGCAAATC
TEE-289 TCCAGTCGATCATCATATAGTCAGCACTTATCATACAC 6903
CAAGCCGTGTGCAAGGAAAGGGAA TACAACCATGAACATGATAGATGGATGGTT TEE-290
TACAGATAAGAAAATTGAGACTCAAGAGTATTACATA 6904
AATTGTTTCAGCTACCACAGCAAAA AATGGTATGGTTGGGAATCAAGCTCAGGG TEE-291
AGCCTATCAAAAAGTGGGCTAAGAATATGAATACACA 6905
ATTCTCAAAAGAAGATATACAAATG GGCAACAAACATATGAAAACATACTCAACATCACTAA
TGATCAGGGAAATG TEE-292 GAAAATGAACAATATGAACAAACAAACAAAATTACTA 6906
CCCTTACGAAAGTACGTGCATTCTA GTATGGTGACAAAAAGGAAAG TEE-293
ACATACGCAAATCAATAAACATAATCCATCACATAAA 6907
CAGAACCAAAGACAAAAATCACATG ATTATCTCAATAGATGCAGAAAAGGCCTTCGAC TEE-294
AAGAGTATCAACAGTAAATTACATTAGCAGAAGAATC 6908
AACAAACATGAAAATAGAAATTATG GTAGCCAAAGAACAG TEE-295
AATCGAATGGAATCAACATCAAACGGAAAAAAACGGA 6909
ATTATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-296
GAAAGGAATAGAATGGAATGGATCGTTATGGAAAGAC 6910
ATCGAATGGGATGGAATTGACTCGAATGGATTGGACT GGAATG
GAACGGACTCGAATGGAATGGACTGGAATG TEE-297
TAAGCAATTTCAGCAGTCTCAGGATACAAAATCAATGT 6911
GCAAAAATCACAAGCATTCTTATACACCAACAACAGA CAAAC AGAGAGCCAAATCG TEE-298
AACGGAATCAAACGGAATTATCGAATGGAATCGAAGA 6912
GAATCATCGAATGGCCACGAATGGAATCATCTAATGG AATGG
AATGGAATAATCCATGGACCCGAATG TEE-299
ACATCAAACGGAATCAAACGGAATTATCGAATGGAAT 6913
CGAAAAGAATCATCGAACGGACTCGAATGGAATCATC TAATGG AATGGAATGGAAG TEE-300
ATCGAATGGAATCAACATCAAACGGAAAAAAACGGAA 6914
TTATCAAATGGAATCGAAGAGAATCATCGAATGGACC TEE-301
GAATAATCATTGAACGGAATCGAATGGAAACATCATC 6915
GAATGGAAACGAATGGAATCATCATCGAATGGAAATG AAAGG AGTCATC TEE-302
CATCAAACGGAATCAAACGGAATTATCGAATGGAATC 6916
GAAAAGAATCATCGAACGGACTCGAATGGAATCATCT AATGGA
ATGGAATGGAAGAATCCATGGACTCGAATG TEE-303
AAACGGAATCAAACGGAATTATCGAATGGAATCGAAG 6917
AGAATCATCGAATGGACTCGAATGGAATCATCTAATG GAATGG AATGGAAGAATCCATGG
TEE-304 ATACACAAATCAATAAATGTAATCCAGCATATAAACA 6918
GAACCAAAGACAAAAACCATATGATTATCTCAATGGA TGCAGA AAAGGCC TEE-305
AATCGAATAGAATCATCGAATGGACTCGAATGGAATC 6919 ATCGAATGTAATGATGGAACAGTC
TEE-306 TGGAATGGAATCATCGCATAGAATCGAATGGAATTAC 6920
CATCGAATGGGATCGAATGGTATCAACATCAAACGCA AAAAAA
CGGAATTATCGAATGGAATCGAAGAGAATCTTCGAAC GGACCCG TEE-307
ATGGAATGGAATGGAATGGAATTAAATGGAATGGAAA 6921
GGAATGGAATCGAATGGAAAGGAATC TEE-308
GTCGAAATGAATAGAATGCAATCATCATCAAATGGAA 6922
TCCAATGGAATCATCATCAAATAGAATCGAATGGAAT CATCAA ATGGAATCGAATGGAGTCATTG
TEE-309 TGGAATTATCGAAAGCAAACGAATAGAATCATCGAAT 6923
GGACTCGAATGGAATCATCGAATGGAATGGAATGGAA CAG TEE-310
AAAGGAATGGAATGCAATGGAATGCAATGGAATGCAC 6924
AGGAATGGAATGGAATGGAATGGAAAGGAATG TEE-311
AATCTAATGGAATCAACATCAAACGGAAAAAAACGGA 6925
ATTATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-312
TACACAACAAAAGAAATACTCAACACAGTAAACAGAC 6926
AACCTTCAGAACAGGAGAAAATATTTGCAAATACATC TAACAA AGGGCTAATATCCAGAATCT
TEE-313 TGCAATCCTAGTCTCAGATAAAACAGACATTAAACCA 6927
ACAAAGATCAAAAGAGACAAAGAAGGCCATTAC TEE-314
GAATCGAATGGAATCAACATCAAACGGAAAAAAACGG 6928
AATTATCGAATGGAATCGAAAAGAATCATCGAATGGA CC TEE-315
AATGGAATCGAATGGAATGCAATCCAATGGAATGGAA 6929
TGCAATGCAATGGAATGGAATCGAACGGAATGCAGTG GAAGG GAATGG TEE-316
GAACACAGAAAAATTTCAAAGGAATAATCAACAGGGA 6930
TTGATAACTAACTGGATTTAGAGAGCCAAGGCAAAGA GAATC AAAGCACAGGGCCTGAGTCGGAG
TEE-317 AGTTGAATAGAACCAATCCGAATGAAATGGAATGGAA 6931
TGGAACGGAATGGAATTGAATGGAATGGAATGGAATG CAATG GA TEE-318
AACTCGATTGCAATGGAATGTAATGTAATGGAATGGA 6932
ATGGAATTAACGCGAATAGAATGGAATGGAATGTAAT GGAACG GAATGGAATG TEE-319
AAGCGGAATAGAATTGAATCATCATTGAATGGAATCG 6933
AGTAGAATCATTGAAATCGAATGGAATCATAGAATGG AATCCA AT TEE-320
AATGGAATCGAAAGGAATAGAATGGAATGGATCGTTA 6934
TGGAAAGATATCGAATGGAATGGAATTGACTCGAATG GAATG GACTGGAATGGAACG TEE-321
TAACGGAATAATCATCGAACAGAATCAAATGGAATCA 6935
TCATTGAATGGAATTGAATGGAATCTTCGAATAGACAT GAATG GACCATCATCG TEE-322
AACGGAATCAAACGGAATTATCGAATGGAATCGAATA 6936
GAATCATCGAACGGACTCGAATGGAATCATCTAATGG AATGGA ATGGAAG TEE-323
ATTGGAATGGAACGGAACAGAACGGAATGGAATGGAA 6937
TAGAATGGAATGGAATGGAATGGTATGGAATGGAATG GAATG GTACG TEE-324
AATCCACAAAGACAACAGAAGAAAAGACAACAGTAG 6938
ACAAGGATGTCAACCACATTTTGGAAGAGACAAGTAA TCAAAC ACATGGCA TEE-325
GAATCGAATGGAATCAACATCAAACGGAAAAAAACGG 6939
AATTATCGAATGGAATCGAAAAGAATCATCGAACGGA CTCGA
ATGGAATCATCTAATGGAATGGAATGGAAGAATCCAT GG TEE-326
AATGGAATCGAATGGAATCATCATCAAATGGAATCTA 6940
ATGGAATCATTGAACGGAATTGGATGGAATCGTCAT TEE-327
CAACATCAAACGGAAAAAAACGGAATTATCGAATGGA 6941
ATCGAAGAGAATCATCGAATGGACC TEE-328
CACAACCAAAGCAATGAAAGAAAAGCACAGACTTATT 6942
GAAATGAAAGTACACACCACAGAATGGGAGCAGGCTC AAGCA AGC TEE-329
ATCAAAGGGAATCAAGCGGAATTATCGAATGGAATCG 6943
AAGAGAATCATCGAATGGACTCGAATGGAATCATGTG ATGGA
ATGGAATGGAATAATCCACGGACT TEE-330
GGAATCGAATGGAATCAATATCAAACGGAGAAAAACG 6944
GAATTATCGAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-331
AGGAATGGACACGAACGGAATGCAATCGAATGGAATG 6945
GAATCTAATAGAAAGGAATTGAATGAAATGGACTGG TEE-332
GGAAGGGAATCAAATGCAACAGAATGTAATGGAATGG 6946
AATGCAATGGAATGCAATGGAATGGAATGGAATGCAA TGGAA TGG
TEE-333 AAATTGGATTGAATCGAATCGAATGGAAAAAATGAAA 6947
TCAAATGAAATTGAATGGAATCGAAATGAATGTAAAC AATGG
AATCCAATGGAATCCAATGGAATCGAATCAAATGGTTT TGAGTGGCGTAAAATG TEE-334
AATGGAAGGGAATGGAATGGAATCGAATCGAATGGAA 6948
CAGAATTCAATGGAATGGAATGGAATGGAATGGAATC GAATG GAATGG TEE-335
GAAAAATCATTGAACGGAATCGAATGGAATCATCATC 6949
GGATGGAAACGAATGGAATCATCATCGAATGGAAATG AAAGG AGTCATC TEE-336
GGAATCGAATGGAATCAACATCAAACGGAGAAAAACG 6950
GAATTATCGAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-337
AAAGAAATGTCACTGCGTATACACACACACGCACATA 6951
CACACACCATGGAATACTACTCAGCTATACAAAGGAA TGAAAT AATCCACAGCCAC TEE-338
GGAATCGAATGGAATCAATATCAAACGGAAAAAAACG 6952
GAATTATCGAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-339
TGAACGGAATCGAATGGAATCATCATCGGATGGAAAC 6953
GAATGGAATCATCATCGAATGGAAATGAAAGGAGTCA TC TEE-340
GAATAGAACGAAATGGAATGGAATGGAATGGAATGGA 6954 AAGGAATGGAATGGAATGGAACG
TEE-341 TGGAATTATCGTCGAATAGAATCGAATGGTATCAACAT 6955
CAAACGGAAAAAAACGGAATTATCGAATGGAATCGAA GAGA
ATCATCGAACGGACTCGAATGGAATCATCTAATGGAA TGGAATGGAATAATCCATGG TEE-342
GACAAAAAGAATCATCATCGAATAGAATCAAATGGAA 6956
TCTTTGAATGGACTCAAAAGGAATATCGTCAAATGGA ATCAAA
AGCCATCATCGAATGGACTGAAATGGAATTATCAAAT GGACTCG TEE-343
AACCAAACCAAGCAAACAAACAAACAGTAAAAACTCA 6957
ATAACAACCAACAAACAGGAAATACCAGGTAATTCAG ATTAT CTAGTTATGTGCCATAGT
TEE-344 GAATGAATTGAATGCAAACATCGAATGGTCTCGAATG 6958
GAATCATCTTCAAATGGAATGGAATGGAATCATCGCAT AGAAT
CGAATGGAATTATCAACGAATGGAATCGAATGGAATC
ATCATCAGATGGAAATGAATGGAATCGTCAT TEE-345
TGGAATGGAATCAAATCGCATGGAATCGAATGGAATA 6959
GAAAAGAATCAAACAGAGTGGAATGGAATGGAATGG AATGGA ATCATGCCGAATGGAATG
TEE-346 AAATGGAATAATGAAATGGAATCGAACGGAATCATCA 6960
TCAAAAGGAACCGAATGAAGTCATTGAATGGAATCAA AGGCA
ATCATGGTCGAATGGAATCAAATGGAAACAGCATTGA
ATAGAATTGAATGGAGTCATCACATGGAATCG TEE-347
GAATTAACCCGAATAGAATGGAATGGAATGGAATGGA 6961
ACAGAACGGAACGGAATGGAATGGAATGGAATGGAAT GGAATG TEE-348
AAGATATACAAGCAGCCAACAAACATACGAAAGAATG 6962
CTCAACATCACTAATCCTCAGAGAAATTTAAATCAAAA CCACA
ATGAGTTACAATCTCATACCAGTCAGAAT TEE-349
AGATAAGTGGATGAACAGATGGACAGATGGATGGATG 6963
GATGGATGGATGGATGGATGCCTGGAAGAAAGAAGAA TGGAT AGTAAGCTGGGTATA TEE-350
AGAATTACAAACCACTGCTCAACAAAATAAAAGAGTA 6964
CACAAACAAATGGAAGAATATTCCATGCTTATGGATA GGAAGA
ATCAATATTGTGAAAATGGCCATACT TEE-351
CATCGAATGGACTCGAATGGAATAATCATTGAACGGA 6965
ATCGAAGGGAATCATCATCGGATGGAAACGAATGGAA TCATCA TCGAATGGAAATG TEE-352
AAAGGAATCAAACGGAATTATCGAATGGAATCGAAAA 6966
GAATCATCGAACGGACTCGAATGGAATCATCTAATGG AATGG
AATGGAAGAATCCATGGACTCGAATG TEE-353
GGATATAAACAAGAAAACAACTAATCACAACTCAATA 6967
TCAAAGTGCAATGATGGTGCAAAATGCAAGTATGGTG GGGAC AGAGAAAGGATGC TEE-354
AACATCAAACGGAAAAAAACGGAAATATCGAATGGAA 6968 TCGAAGAGAATCATCGAATGGACC
TEE-355 TAAAATGGAATCGAATGGAATCAACATCAAATGGAAT 6969
CAAATGGAATCATTGAACGGAATTGAATGGAATCGTC AT TEE-356
AATCATCATCGAATGGAATCGAATGGTATCATTGAATG 6970
GAATCGAATGGAATCATCATCAGATGGAAATGAATGG AATCG TCAT TEE-357
CAATGCGTCAAGCTCAGACGTGCCTCACTACGGCAATG 6971
CGTCAAGCTCAGGCGTGCCTCACTAT TEE-358
TAAGCTGATAAGCAACTTTAGCAAAGTCTCAGGATAC 6972
AAAATCAATGTACAAAAATCACAAGCATTCTTATACAC CAACA ACAGACAGACGGAGAGCCAAA
TEE-359 AATCAAAGAATTGAATCGAATGGAATCATCTAATGTA 6973
CTCGAATGGAATCACCAT TEE-360 ATGAACACGAATGTAATGCAATCCAATAGAATGGAAT
6974 CGAATGGCATGGAATATAAAGAAATGGAATCGAAGAG AATGG
AAACAAATGGAATGGAATTGAATGGAATGGAATTG TEE-361
ATCAAACGGAATCAAACGGAATTATCGAATGGAATCG 6975
AAGAGAATCATCGAACGGACTCGAATGGAATCATCTA ATGGAA TGGGATGG TEE-362
AATGGAAAGGAATCAAATGGAATATAATGGAATGCAA 6976
TGGACTCGAATGGAATGGAATGGAATGGACCCAAATG GAATG GAATGGAATGGAATG TEE-363
GGAATACAACGGAATGGAATCGAAAAAAATGGAAAG 6977
GAATGAAATGAATGGAATGGAATGGAATGGAATGGAT GGGAA TGGAATGGAATGG TEE-364
GAATCAAGCGGAATTATCGAATGGAATCGAAGAGAAT 6978
CATCGAAAGGACTCGAATGGAATCATCTAATGGAATG GAATG GAATAATACACGGACC
TEE-365 AAGATAACCTGTGCCCAGGAGAAAAACAATCAATGGC 6979
AACAAAAGCAGAAACAACACAAATGATACAATTAGCA GACAG AAACATTGAGATTGCTATT
TEE-366 AATGGACTCCAATGGAATAATCATTGAACGGAATCTA 6980
ATGGAATCATCATCGGATGGAAATGAGTGGAATCATC ATCGAA TGGAATCG TEE-367
AATCTATAAACGTAATCCATCACATAAACAGGACCAA 6981
AGAGAAAAACCGCATGATTATCTCAAGAATGCAGAAA AGGCC TEE-368
TAATTGATTCGAAATTAATGGAATTGAATGGAATGCAA 6982
TCAAATGGAATGGAATGTAATGCAATGGAATGTAATA GAATG GAAAGCAATGGAATG TEE-369
AAAGGAATGGACTTGAACAAAATGAAATCGAACGATA 6983
GGAATCGTACAGAACGGAAAGAAATGGAACGGAATG GAATG TEE-370
TGAGCAGGGAACAATGCGGATAAATTTCACAAATACA 6984
ATGTTGAGCAAAAGAAAGACACAAAAGAATACACACA TACAC ACCATATGGGCTAGG TEE-371
AATGGAATCGAACGGAATCATCATCAAACGGAACCGA 6985
ATGGAATCATTGAATGGAATCAAAGGCAATCATGGTC GAATG TEE-372
AATGGAATGGAATGTACAAGAAAGGAATGGAATGAAA 6986
CCGAATGGAATGGAATGGACGCAAAATGAATGGAATG GAAGT CAATGG TEE-373
AACGGAAAAAAACGGAATTATCGAATGGAATCGAAGA 6987 GAATCATCGAATGGACC
TEE-374 GGAATAATCATTGAACGGAATCGAATGGAATCATCAT 6988
CGGATGGAAACGAATGGAATCATCATCGAATGGAAAT GAAAG GAGTCATC TEE-375
GGAACGAAATCGAATGGAACGGAATAGAATAGACTCG 6989
AATGTAATGGATTGCTATGTAATTGATTCGAATGGAAT GGAAT CG TEE-376
TGAAAGGAATAGACTGGAACAAAATGAAATCGAATGG 6990
TAGGAATCATACAGAACAGAAAGAAATGGAACGGAAT GGAATG TEE-377
AACCCGAATAGAATGGAATGGAATGGAATGGAACGGA 6991
ACGGAATGGAATGGAATGGATTGGAATGGAATGGAATG TEE-378
AAAGAGAATCAAATGGAATTGAATCGAATGGAATCGA 6992
ATGGATTGGAAAGGAATAGAATGGAATGGAATGGAAT GGAAT GGAATGGAATG TEE-379
AATGGAATCATCAGTAATGGAATGGAAAGGAATGGAA 6993
AGGACTGGAATGGAATGGAATGGAATGGAATGG TEE-380
GGAACAAAATGAAATCGAACGGTAGGAATCGTACAGA 6994
ACGGAAAGAAATGGAACGGAATGGAATGCACTCAAAT GGAAA
GGAGTCCAATGGAATCGAAAGGAATAGAATGGAATGG TEE-381
AGAATGAGATCAAGCAGTATAATAAAGGAAGAAGTAG 6995
CAAAATTACAACAGAGCAGTGAAATGGATATGCTTTCT GGCA
ATAATTGTGAAAGGTCTGGTAATGAGAAAGTAGCAAC AGCTAGTGGCTGCCAC TEE-382
AACAAATGGAATCAACATCGAATGGAATCGAATGGAA 6996
ACACCATCGAATTGAAACGAATGGAATTATCATGAAA TTGAAA TGGATGGACTCATCATCG
TEE-383 TAACATGCAGCATGCACACACGAATACACAACACACA 6997
AACATGTATGCACGCACACGTGAATACACAACACACA CAAACA
TGCATGCATGCATACATGAATACACAGCACACAAATA TCCAGCAT TEE-384
GAATGGAATCAACATCAAACGGAAAAAAAACGGAATT 6998
ATCGAATGGAATCGAATAGAATCATCGAATGGACC TEE-385
AATCGAATGAAATGGAGTCAAAAGGAATGGAATCGAA 6999
TGGCAAGAAATCGAATGTAATGGAATCGCAAGGAATT GATGT GAACGGAACGGAATGGAAT
TEE-386 AATGGAATTGAACGGAAACATCAGCGAATGGAATCGA 7000
AAGGAATCATCATGGAATAGATTCGAATGGAATGGAA AGGAA TGGAATGGAATG
TEE-387 ATGGAATCAACATCAAACAGAATCAAACGGAATTATC 7001
GAATGGAATCGAAGACAATCATCGAATGGACTCGAAT GGAATC
ATCTAATGGAATGGAATGGAAGAATCCATGGTCTCGA ATGCAATCATCATCG TEE-388
GAATAATCATTGAACGGAATCGAATGGAATCATCTTCG 7002
GATGGAAACGAATGGAATCATCATCGAATGGAAATGA AAGGA GTCATC TEE-389
AATGGACTCGAATGGAATAATCATTGAACGGAATCGA 7003
ATGGAATCATCATCGGATGGAAATGAGTGGAATCATC ATCGAA TGGAATCG TEE-390
AAATGAAATCGAACGGTAGGAATCGTACAGAACGGAA 7004
AGAAATGGAACGGAATGGAATGCAATCGAATGGAAAG GAGTC CAATGGAAGGGAATCGAAT
TEE-391 TACCAAACATTTAAAGAACAAATATCAATCCTACGCA 7005
AACCATTCTGAAACACAGAGATGGAGGATATACAGCG AAACTC ATTCTACATGGCC TEE-392
TATTGGAATGGAATGGAATGGAGTCGAATGGAACGGA 7006
ATGCACTCGAATGGAAGGCAATGCAATGGAATGCACT CAACA
GGAATAGAATGGAATGGAATGGAATGG TEE-393
GGAATTTAATAGAATGTACCCGAATGGAACGGAATGG 7007
AATGGAATTGTATGGCATGGAATGGAA TEE-394
GCAATCCAATAGAATGGAATCGAATGGCATGGAATAT 7008
AAAGAAATGGAATCGAAGAGAATGGAGACAAATGGA ATGGAA TTGAATGGAATGGAATTG
TEE-395 AATGGAATCGAATGGAATCATCATCAAATGGAATCTA 7009
ATGGAATCATTGAACGGAATTAAATGGAATCGTCATC GAATGA
ATTCAATGCAATCAACGAATGGTCTCGAATGGAACCAC TEE-396
AATTGCAAAAGAAACACACATATACACATATAAAACT 7010
CAAGAAAGACAAAACTAACCTATGGTGATAGAAATCA GAAAA
GTACAGTACATTGGTTGTCTTGGTGGG TEE-397
TGACATCATTATTATCAAGAAACATTCTTACCACTGTT 7011
ACCAACTTCCCAACACAGACTATGGAGAGAGAGATAA GACAGA ATAGCATT TEE-398
AAAGAATTGAATTGAATAGAATCACCAATGAATTGAA 7012
TCGAATGGAATCGTCATCGAATGGAATCGAAGGGAAT CATTGG ATGGGCTCA TEE-399
ATCATCGAATGGAATCGAATGGAATCAATATCAAACG 7013
GAAAAAAACGGAATTATCGAATGGAATCGAATAGAAT CATCGA ATGGACC TEE-400
GAATGAAATCGTATAGAATCATCGAATGCAACTGAAT 7014
GGAATCATTAAATGGACTTGAAAGGAATTATTATGGA ATGGAA TTG TEE-401
TAAGCAACTTCAGCAAAGTCTCAGGATACAAAATCAA 7015
TGTGCAAAAATCTCAAGCATTCTTATACACGAACAACA GACAA ACAGAGAGCT TEE-402
ACTCAAAAGGAATTGATTCGAATGGAATAGAATGGCA 7016
AGGAATAGTATTGAATTGAATGGAATGGAATGGACCC AAATG TEE-403
GAATGGAATTTAAAGGAATAGAATGGAAGGAATCGGA 7017
TGGAATGGAATGGAATAGAATGGAGTCGAATGGAATA GAATC GAATGGAATGGCATTG
TEE-404 TGAGAAAATGATGGAAAAGAGGAATAAAACGAAACA 7018
AAACCACAGGAACACAGGTGCATGTGAATGTGCACAG ACAAA
GATACAGGGCGGACTGGGAAGGAAGTTTCTGCACCAG AATTTGGGG TEE-405
AACAAAAAATGAGTCAAGCCTTAAATAAAATCAGAGC 7019
CAAAAAAGAAGACATTACATCTGATAAGACAAAAATT CAAAG GACCATC TEE-406
AACCCAGTGGAATTGAATTGAATGGAATTGAATGGAA 7020
TGGAAAGAATCAATCCGAGTCGAATGGAATGGTATGG AATGGA ATGGCATGGAATCAAC
TEE-407 ATCAACATCAAACGGAAAAAAAACGGAATTATCGAAT 7021
GGAATCGAAGAGAATCATCGAATGGACC TEE-408
AAGGAATGGAATGGTACGGAATAGAATGGAATGGAAC 7022
GAATTGTAATGGAATGGAATTTAATGGAACGGAATGG AATGG AATGGAATCAACG TEE-409
AACGGAATGGAAAGCAATTTAATCAAATGCAATACAG 7023
TGGAATTGAAGGGAATGGAATGGAATGGC TEE-410
AATCGAATGGAACGGAATAGAATAGACTCGAATGTAA 7024
TGGATTGCTATGTAATTGATTCGAATGGAATGGAATCG AATGG
AATGCAATCCAATGGAATGGAATGCAATGCAATGGAA
TGGAATCGAACGGAATGCAGTGGAAGGGAATGG TEE-411
TAGCAACATTTTAGTAACATGATAGAAACAAAACAGC 7025
AACATAGCAATGCAATAGTAACACAACAGCAACATCA TAACAT GGCAGCA TEE-412
AATGGAATCGAAGAGAATGGAAACAAATGGAATGGA 7026
ATTGAATGGAATGGAATTGAATGGAATGGGAAGGAAT GGAGTG TEE-413
AGCAAACAAGTGAATAAACAAGCAAACAAGTGAACA 7027
AGCAAACAAGTGAATAAACAAGCAAACAAGTGAACA AGCAAA
CAAGTGAATAAACAAGCAAACAAGTGAACAAGGAAA CAAGTGAATAAACAAAGGCTCT TEE-414
AATGGAATCAACACGAGTGCAATTGAATGGAATCGAA 7028
TGGAATGGAATGGAATGGAATGAATTCAACCCGAATG GAATG GAAAGGAATGGAATC TEE-415
GAATCGAATGGAATCAACATCAAACGGAAAAAAACGG 7029
AATTATCGAATGGAATCGAAGAGAATCATCGAATGGA CC TEE-416
AACACGAATGTAATGCAATCCAATAGAATGGAATCGA 7030
ATGGCATGGAATATAAAGAAATGGAATCGAAGAGAAT GGAAA
CAAACGGAATGGAATTGAATGGAATGGAATTGAATGG AATGGGAACGAATGGAGTGAAATTG
TEE-417 GAATGGAACGGAATAGAACAGACTCGAATGTAATGGA 7031
TTGCTATGTAATTGATTCGAATGGAATGGAATCGAATG GAATG
CAATCCAATGGAATGGAATGCAATGCAATGGAATGGA ATCGAATGGAATGCAGTGGAAGGGAATGG
TEE-418 GAATCGAATGGAATCAATATCAAACGGAAAAAAACGG 7032
AATTATCGAATGGAATCGAAGAGAATCATCGAATGGA CC TEE-419
ATAAACATCAAACGGAATCAAACGGAATTATCGAATG 7033
GAATCGAAGAGAATAATCGAATGGACTCAAATGGAGT CATCTA
ATGGAATGGTATGGAAGAATCCATGGACTCCAACGCA ATCATCAGCGAATGGAATC TEE-420
AAAAGAAAAGACAAAAGACACCAATTGCCAATACTGA 7034
AATGAAAAAACAGGTAATAACTATTGATCCCATGGAC ATTAA AATGATGTTGAAGGAACACCAC
TEE-421 AATGTCAAGTGGAATCGAGTGGAATCATCGAAAGAAA 7035
TCGAATGGAATCGAAGGGAATCATTGGATGGGCTCAA AT TEE-422
ATCATCGAATGGAATAGAATGGTATCAACATCAAACG 7036
GAGAAAAACGGAATTATCGAATGGAATCGAAGAGAAT CTTCGA ACGGACC TEE-423
GAATGGAATCATCGCATAGAATCGGATGGAATTATCA 7037
TCGAATGGAATCGAATGGTATCAACATCAAACGGAAA AAAACG
GAATTATCGAATGGAATCGAATTGAATCATCGAACGG ACCCG TEE-424
AATGGACTCGAATGGAATAATCATTGAACGGAATCGA 7038
ATGGAATCATCATCGGATGGAAATGAATGGAATAATC CATGGA
CTCGAATGCAATCATCATCGAATGGAATCGAATGGAA TCATCGAATGGACTCG TEE-425
AATGCAATCATCAACTGGCTTCGAATGGAATCATCAAG 7039
AATGGAATCGAATGGAATCATCGAATGGACTC TEE-426
AAGAGACCAATAAGGAATAAGTAAGCAACAAGAGGA 7040
AGGAGAAAAGGGCAAGAGAGATGACCAGAGTT TEE-427
TGGAATCATCATAAAATGGAATCGAATGGAATCAACA 7041
TCAAATGGAATCAAATGGAATCATTGAACGGAATTGA ATGGAA TCGTCAT TEE-428
GGAATCATCGCATAGAATCGAATGGAATTATCATCGA 7042
ATGGAATCGAATGGAATCAACATCAAACGAAAAAAAA CCGGA
ATTATCGAATGGAATCGAAGAGAATCATCGAACGGACC TEE-429
AAATCATCATCGAATGGGATCGAATGGTATCCTTGAAT 7043
GGAATCGAATGGAATCATCATCAGATGGAAATGAATG GAATC GTCAT TEE-430
GGAATGTAATAGAACGGAAAGCAATGGAATGGAACGC 7044
ACTGGATTCGAGTGCAATGGAATCTATTGGAATGGAAT CGAAT
GGAATGGTTTGGCATGGAATGGAC TEE-431
AAACAATGGAAGATAATGGAAAGATATCGAATGGAAT 7045
AGAATGGAATGGAATGGACTCAAATGGAATGGACTTT AATGG AATGG TEE-432
GGAACGAAATCGAATGGAACGGAATAGAATAGACTCG 7046
AATGTAATGGATTGCTATGTAATTGATTCGAATGGAAT GGAAT
CGAATGGAATGCAATCCAATGGAATGGAATGCAATGC
AATGAATGGAATGGAATGGAATGGAATGGAA TEE-433
AAACCGAATGGAATGGAATGGACGCAAAATGAATGGA 7047
ATGGAAGTCAATGGACTCGAAATGAATGGAATGGAAT GGAAT GGAATG TEE-434
GGAATCGAATGGAATCAACATCAAACGGAAAAAAACA 7048
GAATTATCGTATGGAATCGAATAGAATCATCGAATGG ACC TEE-435
CAACCCGAGTGGAATAAAATGGAATGGAATGGAATGA 7049
AATGGAATGGATCGGAATGGAATCCAATGGAATCAAC TGGAA TGGAATGGAATGGAATG
TEE-436 TATCATCGAATGGAATCGAATGGAATCAACATCAAAC 7050
GGAAAAAAACGGAATTATCGAATGGAATCGAAGAGAA TCATC GAATGGACC TEE-437
CGGAATAATCATTGAACGGAATCGAATGGAATCATCA 7051
TCGGATGGAAACGAATGGAATCATCATCGAATGGAAA TGAAAG GAGTCATC TEE-438
CAACACACAGAGATTAAAACAAACAAACAAACAATCC 7052
AGCCCTGACATTTATGAGTTTACAGACTGGTGGAGAGG CAGAG AAG TEE-439
CACTACAAACCACGCTCAAGGCAATAAAAGAACACAA 7053
ACAAATGGAAAAACATTCCATGCTCATGGATGGG TEE-440
AATCGAATGGAATTAACATCAAACGGAAAAAAACGGA 7054
ATTATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-441
TGGAAAAGAATCAAATTGAATGGCATCGAACGGAATG 7055
GGATGGAATGGAATAGACCCAGATGTAATGGACTCGA ATGGA ATG TEE-442
GACTAATATTCAGAATATACAAGGAACTCAAACAACT 7056
CAACAGTAGAAAAAAAAACCTGAATAGACATTTCTCA AAAGAA GACATACAAATGGCC
TEE-443 GGTCCATTCGATGATTCTCTTCGATTCCATTCGATAATT 7057
CCGTTTTTTCCCGTTTGATGTTGATTCC TEE-444
GGAACGAAATCGAATGGAACGGAATAGAATAGACTCG 7058
AATGTAATGGATTGCTATGTAATTGATTCGAATGGAAT GGAAT
CGAATGGAATGCAATCCAATGGAATGGAATGCAATGC
AATGAATGGAATGGAATGGAATGGAATGGA TEE-445
AGCAACTTCAGTAAAGTGTCAGGATACAAAATCAATG 7059
TGCAAAAATCACAAGCATTCTTATACATCAATAACAGA CAAAC AGAGAGCCAAA TEE-446
GAATAATCATTGAACGGAATCGAATGGAATCATCATC 7060
GGATGGAAACGAATGGAATCATCATCGAATGGAAATG AAAGG AGTCATC TEE-447
TAATCATCTTCGAATTGAAAACAAAGCAATCATTAAAT 7061
GTACTCTAACGGAATCATCGAATGGACC TEE-448
GGAATCGAATGGAATCAACATCAAACGGAAAAAAACG 7062
GAATTATCGAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-449
AGAGAAAAGATGATCATGTAACCATTGAAAAGACAAT 7063
GTACAAAACTAATACTAATCACACAGGACCAGAAAGC AATTTA GACCAT TEE-450
AATGGAATCGAATGGAATCAACATCAAACGGAAAAAA 7064
CGGAATTATCGAATGGAATCAAAGAGAATCATCGAAT GGACC TEE-451
AATGGAATTATCATCGAATGGAATCGAATGGAATCAA 7065
CATCAAACGGAAAAAAACGGAATTATCGAATGGAATC GAAGA GAATCATCGAATGGACC
TEE-452 GTCAACACAGGACCAACATAGGACCAACACAGGGTCA 7066
ACACAGGACCAACATAGGACCAACACAGGGTCAACAC AAGAC
CAACATGGGACCAACACAGGGTCAACATAGGACCAAC
ATGGGACCAACACAGGGTCAACACAGGACCAAC TEE-453
GAATCAACTCGATTGCAATCGAATGGAATGGAATGGT 7067
ATTAACAGAATAGAATGGAATGGAATGGAATGGAACG GAACG TEE-454
ACTCGAATGCAATCAACATCAAACGGAATCAAACGGA 7068
ATTATCGAATGGAATCGAAGAGAATCATCGAACGGAC TCGAAT
GGAATCATCTAATGGAATGGAATGG TEE-455
AATGGAATGGAATAATCGACGGACCCGAATGCAATCA 7069
TCATCGTACAGAATCGAATGGAATCATCGAATGGACT GGAATG GAATGG TEE-456
AATACAAACCACTGCTCAACGAAATAAAAGAGGATAC 7070
AAACAAATGGAAGAACATTCTATGCTCATGGGTAGGA TGAATT
CATATCGTGAAAATGGCCATACTGCC TEE-457
AAACACGCAAACACACACACAAGCACACTACCACACA 7071
AGCGGACACACATGCAAACACGCGAACACACACACAT ATACA
CACAAGCACATTACAAAACACAAGCAAACACCAGCAG ACACACAAACACACAAACATACATGG
TEE-458 AATCGAACGGAATCAACATCAAACGGAAAAAAAACGG 7072
AATTATCGAATGGAATCGAAGAGAATCATCGAATGGA CC TEE-459
TAATTGATTCGAATGGAATGGAATAGAATGGAATTGA 7073
ATGGAATGGACCATAATGGATTGGACTTTAATAGAAA GGGCATG TEE-460
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 7074
TACAAAAGTCACAAGCATTCTTATACACCAACAAAAG ACAAAC AGAGAGCC TEE-461
ACATCAAACGGAAAAAAAAAACAAAACGGAATTATCG 7075
AATGGAATCGAAGAGAATCATCGAATGGACC TEE-462
GAAATTCCAATTAAAATGAAATCGACTTATCTTAACAA 7076
ATATAGCAATGCTGACAACACTTCTCCGGATATGGGTA CTGCT TEE-463
ACATCTCACTTTTAGTAATGAACAGATCATTCAGACAG 7077
AAAATTAGCAAAGAAACATCAGAGTTAAACTACACTC TAAAC CAAATGGACCTA TEE-464
GAAGAAAGCATTCATTCAAGACATCTAACTCGTTGATA 7078
TAATGCATACAGTTCAAAATGATTACACTATCATTACA TCTAG GGCTTTC TEE-465
ACACACACATTCAAAGCAGCAATATTTACAACAGCCA 7079
AAAGGTGGAAACAATTGAGCAATTG TEE-466
ATCATCGAATAGAATCGAATGGTATCAACACCAAACG 7080
GAAAAAAACGGAATTATCGAATGGAATCGAAGAGAAT CTTCGA ACGGACC TEE-467
ATCAACATCAAACGGAAAAAACGGAATTATCGAATGG 7081
AATCGAAGAGAATCATCGAACGGACC TEE-468
AATCGAAAGGAATGTCATCGAATGGAATGGACTCAAA 7082
TGGAATAGAATCGGATGGAATGGCATCGAATGGAATG GAATG GAATTGGATGGAC TEE-469
AACATGAACAGTGGAACAATCAGTGAACCAATACAAG 7083
GGTTAAATAAGCTAGCAATTAAAAGCTGTATCACTGGT CTAAA
GATAGAAGATCAAGTAGAAAATCAGCGCAAGAGGAA AGATATACGAAAACTAATGGCC TEE-470
CGAATGGAATCATTATGGAATGGAATGAAATGGAATA 7084
ATCAAATGGAATTGAATGGAATCATCGAATGGAATCG AACAAA
ATCCTCTTTGAATGGAATAAGATGGAATCACCAAATGG AATTG TEE-471
AAGGGAATTGAATAGAATGAATCCGAATGGAATGGAA 7085
TGGAATGGAATGGAATGGAATGGAATGGAATGGAATG GAATG TEE-472
GAATGGAATCGAATCAAATTAAATCAAATGGAATGCA 7086
ATAGAAGGGAATACAATGGAATAGAATGGAATGGAAT GGAAT GGACT TEE-473
AAACGGAATCAAACGGAATTATCGAATGGAATCGAAG 7087
AGAATCATCGAACGGACTCGAATGGAATCATCTAATG GAATG GAATGGAAGAATCCATGGACT
TEE-474 ATGGAATCAACATCAAACGGAAAAAAAAACGGAATTA 7088
TCGAATGGAATCGAAGAGAATCATCGAATGGACCAGA ATGGA ATCATCTAATGGAATGGAATGG
TEE-475 AATGGAATCATCATCGAATGGAATCGAATGGAATCAT 7089
GGAATGGAATCAAATGGAATCAAATGGAATCGAATGG AATGG AATGGAATG TEE-476
AACGGAATCAAACGGAATTACCGAATGGAATCGAATA 7090
GAATCATCGAACGGACTCGAATGGAATCATCTAATGG AATGGA ATGGAAG TEE-477
AAACGGAATCAAACGGAATTATCGAATGGAATCGAAA 7091
AGAATCATCGAACGGACTCGAATGGAATCATCTAATG GAATG GAATGGAAGAATCCATGG
TEE-478 GAATGATACGGAATACAATGGAATGGAACGAAATGAA 7092
ATGGAATGGAATGGAATGGAATGGAATGGAATGG TEE-479
ACAGCAAGAGAGAAATAAAACGACAAGAAAACTACA 7093
AAATGCCTATCAATAGTTACTTTAAATATCAGTGGACC AAATCA
GTGAAACAAAAGACACAGAGTGGC TEE-480
AATGGACTCGAATGGATTAATCATTGAACGGAATCGA 7094
ATGGAATCATCATCGGATGGTAATGAATGGAATCATC ATCGAA TGGAATCGG TEE-481
GAATGGAATCGAAAGGAATGTCATCGAATGGAATGGA 7095
ATGGAACGGAATGGAATCGAATGGAATGGACTCGAAT GGAAT AGAATCGAATGCAATGGCATCG
TEE-482 ATCGAATGGAATCAACATCAGACGGAAAAAAACGGAA 7096
TTATCAAATGGAATCGAAGAGAATCATCGAATGGACC TEE-483
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 7097
TGCAAAAATCAAAAGCATTCTTATGCACCAATAACAG ACACAG AGCCAAAT TEE-484
AATGGAATGGAACGCAATTGAATGGAATGGAATGGAA 7098
CGGAATCAACCTGAGTCAAATGGAATGGAATGGAATG GAATG TEE-485
GGAACGAAATCGAATGGAACGGAATAGAATAGACTCG 7099
AATGTCATGGATTGCTATGTAATTGATTGGAATGGAAT GGAAT CG TEE-486
TAGCAGGAAACAGCAAACTCAAATTAAGTAATTTCAA 7100
GAGCGTATCATCAATGAACTATTTTCAAAGATGTGGGC AAGAT TEE-487
GAATTGAAAGGAATGTATTGGAATAAAATGGAATCGA 7101
ATAGGTTGAAATACCATAGGTTCGAATTGAATGGAAT GGGAGG GACACCAATGGAATTG
TEE-488 AAGCAACTTCAGCAAAGTCTCGGGATACAAAATCAAT 7102
GTGCAAAAATCACAAGCATTCTTATACACCACTAACAG ACAAA TGGAGAGTC TEE-489
GAATGGAATCAACATCAAACGGAAAAAAACGGAATTA 7103
TCGAATGGAATCGAAGAGAATCATCGAATGGACCAGA ATGGA
ATCATCTAATGGAATGGAATGGAATAATCCATGG TEE-490
AAAAGCAATTGGACTGATTTTAAATATACGTGGCAAC 7104
AAGGATAAACTGCTAATGATGGGTTTGCAAATACAGA TCG TEE-491
AATGGAATCAACATCGAACGGAAAAAAACGGAATTAT 7105
CGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-492
AAACGGAATTATCAAATGGAATCGAAGAGAATCATCG 7106
AACGGACTCGAATGGAATCATCTAATGGAATGGAATG GAAG TEE-493
TGCAAGATAACACATTTTAGTTGACACCATTGAAAACA 7107
GTTTTAACCAAGAATATTAGAACCAATGAAGCAGAGA AATCA
AAAGGGTGGATGGAACTGCCAAAGGATG TEE-494
TAGAACAGAATTGAATGGAATGGCATCAAATGGAATG 7108
GAAACGAAAGGAATGGAATTGAATGGACTCAAATGTT ATGGA ATCAAAGGGAATGGACTC
TEE-495 AAGAGAATCATCGAATGGAATCGAATGGAATCAACAT 7109
CAAACGGAAAAAAACGGAATTATCGAATGGAATCGAA GAGAA TCATCGAATGGACC TEE-496
ATCAACATCAAACGGAAAAAAACGGAATTATCGAATG 7110
GAATCGAAGAGAATCATCGAATGGACC TEE-497
GAATCAACATCAAACGGAAAAAAACCGAATTATCGAA 7111
TGGAATCGAAGAGAATCATCGAATGGACC TEE-498
ATCAACATCAAACGGAATCAAACGGAATTATCGAATG 7112
GAATCGAAGAGAATCATCAAATGGACTCGAATGGAAT CATCTA
ATGGAATGGAATGGAAGAATCCATGG TEE-499
ATCGAATGGAATCATTGAATGGAAAGGAATGGAATCA 7113
TCATGGAATGGAAACGAATGGAATCACTGAATGGACT CGAATG GGATCATCA TEE-500
ATTCAGCCTTTAAAAAAAGAAGACAGTCCTGTCATTTG 7114
TGACAATATGAATGAAACAGACATCACATTAAATGAA ATGAG CCAGGCGCAG TEE-501
GAATGAAATGAAATCAAATGGAATGTACATGAATGGA 7115
ATAGAAAAGAATGCATCTTTCTCGAACGGAAGTGCATT GAATG
GAAAGGAATCTACTGGAATGGATTCGAATGGAATGGA ATGGGATGGAATGGTATGG TEE-502
AACATCAAACGGAATCAAACGGAATTATCGAATGGAA 7116
TCGAAGAGAATCATCGAACGGACTCGAATGGAATCAT CTAATG
GAATGGAATGGAAGAATCCATGGACTCGAATGCAATC
ATCATCGAATGAAATCGAATGGAATCATCGAATGGAC TCG TEE-503
ATGGAATTCAATGGAATGGACATGAATGGAATGGACT 7117
TCAATGGAATGGTATCAAATGGAATGGAATTCAGT TEE-504
AATGGAAAGGAATCGAATGGAAGGGAATGAAATTGAA 7118
TCAACAGGAATGGAAGGGAATAGAATAGACGGCAATG GAAT GGACTCG TEE-505
AGCAACTTCAGCAAAGTATCAGGATACAAAATCAATG 7119
TACAAAAATCCCAAGCATTCTTATACACCAACAACAG ACAAAC AGAGAGCC TEE-506
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCGATG 7120
TGCAAAAATCACAAGCATTCTTATACACCAACAACAG ATAAAC AGAGAGCC TEE-507
AACGGAAAAAAAACGGAATTATCGAATGGAATCGAAG 7121
AGAATCATCGAATGGACCAGAATGGAATCATCTAATG GAATG
GAATGGAATAATCCATGGACTCGAATG TEE-508
GGAATCAAACGGAATTATCGAATGGAATCGAAGAGAA 7122
TCATAGAACGGACTCAAATGGAATCATCTAATGGAAT GGAAT GGGAGAATCCATGGACTCGAATG
TEE-509 AATGGAATCAATATCAAACGGAAAAAAACGGAATTAT 7123
CGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-510
AACGGAATCAAACGGAATTATCGAATGGAATCGAAAA 7124
GAATCATCGAACGGACTCGAATGGAATCATCTAATGG AATGG AATGGAAGAATCCATGG
TEE-511 AAACGGAATTATCGAATGGAATCAAAGAGAATCATCG 7125
AATGGCCACGAATGGAATCATATAATGGAATGGAATG GAATA ATCCATGGACC TEE-512
AATGGAATCGAATGGATTGATATCAAATGGAATGGAA 7126
TGGAAGGGAATGGAATGGAATGGAATTGAACCAAATG TAATG GATTTG TEE-513
TAAAAGACGGAACAGATAGAAAGCAGAAAGGAAAGG 7127
TGAATTGCATTACCACTATTCATACTGCCACACACATG ACATTA GGCCAAGTC TEE-514
AATGGAATCGAATGGAACAATCAAATGGACTCCAATG 7128
GAGTCATCTAATGGAATCGAGTGGAATCATCGAATGG ACTCG TEE-515
TAACACATAAACAAACACAGAGACAAAATCTCCGAGA 7129
TGTTAATCTGCTCCAGCAATACAGAACAATTTCTATTA CCAAC AGAATGCTTAATTTTTCTGCCT
TEE-516 GGAATCGAATGGAATCAACATCAAACGGAAAAAAACG 7130
GAATTATCGAATGGAATCAAAGAGAATCATCGAATGG ACC TEE-517
AGAATGGAAAGGAATCGAAACGAAAGGAATGGAGAC 7131 AGATGGAATGGAATG TEE-518
GAATCATCATAAAATGGAATCGAATGGAATCAACATC 7132
AAATGGAATCAAATGGTCTCGAATGGAATCATCTTCAA ATGGA ATGGAATGG TEE-519
AACAACAATGACAAACAAACAACAACGACAAAGACAT 7133
TTATTTGGTTCACAAATCTCCAGGGTGTACAAGAAGCA TGGTG
CCAGCATCTGCTCAGCTTCTGATGAGGGCTCTGGGAAG CTTTTACTC TEE-520
AACGGACTCGAACGGAATATAATGGAATGGAATGGAT 7134
TCGAAAGGAATGGAATGGAATGGACAGGAAAAGAATT GAATG GGATTGGAATGGAATCG
TEE-521 AACATCAAACGAAATCAAACGGAATTATCAAATTGAA 7135
TCGAAGAGAATCATCGAATTGCCACGAATGCAATCAT CTAATG
GTATGGAATGGAATAATCCATGGACCCAGATG TEE-522
AGAAATTAACAGCAAAAGAAGGATGCAGTGCAACTCA 7136
GGACAACACATACAATTCAAGCAACAAATGTATAGTG GCTGG GCACCAAGGATACAG TEE-523
GCAATAAAATCGACTCAGATAGAGAAGAATGCAATGG 7137
AATGGAATGGAATGGAATGGAATGGGATGGAATGGTA TGGAA TGG TEE-524
AATGGACTCGAATGAAATCATCATCAAACGGAATCGA 7138
ATGGAATCATTGAATGGAAAGGATGGGATCATCATGG AATGGA AACGAATGGAATCACTG
TEE-525 CCACATAAAACAAAACTACAAGACAATGATAAAGTTC 7139
ACAACATTAACACAATCAGTAATGGAAAAGCCTAGTC AATGGC AG TEE-526
TGGAATGGAATGGAATGGAATCAAATCGCATGGTAAT 7140
GAATCAAATGGAATCAAATCGAATGGAAATAATGGAA TCGAA
GGGAAACGAATGGAATCGAATTGCACTGATTCTACTG
ACTTCGAGGAAAATGAAATGAAATGCGGTGAAGTGGA ATGG TEE-527
GAATGTTATGAAATCAACTCGAACGGAATGCAATAGA 7141
ATGGAATGGAATGGAATGGAATGGAATGGAATGG TEE-528
AATGGAATCATTGAATGGAATGGAATGGAATCATCAA 7142
AGAAAGGAATCGAAGGGAATCATCGAATGGAATCAAA CGGAA
TCATCGAATGGAATGGAATGGAATG TEE-529
GGAATCAACATCAAACGGAAAAAAAACGGAATTATCG 7143
AATGGAATCGAAGAGAATCATCGAATGGACC TEE-530
GGAATAATCATCATCAAACAGAACCAAATGGAATCAT 7144
TGAATGGAATCAAAGGCAATCATGGTCGAATG TEE-531
GCATAGAATCGAATGGAATTATCATTGAATGGAATCG 7145
AATGGAATCAACATCAAACGGAAAAAAACGGAATTAT CGAATG
GAATCGAAGAGAATCATCGAATGGACCC TEE-532
AATGGAATCGAAGAGAATCATCGAACGGACTCGAATG 7146
GAATCATCTAATGGAATGGAATGGAATAATCCATGGA CCCGAA TG TEE-533
AAATGAATCGAATGGAATTGAATGGAATCAAATAGAA 7147
CAAATGGAATCGAAATGAATCAAATGGAATCGAATCG AATGG AATTGAATGGCATGGAATTG
TEE-534 AGTTAATCCGAATAGAATGGAATGGAATGCAATGGAA 7148
CGGAATGGAACGGAATGGAATGGAATGGAATGGAATG GAATG TEE-535
ATCACAATCACACAACACATTGCACATGCATAACATGC 7149
ACTCACAATACACACACAACACATACACAACACACAT GCAAT
ACAACACAAAACGCAACACAACATATACACAACACAC AGCACACACATGCC TEE-536
AAAGACTTAAACGTTAGACCTAAAACCATAAAAACCC 7150
TAGAGGAAAACCTAGGCATTACCATTCAGGACTTAGG CATGGG CAAGGAC TEE-537
AAAGTCCAAAGATGAACAAAATATCCAGAAGGAAAAC 7151
AAATGCACTTGGGGAGTGGGAAAGAAAACCAAGACTG AGCAA
TGCGTCAAGCTCAGACGTGCCTCACTACG TEE-538
AAACGGAATCAAACGGAATTATCGAATGGAGTCGAAA 7152
AGAATCATCGAACGGACTCGAATGGAATCATCTAATG GAATG GAATGGAAGAATCCATGG
TEE-539 AATTGATTCGAAATTAATGGAATTGAATGGAATGCAAT 7153
CAAATGGAATGGAATGTAATGCAATGGAATGTAATAG AATGG AAAGCAATGGAATG TEE-540
TACAGAACACATGACTCAACAACAGCAGAAAGCATAT 7154
TCTTTTCAAATGCACATGAAACATTATCATGATGGACC AAAT TEE-541
GGAACAAAATGAAATCGAACGGTAGGAATCATACAGA 7155
ACAGAAAGAAATGGAACGGAATGGAATG TEE-542
AACGGAAAAAACGGAATTATCGAATGGAATCGAAGAG 7156
AATCATCGAATGGAATCGAATGGAGTCATCG TEE-543
AATCGAACGGAATCAACATCAAACGGAAAAAAACGGA 7157
ATTATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-544
AGAATGGAATGCAATAGAATGGAATGCAATGGAATGG 7158
AGTCATCCGTAATGGAATGGAAAGGAATGCAATGGAA TGGAA TGGAATGG TEE-545
ATGGAATCAACATCAAACGGAATCAAACGGAATTATC 7159
GAATGGAATCGAAGAGAATCATCGAACGGATTCGAAT GGAATC
ATCTAATGGAATGGAATGGAAGAATCCATGGACTCGA
ATGCAATCATCAGCGAATGGAATCGAATGGAATCATC GAATGG ACTCG TEE-546
GGAATAAAACGGACTCAATAGTAATGGATTGCAATGT 7160
AATTGATTCGATTTCGAATGGAATCGCATGGAATGTAA TGGAA TGGAATGGAATGGAAGGC
TEE-547 AATGGAATCAACATCAAACGGAAAAAAACGGAATTAT 7161
CGTATGGAATCGAAAAGAATTATCGAATGGACC TEE-548
TCAAACGGAAAAAAACGGAATTATCGAATGGAATCGA 7162 AGAGAATCATCGAATGGACC
TEE-549 ACATCAAACGGAATCAAACGGAATTATCGAATGGAAT 7163
CGAAAAGAATCATCGAACGGACTCGAATGGAATCATC TAATGG
AATGGAATGGAAGAATCCATGGACTCGAATG TEE-550
TGGAATCGAATGGAATCAACATCAAACGGAAAAAAAC 7164
GGAATTATCGAATGGAATCGAAGAGAATCATCGAATG GACC TEE-551
AATGGAATCGAATGCAATCATCGAACGGAATCGAATG 7165
GCATCACCGAATGGAATGGAATGGAATGGAATGGAAT GG TEE-552
AGAATTGATTGAATCCAAGTGGAATTGAATGGAATGG 7166
AATGGATTAGAAAGGAATGGAATGGATTGGAATGGAT TGGAAT GGAAAGG TEE-553
AACTGCATCAACTAACAGGCAAAATAACCAGCTAATA 7167
TCATAATGACAGGATTAAATTCACAAATGACAATATTA ACCGT AAATGTAAATGGGCTA
TEE-554 GTAAACAAACAATCAAGCAAGTAAGAACAGAAATAAC 7168
AGCATTTGGCTTTTGAGTTAATGACAAGAACACTCGGC ATGGG
AGCCTGGGTGAGCAAATCACAGATCTTC TEE-555
AAAGGAATGGACTGGAACAAAATGAAATCGAACGGTA 7169
GGAATCGTACAGAACGGACAGAAATGGAACGGCATGG AATGC ACTCG TEE-556
GAATCAACCCGAGCGGAAAGGAATGGAATGGAATGGA 7170
ATCAACACGAATGGAATGGAACGGAATGGAATGGGAT GGGAT GAAATGGAATGG TEE-557
AAGAAATGGAATCGAAGAGAATGGAAACAAACGGAA 7171
TGGAATTGAATGGAATGGAATTGAATGGAATGGGA TEE-558
GACATGCAAACACAACACACAGCACACATGGAACATG 7172
CATCAGACATGCAAACACAACACACATACCACACATG GCATAT GCATCAGACGTGCCTCACTAC
TEE-559 AAAGGAATGCACTCGAATGGAATGGACTTGAATGGAA 7173
TGTCTCCGAATGGAACAGACTCGTATGAAATGGAATC GAATGG
AATGGAATCAAATGGAATTGATTTGAGTGAAATGGAA TCAAATGGAATGGCAACG TEE-560
GGAACAAAATGAAATCGAACGGTAGGAATCGTACAGA 7174
ACGGAAAGAAATGGAACGGAATGGAATGCACTCGAAT GGAAA GGAGTCCAAT TEE-561
AAATTGATTGAAATCATCATAAAATGGAATCGAAGGG 7175
AATCAACATCAAATGGAATCAAATGGAATCATTGAAC GGAATT GAATGGAATCGTCAT
TEE-562 AGAATGGAAAGCAATAGAATGGAACGCACTGGATTCG 7176
AGTGCAATGGAATCAATTGGAATGGAATCGAATGGAA TGGAT TGGCA TEE-563
AACACCAAACGGAAAAAAACGGAATTATCGAATGGAA 7177
TCGAAGAGAATCTTCGAACGGACCCGAATGGGATCAT CTAAT
GGAATGGAATGGAATAATCCATGG TEE-564
AATGGAGACTAATGTAATAGAATCAAATGGAATGGCA 7178
TCGAATGGAATGGACTGGAATGGAATGTGCATGAATG GAATGG AATCGAATGGATTG TEE-565
AAATCGAATGGAACGCAATAGAATAGACTCGAATGTA 7179
ATGGATTGCTATGTAATTGATTCGAATGGAATGGAATC GACTG
GAATGCAATCCAATGGAATGGAATGCAATGCAATGGA
ATGGAATCGAACGGAATGCAGTGGAAGGGAATGG TEE-566
AATCAACAAGGAACTGAAACAAGTAAACAAGAAAAC 7180
AAATAACACCATAAAACATGGGCAAAGGACATAAACA GACATT
TTTCAAAAAAGACATACAAATGGCCGAG TEE-567
AATGGAATCAACATCAAACGGAAAAAAACGGAATTAT 7181
CGAATGGAATCGAAGAGAATCATCGAATGGACCCAGG CTGGT CTTGAACTCC TEE-568
ATTGAATGGGCTAGAATGGAATCATCTTTGAACGGAAT 7182
CAAAGGGAATCATCATCGAATGGAATCGAATGGAAAT GTCAA CG TEE-569
AATGGACTCGAATGGAATCAACATCAAATGGAATCAA 7183
GCGGAATTATCGAATGAAATCGAAGAGAATCATCGAA TGGACT
CGAAAGGAATCATCTAATGGAATGGAATGGAATAATC CATGGACTCGAATGCAATCATCATCG
TEE-570 AAACGGAAAAAAACGGAATTATTGAATGGAATCGAAG 7184
AGAATCTTCGAACGGACCCGAATGGAATCATCTAATG GAATG GAATGGAATAATCCATGG
TEE-571 ACTCGAGTGGAATTGACTGTAACAAAATGGAAAGTAA 7185
CGGATTGGAATCGAATGGAACGGAATGGAATGGAATG GACAT TEE-572
TACAAACTTTAAAAAATGATCAACAGATACACAGTTA 7186
GCAAGAAAGAATTGAGGGCAAAGAATATGCCAGACAA ACTCA
AGAGGAAGATGATGGTAGAGATAGGTCACATTGGAGT GTCA TEE-573
AAATCAACAACAAACGGAAAAAAAAGGAATTATCGAA 7187
TGGAATCAAAGAGAATCATCGAATGGACC TEE-574
AACGGAATCAAACGGAATTATCGAATGGAATCGAAAA 7188
GAATCATCGAACGGACTCGAATGGAATCATCTAATGT AATGGA
ATGGAAGAATCCATGGACTCGAATG TEE-575
AACGGAAAAAAACGGAATTATCGAATGGAATCGAAGA 7189
GAATCATCGAATGGACCAGAATGGAATCATCTAATGG AATGG
AATGGAATAATCCATGGACTCGAATG TEE-576
CAACATCAAACGGAAAAAAACGGAATTATGGAATGGA 7190
ATCGAAGAGAATCATCGAATGGACCCGAATGGAATCA TCTGA
AATATAATAGACTCGAAAGGAATG TEE-577
ATGGAATCGAATGGAATGGACTGGAATGGAATGGATT 7191
CGAATGGAATCGAATGGAACAATATGGAATGGTACCA AATG TEE-578
GAATGGAATCAACATCAAACGGAAAAAAACGGAATTA 7192
TCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-579
AAATGGACTCGAATGGAATCATCATAGAATGGAATCG 7193
AATGCAATGGAATGGAATCTTCCGGAATGGAATGGAA TGGAATGGAATGGAG TEE-580
GAATCATCATAAAATGGAATCGAATGGAATCAACATC 7194
AAATGGAATCAAATGGAATCATTGAACGGAATTGAAT GGAATCGTCAT TEE-581
ATCGAATGGAATCAACATCAAACGGAAAAAAACGGAA 7195
TTATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-582
AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATG 7196
TACAAAAATCACAAGCATTCTTATACACCAATAACAG ACAAACAGAGAGCCAAAA TEE-583
AGAAACAGAAAACAGTCAAACCAATGGGCAATCCATA 7197
TCAGATGCAGTATTATGAACAGAAGTGTAAAGAATGC ACCAGGCACAATGGC TEE-584
GATTGGAACGAAATCGAATGGAACGGAATAGAATAGA 7198
CTCGAATGTAATGGATTGCTATGTAATTGATTCGAATG
GAATGGAATCGAATGGAATGCAATCCAATGGAATGGA ATGCAATGCAATGGAATGG TEE-585
ATGGAATGGAATAATCAACGTACTCGAATGCAATCAT 7199
CATCGTATAGAATCGAATGGAATCATCGAATGGACTC
GAATGGAATAATCATTGAACGGAGTCGAATGGAATCA TCATCGGATGGAAAC TEE-586
AAAGAAATCGAATGGAATCAGTGTCGAATGGAATGGA 7200
ATGGAATCGAAGAATTGAATTGAGTAGAATCGAAGGG AATCATTGGATGGGCTCAAAT TEE-587
AGAAAAGATAACTCGATTAACAAATGAACAAACACCT 7201
GAATACACAAGTCTCAAAAGAAGACATAAAAATGGCC AAC TEE-588
ATGGAATCAACATCAAACGGAATCACACGGAATTATC 7202
GAATGGAATCGAAAAGAATCATCGAACGGACTCGAAT GGAATCATCTAATGGAATGGAATGGAAG
TEE-589 AATGGAATCAACATCAAACGGAATCAAGCGAAATTAT 7203
CGAATGGAATCGAAGAGAATCATCGAATGGACTCGAA TGGAATCATCTAATGGAATGGAATGGGAT
TEE-590 AAACACAGTACAAATACTAATTCAAATCAAACTTACTC 7204
AAAGTCATAATCAAACATGCCAGACGGGCTGAGGGGC AGCATTA TEE-591
GGAATCGAGTGGAATCATCGAAAGAAATCGAATGGAA 7205
TCATTGTCGAATGGAATGGAATGGAATCAAAGAATGG AATCGAAGGGAATCATTGGATGGGCT
TEE-592 AAAGAAAGACAGAGAACAAACGTAATTCAAGATGACT 7206
GTTTACATATCCAAGAACATTAGATGGTCAAAGACTTT
AAGAAGGAATACATTCAAAGGCAAAAAGTCACTTACT GATTTTGGTGGAGTTTGCCACATGGAC
TEE-593 GAAAGGAATCATCATTGAATGCAATCACATGGAATCA 7207
TCACAGAATGGAATCGTACGGAATCATCATCGAATGG
AATTGAATGGAATCATCAATTGGACTCGAATGGAATC
ATCAAATGGAATCGATTGGAAGTGTCAAATGGACTCG TEE-594
CAATCAGAGCGGACACAAACAAATTGCATGGGAAGAA 7208 TCAATATCGTGAAAATGGCC
TEE-595 CAGCGCACCACAGCACACACAGTATACACATGACCCA 7209
CAATACACACAACACACAACACATTCACACACCAC TEE-596
GCAAACAGAATTCAACACTACATTAGAACGATCATTC 7210
ATCACGACCTAGTAGGATGTTTTTCCTGGGATGCAAGG ATGGTTCAACAT TEE-597
CAATCAAAACAGCAATGAGATACCATTTTACACCAATC 7211
AAAATGGCTACTAAAAAGTCAAAAGCAAATGCC TEE-598
TGGAATAGAATGGAATCAATGTTAAGTGGAATCGAGT 7212
GGAATCATCGAAAGAAATCGAATGGAATCATTGTCGA ATGGTATGGAATGGAATCA TEE-599
AATGGAATGGAATCATCGCATAGAATGGAATGGAATT 7213
ATCATCGAATTGAATCGAATGGTATCAACATCAAACG
GAAAAAAACGGAAATATCGAATGGAATCGAAGAGAAT CATCGAACGGACC TEE-600
GAAAAACAAAACAAAACAAACAAACAAACAATCAAA 7214
AAAGTGGTAGCAGAAACCAGAAAGTCCATGTATATAG CTAATTGGCCTGGTTGT TEE-601
AGACCTTTCTCAGAAGACACACAAATTGCCAACAGGT 7215
ATATGAAAAAATGTTCAATATCACTAATCATCAGGGCG ATGCC TEE-602
CATGGAATCGAATGGAATTATCATCGAATGGAATCGA 7216
ATGGTACCAACACCAAACGGAAAAAAACGGAATTATC
GAATGGAATCGAAGAGAATCTTCGAACGGACC TEE-603
AGAGCAGAAACAAATGGAATTGAAATGAAGACAACA 7217
ATCAAAAGCATCAATGAAATGAAAAGTTGGGTTTTGG AAGAGAGAAACAAT
TEE-604 ACACAAACACACACACACACACACACACACACACACA 7218
CACACACACACACACACACACACACACACATAC TEE-605
AACAAACAAATGAGATGATTTCAGATAGTGATAAACA 7219
CTATAACATAATTAATTCGTGCCAATCAGAGCATAACA
GTGGTGTGGTGGCTGTGGAACAGATAGCAGAC TEE-606
AATGGAATCGAGTGGAATGGAAGGCAATGGAATAGAA 7220
TGGAATGGAATCGAAAGGAACGGAATGGAATGGAATG GAATG TEE-607
AGAAATGGAATCGGAGAGAATGGAAACAAATGGAAT 7221
GGAATTGAATGGAATGGAATTGAATGGAATGGGAACG TEE-608
AAGAGAACTGCAAAACACTGCTCAAAGAAATCAGAGA 7222
TGACAAAAACACATGGAAAAACGTTTCATGCTCATGG ATTGGAAGACTTA TEE-609
AATCAACACGAATAGAATGGAACGGAATGGAATGGAA 7223
TGGAATGGAATGGAATGGAGTGGAATGGAACAGAATG GAGTGGAAT TEE-610
AACATCAAACGAAATCAAACGGAATTATCAAATTGAA 7224
TCGAAGAGAATCATCGAATTGCCACGAATGCAACCAT
CTAATGGTATGGAATGGAATAATCCATGGACCCAGATG TEE-611
CGGAATTATCATCGAATGTAATCGAATGGAATCAACAT 7225
CAAACGGAAAAAAACGGAATTATCGAATGGAATCGAA GAGAATCATCGAATGGACC TEE-612
TGGACACACACGAACACACACCTACACACACGTGGAC 7226
ACACACGGACACATGGACACACACGAACACATGGACA
CACACACGGGGACACACACAGACACACACAGAGACAC ACACGGACACATGG TEE-613
ATCAAACGGAATCAAACGGAATTATCGAATGGAATCG 7227
AAGAGAATCATCGAATGGACTCGAATGGAATCATCTA ATGGAATGGAATGGAAGAATCCATGG
TEE-614 AAATGGAATGGAATGCACTTGAAAGGAATAGACTGGA 7228
ACAAAATGAAATCGAACGGTAGGAATCATACAGAACA GAAAGAAATGGAACGGAATGGAATG
TEE-615 ACCACACACAAAATACACCACACACCACACACACACC 7229
ACACACTATACACACACCACACACCACACAC TEE-616
AAAGAAATAGAAGGGAGTTGAACAGAATCGAATGGA 7230
ATCGAATCAAATGGAATCGAATGGCATCAAATGGAAT CGAATGGAATGTGGTGAAGTGGATTGG
TEE-617 GGAATCATCATAAAATGGAATCGAATGGAATCATCAT 7231
CAAATGGAATCAAATGGAATCATTGAACGGAATTGAA TGGAATCGTCAT TEE-618
AAAGATCAATGTACAAAAATCAGCAGCATTTCTATAA 7232
ACCAACAATGTCCAGGCTGAGAGAGAAATCAAGAAAA CAATTC TEE-619
TGGAATGGAATGGAATGAAATAAACACGAATAGAATG 7233
GAACGGAATGGAACGGAATGGAATGGAATGGAATGG AAAG TEE-620
TAATCAGCACAATCAACTGTAGTCACAAAACAAATAG 7234
TAACGCAATGATAAAGAAACAGAGAACTAGTTCAAAT AAACATGATAAGATGGGG TEE-621
AAGCGGAATTATCAAATGGAATCGAAGAGAATGGAAA 7235
CAAATGGAATGGAATTGAATGGAATGGAATTGAATGG AATG TEE-622
AATGGAATCAACATCAAACGGAAAAAAACGGAATTAT 7236
CGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-623
ACTTGAATCGAATGGAAAGGAATTTAATGAACTTAAA 7237
TCGAATGGAATATAATGGTATGGAATGGACTCATGGA ATGGAATGGAAAGGAATC TEE-624
TGGAATCATCATCGAAAGCAAGCGAATGGAATCATCA 7238
AATGGAAACGAATGGAATCATCGAATGGACTCGGATG GAATTGTTGAATGGACT TEE-625
TGGAATCAACATCAAACGGAAAAAAACGGAATTATCG 7239
AATGGAATCGAAGAGAATCATCGAATGGACC TEE-626
TAAGTGAATTGAATAGAATCAATCTGAATGTAATGAA 7240
ATGGAATGGAACGGAATGGAATGGAATGGAATGGAAT GGAATGGAATGG TEE-627
AGGAAAATTTAATCAGCAGGAATAGAAACACACTTGA 7241
GAAATCCATGTGGAATGAAAAGAGAATGGCTGAGCAG CAACAGATTGTCAAAAAGGAAATC
TEE-628 AACATCAAACGGAAAAAAAACGGAATTATCGAATGGA 7242
ATCGAAGAGAATCATCGAATGGACC TEE-629
TAATTGAGAATAAGCATTCCAGTGGAAAAAAAACTAA 7243
ACAATTTGTTGTAAAACATCCTTAAAAGCATCAGAAAG
TTAATACAGCAATGAAGAATTACAGGACCAAATTAAG AATGGTATGGAAGCCTGTTA TEE-630
TATCATCGAATGGAATCGAATGGAATCAACATCAAAC 7244
GGAAAAAAACGGAATTATCGAATTGAATCGAAGAGAA TCATCGAATGGACC TEE-631
AGCAAAACAAACACAATCTGTCGTTCATGGTACTACG 7245
ACATACTGGGAGAGATATTCAAATGATCACACAAAAC AACATG TEE-632
AAGGATTCGAATGGAATGAAAAAGAATTGAATGGAAT 7246
AGAACAGAATGGAATCAAATCGAATGAAATGGAGTGG AATAGAAAGGAATGGAATG TEE-633
AACGGAATCAAACGGAATTATCGAATGGAATCGAAGA 7247
GAATCATCGAACGGACTCGAATGGAATCATCTAATGG
AATGGAATGGAAGAATCCATGGACTCGAATGCAATCA
TCATCGAATGGAATCGAACGGAATCATCGAATGGCC TEE-634
AATCAACTAGATGTCAATGGAATGCAATGGAATAGAA 7248
TGGAATGGAATTAACACGAATAGAATGGAATGGAATG GAATGGAATGG TEE-635
AATGGACTCGAATGGAATAATCATTGAACGGAATCGA 7249
ATGGAATCATCATCGGATGGAAATGAATGGAATCATC ATCGCATGGAATCG TEE-636
GAATGGAATGATACGGAATAGAATGGAATGGAACGAA 7250
ATGGAATTGAAAGGAAAGGAATGGAATGGAATGGAAT GG TEE-637
AATCATCATCGAATGGAATCGAATGGTATCATTGAGTG 7251
GAATCGAATGGAATCATCATCAGATGGAAATGAATGG AATCGTCAT TEE-638
GAATCAAATCAATGGAATCAAATCAAATGGAATGGAA 7252
TGGAATTGTATGGAATGGAATGGCATGG TEE-639
TAATGCAGTCCAATAGAATGGAATCGAATGGCATGGA 7253
ATATAAAGAAATGGAATCGAAGAGAATGGGAACAAAT
GGAATGGAATTGAGTGGAATGGAATTGAATGGAATGG GAACGAATGGAGTG TEE-640
AACATCAAACGGAAAAAAACGGAATTATCGAATGGAA 7254 TCGAAGAGAATCATCGAATGGACC
TEE-641 ATCAAAAGGAACGGAATGGAATGGAATGGAATGGAAT 7255
GGAATGGAATGGAATGGAATGAAATCAACCCGAATGG AATGGATTGGCATAGAGTGGAATGG
TEE-642 GCCAACAATCATATGAGAAAAAGCTCAACATCACTGA 7256
TCATTTCAGGAATGCAAATCAAAACCACAATGAGATA CTATCA TEE-643
AATCAAATGGAATGAAATCGAATGGAATTGAATCGAA 7257
TGGAATGCAATAGAATGTCTTCAAATGGAATCGAATG GAAATTGGTGAAGTGGACGGGAGTG
TEE-644 TAATGGAATCAACATCAAACGGAAAAAAACGGAATTA 7258
TCGAATGCAATCGAAGAGAATCATCGAATGGACC TEE-645
AGCAACTTCAGCAAAGTCTCAGCATACAAAATCAATG 7259
TGCAAAAATCACACGCATTCCTATACACCAATAACAG ACAAACAGAGAGCC TEE-646
GAATCAAATGGAATGGACTGTAATGGAATGGATTCGA 7260
ATGGAATCGAATGGAGTGGACTCAAATGGAATG TEE-647
AACAAGTGGACGAAGGATATGAACAGACACTTCTCAA 7261
GACATTTATGCAGCCAACAGACACACGAAAAAATGCT CATCATCACTGGCCATCAG TEE-648
AAACGGAAAAAAACGGAATTATCGAATGGAATCGAAT 7262 AGAATCATCGAATGGACC
TEE-649 TGGAACCGAACAAAGTCATCACCGAATGGAATTGAAA 7263
TGAATCATAATCGAATGGAATCAAATGGCATCTTCGAA
TTGACTCGAATGCAATCATCCACTGGGCTT TEE-650
AACGGAATCACGCGGAATTATCGAATGGAATCGAAGA 7264
GAATCATCGAATGGACTCGAATGGAATCATCTAATGG AATGGAATGG TEE-651
GGAATCAACTCGATTGCAATGGAATGCAATGGAAAGG 7265
AATGGAATGCAATTAAAGCGAATAGAATGGAATGGAA TGGAATGGAACGGAATGGAATG
TEE-652 AAAACAAACAACAACGACAAATCATGAGACCAGAGTT 7266
AAGAAACAATGAGACCAGGCTGGGTGTGGTG TEE-653
AATCGAAAGGAATGCAATATTATTGAACAGAATCGAA 7267
AAGAATGGAATCAAATGGAATGGAACAGAGTGGAATG GACTGC TEE-654
AAGGAATCGAATGGAAGTGAATGAAATTGAATCAACA 7268
GGAATGGAAGGGAATAGAATAGACTGTAATGGAATGG ACTCG TEE-655
AACCCGAGTGCAATAGAATGGAATCGAATGGAATGGA 7269
ATGGAATGGAATGGAATGGAATGGAGTC TEE-656
GAATGGAATTGAAAGGAATGGAATGCAATGGAATGGA 7270
ATGGGATGGAATGGAATGCAATGGAATCAACTCGATT GCAATG TEE-657
GAAAAAAACGGAATTATCGAATTGAATCAAATAGAAT 7271
CATCGAACGGACCAAAATGGAATCATCTAATGGAATG GAATGGAATAATCCATGGACTCTAATG
TEE-658 TGGAATCATCTAATGGAATGGAATGGAATAATCCATG 7272
GACTCGAATGCAATCATCATAAAATGGAATCGAATGG
AATCAACATCAAATGGAATCAAATGGGATCATTGAAC GGAATTGAATGGAATCGTCAT TEE-659
GAAAAAAACGGAATTATCGAATTGAATCGAATAGAAT 7273
CATCGAACGGACCAGAATGGAATCATCTAATGGAATG GAATGGAATAATCCATGGACTCGAATG
TEE-660 AACCACTGCTTAAGGAAATAAGAGAGAACACAAACAA 7274
ATGGAAAAACGTTCCATGCTCATGGATAGGAGAATCA ATATCGTGAAAATGGCC TEE-661
TATCGAATGGAATGGAAAGGAGTGGAGTAGACTCGAA 7275
TAGAATGGACTGGAATGAAATAGATTCGAATGGAATG GAATGGAATGAAGTGGACTCG TEE-662
GTATCAACATCAAACGGAAAAAAACGGAATTATCGAA 7276
TGGAATCATCTAATGGAATGGAATGGAATAATCCATG GACTCGAATG TEE-663
TAAATGGAGACATCATTGAATACAATTGAATGGAATC 7277
ATCACATGGAATCGAATGGAATCATCGTAAATGCAAT CAAGTGGAATCAT TEE-664
GAATGGAATTGAAAGGTATCAACACCAAACGGAAAAA 7278
AAAACGGAATTATCGAATGGAATCGAAGAGAATCATC GAACGGACC TEE-665
AGCAATTTCAGCAAAGTCTCAGGATACAAAATCAATG 7279
TACAAATTCACAAGCATTCTTATGGACCAACAACAG TEE-666
GGAATCGAATGGCATCAACATCAAACGGAAAAAAACG 7280
GAATTATCGAATGGAATCGAATGGAATCATC TEE-667
AAACAAAACACAGAAATGCAAAGACAAAACATAAAA 7281
CGCAGCCATAAAGGACATATTTTAGATAACTGGGGAA ATTTGTATGGGCTGTGT TEE-668
AATGGAATCAACATCAAACGGAATCAAACGGAATTAT 7282
CGAATGGAATCGAAGAGAATCATCGAACGGACTCGAA TGGAATCATCTAATGGAATGGAATGGAAG
TEE-669 AATCGAATGGAATCAGCATCAAACGGAAAAAAACGGA 7283
ATTATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-670
AAACGGAATTATAGAATGGACTGGAAGAGAATCATCG 7284
AACGGACTAGAATGGAATCATCTAATCGAATGGAATG GAACAATCCATGGTCTAGCA TEE-671
TGAACAGAGAATTGGACAAAACGCACAAAGTAAAGAA 7285
AAAGAATGAAGCAACAAAAGCAGAGATTTATTGAAAA
CAAAAGTACACACCACACAGGGTGGGAGTGG TEE-672
ATCATAACGACAAGAACAAATTCACACACAACAATAT 7286
TAACTTCAAATCCAAATGGGTTAAATGCTCCAATTAAA GGATGCAGACGGGCAAATTGGATA
TEE-673 ATCATAACGACAAGAACAAATTCACACACAACAATAT 7287
TCACTTCAAATCCAAATGGGTTAAATGCTCCAATTAAA GGATGCAGACGGGCAAATTGGATA
TEE-674 GAATGGAATCGAATGGATTGATATCAACTGGAATGGA 7288
ATGGAAGGGAATGGAATGGAATGGAATTGAACCAAAT GTAATGACTTGAATGGAATG TEE-675
GAATCAACATCAAACGGAAAAAAACGGAATTATCGAA 7289
TGGAATCGAAGAGAATCATCGAATGGACC TEE-676
GGAATCAACATCAAACGGAAAAAAACGGAATTATCGA 7290
ATGGAATCGAAGAGAATCATCGAATGGACC TEE-677
ATGGAATCAACATCAAACGGAATCAAACGGAATTATC 7291
GAATGGAATCAAAGAGAATCATCGAACGGACTCGAAT
GGAATCATCTAATGGAATGGAATGGAAGAATCCATGG ACTCGAATGCAATCATCATCGAAT
TEE-678 GGAATGGAATGGAATGGAGCCGAATGGAATGGAATGT 7292 ACTCAAATGGAATGC
TEE-679 AAAACACCTAGGAATACAGATAACAAGGGACATTAAC 7293
TACCTCTTAAAGAGAACTACAAACCACTGCTCAAGGA
AATGAGAGAGGACACAAACACATGGAAAAACATTCCA
TCCTCATGGATAGGAAGAATCAATATTGTGAAAATGG CC TEE-680
AACACGACTTTGAGAAGAGTAAGTGATTGTTAATTAA 7294
AGCAAGAGAATTATTGATGTATCACAGTCATGAGAAA TATTGGAAGGAATATGGTCCATAC
TEE-681 ACACATATCAAACAAACAAAAGCAATTGACTATCTAG 7295
AAATGTCTGGGAAATGGCAAGATATTACA TEE-682
GGAATCATCATATAATGGAATCGAATGGAATCAACAT 7296
CAAATGGAATCAAATGGAATCATTGAACGGAATTGAA TGGAATCGTCAT TEE-683
AATGGAATCAACATCAAACGGAATCAAATGGAATTAT 7297
CGAATGGAATCGAAGAGAATCATCGAATTGTCACGAA
TGGAATCATCTAATGGAATGGAATGGAATAATCCATG
GCCCCTATGCAATGGACTCGAATGAAATCATCATCAAA
CAGAATCGAATGGAATCATCTAATGGAATGGAATGGC ATAATCCATGGACTCGAATG TEE-684
TAAAATGAAACAAATATACAACACGAAGGTTATCACC 7298
AGAAATATGCCAAAACTTAAATATGAGAATAAGACAG TCTCAGGGGCCACAGAG TEE-685
AAAATACAGCGTTATGAAAAGAATGAACACACACACA 7299 CACACACACACACAGAAAATGT
TEE-686 CAAACAAATAGGTACCAAACAAATAACAACATAAACC 7300
TGACAACACACTTATTTACAAGAGACATCCCTTATATG
AAAGGGTACAGAAAAGTCGATGGTAAGATGATGGGGA AAGGTATACCAACCACTAGCAGAAGG
TEE-687 TGGAATCGAATGGAATCAATATCAAACGGAAAAAAAC 7301
GGAATTATCGAATGGAATCGAAAAGAATCATCGAATG GGCCCGAATGGAATCATCT TEE-688
ACAAATGGAATCAACAACGAATGGAATCGAATGGAAA 7302
CGCCATCGAAAGGAAACGAATGGAATTATCATGAAAT TGAAATGGATG TEE-689
AATCAATAAATGTAAACCAGCATATAAACAGAACCAA 7303
CGACAAAAACCACATGATTATCTCAATAGATGCAGAA AAGGCC TEE-690
AAAATAAACGCAAATTAAAATCACAAGATACCAACAC 7304
ATTCCCACGGCTAAGTACGAAGAACAAGGGCGAATGG TCAGAATTAAGCTCAAACCT TEE-691
CAACATCAAACGGAATCAAACGGAATTATCGAATGGA 7305
ATCGAAGAGAATCATCGAATGGACTCGAATGGAATCA TCTAATGGAATGGAATGGAAG TEE-692
ACATCAAACGGAAAAAAACGGAATTATCGAATGGAAT 7306 CGAAGAGAATCATCGAATGGACC
TEE-693 AATGGACTCGAATAGAATTGACTGGAATGGAATGGAC 7307
TCGAATGGAATGGAATGGAATGGAAGGGACTCG TEE-694
AAGAAAGACAGAGAACAAACGTAATTCAAGATGACTG 7308
ATTACATATCCAAGAACATTAGATGGTCAAAGACTTTA
AGAAGGAATACATTCAAAGGCAAAACGTCACTTACTG ATTTTGGTGGAGTTTGCCACATGGAC
TEE-695 GAATGGAATCGAATGGAATGAACATCAAACGGAAAAA 7309
AACGGAATTATCGAATGGAATCAAAGAGAATCATCGA ATGGACCCG TEE-696
ATGGACTCGAATGTAATAATCATTGAACGGAATCGAA 7310
TGGAATCATCATCGGATGGAAACGAATGGAATCATCA TCGAATGGAATCGAATGGGATC
TEE-697 GAAATGGAATGGAAAGGAATAAAATCAAGTGAAATTG 7311
GATGGAATGGATTGGAATGGATTGGAATG TEE-698
AAACGGAAAAAAAACGGAATTATCGAATGGAATCGAA 7312
GAGAATCATCGAACGAACCAGAATGGAATCATCTAAT GGAATGGAATGGAATAATCCATGG
TEE-699 ATTAACCCGAATAGAATGGAATGGAATGGAATGGAAC 7313
GGAACGGAATGGAATGGAATGGAATGGAATGGAATGG ATCG TEE-700
AACATCAAACGGAAAAAAACGGAATTATCGTATGGAA 7314 TCGAAGAGAATCATCGAATGGACC
TEE-701 GAATAGAATTGAATCATCATTGAATGGAATCGAGTAG 7315
AATCATTGAAATCGAATGGAATCATCATCGAATGGAA TTGGGTGGAATC TEE-702
CACCGAATAGAATCGAATGGAACAATCATCGAATGGA 7316
CTCAAATGGAATTATCCTCAAATGGAATCGAATGGAAT TATCG TEE-703
AATGCAATCGAATAGAATCATCGAATAGACTCGAATG 7317
GAATCATCGAATGGAATGGAATGGAACAGTC TEE-704
AAATCATCATCGAATGGAATCGAATGGTATCATTGAAT 7318
GGAATCGAATGGAATCATCATCAGATGGAAATGAATG GAATCGTCAT TEE-705
GAATGGAATCGAAAGGAATAGAATGGAATGGATCGTT 7319
ATGGAAAGACATCGAATGGAATGGAATTGACTCGAAT GGAATGGACTGGAATGGAACG
Example 46
In Vitro Expression of Modified Nucleic Acids with miR-122
[1086] MicroRNA controls gene expression through the translational
suppression and/or degradation of target messenger RNA. The
expression of G-CSF mRNA and Factor IX mRNA with human or mouse
alpha-globin 3' untranslated regions (UTRs) were down regulated in
human primary hepatocytes using miR-122 sequences in the 3'UTR.
[1087] Primary human hepatocytes were seeded at a density of 350000
per well in 500 ul cell culture medium (InVitro GRO medium from
Celsis, Chicago, Ill.).
[1088] G-CSF mRNA having a human alpha-globin 3'UTR (G-CSF Hs3'UTR;
mRNA sequence shown in SEQ ID NO: 7320; polyA tail of approximately
140 nucleotides not shown in sequence; 5' cap, Cap1) or a mouse
alpha-globin 3'UTR (G-CSF Mm3'UTR; mRNA sequence shown in SEQ ID
NO: 7321; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1) were fully modified with 5-methylcytidine
and 1-methylpseudouridine. G-CSF mRNA containing a human 3'UTR
having a miR-122 sequence in the 3'UTR (G-CSF Hs3'UTR miR-122; mRNA
sequence shown in SEQ ID NO: 7322; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1), or a miR-122 seed
sequence in the 3'UTR (G-CSF Hs3'UTR miR-122 seed; mRNA sequence
shown in SEQ ID NO: 7323; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) or a miR-122
sequence without the seed sequence in the 3'UTR (G-CSF Hs3'UTR
miR-122 seedless; mRNA sequence shown in SEQ ID NO: 7324; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1) were fully modified with 5-methylcytidine and
1-methylpseudouridine. G-CSF mRNA containing a mouse 3'UTR having a
miR-122 sequence in the 3'UTR (G-CSF Mm3'UTR miR-122; mRNA sequence
shown in SEQ ID NO: 7325; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1), or a miR-122 seed
sequence in the 3'UTR (G-CSF Mm3'UTR miR-122 seed; mRNA sequence
shown in SEQ ID NO: 7326; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) or a miR-122
sequence without the seed sequence in the 3'UTR (G-CSF Mm3'UTR
miR-122 seedless; mRNA sequence shown in SEQ ID NO: 7327; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1) were fully modified with 5-methylcytidine and
1-methylpseudouridine.
[1089] Factor IX mRNA having a human alpha-globin 3'UTR (Factor IX
Hs3'UTR; mRNA sequence shown in SEQ ID NO: 7328; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1)
or a mouse alpha-globin 3'UTR (Factor IX Mm3'UTR; mRNA sequence
shown in SEQ ID NO: 7329; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) were fully
modified with 5-methylcytidine and 1-methylpseudouridine. Factor IX
mRNA containing a human 3'UTR having a miR-122 sequence in the
3'UTR (Factor IX Hs3'UTR miR-122; mRNA sequence shown in SEQ ID NO:
7330; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1), or a miR-122 seed sequence in the 3'UTR
(Factor IX Hs3'UTR miR-122 seed; mRNA sequence shown in SEQ ID NO:
7331; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1) or a miR-122 sequence without the seed
sequence in the 3'UTR (Factor IX Hs3'UTR miR-122 seedless; mRNA
sequence shown in SEQ ID NO: 7332; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) were fully
modified with 5-methylcytidine and 1-methylpseudouridine. Factor IX
mRNA containing a mouse 3'UTR having a miR-122 sequence in the
3'UTR (Factor IX Mm3'UTR miR-122; mRNA sequence shown in SEQ ID NO:
7333; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1), or a miR-122 seed sequence in the 3'UTR
(Factor IX Mm3'UTR miR-122 seed; mRNA sequence shown in SEQ ID NO:
7334; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1) or a miR-122 sequence without the seed
sequence in the 3'UTR (Factor IX Mm3'UTR miR-122 seedless; mRNA
sequence shown in SEQ ID NO: 7335; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) were fully
modified with 5-methylcytidine and 1-methylpseudouridine.
[1090] Each G-CSF or Factor IX mRNA sequence was tested at a
concentration of 500 ng per well in 24 well plates. 24, 48 and 72
hours after transfection, the expression of protein was measured by
ELISA. The protein levels for G-CSF are shown in Table 36 and the
protein levels for Factor IX are shown in Table 37.
TABLE-US-00037 TABLE 36 G-CSF Protein Expression Protein Expression
(ng/ml) Description 24 Hours 48 Hours 72 Hours G-CSF Hs3'UTR 43.9
18.8 5.7 G-CSF Hs3'UTR miR-122 6.9 0.7 0.12 G-CSF Hs3'UTR miR-122
seed 48.5 25.6 8.2 G-CSF Hs3'UTR miR-122 seedless 31.7 11.7 3.4
G-CSF Mm3'UTR 84.9 100.4 21.3 G-CSF Mm3'UTR miR-122 24.0 3.03 0.8
G-CSF Mm3'UTR miR-122 seed 115.8 96.4 19.2 G-CSF Mm3'UTR miR-122
seedless 113.1 92.9 18.9
TABLE-US-00038 TABLE 37 Factor IX Protein Expression Protein
Expression (ng/ml) Description 24 Hours 48 Hours 72 Hours G-CSF
Hs3'UTR 43.9 18.8 5.7 G-CSF Hs3'UTR miR-122 6.9 0.7 0.12 G-CSF
Hs3'UTR miR-122 seed 48.5 25.6 8.2 G-CSF Hs3'UTR miR-122 seedless
31.7 11.7 3.4 G-CSF Mm3'UTR 84.9 100.4 21.3 G-CSF Mm3'UTR miR-122
24.0 3.03 0.8 G-CSF Mm3'UTR miR-122 seed 115.8 96.4 19.2 G-CSF
Mm3'UTR miR-122 seedless 113.1 92.9 18.9
Example 47
In Vitro Expression of Modified Nucleic Acid with Mir-142 or
miR-146 Binding Sites
[1091] HeLa and RAW264 cells were seeded at a density of 17000 and
80000 per well respectively, in 100 ul cell culture medium
(DMEM+10% FBS).
[1092] G-CSF mRNA (G-CSF; mRNA sequence shown in SEQ ID NO: 6595;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1) was fully modified with 5-methylcytidine and
1-methylpseudouridine.
[1093] G-CSF mRNA having a miR-142-3p sequence in the 3'UTR (G-CSF
miR-142-3p; mRNA sequence shown in SEQ ID NO: 6634; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1),
or a miR-142-3p seed sequence in the 3'UTR (G-CSF miR-142-3p seed;
mRNA sequence shown in SEQ ID NO: 6636; polyA tail of approximately
140 nucleotides not shown in sequence; 5' cap, Cap1) or a
miR-142-3p sequence without the seed sequence in the 3'UTR (G-CSF
miR-142-3p seedless; mRNA sequence shown in SEQ ID NO: 6638; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1) were fully modified with 5-methylcytidine and
1-methylpseudouridine.
[1094] G-CSF mRNA having a miR-142-5p sequence in the 3'UTR (G-CSF
miR-142-5p; mRNA sequence shown in SEQ ID NO: 6628; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1),
or a miR-142-5p seed sequence in the 3'UTR (G-CSF miR-142-5p seed;
mRNA sequence shown in SEQ ID NO: 6630; polyA tail of approximately
140 nucleotides not shown in sequence; 5' cap, Cap1) or a
miR-142-5p sequence without the seed sequence in the 3'UTR (G-CSF
miR-142-5p seedless; mRNA sequence shown in SEQ ID NO: 6632; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1) were fully modified with 5-methylcytidine and
1-methylpseudouridine.
[1095] G-CSF mRNA having a miR-146a sequence in the 3'UTR (G-CSF
miR-146a; mRNA sequence shown in SEQ ID NO: 6640; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1),
or a miR-146a seed sequence in the 3'UTR (G-CSF miR-146a seed; mRNA
sequence shown in SEQ ID NO: 6642; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) or a miR-146a
sequence without the seed sequence in the 3'UTR (G-CSF miR-146a
seedless; mRNA sequence shown in SEQ ID NO: 6644; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1)
were fully modified with 5-methylcytidine and
1-methylpseudouridine.
[1096] Each G-CSF mRNA sequence was tested at a concentration of
500 ng per well in 24 well plates for each cell type. 24 hours
after transfection, the expression of protein was measured by ELISA
and the protein levels are shown in Table 38. The G-CSF sequences
with a miR-142-3p sequence in the 3'UTR down regulated G-CSF
expression in RAW264 cells whereas the G-CSF sequences with a
miR-142-5p or miR-146a sequence in the 3'UTR did not down regulate
G-CSF expression.
TABLE-US-00039 TABLE 38 G-CSF Expression HeLa Cells RAW264 Cells
Protein Expression Protein Expression Description (ng/ml) (ng/ml)
G-CSF 243.5 173.7 G-CSF miR-142-3p 309.1 67.6 G-CSF miR-142-3p seed
259.8 178.1 G-CSF miR-142-3p seedless 321.7 220.2 G-CSF miR-142-5p
291.8 223.3 G-CSF miR-142-5p seed 261.3 233.1 G-CSF miR-142-5p
seedless 330.2 255.1 G-CSF miR-146a 272.6 125.2 G-CSF miR-146a seed
219.4 138.3 G-CSF miR-146a seedless 217.7 132.8
Example 48
Effect of Kozak Sequence on Expression of Modified Nucleic
Acids
[1097] HeLa cells were seeded at a density of 17000 per well in 100
ul cell culture medium (DMEM+10% FBS). G-CSF mRNA having an IRES
sequence and Kozak sequence (G-CSF IRES Kozak; mRNA sequence shown
in SEQ ID NO: 7336; polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1), G-CSF mRNA having an IRES
sequence but not a Kozak sequence (G-CSF IRES; mRNA sequence shown
in SEQ ID NO: 7337; polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1), G-CSF mRNA without an IRES or
Kozak sequence (GCSF no Kozak; mRNA sequence shown in SEQ ID NO:
7338; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1) or a G-CSF sequence having a Kozak sequence
(G-CSF Kozak; mRNA sequence shown in SEQ ID NO: 7339; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1)
were fully modified with fully modified with 5-methylcytidine and
1-methylpseudouridine and tested at a concentration of 75 ng per
well in 24 well plates. 24 hours after transfection, the expression
of G-CSF was measured by ELISA, and the results are shown in Table
39.
TABLE-US-00040 TABLE 39 G-CSF expression Description Protein
Expression (ng/ml) G-CSF IRES Kozak 2.01 G-CSF IRES 1.64 G-CSF no
Kozak 795.53 G-CSF Kozak 606.28
Example 49
MALAT1 Constructs
[1098] Modified mRNA encoding G-CSF or mCherry with a human or
mouse MALAT1 sequence and their corresponding cDNA sequences are
shown below in Table 40. In Table 40, the start codon of each
sequence is underlined and the MALAT1 sequences are bolded.
TABLE-US-00041 TABLE 40 MALAT1 Constructs SEQ ID Sequence NO: G-CSF
Optimized G-CSF cDNA sequence containing 7340 with a T7 polymerase
site, kozak sequence, and Mouse a Mouse MALAT1 sequence (bold):
MALAT1 TAATACGACTCACTATA sequence
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATA TAAGAGCCACC
ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTA
TGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGG
ACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCAT
CGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAG
GTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAG
AGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGA
GGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCC
TGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA
GTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGT
TCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAAT
CTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAG
CTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGC
AGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCC
CACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTT
CAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACC
TTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGA CATCTTGCGCAGCCG TGATAATAG
GATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCC
TGAGAAAACAACCTTTTGTTTTCTCAGGTTTTGCTT
TTTGGCCTTTCCCTAGCTTTAAAAAAAAAAAAGCAA
AAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTA GA mRNA sequence
(transcribed): 7341 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU
AUAAGAGCCACC AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUU
AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUC
UGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCC
UCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUG
GAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCA
CUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGC
CAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUG
GGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCG
CAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUC
CACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAA
GCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACG
CUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCA
ACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUG
GCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCG
GCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGA
GUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAA
GUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG UGAUAAUAG
GAUUCGUCAGUAGGGUUGUAAAGGUUUUUCUUUU
CCUGAGAAAACAACCUUUUGUUUUCUCAGGUUUUG
CUUUUUGGCCUUUCCCUAGCUUUAAAAAAAAAAAA
GCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCG GC mCherry Optimized mCherry
cDNA sequence containing a T7 7342 with polymerase site, kozak
sequence, and a Mouse Mouse MALAT1 sequence (bold):
TAATACGACTCACTATA MALAT1 GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATA
sequence TAAGAGCCACC ATGGTATCCAAGGGGGAGGAGGACAACATGGCGATC
ATCAAGGAGTTCATGCGATTCAAGGTGCACATGGAAG
GTTCGGTCAACGGACACGAATTTGAAATCGAAGGAGA
GGGTGAAGGAAGGCCCTATGAAGGGACACAGACCGC
GAAACTCAAGGTCACGAAAGGGGGACCACTTCCTTTC
GCCTGGGACATTCTTTCGCCCCAGTTTATGTACGGGTC
CAAAGCATATGTGAAGCATCCCGCCGATATTCCTGAC
TATCTGAAACTCAGCTTTCCCGAGGGATTCAAGTGGG
AGCGGGTCATGAACTTTGAGGACGGGGGTGTAGTCAC
CGTAACCCAAGACTCAAGCCTCCAAGACGGCGAGTTC
ATCTACAAGGTCAAACTGCGGGGGACTAACTTTCCGT
CGGATGGGCCGGTGATGCAGAAGAAAACGATGGGAT
GGGAAGCGTCATCGGAGAGGATGTACCCAGAAGATG
GTGCATTGAAGGGGGAGATCAAGCAGAGACTGAAGTT
GAAAGATGGGGGACATTATGATGCCGAGGTGAAAAC
GACATACAAAGCGAAAAAGCCGGTGCAGCTTCCCGGA
GCGTATAATGTGAATATCAAGTTGGATATTACTTCACA
CAATGAGGACTACACAATTGTCGAACAGTACGAACGC
GCTGAGGGTAGACACTCGACGGGAGGCATGGACGAG TTGTACAAA TGATAATAG
GATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCC
TGAGAAAACAACCTTTTGTTTTCTCAGGTTTTGCTT
TTTGGCCTTTCCCTAGCTTTAAAAAAAAAAAAGCAA
AAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTA GA mRNA sequence
(transcribed): 7343 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU
AUAAGAGCCACC AUGGUAUCCAAGGGGGAGGAGGACAACAUGGCGAUC
AUCAAGGAGUUCAUGCGAUUCAAGGUGCACAUGGAA
GGUUCGGUCAACGGACACGAAUUUGAAAUCGAAGGA
GAGGGUGAAGGAAGGCCCUAUGAAGGGACACAGACC
GCGAAACUCAAGGUCACGAAAGGGGGACCACUUCCU
UUCGCCUGGGACAUUCUUUCGCCCCAGUUUAUGUAC
GGGUCCAAAGCAUAUGUGAAGCAUCCCGCCGAUAUU
CCUGACUAUCUGAAACUCAGCUUUCCCGAGGGAUUC
AAGUGGGAGCGGGUCAUGAACUUUGAGGACGGGGG
UGUAGUCACCGUAACCCAAGACUCAAGCCUCCAAGA
CGGCGAGUUCAUCUACAAGGUCAAACUGCGGGGGAC
UAACUUUCCGUCGGAUGGGCCGGUGAUGCAGAAGAA
AACGAUGGGAUGGGAAGCGUCAUCGGAGAGGAUGU
ACCCAGAAGAUGGUGCAUUGAAGGGGGAGAUCAAGC
AGAGACUGAAGUUGAAAGAUGGGGGACAUUAUGAU
GCCGAGGUGAAAACGACAUACAAAGCGAAAAAGCCG
GUGCAGCUUCCCGGAGCGUAUAAUGUGAAUAUCAAG
UUGGAUAUUACUUCACACAAUGAGGACUACACAAUU
GUCGAACAGUACGAACGCGCUGAGGGUAGACACUCG ACGGGAGGCAUGGACGAGUUGUACAAA
UGAUAAUAG GAUUCGUCAGUAGGGUUGUAAAGGUUUUUCUUUU
CCUGAGAAAACAACCUUUUGUUUUCUCAGGUUUUG
CUUUUUGGCCUUUCCCUAGCUUUAAAAAAAAAAAA
GCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCG GC G-CSF Optimized G-CSF cDNA
sequence containing a T7 7344 with polymerase site, kozak sequence,
and a Human Human MALAT1 sequence (bold): TAATACGACTCACTATA MALAT1
GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATA sequence TAAGAGCCACC
ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTA
TGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGG
ACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCAT
CGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAG
GTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAG
AGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGA
GGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCC
TGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA
GTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGT
TCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAAT
CTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAG
CTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGC
AGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCC
CACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTT
CAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACC
TTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGA CATCTTGCGCAGCCG TGATAATAG
TGCTCTTCAGTAGGGTCATGAAGGTTTTTCTTTTCC
TGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGC
TTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGC
AAAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTC TAGA mRNA sequence
(transcribed): 7345 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU
AUAAGAGCCACC AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUU
AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUC
UGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCC
UCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUG
GAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCA
CUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGC
CAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUG
GGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCG
CAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUC
CACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAA
GCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACG
CUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCA
ACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUG
GCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCG
GCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGA
GUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAA
GUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG UGAUAAUAG
UGCUCUUCAGUAGGGUCAUGAAGGUUUUUCUUUUC
CUGAGAAAACAACACGUAUUGUUUUCUCAGGUUUU
GCUUUUUGGCCUUUUUCUAGCUUAAAAAAAAAAAA
AGCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGC GGC mCherry Optimized mCherry
cDNA sequence containing a T7 7346 with polymerase site, kozak
sequence, and a Human Human MALAT1 sequence (bold):
TAATACGACTCACTATA MALAT1 GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATA
sequence TAAGAGCCACC ATGGTATCCAAGGGGGAGGAGGACAACATGGCGATC
ATCAAGGAGTTCATGCGATTCAAGGTGCACATGGAAG
GTTCGGTCAACGGACACGAATTTGAAATCGAAGGAGA
GGGTGAAGGAAGGCCCTATGAAGGGACACAGACCGC
GAAACTCAAGGTCACGAAAGGGGGACCACTTCCTTTC
GCCTGGGACATTCTTTCGCCCCAGTTTATGTACGGGTC
CAAAGCATATGTGAAGCATCCCGCCGATATTCCTGAC
TATCTGAAACTCAGCTTTCCCGAGGGATTCAAGTGGG
AGCGGGTCATGAACTTTGAGGACGGGGGTGTAGTCAC
CGTAACCCAAGACTCAAGCCTCCAAGACGGCGAGTTC
ATCTACAAGGTCAAACTGCGGGGGACTAACTTTCCGT
CGGATGGGCCGGTGATGCAGAAGAAAACGATGGGAT
GGGAAGCGTCATCGGAGAGGATGTACCCAGAAGATG
GTGCATTGAAGGGGGAGATCAAGCAGAGACTGAAGTT
GAAAGATGGGGGACATTATGATGCCGAGGTGAAAAC
GACATACAAAGCGAAAAAGCCGGTGCAGCTTCCCGGA
GCGTATAATGTGAATATCAAGTTGGATATTACTTCACA
CAATGAGGACTACACAATTGTCGAACAGTACGAACGC
GCTGAGGGTAGACACTCGACGGGAGGCATGGACGAG TTGTACAAA TGATAATAG
TGCTCTTCAGTAGGGTCATGAAGGTTTTTCTTTTCC
TGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGC
TTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGC
AAAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTC TAGA mRNA sequence
(transcribed): 7347 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU
AUAAGAGCCACC AUGGUAUCCAAGGGGGAGGAGGACAACAUGGCGAUC
AUCAAGGAGUUCAUGCGAUUCAAGGUGCACAUGGAA
GGUUCGGUCAACGGACACGAAUUUGAAAUCGAAGGA
GAGGGUGAAGGAAGGCCCUAUGAAGGGACACAGACC
GCGAAACUCAAGGUCACGAAAGGGGGACCACUUCCU
UUCGCCUGGGACAUUCUUUCGCCCCAGUUUAUGUAC
GGGUCCAAAGCAUAUGUGAAGCAUCCCGCCGAUAUU
CCUGACUAUCUGAAACUCAGCUUUCCCGAGGGAUUC
AAGUGGGAGCGGGUCAUGAACUUUGAGGACGGGGG
UGUAGUCACCGUAACCCAAGACUCAAGCCUCCAAGA
CGGCGAGUUCAUCUACAAGGUCAAACUGCGGGGGAC
UAACUUUCCGUCGGAUGGGCCGGUGAUGCAGAAGAA
AACGAUGGGAUGGGAAGCGUCAUCGGAGAGGAUGU
ACCCAGAAGAUGGUGCAUUGAAGGGGGAGAUCAAGC
AGAGACUGAAGUUGAAAGAUGGGGGACAUUAUGAU
GCCGAGGUGAAAACGACAUACAAAGCGAAAAAGCCG
GUGCAGCUUCCCGGAGCGUAUAAUGUGAAUAUCAAG
UUGGAUAUUACUUCACACAAUGAGGACUACACAAUU
GUCGAACAGUACGAACGCGCUGAGGGUAGACACUCG ACGGGAGGCAUGGACGAGUUGUACAAA
UGAUAAUAG UGCUCUUCAGUAGGGUCAUGAAGGUUUUUCUUUUC
CUGAGAAAACAACACGUAUUGUUUUCUCAGGUUUU
GCUUUUUGGCCUUUUUCUAGCUUAAAAAAAAAAAA
AGCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGC GGC
[1099] These modified mRNA sequences can include at least one
chemical modification described herein. The G-CSF or mCherry
modified mRNA sequence can be formulated, using methods described
herein and/or known in the art, prior to transfection and/or
administration.
[1100] The modified mRNA sequence encoding G-CSF or mCherry can be
transfected in vitro to various cell types such as HEK293, HeLa,
PBMC and BJ fibroblast and those described in Table 25 of
co-pending U.S. Provisional Application No. 61/839,903, filed Jun.
27, 2013, the contents of which are herein incorporated by
reference in its entirety, using methods disclosed herein and/or
are known in the art. The cells are then analyzed using methods
disclosed herein and/or are known in the art to determine the
concentration of G-CSF or mCherry and/or the cell viability.
Example 50
Oncology-Related Targets
[1101] Septin 4 may be an oncology-related polypeptide of interest.
Shown in Table 41, in addition to the name and description of the
gene encoding the oncology-related polypeptide of interest, are the
ENSEMBL Transcript ID (ENST), the ENSEMBL Protein ID (ENSP), each
present where applicable, and when available the optimized sequence
ID (OPT. SEQ ID).
TABLE-US-00042 TABLE 41 Oncology-Related Targets Target Trans.
Prot. OPT. Descrip- ENST SEQ ENSP SEQ SEQ Target tion ID ID NO ID
ID NO ID NO SEPT4 septin 4 317256 7348 321071 7355 7363, 7368,
7375, 7382, 7389, 7396 SEPT4 septin 4 317268 7349 321674 7356 7364,
7369, 7376, 7383, 7390, 7397, 7403-7489 SEPT4 septin 4 393086 7350
376801 7357 7370, 7377, 7384, 7391, 7398 SEPT4 septin 4 412945 7351
414779 7358 7365, 7371, 7378, 7385, 7392, 7399 SEPT4 septin 4
426861 7352 402348 7359 7366, 7372, 7379, 7386, 7393, 7400 SEPT4
septin 4 457347 7353 402000 7360 7367, 7373, 7380, 7387, 7394, 7401
SEPT4 septin 4 583114 7354 463768 7361 7374, 7381, 7388, 7395, 7402
SEPT4 septin 4 7362
Example 51
Confirmation and of Peptide Identity from Chemically Modified
mRNA
[1102] Cell lysates containing protein produced from: (a)
apoptosis-inducing factor 1, mitochondrial, short isoform (AIFsh;
gene name AIFM1) modified mRNA (mRNA sequence shown in SEQ ID NO.
6617 (Table 42); polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1); (b) copper metabolism (Murr1)
domain containing 1 (COMMD1) modified mRNA (mRNA sequence shown in
SEQ ID NO. 7491 (Table 42); polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1); (c) septin 4
(SEPT4) modified mRNA (mRNA sequence shown in SEQ ID NO. 7362
(Table 42); polyA tail of approximately 140 nucleotides not shown
in sequence; 5' cap, Cap1); and (d) diablo, IAP-binding
mitochondrial protein (DIABLO) modified mRNA (mRNA sequence shown
in SEQ ID NO. 7494 (Table 42); polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1); all fully
modified with 5-methylcytidine and pseudouridine (5 mC and pU),
fully modified with 5-methylcytidine and 1-methylpseudouridine (5
mC and 1 mpU), modified where 25% of uridine modified with
2-thiouridine and 25% of cytidine modified with 5-methylcytidine
(s2U and 5 mC), fully modified with pseudouridine (pU), or fully
modified with 1-methylpseudouridine (1 mpU) were evaluated using
the LC-MS/MS with quantitative LC-MRM as described in Example
31.
TABLE-US-00043 TABLE 42 Target Sequences SEQ ID Description
Sequence NO: AIFsh GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAU 6617
AAGAGCCACCAUGGAAAAAGUCAGACGAGAGGGGGUU
AAGGUGAUGCCCAAUGCUAUUGUGCAAUCCGUUGGAG
UCAGCAGUGGCAAGUUACUUAUCAAGCUGAAAGACGG
CAGGAAGGUAGAAACUGACCACAUAGUGGCAGCUGUG
GGCCUGGAGCCCAAUGUUGAGUUGGCCAAGACUGGUG
GCCUGGAAAUAGACUCAGAUUUUGGUGGCUUCCGGGU
AAAUGCAGAGCUACAAGCACGCUCUAACAUCUGGGUG
GCAGGAGAUGCUGCAUGCUUCUACGAUAUAAAGUUGG
GAAGGAGGCGGGUAGAGCACCAUGAUCACGCUGUUGU
GAGUGGAAGAUUGGCUGGAGAAAAUAUGACUGGAGCU
GCUAAGCCGUACUGGCAUCAGUCAAUGUUCUGGAGUG
AUUUGGGCCCCGAUGUUGGCUAUGAAGCUAUUGGUCU
UGUGGACAGUAGUUUGCCCACAGUUGGUGUUUUUGCA
AAAGCAACUGCACAAGACAACCCCAAAUCUGCCACAGA
GCAGUCAGGAACUGGUAUCCGAUCAGAGAGUGAGACA
GAGUCCGAGGCCUCAGAAAUUACUAUUCCUCCCAGCAC
CCCGGCAGUUCCACAGGCUCCCGUCCAGGGGGAGGACU
ACGGCAAAGGUGUCAUCUUCUACCUCAGGGACAAAGU
GGUCGUGGGGAUUGUGCUAUGGAACAUCUUUAACCGA
AUGCCAAUAGCAAGGAAGAUCAUUAAGGACGGUGAGC
AGCAUGAAGAUCUCAAUGAAGUAGCCAAACUAUUCAA
CAUUCAUGAAGACUGAUAAUAGGCUGGAGCCUCGGUG
GCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCU
CCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAA UAAAGUCUGAGUGGGCGGC
MEKVRREGVKVMPNAIVQSVGVSSGKLLIKLKDGRK 7490
VETDHIVAAVGLEPNVELAKTGGLEIDSDFGGFRVNA
ELQARSNIWVAGDAACFYDIKLGRRRVEHHDHAVVS
GRLAGENMTGAAKPYWHQSMFWSDLGPDVGYEAIG
LVDSSLPTVGVFAKATAQDNPKSATEQSGTGIRSESET
ESEASEITIPPSTPAVPQAPVQGEDYGKGVIFYLRDKV
VVGIVLWNIFNRMPIARKIIKDGEQHEDLNEVAKLFNI HED COMMD1
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 7491
UAUAAGAGCCACCAUGGCGGCGGGCGAGCUUGAG
GGUGGCAAACCCCUGAGCGGGCUGCUGAAUGCGC
UGGCCCAGGACACUUUCCACGGGUACCCCGGCAUC
ACAGAGGAGCUGCUACGGAGCCAGCUAUAUCCAG
AGGUGCCACCCGAGGAGUUCCGCCCCUUUCUGGCA
AAGAUGAGGGGGAUUCUUAAGUCUAUUGCGUCUG
CAGACAUGGAUUUCAACCAGCUGGAGGCAUUCUU
GACUGCUCAAACCAAAAAGCAAGGUGGGAUCACA
UCUGACCAAGCUGCUGUCAUUUCCAAAUUCUGGA
AGAGCCACAAGACAAAAAUCCGUGAGAGCCUCAU
GAACCAGAGCCGCUGGAAUAGCGGGCUUCGGGGC
CUGAGCUGGAGAGUUGAUGGCAAGUCUCAGUCAA
GGCACUCAGCUCAAAUACACACACCUGUUGCCAU
UAUAGAGCUGGAAUUAGGCAAAUAUGGACAGGAA
UCUGAAUUUCUGUGUUUGGAAUUUGAUGAGGUCA
AAGUCAACCAAAUUCUGAAGACGCUGUCAGAGGU
AGAAGAAAGUAUCAGCACACUGAUCAGCCAGCCU
AACUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUC
CCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAU AAAGUCUGAGUGGGCGGC
MAAGELEGGKPLSGLLNALAQDTFHGYPGITEELLRS 7492
QLYPEVPPEEFRPFLAKMRGILKSIASADMDFNQLEAF
LTAQTKKQGGITSDQAAVISKFWKSHKTKIRESLMNQ
SRWNSGLRGLSWRVDGKSQSRHSAQIHTPVAIIELELG
KYGQESEFLCLEFDEVKVNQILKTLSEVEESISTLISQPN SEPT4
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 7362
UAUAAGAGCCACCAUGGACCGUUCACUGGGAUGG
CAAGGGAAUUCUGUCCCUGAGGACAGGACUGAAG
CUGGGAUCAAGCGUUUCCUGGAGGACACCACGGA
UGAUGGAGAACUGAGCAAGUUCGUGAAGGAUUUC
UCAGGAAAUGCGAGCUGCCACCCACCAGAGGCUA
AGACCUGGGCAUCCAGGCCCCAAGUCCCGGAGCCA
AGGCCCCAGGCCCCGGACCUCUAUGAUGAUGACCU
GGAGUUCAGACCCCCCUCGCGGCCCCAGUCCUCUG
ACAACCAGCAGUACUUCUGUGCCCCAGCCCCUCUC
AGCCCAUCUGCCAGGCCCCGCAGCCCAUGGGGCAA
GCUUGAUCCCUAUGAUUCCUCUGAGGAUGACAAG
GAGUAUGUGGGCUUUGCAACCCUCCCCAACCAAG
UCCACCGAAAGUCCGUGAAGAAAGGCUUUGACUU
UACCCUCAUGGUGGCAGGAGAGUCUGGCCUGGGC
AAAUCCACACUUGUCAAUAGCCUCUUCCUCACUG
AUCUGUACCGGGACCGGAAACUUCUUGGUGCUGA
AGAGAGGAUCAUGCAAACUGUGGAGAUCACUAAG
CAUGCAGUGGACAUAGAAGAGAAGGGUGUGAGGC
UGCGGCUCACCAUUGUGGACACACCAGGUUUUGG
GGAUGCAGUCAACAACACAGAGUGCUGGAAGCCU
GUGGCAGAAUACAUUGAUCAGCAGUUUGAGCAGU
AUUUCCGAGACGAGAGUGGCCUGAACCGAAAGAA
CAUCCAAGACAACAGGGUGCACUGCUGCCUGUAC
UUCAUCUCACCCUUCGGCCAUGGGCUCCGGCCAUU
GGAUGUUGAAUUCAUGAAGGCCCUGCAUCAGCGG
GUCAACAUCGUGCCUAUCCUGGCUAAGGCAGACA
CACUGACACCUCCCGAAGUGGACCACAAGAAACGC
AAAAUCCGGGAGGAGAUUGAGCAUUUUGGAAUCA
AGAUCUAUCAAUUCCCAGACUGUGACUCUGAUGA
GGAUGAGGACUUCAAAUUGCAGGACCAAGCCCUA
AAGGAAAGCAUCCCAUUUGCAGUAAUUGGCAGCA
ACACUGUAGUAGAGGCCAGAGGGCGGCGAGUUCG
GGGUCGACUCUACCCCUGGGGCAUCGUGGAAGUG
GAAAACCCAGGGCACUGCGACUUUGUGAAGCUGA
GGACAAUGCUGGUACGUACCCACAUGCAGGACCU
GAAGGAUGUGACACGGGAGACACAUUAUGAGAAC
UACCGGGCACAGUGCAUCCAGAGCAUGACCCGCCU
GGUGGUGAAGGAACGGAAUCGCAACAAACUGACU
CGGGAAAGUGGUACCGACUUCCCCAUCCCUGCUG
UCCCACCAGGGACAGAUCCAGAAACUGAGAAGCU
UAUCCGAGAGAAAGAUGAGGAGCUGCGGCGGAUG
CAGGAGAUGCUACACAAAAUACAAAAACAGAUGA
AGGAGAACUAUUGAUAAUAGGCUGGAGCCUCGGU
GGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGC
CCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGU CUUUGAAUAAAGUCUGAGUGGGCGGC
MDRSLGWQGNSVPEDRTEAGIKRFLEDTTDDGELSKF 7493
VKDFSGNASCHPPEAKTWASRPQVPEPRPQAPDLYDD
DLEFRPPSRPQSSDNQQYFCAPAPLSPSARPRSPWGKL
DPYDSSEDDKEYVGFATLPNQVHRKSVKKGFDFTLM
VAGESGLGKSTLVNSLFLTDLYRDRKLLGAEERIMQT
VEITKHAVDIEEKGVRLRLTIVDTPGFGDAVNNTECW
KPVAEYIDQQFEQYFRDESGLNRKNIQDNRVHCCLYF
ISPFGHGLRPLDVEFMKALHQRVNIVPILAKADTLTPP
EVDHKKRKIREEIEHFGIKIYQFPDCDSDEDEDFKLQD
QALKESIPFAVIGSNTVVEARGRRVRGRLYPWGIVEV
ENPGHCDFVKLRTMLVRTHMQDLKDVTRETHYENY
RAQCIQSMTRLVVKERNRNKLTRESGTDFPIPAVPPGT
DPETEKLIREKDEELRRMQEMLHKIQKQMKENY Diablo,
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 7494 IAP-binding
UAUAAGAGCCACCAUGGCGGCUCUGAAGAGUUGG mitochondrial
CUGUCGCGCAGCGUAACUUCAUUCUUCAGGUACA protein
GACAGUGUUUGUGUGUUCCUGUUGUGGCUAACUU (DIABLO)
UAAGAAGCGGUGUUUCUCAGAAUUGAUAAGACCA
UGGCACAAAACUGUGACGAUUGGCUUUGGAGUAA
CCCUGUGUGCGGUUCCUAUUGCACAGAAAUCAGA
GCCUCAUUCCCUUAGUAGUGAAGCAUUGAUGAGG
AGAGCAGUGUCUUUGGUAACAGAUAGCACCUCUA
CCUUUCUCUCUCAGACCACAUAUGCGUUGAUUGA
AGCUAUUACUGAAUAUACUAAGGCUGUUUAUACC
UUAACUUCUCUUUACCGACAAUAUACAAGUUUAC
UUGGGAAAAUGAAUUCAGAGGAGGAAGAUGAAGU
GUGGCAGGUGAUCAUAGGAGCCAGAGCUGAGAUG
ACUUCAAAACACCAAGAGUACUUGAAGCUGGAAA
CCACUUGGAUGACUGCAGUUGGUCUUUCAGAGAU
GGCAGCAGAAGCUGCAUAUCAAACUGGCGCAGAU
CAGGCCUCUAUAACCGCCAGGAAUCACAUUCAGC
UGGUGAAACUGCAGGUGGAAGAGGUGCACCAGCU
CUCCCGGAAAGCAGAAACCAAGCUGGCAGAAGCA
CAGAUAGAAGAGCUCCGUCAGAAAACACAGGAGG
AAGGGGAGGAGCGGGCUGAGUCGGAGCAGGAGGC
CUACCUGCGUGAGGAUUGAUAAUAGGCUGGAGCC
UCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCC
CCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC
MAALKSWLSRSVTSFFRYRQCLCVPVVANFKKRCFSE 1986
LIRPWHKTVTIGFGVTLCAVPIAQKSEPHSLSSEALMR
RAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLY
RQYTSLLGKMNSEEEDEVWQVIIGARAEMTSKHQEY
LKLETTWMTAVGLSEMAAEAAYQTGADQASITARNH
IQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEE GEERAESEQEAYLRED
[1103] Peptide fragments identified for the evaluated proteins are
shown in Table 43.
TABLE-US-00044 TABLE 43 Protein and Peptide Fragment Sequences
Peptide 5mC 5mC s2U Fragment and and and SEQ ID NO pU 1mpU 5mC pU
1mpU AIFM1 DGEQHEDLNEV 7495 -- -- -- -- YES AK TGGLEIDSDFGG 7496
YES -- -- YES YES FR COMMD1 ESLMNQSR 7497 YES YES YES YES YES
HSAQIHTPVAIIE 7498 -- -- YES YES YES LELGK WNSGLR 7499 -- YES YES
YES YES SEPT4 ESGTDFPIPAVPP 7500 YES YES YES YES YES GTDPETEK
FLEDTTDDGELSK 7501 YES YES YES YES YES HAVDIEEK 7502 YES YES YES
YES YES DIABLO AVYTLTSLYR 7503 YES YES YES YES YES LAEAQIEELR 7504
YES YES YES YES YES NHIQLVK 7505 YES YES YES YES YES
Example 52
Detection of C.A. Caspase 3 and C.A Caspase 6
[1104] Human lung cancer A549 cells were plated in 6-wells, and
transfected with Lipofectamine 2000 (Life Technologies) and 5 .mu.g
of constitutively active (C.A.) caspase 3 mRNA (mRNA sequence shown
in SEQ ID NO: 6619 (Table 44); polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) or constitutively
active (C.A.) caspase 6 mRNA (mRNA sequence shown in SEQ ID NO:
7506 (Table 44); polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1) fully modified with
5-methylcytidine and 1-methylpseudouridine (5 mC and 1 mpU) or
fully modified with 1-methylpseudouridine (1 mpU). Cells were
harvested 7-10 hours post-transfection and lysed in RIPA buffer
containing a protease inhibitor cocktail (Roche, Indianapolis,
Ind.). 20 .mu.g of cell lysate per lane was run for Western
blotting to detect endogenous and introduced caspase 3; endogenous
and introduced caspase 6; the caspase 3 downstream-substrate PARP;
and the caspase 6 downstream-substrate lamin A/C. Compared to
control lysate, higher levels of cleaved caspase 3 and cleaved
caspase 6 were detected in C.A. caspase 3 and C.A. caspase 6
modified mRNA transfected cells, respectively. As shown in FIG. 7,
cleavage of the downstream substrates PARP and lamin A/C were
detected in cells treated with C.A. caspase 3 modified mRNA (FIG.
7A) and C.A. caspase 6 modified mRNAs (FIG. 7B). The 5 lanes of the
Westerns shown in FIG. 7A and 7B contain lysate from the following:
1) untransfected HeLa cells, 2) untransfected A549, 3) A549
lipofectamine alone control, 4) A549 transfected with 5 mC, 1 mpU
modified mRNA, and 5) A549 transfected with 1 mpU modified
mRNA.
TABLE-US-00045 TABLE 44 C.A. Caspase Sequences Description Sequence
SEQ ID NO: C.A. GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUA 6619 caspase 3
UAAGAGCCACCAUGAUUGAGACAGACAGUGGUGUUGA
UGAUGACAUGGCGUGUCAUAAAAUACCAGUGGAGGCC
GACUUCUUGUAUGCAUACUCCACAGCACCUGGUUAUU
AUUCUUGGCGAAAUUCAAAGGAUGGCUCCUGGUUCAU
CCAGUCGCUUUGUGCCAUGCUGAAACAGUAUGCCGAC
AAGCUUGAAUUUAUGCACAUUCUUACCCGGGUUAACC
GAAAGGUGGCAACAGAAUUUGAGUCCUUUUCCUUUGA
CGCUACUUUUCAUGCAAAGAAACAGAUUCCAUGUAUU
GUUUCCAUGCUCACAAAAGAACUCUAUUUUUAUCACG
AUGAAGUUGAUGGGGGAUCCCCCAUGGAGAACACUGA
AAACUCAGUGGAUUCAAAAUCCAUUAAAAAUUUGGA
ACCAAAGAUCAUACAUGGAAGCGAAUCAAUGGACUCU
GGAAUAUCCCUGGACAACAGUUAUAAAAUGGAUUAUC
CUGAGAUGGGUUUAUGUAUAAUAAUUAAUAAUAAGA
AUUUUCAUAAGAGCACUGGAAUGACAUCUCGGUCUGG
UACAGAUGUCGAUGCAGCAAACCUCAGGGAAACAUUC
AGAAACUUGAAAUAUGAAGUCAGGAAUAAAAAUGAU
CUUACACGUGAAGAAAUUGUGGAAUUGAUGCGUGAU
GUUUCUAAAGAAGAUCACAGCAAAAGGAGCAGUUUU
GUUUGUGUGCUUCUGAGCCAUGGUGAAGAAGGAAUA
AUUUUUGGAACAAAUGGACCUGUUGACCUGAAAAAA
AUAACAAACUUUUUCAGAGGGGAUCGUUGUAGAAGU
CUAACUGGAAAACCCAAACUUUUCAUUAUUCAGGCCU
GCCGUGGUACAGAACUGGACUGUGGCAUUGAGACAGA
CUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUU
GCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCU
GCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC
MIETDSGVDDDMACHKIPVEADFLYAYSTAPGYYSW 2484
RNSKDGSWFIQSLCAMLKQYADKLEFMHILTRVNRK
VATEFESFSFDATFHAKKQIPCIVSMLTKELYFYHDE
VDGGSPMENTENSVDSKSIKNLEPKIIHGSESMDSGIS
LDNSYKMDYPEMGLCIIINNKNFHKSTGMTSRSGTD
VDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVS
KEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNF
FRGDRCRSLTGKPKLFIIQACRGTELDCGIETD C.A.
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAA 7506 caspase 6
AUAUAAGAGCCACCAUGGUAGAAAUAGAUGCAGC
CUCCGUUUACACGCUGCCUGCUGGAGCUGACUUC
CUCAUGUGUUACUCUGUUGCAGAAGGAUAUUAU
UCUCACCGGGAAACUGUGAACGGCUCAUGGUACA
UUCAAGAUUUGUGUGAGAUGUUGGGAAAAUAUG
GCUCCUCCUUAGAGUUCACAGAACUCCUCACACU
GGUGAACAGGAAAGUUUCUCAGCGCCGAGUGGAC
UUUUGCAAAGACCCAAGUGCAAUUGGAAAGAAGC
AGGUUCCCUGUUUUGCCUCAAUGCUAACUAAAAA
GCUGCAUUUCUUUCCAAAAUCUAAUCUCGAGCAC
CACCACCACCACCACGUUGAAAUUGAUGGGGGAU
CCCCCAUGAGCUCGGCCUCGGGGCUCCGCAGGGG
GCACCCGGCAGGUGGGGAAGAAAACAUGACAGAA
ACAGAUGCCUUCUAUAAAAGAGAAAUGUUUGAU
CCGGCAGAAAAGUACAAAAUGGACCACAGGAGGA
GAGGAAUUGCUUUAAUCUUCAAUCAUGAGAGGU
UCUUUUGGCACUUAACACUGCCAGAAAGGCGGGG
CACCUGCGCAGAUAGAGACAAUCUUACCCGCAGG
UUUUCAGAUCUAGGAUUUGAAGUGAAAUGCUUU
AAUGAUCUUAAAGCAGAAGAACUACUGCUCAAAA
UUCAUGAGGUGUCAACUGUUAGCCACGCAGAUGC
CGAUUGCUUUGUGUGUGUCUUCCUGAGCCAUGGC
GAAGGCAAUCACAUUUAUGCAUAUGAUGCUAAA
AUCGAAAUUCAGACAUUAACUGGCUUGUUCAAAG
GAGACAAGUGUCACAGCCUGGUUGGAAAACCCAA
GAUAUUUAUCAUCCAGGCAUGUCGGGGAAACCAG
CACGAUGUGCCAGUCAUUCCUUUGGAUGUAGUAG
AUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCU
UCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCC
CCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAU AAAGUCUGAGUGGGCGGC
MVEIDAASVYTLPAGADFLMCYSVAEGYYSHRETVN 2486
GSWYIQDLCEMLGKYGSSLEFTELLTLVNRKVSQRR
VDFCKDPSAIGKKQVPCFASMLTKKLHFFPKSNLEHH
HHHHVEIDGGSPMSSASGLRRGHPAGGEENMTETDA
FYKREMFDPAEKYKMDHRRRGIALIFNHERFFWHLT
LPERRGTCADRDNLTRRFSDLGFEVKCFNDLKAEELL
LKIHEVSTVSHADADCFVCVFLSHGEGNHIYAYDAKI
EIQTLTGLFKGDKCHSLVGKPKIFIIQACRGNQHDVPV IPLDVVD
Example 53
Expression of Modified C.A. Caspase 3 and C.A. Caspase 6 mRNA
[1105] The activity of cultured human lung adenocarcinoma A549
cells was evaluated through the measurement of formazan converted
by mitochondrial dehydrogenases from WST-1 substrate (Roche,
Indianapolis, Ind.). 7500 cells per 96-well were treated with a
single dose of varying amounts of Lipofectamine 2000-lipoplexed
constitutively active (C.A.) caspase 3 mRNA (mRNA sequence shown in
SEQ ID NO: 6619 (Table 44); polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1) or constitutively
active (C.A.) caspase 6 mRNA (mRNA sequence shown in SEQ ID NO:
7506 (Table 44); polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1) fully modified with
5-methylcytidine and 1-methylpseudouridine (5 mC and 1 mpU) or
fully modified with 1-methylpseudouridine (1 mpU) or a control
proteins (eGFP (mRNA sequence shown in SEQ ID NO: 7507; polyA tail
of approximately 140 nucleotides not shown in sequence; 5' cap,
Cap1; fully modified with 5-methylcytidine and
1-methylpseudouridine (5 mC and 1 mpU) or fully modified with
1-methylpseudouridine (1 mpU)) and luciferase (mRNA sequence shown
in SEQ ID NO: 7508; polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1; fully modified with
5-methylcytidine and 1-methylpseudouridine (5 mC and 1 mpU) or
fully modified with 1-methylpseudouridine (1 mpU))). Cellular
activity was measured in cultured cells 1 day after mRNA treatment
according to the WST-1 manufacturer's protocol, and is plotted as a
450 nm absorbance reading of the converted formazan that had been
corrected for background signal. As shown in Table 45, increasing
amounts of transfected C.A. caspase mRNA (0 ng, 2 ng, 10 ng, 50 ng
and 250 ng) markedly inhibited the optical density (OD) signal (as
a readout of cellular activity) compared to controls. Similar
results were obtained in human lung adenocarcinoma H441 cells and
human cervical cancer HeLa cells.
TABLE-US-00046 TABLE 45 Cellular Activity Amount of mRNA WST-1 OD
mean Description (ng) (450-690 nm) C.A. caspase 3 0 2.46 (1mpU and
5mC) 2 1.93 10 1.05 50 0.31 250 0.04 C.A. caspase 6 0 2.49 (1mpU
and 5mC) 2 2.41 10 1.64 50 0.75 250 0.30 eGFP 0 2.37 (1mpU and 5mC)
2 2.36 10 2.19 50 1.84 250 1.84 Luciferase 0 2.26 (1mpU and 5mC) 2
1.93 10 2.00 50 1.94 250 1.87 C.A. caspase 3 0 2.62 (1mpU) 2 2.35
10 1.80 50 0.91 250 0.17 C.A. caspase 6 0 2.17 (1mpU) 2 2.34 10
2.01 50 1.29 250 0.42 eGFP 0 2.56 (1mpU) 2 2.67 10 2.86 50 2.72 250
2.38 Luciferase 0 2.12 (1mpU) 2 2.56 10 2.68 50 2.64 250 2.21
Example 54
MYC Inhibitors Modified mRNA
[1106] Human hepatocellular carcinoma Hep3B cells were plated in a
6-well plate at a seeding density of 3.times.10.sup.6 cells/well
and Lipofectamine 2000-transfected with mRNAs fully modified with
5-methylcytidine and 1-methylpseudouridine (5 mC and 1 mpU) or
fully modified with 1-methylpseudouridine (1 mpU) designed to
encode the following: fluorescent protein mCherry (mRNA sequence
shown in SEQ ID NO: 6602; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1), non-translatable
Factor IX (mRNA sequence shown in SEQ ID NO: 7509; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1),
full length wildtype C-MYC (mRNA sequence shown in SEQ ID NO: 7510;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1), MYC inhibitor A (mRNA sequence shown in SEQ ID NO:
7511 (Table 46); polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1), MYC inhibitor B (mRNA sequence
shown in SEQ ID NO: 7513 (Table 46); polyA tail of approximately
140 nucleotides not shown in sequence; 5' cap, Cap1), MYC inhibitor
C (mRNA sequence shown in SEQ ID NO: 7418 (Table 46); polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1)
and MYC inhibitor D (mRNA sequence shown in SEQ ID NO: 7515 (Table
46); polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1). Cells were collected 8 hours
post-transfection and lysates were made using RIPA lysis buffer
including a protease inhibitor cocktail (Roche, Indianapolis,
Ind.). Equal amounts of lysate determined by BCA assay were
resolved by SDS-PAGE through 4-12% BIS-TRIS gels, transferred to
nitrocellulose blots and probed with appropriate primary and
secondary antibodies. Western blot analyses revealed positive
expression of the 4 modified mRNA MYC inhibitors, as well as full
length C-MYC, in Hep3B cells.
TABLE-US-00047 TABLE 46 MYC Inhibitor Sequences Description
Sequence SEQ ID NO: MYC GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAA 7511
inhibitor A AUAUAAGAGCCACCAUGACCGAAGAAAACGUCAA
GAGAAGAACCCAUAAUGUCCUCGAGCGCCAGCGG
CGCAAUGAGCUCAAGCGCAGCUUCUUUGCACUCA
GGGACCAAAUUCCAGAGUUGGAGAACAACGAAAA
GGCCCCGAAGGUGGUGAUCCUUAAGAAGGCGACU
GCCUACAUCCUGUCGGUGCAGGCUGAGACUCAAA
AGCUGAUCUCCGAAAUCGAUCUGCUCCGGAAACA
GAACGAACAACUGAAACACAAACUGGAACAGCUG
CGGAAUUCAUGCGCGUGAUAAUAGGCUGGAGCCU
CGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCC
CCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC
MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELE 7512
NNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQ NEQLKHKLEQLRNSCA MYC
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAA 7513 inhibitor B
AUAUAAGAGCCACCAUGACCGAAGAAAACGUCAA
GAGAAGAACCCAUAAUGUCCUCGAGCGCCAGCGG
CGCAAUGAGCUCAAGCGCAGCUUCUUUGCACUCA
GGGACCAAAUUCCAGAGUUGGAGAACAACGAAAA
GGCCCCGAAGGUGGUGAUCCUUAAGAAGGCGACU
GCCUACAUCCUGUCGGUGCAGGCUGAGAAUCAAA
AGCUGAUCUCCGAAAUCGAUCUGCUCCGGAAACA
GAACGAACAACUGAAACACAAACUGGAACAGCUG
CGGAAUUCAUGCGCGUGAUAAUAGGCUGGAGCCU
CGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCC
CCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC
MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELE 7514
NNEKAPKVVILKKATAYILSVQAENQKLISEIDLLRK QNEQLKHKLEQLRNSCA MYC
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAA 7515 inhibitor C
AUAUAAGAGCCACCAUGAGCGCCGCUGAUAAGCG
GGCUCACCACAAUGCGUUGGAGAGGAAGAGGCGC
GACCACAUCAAAGACUCGUUCCAUUCACUCCGGG
ACUCCGUGCCGUCGCUGCAAGGAGAAAAAGCCUC
CCGGGCACAGAUCCUCGACAAGGCGACUGAGUAC
AUUCAGUACAUGCGCCGCAAGAACCACACCCAUC
AGCAAGAUAUCGACGAUCUUAAGAGACAGAACGC
GCUGCUGGAACAACAGGUCCGCGCACUGGAAAAG
GCCAGAAGCUCAGCCUGAUAAUAGGCUGGAGCCU
CGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCC
CCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC
MSAADKRAHHNALERKRRDHIKDSFHSLRDSVPSLQ 7516
GEKASRAQILDKATEYIQYMRRKNHTHQQDIDDLKR QNALLEQQVRALEKARSSA MYC
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUA 6621 inhibitor D
UAAGAGCCACCAUGACCGAAGAAAACGUCAAGAGAAG
AACCCAUAAUGUCCUCGAGCGCCAGCGGCGCAAUGAG
CUCAAGCGCAGCUUCUUUGCACUCAGGGACCAAAUUC
CAGAGUUGGAGAACAACGAAAAGGCCCCGAAGGUGGU
GAUCCUUAAGAAGGCGACUGCCUACAUCCUGUCGGUG
CAGGCUGAGACUCAAAAGCUGAUCUCCGAAAUCGAUC
UGCUCCGGAAACAGAACGAACAACUGAAACACAAACU
GGAACAGCUGCGGAAUUCAUGCUGAUAAUAGGCUGGA
GCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGU
GGUCUUUGAAUAAAGUCUGAGUGGGCGGC MTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELE
7517 NNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQ NEQLKHKLEQLRNSC
Example 55
Expression of MYC Inhibitors Modified mRNA
[1107] Hep3B cells were plated in a 96-well plate at a seeding
density of 2500 cells/well and Lipofectamine 2000-transfected with
0, 0.2 nM, 0.7 nM, 2 nM or 6 nM of modified mRNAs fully modified
with 1-methylpseudouridine (1 mpU) designed to encode the
following: mCherry (mRNA sequence shown in SEQ ID NO: 6602; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1), non-translatable Factor IX (mRNA sequence shown in SEQ
ID NO: 7509; polyA tail of approximately 140 nucleotides not shown
in sequence; 5' cap, Cap1), full length wildtype C-MYC (mRNA
sequence shown in SEQ ID NO: 7510; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1), MYC inhibitor A
(mRNA sequence shown in SEQ ID NO: 7511; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1),
MYC inhibitor B (mRNA sequence shown in SEQ ID NO: 7513; polyA tail
of approximately 140 nucleotides not shown in sequence; 5' cap,
Cap1), MYC inhibitor C (mRNA sequence shown in SEQ ID NO: 7515;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1) and MYC inhibitor D (mRNA sequence shown in SEQ ID
NO: 6621; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1). Cellular activity was measured 48 hours
post-transfection with the use of WST-1 according to manufacturer's
instructions (Roche, Indianapolis, Ind.). Absorbance readings were
taken at 450 nm 4 hours after the addition of WST-1, and
background-corrected results are shown in Table 47. The three
highest concentrations of each of the inhibitors (MYC inhibitor A,
MYC inhibitor B, MYC inhibitor C and MYC inhibitor D) reduced
absorbance signal compared to the controls.
TABLE-US-00048 TABLE 47 Cellular Activity Amount of mRNA WST-1 OD
mean Description (nM) (450-690 nm) MYC inhibitor A 0 0.45 0.2 0.42
0.7 0.09 2 0.12 6 0.05 MYC inhibitor B 0 0.67 0.2 0.69 0.7 0.24 2
0.09 6 0.05 MYC inhibitor C 0 0.73 0.2 0.73 0.7 0.34 2 0.09 6 0.04
MYC inhibitor D 0 0.74 0.2 0.68 0.7 0.32 2 0.14 6 0.07 mCherry 0
0.66 0.2 0.66 0.7 0.62 2 0.60 6 0.51 Non-translatable 0 0.65 FIX
0.2 0.65 0.7 0.61 2 0.62 6 0.49 Wild-Type MYC 0 0.58 0.2 0.51 0.7
0.51 2 0.51 6 0.46
Example 56
In Vivo Expression of Modified mRNA
[1108] A. BALB/C Nuce Mice
[1109] BALB/c nude mice were injected intravenously with 0.1 mg/kg
luciferase modified mRNA without a miR-122 binding site
("non-targeted mRNA"; mRNA sequence shown in SEQ ID NO: 7518; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1; fully modified with 5-methylcytidine and
1-methylpeudouridine) formulated in a lipid nanoparticle described
in Table 48 or luciferase modified mRNA with a miR-122 binding site
in the 3'UTR ("miR-122 targeted mRNA"; mRNA sequence shown in SEQ
ID NO: 7519; polyA tail of approximately 140 nucleotides not shown
in sequence; 5' cap, Cap1; fully modified with 5-methylcytidine and
1-methylpeudouridine) formulated in a lipid nanoparticle described
in Table 49.
TABLE-US-00049 TABLE 48 Lipid Nanoparticle for Non-targeted mRNA
LNP Luciferase: non-targeted mRNA Lipid DLin-KC2-DMA Lipid/RNA
wt/wt 20 Mean size 73.3 nm PDI: 0.06
TABLE-US-00050 TABLE 49 Lipid Nanoparticle for Targeted mRNA LNP
Luciferase: targeted mRNA Lipid DLin-KC2-DMA Lipid/RNA wt/wt 20
Mean size 70.6 nm PDI: 0.08
[1110] 24 hours post-treatment, animals were anesthetized, injected
with the luciferase substrate D-luciferin and the bioluminescence
imaging (BLI) from living animals was evaluated in an IVIS imager
15 minutes later. Signals were obtained from animals injected with
non-targeted mRNA and from miR-122 targeted mRNA, and presented in
Table 50. The total light signal produced from livers of animals
treated with miR 122 targeted mRNA is 29.times. lower than
non-targeted mRNA, showing that the engineered element in the 3'UTR
may inhibit protein expression in normal tissue.
TABLE-US-00051 TABLE 50 In vivo expression of modified mRNA
modulated by an engineered miR122 binding site Luciferase signal
from liver Description (photons/sec) Non-targeted mRNA 7.9 .times.
10.sup.7 miR-122 targted mRNA 2.7 .times. 10.sup.6
[1111] B. BALB/c Nude Mice with Hepatocellular Carcinoma Hep3B
Cells
[1112] BALB/c nude mice were intrahepatically implanted with
2.times.10.sup.6 hepatocellular carcinoma Hep3B cells and resulting
orthotopic tumors allowed to grow for 24 days. Tumor-bearing mice
were then intravenously injected with 0.1 mg/kg luciferase modified
mRNA without a miR-122 binding site ("non-targeted mRNA"; mRNA
sequence shown in SEQ ID NO: 7518; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytidine and 1-methylpeudouridine) or luciferase
modified mRNA with a miR-122 binding site in the 3'UTR ("miR-122
targeted mRNA"; mRNA sequence shown in SEQ ID NO: 7519; polyA tail
of approximately 140 nucleotides not shown in sequence; 5' cap,
Cap1; fully modified with 5-methylcytidine and
1-methylpeudouridine) formulated in a lipid nanoparticle described
in Table 45 (above). 24 hr post-treatment animals were
anesthetized, injected with the luciferase substrate D-luciferin
and bioluminescence imaging (BLI) from living animals was evaluated
in an IVIS imager 20 minutes later. Signal from orthotopic tumors
compared to adjacent normal liver was quantified, and
miR-122-targeted mRNA systemically delivered via lipid
nanoparticles achieved over 2-fold enrichment in tumor compared to
normal liver.
Example 57
Modified Nucleic Acids with a Mir-122 Sequence
[1113] A. HeLa Cells
[1114] HeLa cells were seeded at a density of 15,000 per well in
100 ul cell culture medium (DMEM+10% FBS). G-CSF mRNA having a
miR-122 sequence in the 3'UTR (G-CSF miR122; mRNA sequence shown in
SEQ ID NO: 7325; polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap,Cap 1; fully modified with
5-methylcytosine and 1-methylpseudouridine) or G-CSF mRNA having a
miR-122 sequence without the seed sequence in the 3'UTR (G-CSF
seedless; mRNA sequence shown in SEQ ID NO: 7327; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine)
were transfected with 0.3 ul per well of Lipofectamine 2000 at a
concentration of 75 ng of mRNA per well in 96 well plates. The
supernatant was collected between 16-18 hours after transfection
and expression of G-CSF was measured by ELISA, and the results are
shown in Table 51.
TABLE-US-00052 TABLE 51 G-CSF Expression in HeLa Protein Expression
Description (ng/ml) G-CSF miR122 292.1 G-CSF seedless 335.7
[1115] B. Primary Human and Rat Hepatocytes
[1116] Primary human or rat hepatocytes cells were seeded at a
density of 350,000 cells per well in 500 ul cell culture medium
(InvitroGRO CP and InVitroGRO HI Medium+2.2% Torpedo Antibiotic
Mix). G-CSF mRNA having a miR-122 sequence in the 3'UTR (G-CSF
miR122; mRNA sequence shown in SEQ ID NO: 7520; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine) or
G-CSF mRNA having a miR-122 sequence without the seed sequence in
the 3'UTR (G-CSF seedless; mRNA sequence shown in SEQ ID NO: 7521;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine) were transfected with 1 ul per well of
Lipofectamine 2000 at a concentration of 500 ng of mRNA per well in
24 well plates for the primary human hepatocytes and the primary
rat hepatocytes. The supernatant was collected between 16-18 hours
after transfection and expression of G-CSF was measured by ELISA,
and the results are shown in Table 52. The mir-122 binding site
sequence in the mRNA dampened the G-CSF protein expression in the
primary hepatocytes.
TABLE-US-00053 TABLE 52 G-CSF Expression in Hepatocytes Primary
Human Primary Rat Hepatocytes Hepatocytes Protein Expression
Protein Expression Description (ng/ml) (ng/ml) G-CSF miR122 116 26
G-CSF seedless 463 85
Example 58
Time Course of Modified Nucleic Acids with a Mir-122 Sequence
[1117] A. HeLa Cells
[1118] HeLa cells were seeded at a density of 17,000 per well in
100 ul cell culture medium (DMEM+10% FBS). G-CSF mRNA without a
miR-122 sequence in the 3'UTR (G-CSF; mouse 3' UTR mRNA sequence
shown in SEQ ID NO: 7321; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine; human 3'UTR mRNA
sequence shown in SEQ ID NO: 7320; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine), G-CSF mRNA having
a miR-122 sequence in the 3'UTR (G-CSF miR122; mouse 3' UTR mRNA
sequence shown in SEQ ID NO: 7325; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine; human 3'UTR mRNA
sequence shown in SEQ ID NO: 7322; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine), G-CSF mRNA having
a miR-122 seed sequence in the 3'UTR (G-CSF seed; mouse 3' UTR mRNA
sequence shown in SEQ ID NO: 7326; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine; human 3'UTR mRNA
sequence shown in SEQ ID NO: 7323; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine), G-CSF mRNA having
a miR-122 sequence without the seed sequence in the 3'UTR (G-CSF
seedless; mouse 3' UTR mRNA sequence shown in SEQ ID NO: 7327;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7324; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), Factor IX mRNA without a miR-122 sequence
in the 3'UTR (FIX; mouse 3' UTR mRNA sequence shown in SEQ ID NO:
7329; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7328; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), Factor IX mRNA having a miR-122 sequence in
the 3'UTR (FIX miR122; mouse 3' UTR mRNA sequence shown in SEQ ID
NO: 7333; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7330; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), Factor IX mRNA having a miR-122 seed
sequence in the 3'UTR (FIX seed; mouse 3' UTR mRNA sequence shown
in SEQ ID NO: 7334; polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1; fully modified with
5-methylcytosine and 1-methylpseudouridine; human 3'UTR mRNA
sequence shown in SEQ ID NO: 7331; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine) or Factor IX mRNA
having a miR-122 sequence without the seed sequence in the 3'UTR
(FIX seedless; mouse 3' UTR mRNA sequence shown in SEQ ID NO: 7335;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7332; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine) were transfected with 0.3 ul per well of
Lipofectamine 2000 at a concentration of 75 ng of mRNA per well in
96 well plates. The supernatant was collected between 16-18 hours
after transfection, expression of G-CSF or Factor IX was measured
by ELISA, and the results are shown in Table 53.
TABLE-US-00054 TABLE 53 Expression in HeLa Protein Expression Mm
Protein Expression Description 3'UTR (ng/ml) Hs 3'UTR (ng/ml) G-CSF
271.72 69.4 G-CSF miR122 305.36 68.8 G-CSF seed 209.5 98.0 G-CSF
seedless 243.2 80.9 FIX 249.8 131.6 FIX mir122 204.6 55.4 FIX seed
290.05 127.6 FIX seedless 180.9 31.6
[1119] B. Primary Human and Rat Hepatocytes
[1120] Primary human or rat hepatocytes cells were seeded at a
density of 350,000 cells per well in 500 ul cell culture medium
(InvitroGRO CP and InVitroGRO HI Medium+2.2% Torpedo Antibiotic).
G-CSF mRNA without a miR-122 sequence in the 3'UTR (G-CSF; mouse 3'
UTR mRNA sequence shown in SEQ ID NO: 7321; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine;
human 3'UTR mRNA sequence shown in SEQ ID NO: 7320; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine),
G-CSF mRNA having a miR-122 sequence in the 3'UTR (G-CSF miR122;
mouse 3' UTR mRNA sequence shown in SEQ ID NO: 7325; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine;
human 3'UTR mRNA sequence shown in SEQ ID NO: 7322; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine),
G-CSF mRNA having a miR-122 seed sequence in the 3'UTR (G-CSF seed;
mouse 3' UTR mRNA sequence shown in SEQ ID NO: 7326; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine;
human 3'UTR mRNA sequence shown in SEQ ID NO: 7323; polyA tail of
approximately 140 nucleotides not shown in sequence; 5' cap, Cap1;
fully modified with 5-methylcytosine and 1-methylpseudouridine),
G-CSF mRNA having a miR-122 sequence without the seed sequence in
the 3'UTR (G-CSF seedless; mouse 3' UTR mRNA sequence shown in SEQ
ID NO: 7327; polyA tail of approximately 140 nucleotides not shown
in sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7324; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), Factor IX mRNA without a miR-122 sequence
in the 3'UTR (FIX; mouse 3' UTR mRNA sequence shown in SEQ ID NO:
7329; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7328; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), Factor IX mRNA having a miR-122 sequence in
the 3'UTR (FIX miR122; mouse 3' UTR mRNA sequence shown in SEQ ID
NO: 7333; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7330; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), Factor IX mRNA having a miR-122 seed
sequence in the 3'UTR (FIX seed; mouse 3' UTR mRNA sequence shown
in SEQ ID NO: 7334; polyA tail of approximately 140 nucleotides not
shown in sequence; 5' cap, Cap1; fully modified with
5-methylcytosine and 1-methylpseudouridine; human 3'UTR mRNA
sequence shown in SEQ ID NO: 7331; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine) or Factor IX mRNA
having a miR-122 sequence without the seed sequence in the 3'UTR
(FIX seedless; mouse 3' UTR mRNA sequence shown in SEQ ID NO: 7335;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine; human 3'UTR mRNA sequence shown in SEQ ID
NO: 7332; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine) were transfected with 1 ul per well of
Lipofectamine 2000 at a concentration of 500 ng per well in 24 well
plates for the primary human hepatocytes and the primary rat
hepatocytes. The supernatant was collected at 24 hours, 48 hours
and 72 hours after transfection, expression of G-CSF and Factor IX
was measured by ELISA, and the results are shown in Table 54. The
mir-122 binding site sequence in the mRNA dampened the G-CSF and
Factor IX protein expression in the primary hepatocytes.
TABLE-US-00055 TABLE 54 G-CSF Expression in Hepatocytes Primary
Human Primary Human Hepatocytes Hepatocytes Protein Expression
Protein Expression (ng/ml) (ng/ml) Description Time Point Mm 3'UTR
Hs 3'UTR G-CSF 24 hours 43.9 84.9 48 hours 18.8 100.4 72 hours 5.7
21.3 G-CSF miR122 24 hours 6.9 24.0 48 hours .7 3.03 72 hours .12
.88 G-CSF seed 24 hours 48.5 115.8 48 hours 25.6 96.4 72 hours 8.2
19.2 G-CSF seedless 24 hours 31.7 113.1 48 hours 11.7 92.9 72 hours
3.4 18.9 FIX 24 hours 90.8 63.2 48 hours 159.6 124.8 72 hours 70.5
44.3 FIX mir122 24 hours 11.8 15.9 48 hours 5.0 4.4 72 hours 1.0 .4
FIX seed 24 hours 77.2 60.2 48 hours 115.0 63.0 72 hours 41.7 20.1
FIX seedless 24 hours 69.3 53.7 48 hours 123.8 75.0 72 hours 49.0
24.5
Example 59
Time Course of Modified Nucleic Acids with a Mir-122 Sequence in
Cancer Cells
[1121] A. Base Level of miR-122
[1122] The base level of mir-122 in Human hepatocytes, rat
hepatocytes, human hepatocellular carcinoma cells (Hep3B) and HeLa
cells were determined by TAQMAN.RTM. analysis using the
manufacturers protocol. The levels were normalized to U6 and the
results are shown in Table 55.
TABLE-US-00056 TABLE 55 miR-122 Levels in Various Cell Types
miR-122 level Cell Type (normalized to U6) Human Hepatocytes 16.8
Rat Hepatocytes 10.9 Hep3B 0 HeLa 0
[1123] B. Primary Human Hepatocytes and Hep3B Cells
[1124] Primary human hepatocytes were seeded at a density of 50,000
cells per well in 100 ul cell culture medium (InvitroGRO CP and
InVitroGRO HI Medium+2.2% Torpedo Antibiotic Mix) and Hep3B cells
were seeded at a density of 20,000 cells per well in 100 ul cell
culture medium MEM+10% FBS. G-CSF mRNA without a miR-122 sequence
in the 3'UTR (G-CSF; mRNA sequence shown in SEQ ID NO: 7320; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), G-CSF mRNA having a miR-122 sequence in the
3'UTR (G-CSF miR122; mRNA sequence shown in SEQ ID NO: 7322; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), G-CSF mRNA having a miR-122 seed sequence
in the 3'UTR (G-CSF seed; mRNA sequence shown in SEQ ID NO: 7323;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine) or G-CSF mRNA having a miR-122 sequence
without the seed sequence in the 3'UTR (G-CSF seedless; mRNA
sequence shown in SEQ ID NO: 7324; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine) were transfected
with 0.3 ul per well of Lipofectamine 2000 at a concentration of 75
ng of mRNA per well in 96 well plates for the primary human
hepatocytes and the Hep3B cells. The supernatant was collected at
24 hours, 48 hours and 72 hours after transfection, expression of
G-CSF was measured by ELISA, and the results are shown in Table 56.
The mir-122 binding site sequence in the mRNA dampened the G-CSF
protein expression in the primary human hepatocytes but not in the
Hep3B cells.
TABLE-US-00057 TABLE 56 G-CSF Expression Primary Human Hepatocytes
Hep3B Protein Expression Protein Expression (ng/ml) (ng/ml)
Description Time Point Hs 3'UTR Hs 3'UTR G-CSF 24 hours 76 55 48
hours 12 33 72 hours 6 10 G-CSF miR122 24 hours 32 37 48 hours 1 27
72 hours 0 6 G-CSF seed 24 hours 75 39 48 hours 11 28 72 hours 4 6
G-CSF seedless 24 hours 79 49 48 hours 15 35 72 hours 6 9
Example 60
Time Course of Modified Nucleic Acids with a Mir-142 3p
Sequence
[1125] A. Base Level of miR-143 3p
[1126] The base level of miR-142 3p in RAW264.7 cells and HeLa
cells were determined by TAQMAN.RTM. analysis using the
manufacturer's protocol. The levels were normalized to U6 and the
results are shown in Table 57.
TABLE-US-00058 TABLE 57 miR-142 3p Levels in Various Cell Types
miR-122 level Cell Type (normalized to U6) Human Hepatocytes 16.8
Rat Hepatocytes 10.9 Hep3B 0 HeLa 0
[1127] B. HeLa and RAW264.7 Cells
[1128] HeLa cells were seeded at a density of 17,000 per well in
100 ul cell culture medium DMEM+10% FBS and RAW264.7 cells were
seeded at a density of 200,000 per well in 100 ul cell culture
medium DMEM+10% FBS. G-CSF mRNA without a miR-142 3p sequence in
the 3'UTR (G-CSF; mRNA sequence shown in SEQ ID NO: 7522; polyA
tail of approximately 140 nucleotides not shown in sequence; 5'
cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), G-CSF mRNA having a miR-142 3p sequence in
the 3'UTR (G-CSF miR142 3p; mRNA sequence shown in SEQ ID NO: 7523;
polyA tail of approximately 140 nucleotides not shown in sequence;
5' cap, Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine), G-CSF mRNA having a miR-142 3p seed
sequence in the 3'UTR (G-CSF seed; mRNA sequence shown in SEQ ID
NO: 7524; polyA tail of approximately 140 nucleotides not shown in
sequence; 5' cap, cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine) or G-CSF mRNA having a miR-142 3p sequence
without the seed sequence in the 3'UTR (G-CSF seedless; mRNA
sequence shown in SEQ ID NO: 7525; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine) were transfected
with 0.3 ul per well of Lipofectamine 2000 at a concentration of 75
ng of mRNA per well in 96 well plates for HeLa or with 1 ul per
well of Lipofectamine 2000 at a concentration of 250 ng of mRNA per
well in 24 well plates for RAW264.7 cells. The supernatant was
collected 16-18 hours after transfection, expression of G-CSF was
measured by ELISA, and the results are shown in Table 58. miR-142
3p sites in G-CSF were shown to down-regulate G-CSF expression in
RAW264.7 cells.
TABLE-US-00059 TABLE 58 Expression HeLa Protein RAW264.7 Protein
Description Expression (ng/ml) Expression (ng/ml) G-CSF 243.5 124.8
G-CSF miR142 3p 309.1 42.8 G-CSF seed 259.8 148.1 G-CSF seedless
321.7 185.2
[1129] C. Time Course in RAW264.7 Cells
[1130] RAW264.7 cells were seeded at a density of 60,000 cells per
well in 100 ul cell culture medium (DMEM+10% FBS). G-CSF mRNA
without a miR-142 3p sequence in the 3'UTR (G-CSF; mRNA sequence
shown in SEQ ID NO: 7522; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine), G-CSF mRNA having
a miR-142 3p sequence in the 3'UTR (G-CSF miR142 3p; mRNA sequence
shown in SEQ ID NO: 7523; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine), G-CSF mRNA having
a miR-142 3p seed sequence in the 3'UTR (G-CSF seed; mRNA sequence
shown in SEQ ID NO: 7524; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine) or G-CSF mRNA
having a miR-142 3p sequence without the seed sequence in the 3'UTR
(G-CSF seedless; mRNA sequence shown in SEQ ID NO: 7525; polyA tail
of approximately 140 nucleotides not shown in sequence; 5' cap,
Cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine) were transfected with 0.3 ul per well of
Lipofectamine 2000 at a concentration of 75 ng of mRNA per well in
96 well plates. The supernatant was collected at 24 hours, 48 hours
and 72 hours after transfection, expression of G-CSF was measured
by ELISA, and the results are shown in Table 59. The mir-142 3p
binding site sequence in the mRNA showed a strong suppression of
G-CSF expression in RAW264.7 cells over time.
TABLE-US-00060 TABLE 59 G-CSF Expression RAW264.7 Cells Description
Time Point Protein Expression (ng/ml) G-CSF 24 hours 133.5 48 hours
69.7 72 hours 2.1 G-CSF miR142 3p 24 hours 60.1 48 hours 9.2 72
hours .3 G-CSF seed 24 hours 244.9 48 hours 68.9 72 hours 2.3 G-CSF
seedless 24 hours 250.2 48 hours 95.9 72 hours 3.0
[1131] D. miR-142 3p in PBMC
[1132] Peripheral blood mononuclear cells (PBMCs) were seeded at a
density of 150,000 cells per well in 100 ul cell culture medium
(Opti-MEM and after transfection add 10% FBS). G-CSF mRNA having a
miR-142 3p sequence in the 3'UTR (G-CSF miR142 3p; mRNA sequence
shown in SEQ ID NO: 7523; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, Cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine), G-CSF mRNA having
a miR-142 3p seed sequence in the 3'UTR (G-CSF seed; mRNA sequence
shown in SEQ ID NO: 7524; polyA tail of approximately 140
nucleotides not shown in sequence; 5' cap, cap1; fully modified
with 5-methylcytosine and 1-methylpseudouridine) or G-CSF mRNA
having a miR-142 3p sequence without the seed sequence in the 3'UTR
(G-CSF seedless; mRNA sequence shown in SEQ ID NO: 7525; polyA tail
of approximately 140 nucleotides not shown in sequence; 5' cap,
cap1; fully modified with 5-methylcytosine and
1-methylpseudouridine) were transfected in triplicate with 0.4 ul
per well of Lipofectamine 2000 at a concentration of 500 ng of mRNA
per well in 96 well plates for 2 or 3 donors. The supernatant was
collected at 24 hours after transfection and the expression of
G-CSF was measured by ELISA. The results for the 2 donors are shown
in Table 60 and the results for the 3 donors are shown in Table 61.
The mir-142 3p binding site sequence in the mRNA was shown to down
regulate G-CSF expression in human PBMC.
TABLE-US-00061 TABLE 60 Expression PBMC (2 donors) Protein
Expression Description (ng/ml) G-CSF miR142 3p 5.09 G-CSF seed
10.06 G-CSF seedless 9.38
TABLE-US-00062 TABLE 61 Expression PBMC (3 donors) Protein
Expression Description (ng/ml) G-CSF miR142 3p 7.48 G-CSF seed
13.40 G-CSF seedless 13.98
OTHER EMBODIMENTS
[1133] It is to be understood that the words which have been used
are words of description rather than limitation, and that changes
may be made within the purview of the appended claims without
departing from the true scope and spirit of the invention in its
broader aspects.
[1134] While the present invention has been described at some
length and with some particularity with respect to the several
described embodiments, it is not intended that it should be limited
to any such particulars or embodiments or any particular
embodiment, but it is to be construed with references to the
appended claims so as to provide the broadest possible
interpretation of such claims in view of the prior art and,
therefore, to effectively encompass the intended scope of the
invention.
[1135] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, section headings, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140206852A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140206852A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References