U.S. patent application number 16/930720 was filed with the patent office on 2021-08-05 for engineered nucleic acids and methods of use thereof.
This patent application is currently assigned to ModernaTX, Inc.. The applicant listed for this patent is ModernaTX, Inc.. Invention is credited to Kenechi Ejebe, Sayda M. Elbashir, Jason P. Schrum, Gregory J. Sieczkiewicz.
Application Number | 20210236655 16/930720 |
Document ID | / |
Family ID | 1000005523427 |
Filed Date | 2021-08-05 |
United States Patent
Application |
20210236655 |
Kind Code |
A1 |
Schrum; Jason P. ; et
al. |
August 5, 2021 |
Engineered Nucleic Acids and Methods of Use Thereof
Abstract
Provided are compositions and methods for delivering biological
moieties such as modified nucleic acids into cells to modulate
protein expression. Such compositions and methods include the use
of modified messenger RNAs, and are useful for production of
proteins.
Inventors: |
Schrum; Jason P.;
(Somerville, MA) ; Sieczkiewicz; Gregory J.;
(Hopkinton, MA) ; Ejebe; Kenechi; (Cambridge,
MA) ; Elbashir; Sayda M.; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
ModernaTX, Inc.
Cambridge
MA
|
Family ID: |
1000005523427 |
Appl. No.: |
16/930720 |
Filed: |
July 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15611490 |
Jun 1, 2017 |
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16930720 |
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14535484 |
Nov 7, 2014 |
9701965 |
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15611490 |
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13252049 |
Oct 3, 2011 |
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14535484 |
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61404413 |
Oct 1, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2887 20130101;
C12N 15/67 20130101; C12N 2310/3341 20130101; C12N 2310/335
20130101; C12N 15/102 20130101; C07H 21/02 20130101; C12N 15/1138
20130101; C07K 2317/24 20130101; A61K 48/0066 20130101; G01N 33/559
20130101; C07K 16/00 20130101; C12N 5/0602 20130101; C12N 15/1136
20130101; C12N 15/11 20130101; C12P 21/00 20130101; C07H 19/10
20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/113 20060101 C12N015/113; C07H 21/02 20060101
C07H021/02; C07H 19/10 20060101 C07H019/10; C12P 21/00 20060101
C12P021/00; C12N 5/071 20060101 C12N005/071; C12N 15/10 20060101
C12N015/10; C12N 15/11 20060101 C12N015/11; C07K 16/00 20060101
C07K016/00; C07K 16/28 20060101 C07K016/28; G01N 33/559 20060101
G01N033/559; C12N 15/67 20060101 C12N015/67 |
Claims
1-4. (canceled)
5. A method of producing an immunoglobulin protein, comprising
delivering two modified mRNA constructs in vivo so as to cause
intracellular translation of the mRNA constructs into heavy and
light chains of an immunoglobulin protein, wherein the modified
mRNA constructs are capable of evading an innate immune response of
a cell into which they are introduced, and wherein the modified
mRNA constructs are substantially devoid of cytidine or uracil
nucleotides.
6. The method of claim 5, wherein the modified mRNA constructs are
substantially devoid of cytidine nucleotides.
7. The method of claim 5, wherein the modified mRNA constructs are
substantially devoid of uracil nucleotides.
8. The method of claim 6, wherein the modified mRNA constructs
comprise 5-methyl-cytidine.
9. The method of claim 7, wherein the modified mRNA constructs
comprise pseudouridine.
10. The method of claim 7, wherein the modified mRNA constructs
comprise 1-methyl-pseudouridine.
11. A kit for immunoglobulin protein production, comprising a first
isolated nucleic acid comprising i) a translatable region encoding
the immunoglobulin protein and ii) a nucleic acid modification,
wherein the first nucleic acid is capable of evading an innate
immune response of a cell into which the first isolated nucleic
acid is introduced, wherein the translatable region is
substantially devoid of cytidine and uracil nucleotides, and
packaging and instructions therefor.
12. The kit of claim 11, wherein the immunoglobulin protein
comprises a polypeptide selected from the group consisting of a
full-length antibody, a heavy chain polypeptide, a light chain
polypeptide, an Fab domain, or a single chain variable fragment
(ScFv) polypeptide, and the first isolated nucleic acid comprises a
messenger ribonucleic acid comprising 5-methyl-cytidine and
pseudouridine.
13. A mammalian cell generated by use of the kit of claim 11.
14. An isolated immunoglobulin protein produced from a production
cell comprising a first isolated nucleic acid comprising i) a
translatable region encoding the immunoglobulin protein and ii) a
nucleic acid modification, wherein the first nucleic acid is
capable of evading an innate immune response of the cell, wherein
the translatable region is substantially devoid of either cytidine
or uracil nucleotides or the combination of cytidine and uracil
nucleotides.
15. A pharmaceutical preparation comprising an effective amount of
a first nucleic acid comprising i) a translatable region encoding
an immunoglobulin protein and ii) a nucleic acid modification,
wherein the first nucleic acid exhibits reduced degradation by a
cellular nuclease and is capable of evading an innate immune
response of a cell into which the first nucleic acid is introduced,
wherein the translatable region is substantially devoid of cytidine
and uracil nucleotides.
16. A method of producing a heterologous protein of interest in a
cell, comprising the step providing a target cell capable of
protein translation; and introducing into the target cell a
composition comprising a first isolated nucleic acid comprising a
translatable region encoding the heterologous protein of interest
and a nucleoside modification, under conditions such that the
protein of interest is produced in the cell.
17. The method of claim 16, wherein the protein of interest is an
immunoglobulin protein.
18. A method of increasing the production of a recombinantly
expressed protein of interest in a cell, comprising the step:
providing a target cell comprising a heterologous nucleic acid
encoding the protein of interest; and introducing into the target
cell a composition comprising a first isolated nucleic acid
comprising a translatable region encoding a translation effector
protein and a nucleoside modification under conditions such that
the effector protein is produced in the cell, thereby increasing
the production of the recombinantly expressed protein in the
cell.
19. The method of claim 18, wherein the protein of interest is an
immunoglobulin protein.
20. A method for modulating the level of a protein of interest in a
target cell, comprising the steps of: modulating the activity of at
least one translation effector molecule in the target cell, wherein
the modulation comprises introducing into the target cell a first
isolated nucleic acid comprising a translatable region encoding the
translation effector protein and a nucleoside modification; and
culturing the cell.
21. A kit for protein production, comprising a first isolated
nucleic acid encoding a translatable region encoding a protein,
wherein the first nucleic acid comprises a nucleic acid
modification, wherein the first nucleic acid displays decreased
degradation in a cell into which the first isolated nucleic acid is
introduced as compared to a nucleic acid not comprising a nucleic
acid modification, and packaging and instructions therefor.
22. A kit for protein production, comprising a first isolated
nucleic acid encoding a translatable region encoding a protein,
wherein the first nucleic acid comprises a nucleic acid
modification, wherein the first nucleic acid is more stable in a
cell into which the first isolated nucleic acid is introduced as
compared to a nucleic acid not comprising a nucleic acid
modification, and packaging and instructions therefor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 13/252,049, filed Oct. 3, 2011, entitled
Engineered Nucleic Acids and Methods of Use Thereof which claims
the benefit of U.S. Provisional Application No. 61/404,413, filed
Oct. 1, 2010, the contents of each of which is incorporated herein
by reference in its entirety.
REFERENCE TO THE SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled M008USCON2SEQLST.tx created on Nov. 4, 2014 which is
57,758 bytes in size. The information in electronic format of the
sequence listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Naturally occurring R NAs are synthesized from four basic
ribonucleotides: ATP, CTP, UTP and GTP, but may contain
post-transcriptionally modified nucleotides. Further, approximately
one hundred different nucleoside modifications have been identified
in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA
Modification Database: 1999 update. Nucl Acids Res 27: 196-197).
The role of nucleoside modifications on the immuno-stimulatory
potential and on the translation efficiency of RNA, however, is
unclear.
[0004] There are multiple problems with prior methodologies of
effecting protein expression. For example, heterologous DNA
introduced into a cell can be inherited by daughter cells (whether
or not the heterologous DNA has integrated into the chromosome) or
by offspring. Introduced DNA can integrate into host cell genomic
DNA at some frequency, resulting in alterations and/or damage to
the host cell genomic DNA. In addition, multiple steps must occur
before a protein is made. Once inside the cell, DNA must be
transported into the nucleus where it is transcribed into RNA. The
RNA transcribed from DNA must then enter the cytoplasm where it is
translated into protein. This need for multiple processing steps
creates lag times before the generation of a protein of interest.
Further, it is difficult to obtain DNA expression in cells;
frequently DNA enters cells but is not expressed or not expressed
at reasonable rates or concentrations. This can be a particular
problem when DNA is introduced into cells such as primary cells or
modified cell lines.
[0005] There is a need in the art for biological modalities to
address the modulation of intracellular translation of nucleic
acids.
[0006] Unless explained otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
herein. The materials, methods, and examples are illustrative only
and not intended to be limiting. Other features of the disclosure
are apparent from the following detailed description and the
claims.
SUMMARY OF THE INVENTION
[0007] Described herein are methods of producing proteins,
polypeptides, and peptides. For example, the method includes
introducing a nucleic acid (e.g., a modified nucleic acid described
herein) encoding a protein, polypeptide, or peptide of interest in
to a cell (e.g., a human cell), under conditions that the protein,
polypeptide, or peptide of interest is produced (e.g., translated)
in the cell. In some embodiments, the nucleic acid comprises one or
more nucleoside modifications (e.g., one or more nucleoside
modifications described herein). In some embodiments, the nucleic
acid is capable of evading an innate immune response of a cell into
which the nucleic acid is introduced. In some embodiments, the
protein, polypeptide, or peptide is a therapeutic protein described
herein. In some embodiments, the protein, polypeptide, or peptide
comprises one or more post-translational modifications (e.g.,
post-translational modifications present in human cells).
Compositions and kits for protein production are also described
herein. Further described herein are cells and cultures with
altered protein levels (e.g., generated by a method described
herein).
[0008] In one aspect, the disclosure features a method of producing
a protein (e.g., a heterologous protein) of interest in a cell, the
method comprising the steps: (i) providing a target cell capable of
protein translation; and (ii) introducing into the target cell a
composition comprising a first isolated nucleic acid comprising a
translatable region encoding the protein of interest and a
nucleoside modification, under conditions such that the protein of
interest is produced in the cell In some embodiments, the method
further comprises the step of substantially purifying the protein
of interest from the cell. In some embodiments, the protein of
interest is a secreted protein.
[0009] In another aspect, the disclosure features a method of
producing a protein (e.g., a heterologous protein) of interest in a
cell, the method comprising the steps: (i) providing a target cell
capable of protein translation; and (ii) introducing into the
target cell a composition comprising: (a) a first isolated nucleic
acid comprising a translatable region encoding the protein of
interest and a nucleoside modification; and (b) a second nucleic
acid comprising an inhibitory nucleic acid, under conditions such
that the protein of interest is produced in the cell. In some
embodiments, the method further comprises the step of substantially
purifying the protein of interest from the cell. In some
embodiments, the protein of interest is a secreted protein.
[0010] In one aspect, the disclosure features a method of
increasing the production of a recombinantly expressed protein of
interest in a cell, comprising the steps: (i) providing a target
cell comprising a recombinant nucleic acid encoding the protein of
interest; and (ii) introducing into the target cell a composition
comprising a first isolated nucleic acid comprising a translatable
region encoding a translation effector protein and a nucleoside
modification under conditions such that the effector protein is
produced in the cell, thereby increasing the production of the
recombinantly expressed protein in the cell.
[0011] In some embodiments, the target cell is a mammalian cell. In
some embodiments, the target cell is a yeast cell. In some
embodiments, the target cell is a bacterial cell, an insect cell,
or a plant cell. In some embodiments, the protein of interest is a
secreted protein. In some embodiments, the protein of interest is a
transmembrane protein. In some embodiments, the protein of interest
is an antibody or an antigen-binding fragment thereof. In some
embodiments, the protein of interest is a growth factor or
cytokine. In some embodiments, the protein of interest is a peptide
or peptidomimetic. In some embodiments, the translation effector
protein is ceramide transfer protein (CERT). In some embodiments,
the translation effector protein is translated in the target cell
in an amount effective to increase efficiency of translation of the
recombinantly expressed protein. In some embodiments, the
translation effector protein is translated in the target cell in an
amount effective to reduce efficiency of translation of proteins in
the cell other than the recombinantly expressed protein. In some
embodiments, the translation effector protein is translated in the
target cell in an amount effective to reduce formation of inclusion
bodies containing the recombinantly expressed protein. In some
embodiments, the translation effector protein is translated in the
target cell in an amount effective to reduce intracellular
degradation of the recombinantly expressed protein. In some
embodiments, the translation effector protein is translated in the
target cell in an amount effective to increase secretion of the
recombinantly expressed protein.
[0012] In another aspect, the disclosure features a method for
altering the level of a protein of interest in a target cell, the
method comprising the steps of: (i) modulating the activity of at
least one translation effector molecule in the target cell; and
(ii) culturing the cell. In some embodiments, the target cell does
not contain a recombinant nucleic acid. In some embodiments, the
method further comprises the step of isolating the protein of
interest.
[0013] In another aspect, the disclosure features a method for
modulating the level of a protein of interest in a target cell,
comprising the steps of: i) modulating the activity of at least one
translation effector molecule in the target cell, wherein the
modulation comprises introducing into the target cell a first
isolated nucleic acid comprising a translatable region encoding the
translation effector protein and a nucleoside modification; and ii)
culturing the cell.
[0014] In one aspect, the disclosure features an animal cell (e.g.,
a mammalian cell) with an altered protein level, generated by the
steps of: (i) introducing into the cell an effective amount of a
first isolated nucleic acid comprising a translatable region
encoding a translation effector protein and a nucleoside
modification; and (ii) culturing the cell. In some embodiments, the
effective amount of the first isolated nucleic acid introduced into
the cell is titrated against a desired amount of protein translated
from the translatable region.
[0015] In one aspect, the disclosure features a high density
culture comprising a plurality of the cells described herein. In
some embodiments, the culture comprises a batch process. In some
embodiments, the culture comprises a continuous feed process.
[0016] In one aspect, the disclosure features a composition for
protein production, the composition comprising a first isolated
nucleic acid comprising a translatable region and a nucleoside
modification, wherein the nucleic acid exhibits reduced degradation
by a cellular nuclease, and a mammalian cell suitable for
translation of the translatable region of the first nucleic acid.
In some embodiments, the mammalian cell comprises a recombinant
nucleic acid.
[0017] In another aspect, the disclosure features a composition for
protein production, the composition comprising: (i) a first
isolated nucleic acid comprising a translatable region and a
nucleoside modification, wherein the nucleic acid exhibits reduced
degradation by a cellular nuclease; (ii) a second nucleic acid
comprising an inhibitory nucleic acid; and (iii) a mammalian cell
suitable for translation of the translatable region of the first
nucleic acid, wherein the mammalian cell comprises a target nucleic
acid capable of being acted upon by the inhibitory nucleic acid. In
some embodiments, the mammalian cell comprises a recombinant
nucleic acid.
[0018] In one aspect, the disclosure features a kit for protein
production, the kit comprising a first isolated nucleic acid
comprising a translatable region and a nucleic acid modification,
wherein the nucleic acid is capable of evading an innate immune
response of a cell into which the first isolated nucleic acid is
introduced, and packaging and instructions therefor.
[0019] In another aspect, the disclosure features a kit for protein
production, the kit comprising: (i) a first isolated nucleic acid
comprising a translatable region, provided in an amount effective
to produce a desired amount of a protein encoded by the
translatable region when introduced into a target cell; (ii) a
second nucleic acid comprising an inhibitory nucleic acid, provided
in an amount effective to substantially inhibit the innate immune
response of the cell; and (iii) packaging and instructions
therefor.
[0020] In yet another aspect, the disclosure features a kit for
protein production, the kit comprising a first isolated nucleic
acid comprising a translatable region and a nucleoside
modification, wherein the nucleic acid exhibits reduced degradation
by a cellular nuclease, and packaging and instructions
therefor.
[0021] In one aspect, the disclosure features a kit for protein
production, the kit comprising a first isolated nucleic acid
comprising a translatable region and at least two different
nucleoside modifications, wherein the nucleic acid exhibits reduced
degradation by a cellular nuclease, and packaging and instructions
therefor.
[0022] In another aspect, the disclosure features a kit for protein
production, the kit comprising: (i) a first isolated nucleic acid
comprising a translatable region; (ii) a second nucleic acid
comprising an inhibitory nucleic acid; and (iii) packaging and
instructions therefor.
[0023] In yet another aspect, the disclosure features a kit for
protein production, the kit comprising: (i) a first isolated
nucleic acid comprising a translatable region and at least one
nucleoside modification, wherein the nucleic acid exhibits reduced
degradation by a cellular nuclease; (ii) a second nucleic acid
comprising an inhibitory nucleic acid; and (iii) packaging and
instructions therefor.
[0024] In one aspect, the disclosure features a kit for protein
production, comprising a first isolated nucleic acid encoding a
translatable region encoding a protein, wherein the first nucleic
acid comprises a nucleic acid modification, wherein the first
nucleic acid displays decreased degradation in a cell into which
the first isolated nucleic acid is introduced as compared to a
nucleic acid not comprising a nucleic acid modification, and
packaging and instructions therefor.
[0025] In another aspect, the disclosure features a kit for protein
production, comprising a first isolated nucleic acid encoding a
translatable region encoding a protein, wherein the first nucleic
acid comprises a nucleic acid modification, wherein the first
nucleic acid displays is more stable in a cell into which the first
isolated nucleic acid is introduced as compared to a nucleic acid
not comprising a nucleic acid modification, and packaging and
instructions therefor.
[0026] In one aspect, the disclosure features a kit for
immunoglobulin protein production, comprising a first isolated
nucleic acid comprising i) a translatable region encoding the
immunoglobulin protein and ii) a nucleic acid modification, wherein
the first nucleic acid is capable of evading an innate immune
response of a cell into which the first isolated nucleic acid is
introduced, wherein the translatable region is substantially devoid
of cytidine and uracil nucleotides, and packaging and instructions
therefor.
[0027] In another aspect, the disclosure features a mammalian cell
generated by use of a kit described herein.
[0028] In yet another aspect, the disclosure features an isolated
immunoglobulin protein produced from a production cell comprising a
first isolated nucleic acid comprising i) a translatable region
encoding the immunoglobulin protein and ii) a nucleic acid
modification, wherein the first nucleic acid is capable of evading
an innate immune response of the cell, wherein the translatable
region is substantially devoid of either cytidine or uracil
nucleotides or the combination of cytidine and uracil
nucleotides.
[0029] In one aspect, the disclosure features a pharmaceutical
preparation comprising an effective amount of a protein described
herein.
[0030] In another aspect, the disclosure features a pharmaceutical
preparation comprising an effective amount of a first nucleic acid
comprising i) a translatable region encoding an immunoglobulin
protein and ii) a nucleic acid modification, wherein the first
nucleic acid exhibits reduced degradation by a cellular nuclease
and is capable of evading an innate immune response of a cell into
which the first nucleic acid is introduced, wherein the
translatable region is substantially devoid of cytidine and uracil
nucleotides.
[0031] Embodiments of the aforesaid methods, cells, cultures,
compositions, preparations, and kits may include one or more of the
following features:
[0032] In some embodiments, the first isolated nucleic acid
comprises messenger RNA (mRNA). In some embodiments, the mRNA
comprises at least one nucleoside selected from the group
consisting of pyridin-4-one ribonucleoside, 5-aza-uridine,
2-thio-5-aza-midine, 2-thiouridine, 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,
5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyluridine, 1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine, 1-taulinomethyl-4-thio-uridine,
5-methyl-uridine, 1-methyl-pseudouridine,
4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine,
1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudomidine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydromidine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA
comprises at least one nucleoside selected from the group
consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine,
N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine,
5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,
pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA
comprises at least one nucleoside selected from the group
consisting of 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diarminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine, 2-meth
ylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine. In some embodiments, mRNA comprises at least one
nucleoside selected from the group consisting of inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 depicts bar graphs of an Enzyme-linked immunosorbent
assay (ELISA) detection of Human G-CSF of in vitro transfected
Chinese Hamster Ovary with modRNA encoding human G-CSF at 12 and 24
hours post-transfection.
[0034] FIG. 2 depicts bar graphs of an Enzyme-linked immunosorbent
assay (ELISA) for Human IgG of in vitro transfected Chinese Hamster
Ovary cells with the Heavy and Light chains of modRNA encoding
Trastuzumab at 12, 24, and 36 hours post-transfection.
[0035] FIG. 3 depicts bar graphs of an Enzyme-linked immunosorbent
assay (ELISA) for detection of Human IgG of in vitro transfected
Human Embryonic Kidneys cells (HEK293) with Heavy and Light chains
of modRNA encoding Trastuzumab at 36 hours post-transfection. R
1,R2,R3 are triplicate transfection experiments performed in a
24-well plate and normalized to untreated samples.
[0036] FIG. 4 depicts an image of a western blot detection of in
vitro transfected Chinese Hamster Ovary cells with the Heavy and
Light chains of modRNA encoding Trastuzumab at 24 hours
post-transfection. HC and LC indicate the Heavy Chain and Light
Chain of Trastuzumab respectively.
[0037] FIG. 5 depicts images from cell immune-staining of in
vitro-transfected Chinese Hamster Ovary cells with the Heavy and
Light chains of modRNA encoding both Trastuzumab and Rituximab at
13 hours post-transfection.
[0038] FIG. 6 depicts images of a binding immunoblot assay of
modRNA encoding Trastuzumab and Rituximab. The black boxes display
the protein of interest.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Methods of producing proteins, polypeptides, and peptides
are described herein. The disclosure provides, at least in pmt,
methods of producing a protein, polypeptide, or peptide (e.g., a
heterologous protein) of interest in a cell, methods increasing the
production of a protein, polypeptide, or peptide (e.g., a
recombinantly expressed protein) of interest in a cell, and methods
of altering the level of a protein, polypeptide, or peptide of
interest in a cell. For example, the methods can include the step
of introducing a nucleic acid (e.g., a modified nucleic acid
described herein) encoding a protein, polypeptide, or peptide of
interest into a cell (e.g., a human cell), under conditions that
the protein, polypeptide, or peptide of interest is produced (e.g.,
translated) in the cell. In some embodiments, the nucleic acid
comprises one or more nucleoside modifications (e.g., one or more
nucleoside modifications described herein). In some embodiments,
the nucleic acid is capable of evading an innate immune response of
a cell into which the nucleic acid is introduced, thus increasing
the efficiency of protein production in the cell. In some
embodiments, the protein is a therapeutic protein described herein.
In some embodiments, the protein comprises one or more
post-translational modifications (e.g., post-translational
modifications present in human cells). Compositions and kits for
protein production are also described herein. Further described
herein are cells and cultures with altered protein levels (e.g.,
generated by a method described herein).
[0040] In general, exogenous nucleic acids, particularly viral
nucleic acids, introduced into cells induce an innate immune
response, resulting in interferon (IFN) production and cell death.
However, it is of great interest for recombinant protein production
to deliver a nucleic acid, e.g., a ribonucleic acid (RNA) inside a
cell, e.g., in cell culture, in vitro, in vivo, or ex vivo, such as
to cause intracelluar translation of the nucleic acid and
production of the encoded protein. Provided herein in part are
nucleic acids encoding useful polypeptides capable of modulating a
cell's function and/or activity, and methods of making and using
these nucleic acids and polypeptides. As described herein, these
nucleic acids are capable of reducing the innate immune activity of
a population of cells into which they are introduced, thus
increasing the efficiency of protein production in that cell
population. Further, one or more additional advantageous activities
and/or properties of the nucleic acids and proteins of the
invention are described.
Methods of Protein Production.
[0041] The methods provided herein are useful for enhancing protein
product yield in a cell culture process. In a cell culture
containing a plurality of host cells, introduction of the modified
mRNAs described herein results in increased protein production
efficiency relative to a corresponding unmodified nucleic acid.
Such increased protein production efficiency can be demonstrated,
e.g., by showing increased cell transfection, increased protein
translation from the nucleic acid, decreased nucleic acid
degradation, and/or reduced innate immune response of the host
cell. Protein production can be measured by ELISA, and protein
activity can be measured by various functional assays known in the
mt. The protein production may be generated in a continuous or a
fed-batch process.
Cell Culture and Growth.
[0042] In the methods of the disclosure, the cells are cultured.
Cells may be cultured in suspension or as adherent cultures. Cells
may be cultured in a variety of vessels including, for example,
bioreactors, cell bags, wave bags, culture plates, flasks,
hyperflasks and other vessels well known to those of ordinary skill
in the art. Cells may be cultured in IMDM (Invitrogen, Catalog
number 12440-53) or any other suitable media including chemically
defined media formulations. Ambient conditions suitable for cell
culture, such as temperature and atmospheric composition, are also
well known to those skilled in the art. The methods of the
disclosure may be used with any cell that is suitable for use in
protein production. In one embodiment, the cells are selected from
the group consisting of animal cells (e.g., mammalian cells),
bacterial cells, plant, microbial, algal, and fungal cells. In some
embodiments, the cells are mammalian cells, such human, mouse, rat,
goat, horse, rabbit, hamster or cow cells. For instance, the cells
may be from an y established cell line, including but not limited
to HeLa, NSO, SP2/0, HEK 293T, Vero, Caco, Caco-2, MDCK, COS-1,
COS-7, K562, Jurkat, CHO-K1, DG44, CHOK1SV, CHO-S, Huvec, CV-1,
HuH-7, NIH3T3, HEK293, 293, A549, HepG2, IMR-90, MCF-7, U-205,
Per.C6, SF9, SF21, or Chinese Hamster Ovary (CHO) cells. In certain
embodiments, the cells are fungal cells, such as cells selected
from the group consisting of: Chrysosporium cells, Aspergillus
cells, Trichoderma cells, Dictyostelium cells, Candida cells,
Saccharomyces cells, Schizosaccharomyces cells, and Penicillium
cells. In certain other embodiments, the cells are bacterial cells,
such as E. coli, B. subtilis, or BL21 cells. Primary and secondary
cells to be transfected by the present method can be obtained from
a variety of tissues and include all cell types which can be
maintained in culture. For example, primary and secondary cells
which can be transfected by the present method include fibroblasts,
keratinocytes, epithelial cells (e.g., mammary epithelial cells,
intestinal epithelial cells), endothelial cells, glial cells,
neural cells, formed elements of the blood (e.g., lymphocytes, bone
marrow cells), muscle cells and precursors of these somatic cell
types. Primary cells can be obtained from a donor of the same
species or another species (e.g., mouse, rat, rabbit, cat, dog,
pig, cow, bird, sheep, goat, horse).
[0043] The cells of the present disclosure are useful for in vitro
production of therapeutic products which can be purified and
delivered by conventional routes of administration. With or without
amplification, these cells can be subject to large-scale
cultivation for harvest of intracellular or extracellular protein
products.
Methods of Cellular Nucleic Acid Delivery.
[0044] Methods of the present disclosure enhance nucleic acid
delivery into a cell population, in vivo, ex vivo, or in culture.
For example, a cell culture containing a plurality of host cells
(e.g., eukaryotic cells such as yeast or mammalian cells) is
contacted with a composition that contains an enhanced nucleic acid
having at least one nucleoside modification and, optionally, a
translatable region. The composition also generally contains a
transfection reagent or other compound that increases the
efficiency of enhanced nucleic acid uptake into the host cells. The
enhanced nucleic acid exhibits enhanced retention in the cell
population, relative to a corresponding unmodified nucleic acid.
The retention of the enhanced nucleic acid is greater than the
retention of the unmodified nucleic acid. In some embodiments, it
is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200%, or more
than 200% greater than the retention of the unmodified nucleic
acid. Such retention advantage may be achieved by one round of
transfection with the enhanced nucleic acid, or may be obtained
following repeated rounds of transfection.
Introduction of Modified or Transient RNAs into Cells for Protein
Production.
[0045] Transiently transfected cells may be generated by methods of
transfection, electroporation, cationic agents, polymers, or
lipid-based delivery molecules well known to those of ordinary
skill in the art. The modified transient RNAs can be introduced
into the cultured cells in either traditional batch like steps or
continuous flow through steps if appropriate. The methods and
compositions of the present disclosure may be used to produce cells
with increased production of one or more protein of interest. Cells
can be transfected or otherwise introduced with one or more RNA.
The cells may be transfected with the two or more RNA constructs
simultaneously or sequentially. In certain embodiments, multiple
rounds of the methods described herein may be used to obtain cells
with increased expression of one or more RNAs or proteins of
interest. For example, cells may be transfected with one or more
RNA constructs that encode an RNA or protein of interest and
isolated according to the methods described herein. The isolated
cells may then be subjected to further rounds of transfection with
one or more other RNA that encode an RNA or protein of interest and
isolated once again. This method is useful, for example, for
generating cells with increased expression of a complex of
proteins, RNAs or proteins in the same or related biological
pathway, RNAs or proteins that act upstream or downstream of each
other, RNAs or proteins that have a modulating, activating or
repressing function to each other, RNAs or proteins that are
dependent on each other for function or activity, or RNAs or
proteins that share homology (e.g., sequence, structural, or
functional homology). For example, this method may be used to
generate a cell line with increased expression of the heavy and
light chains of an immunoglobulin protein (e.g., IgA, IgD, IgE,
IgG, and IgM) or antigen-binding fragments thereof. The
immunoglobulin proteins may be fully human, humanized, or chimeric
immunoglobulin proteins. An RNA that is transfected into a cell of
the disclosure may comprise a sequence that is an RNA encoding a
protein of interest. Any protein may be produced according to the
methods described herein. Examples of proteins that may be produced
according the methods of the disclosure include, without
limitation, peptide hormones (e.g., insulin), glycoprotein hormones
(e.g., erythropoietin), antibiotics, cytokines, enzymes, vaccines
(e.g., HIV vaccine, HPV vaccine, HBV vaccine), anticancer
therapeutics (e.g., Muc1), and therapeutic antibodies. In a
particular embodiment the RNA encodes an immunoglobulin protein or
an antigen-binding fragment thereof, such as an immunoglobulin
heavy chain, an immunoglobulin light chain, a single chain Fv, a
fragment of an antibody, such as Fab, Fab', or (Fab').sub.2, or an
antigen binding fragment of an immunoglobulin. In a specific
embodiment, the RNA encodes erythropoietin. In another specific
embodiment, the RNA encodes one or more immunoglobulin proteins, or
fragments thereof, that bind to and, optionally, antagonize or
agonize a cell surface receptor: the epidermal growth factor
receptor (EGFR), HER2, or c-ErbB-1, such as Erbitux.TM.
(cetuximab).
Isolation or Purification of Proteins.
[0046] The methods described herein can further comprise the step
of isolating or purifying the proteins, polypeptides, or peptides
produced by the methods described herein. Those of ordinary skill
in the art can easily make a determination of the proper manner to
purify or isolate the protein of interest from the cultured cells.
Generally, this is done through a capture method using affinity
binding or non-affinity purification. If the protein of interest is
not secreted by the cultured cells, then a lysis of the cultured
cells would be performed prior to purification or isolation as
described above. One can use unclarified cell culture fluid
containing the protein of interest along with cell culture media
components as well as cell culture additives, such as anti-foam
compounds and other nutrients and supplements, cells, cellular
debris, host cell proteins, DNA, viruses and the like in the
present disclosure. Moreover, the process can be conducted, if
desired, in the bioreactor itself. The fluid may either be
preconditioned to a desired stimulus such as pH, temperature or
other stimulus characteristic or the fluid can be conditioned upon
addition of the polymer(s) or the polymer(s) can be added to a
carrier liquid that is properly conditioned to the required
parameter for the stimulus condition required for that polymer to
be solubilized in the fluid. The polymer(s) is allowed to circulate
thoroughly with the fluid and then the stimulus is applied (change
in pH, temperature, salt concentration, etc) and the desired
protein and polymer(s) precipitate out of solution. The polymer and
desired protein(s) is separated from the rest of the fluid and
optionally washed one or more times to remove any trapped or
loosely bound contaminants. The desired protein is then recovered
from the polymer(s) such as by elution and the like. Typically, the
elution is done under a set of conditions such that the polymer
remains in its solid (precipitated) form and retains any impurities
to it during the selective elution of the desired protein.
Alternatively, the polymer and protein as well as any impurities
can be solubilized in a new fluid such as water or a buffered
solution and the protein be recovered by a means such as affinity,
ion exchange, hydrophobic, or some other type of chromatography
that has a preference and selectivity for the protein over that of
the polymer or impurities. The eluted protein i s then recovered
and if desired subjected to additional processing steps, either
traditional batch like steps or continuous flow through steps if
appropriate.
[0047] Additionally, it is useful to optimize the expression of a
specific polypeptide in a cell line or collection of cell lines of
potential interest, particularly an engineered protein such as a
protein variant of a reference protein having a known activity. In
one embodiment, provided is a method of optimizing expression of an
engineered protein in a target cell, by providing a plurality of
target cell types, and independently contacting with each of the
plurality of target cell types a modified mRNA encoding an
engineered polypeptide. Additionally, culture conditions may be
altered to increase protein production efficiency. Subsequently,
the presence and/or level of the engineered polypeptide in the
plurality of target cell types is detected and/or quantitated,
allowing for the optimization of an engineered polypeptide's
expression by selection of an efficient target cell and cell
culture conditions relating thereto. Such methods are particularly
useful when the engineered polypeptide contains one or more
post-translational modifications or has substantial tertiary
structure, situations which often complicate efficient protein
production.
[0048] "Proteins of interest" or "desired proteins" include those
provided herein and fragments, mutants, variants, and alterations
thereof. Especially, desired proteins/polypeptides or proteins of
interest are for example, but not limited to insulin, insulin-like
growth factor, human growth hormone (hGH), tissue plasminogen
activator (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,
TNF-related apoptosis-inducing ligand (TRAIL); granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), macrophage colony-stimulating
factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and
vascular endothelial growth factor (VEGF). Also included is the
production of erythropoietin or any other hormone growth factors.
The method according to the disclosure can also be advantageously
used for production of antibodies or fragments thereof. Such
fragments include e.g., Fab fragments (Fragment antigen-binding).
Fab fragments consist of the variable regions of both chains which
are held together by the adjacent constant region. These may be
formed by protease digestion, e.g., with papain, from conventional
antibodies, but similar Fab fragments may also be produced in the
mean time by genetic engineering. Further antibody fragments
include F(ab')2 fragments, which may be prepared by proteolytic
cleaving with pepsin.
[0049] The protein of interest is typically recovered from the
culture medium as a secreted polypeptide, or it can be recovered
from host cell lysates if expressed without a secretory signal. It
is necessary to purify the protein of interest from other
recombinant proteins and host cell proteins in a way that
substantially homogenous preparations of the protein of interest
are obtained. As a first step, cells and/or particulate cell debris
are removed from the culture medium or lysate. The product of
interest thereafter is purified from contaminant soluble proteins,
polypeptides and nucleic acids, for example, by fractionation on
immunoaffinity or ion-exchange columns, ethanol precipitation,
reverse phase HPLC, Sephadex chromatography, chromatography on
silica or on a cation exchange resin such as DEAE. In general,
methods teaching a skilled person how to purify a protein
heterologous expressed by host cells, are well known in the art.
Such methods are for example described by (Harris and Angal,
Protein Purification Methods: A Practical Approach, Oxford
University Press, 1995) or (Robert Scopes, Protein Purification:
Principles and Practice, Springer, 1988).
[0050] Methods of the present disclosure enhance nucleic acid
delivery into a cell population, in vivo, ex vivo, or in culture.
For example, a cell culture containing a plurality of host cells
(e.g., eukaryotic cells such as yeast or mammalian cells) is
contacted with a composition that contains an enhanced nucleic acid
having at least one nucleoside modification and, optionally, a
translatable region. The composition also generally contains a
transfection reagent or other compound that increases the
efficiency of enhanced nucleic acid uptake into the host cells. The
enhanced nucleic acid exhibits enhanced retention in the cell
population, relative to a corresponding unmodified nucleic acid.
The retention of the enhanced nucleic acid is greater than the
retention of the unmodified nucleic acid. In some embodiments, it
is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200%, or more
than 200% greater than the retention of the unmodified nucleic
acid. Such retention advantage may be achieved by one round of
transfection with the enhanced nucleic acid, or may be obtained
following repeated rounds of transfection.
[0051] In some embodiments, the enhanced nucleic acid is delivered
to a target cell population with one or more additional nucleic
acids. Such delivery may be at the same time, or the enhanced
nucleic acid is delivered prior to delivery of the one or more
additional nucleic acids. The additional one or more nucleic acids
may be modified nucleic acids or unmodified nucleic acids. It is
understood that the initial presence of the enhanced nucleic acids
does not substantially induce an innate immune response of the cell
population and, moreover, that the innate immune response will not
be activated by the later presence of the unmodified nucleic acids.
In this regard, the enhanced nucleic acid may not itself contain a
translatable region, if the protein desired to be present in the
target cell population is translated from the unmodified nucleic
acids.
Antagonist Protein Expression.
[0052] Methods and compositions described herein can be used to
produced proteins that are capable of attenuating or blocking the
endogenous agonist biological response and/or antagonizing a
receptor or signaling molecule in a mammalian subject. For example,
IL-12 and IL-23 receptor signaling is enhanced in chronic
autoimmune disorders such as multiple sclerosis and inflammatory
diseases such as rheumatoid arthritis, psoriasis, lupus
erythematosus, ankylosing spondylitis and Crohn's disease (Kikly K,
Liu L, Na S, Sedgwick JD (2006) Curr. Opin. Immunol. 18 (6):
670-5). In another embodiment, a nucleic acid encodes an antagonist
for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5 are
required for HIV entry into host cells (Arenzana-Seisdedos F et al,
(1996) Nature. October 3; 383 (6599):400).
[0053] Targeting Moieties. In embodiments of the disclosure,
modified nucleic acids are provided to express a protein-binding
partner or a receptor on the surface of the cell, which functions
to target the cell to a specific tissue space or to interact with a
specific moiety, either in vivo or in vitro. Suitable
protein-binding partners include antibodies and functional
fragments thereof, scaffold proteins, or peptides. Additionally,
modified nucleic acids can be employed to direct the synthesis and
extracellular localization of lipids, carbohydrates, or other
biological moieties.
[0054] Permanent Gene Expression Silencing. A method for
epigenetically silencing gene expression in a mammalian subject,
comprising a nucleic acid where the translatable region encodes a
polypeptide or polypeptides capable of directing sequence-specific
histone H3 methylation to initiate heterochromatin formation and
reduce gene transcription around specific genes for the purpose of
silencing the gene. For example, a gain-of-function mutation in the
Janus Kinase 2 gene is responsible for the family of
Myeloproliferative Diseases.
[0055] Mechanism details. Fission yeast require two RNAi complexes
for siRNA-mediated heterochromatin assembly: the RNA-induced
transcriptional silencing (RITS) complex and the RNA-directed RNA
polymerase complex (RDRC) (Motamedi et al. Cell 2004, 119,
789-802). In fission yeast, the RITS complex contains the siRNA
binding Argonaute family protein Ago1, a chromodomain protein Chp1,
and Tas3. The fission yeast RDRC complex is composed of an
RNA-dependent RNA Polymerase Rdp1, a putative RNA helicase Hrr1,
and a polyA polymerase family protein Cid12. These two complexes
require the Dicer ribonuclease and Clr4 histone H3
methyltransferase for activity. Together, Ago1 binds siRNA
molecules generated through Dicer-mediated cleavage of Rdp1
co-transcriptionally generated dsRNA transcripts and allows for the
sequence-specific direct association of Chp1, Tas3, Hrr1, and Clr4
to regions of DNA destined for methylation and histone modification
and subsequent compaction into transcriptionally silenced
heterochromatin. While this mechanism functions in cis-with
centromeric regions of DNA, sequence-specific trans silencing is
possible through co-transfection with double-stranded siRNAs for
specific region s of DNA and concomitant RNAi-directed silencing of
the siRNA ribonuclease Eli1 (Buhler et al. Cell 2006, 125,
873-886).
Production of Polypeptide Variants.
[0056] Methods and compositions described herein can be used for
production of polypeptide variants. Provided herein are nucleic
acids that encode variant polypeptides, which have a certain
identity with a reference polypeptide sequence. 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,
Prut 1, Griffin, A. M., and Gtiffin, 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
Calillo et al., SIAM J. Applied Math. 48, 1073 (1988).
[0057] In some embodiments, the polypeptide variant has the same or
a similar activity as the reference polypeptide. Alternatively, the
variant has an altered activity (e.g., increased or decreased)
relative to a reference polypeptide. Generally, variants of a
particular polynucleotide or polypeptide of the disclosure 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% or more
sequence identity to that particular reference polynucleotide or
polypeptide as determined by sequence alignment programs and
parameters described herein and known to those skilled in the
art.
[0058] 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 this disclosure. For
example, provided herein is any protein fragment of a reference
protein (meaning a polypeptide sequence at least one amino acid
residue shorter than a reference polypeptide sequence but otherwise
identical) about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or
greater than 100 amino acids in length. In another example, any
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 disclosure. In certain embodiments,
a protein sequence to be utilized in accordance with the disclosure
includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in
any of the sequences provided or referenced herein.
Production of Polypeptide Libraries
[0059] Methods and compositions described herein can be used for
production of polypeptide libraries. Provided herein are
polynucleotide libraries containing nucleoside modifications,
wherein the polynucleotides individually contain a first nucleic
acid sequence encoding a polypeptide, such as an antibody, protein
binding partner, scaffold protein, and other polypeptides known in
the art. Typically, the polynucleotides are mRNA in a form suitable
for direct introduction into a target cell host, which in turn
synthesizes the encoded polypeptide.
[0060] In certain embodiments, multiple variants of a protein, each
with different amino acid modification(s), are produced and tested
to determine the best valiant in terms of pharmacokinetics,
stability, biocompatibility, and/or biological activity, or a
biophysical property such as expression level. Such a library may
contain about 10, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, or over 10.sup.9 possible variants
(including substitutions, deletions of one or more residues, and
insertion of one or more residues).
Production of Polypeptide-Nucleic Acid Complexes
[0061] Methods and compositions described herein can be used for
production of polypeptide-nucleic acid complexes. Proper protein
translation involves the physical aggregation of a number of
polypeptides and nucleic acids associated with the mRNA. Provided
by the disclosure are protein-nucleic acid complexes, containing a
translatable mRNA having one or more nucleoside modifications
(e.g., at least two different nucleoside modifications) and one or
more polypeptides bound to the mRNA. Generally, the proteins are
provided in an amount effective to prevent or reduce an innate
immune response of a cell into which the complex is introduced.
Production of Untranslatable Modified Nucleic Acids.
[0062] Methods and compositions described herein can be used for
production of untranslatable modified nucleic acids. As described
herein, provided are mRNAs having sequences that are substantially
not translatable. Such mRNA is effective as a vaccine when
administered to a mammalian subject.
[0063] Also provided are modified nucleic acids that contain one or
more noncoding regions. Such modified nucleic acids are generally
not translated, but are capable of binding to and sequestering one
or more translational machinery component such as a ribosomal
protein or a transfer RNA (tRNA), thereby effectively reducing
protein expression in the cell. The modified nucleic acid may
contain a small nucleolar RNA (sno-RNA), microRNA (miRNA), small
interfering RNA (siRNA), small hairpin RNA (shRNA), or
Piwi-interacting RNA (piRNA).
Modified Nucleic Acids.
[0064] This disclosure provides methods of producing proteins using
nucleic acids, including RNAs such as messenger RNAs (mRNAs) that
contain one or more modified nucleosides (telmed "modified nucleic
acids"), which have useful properties including the lack of a
substantial induction of the innate immune response of a cell into
which the mRNA is introduced. Because these modified nucleic acids
enhance the efficiency of protein production, intracellular
retention of nucleic acids, and viability of contacted cells, as
well as possess reduced immunogenicity, these nucleic acids having
these properties are termed "enhanced nucleic acids" herein.
[0065] The term "nucleic acid," in its broadest sense, includes any
compound and/or substance that is or can be incorporated into an
oligonucleotide chain. Exemplary nucleic acids for use in
accordance with the present disclosure include, but are not limited
to, one or more of DNA, RNA including messenger mRNA (mRNA),
hybrids thereof, RNA interference (RNAi)-inducing agents, RNAi
agents, small interfering RNAs (siRNAs), small hairpin RNAs
(shRNAs), microRNAs (miRNAs), antisense RNAs, ribozymes, catalytic
DNA, RNAs that induce triple helix formation, aptamers, vectors,
etc., described in detail herein.
[0066] Provided are modified nucleic acids containing a
translatable region and one, two, or more than two different
nucleoside modifications. In some embodiments, the modified nucleic
acid exhibits reduced degradation in a cell into which the nucleic
acid is introduced, relative to a corresponding unmodified nucleic
acid. For example, the degradation rate of the modified nucleic
acid is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, or greater than 90%, compared to the degradation rate of the
corresponding unmodified nucleic acid. Exemplary nucleic acids
include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs),
threose nucleic acids (TNAs), glycol nucleic acids (GNAs), or a
hybrid thereof. In typical embodiments, the modified nucleic acid
includes messenger RNAs (mRNAs). As described herein, the nucleic
acids of the disclosure do not substantially induce an innate
immune response of a cell into which the mRNA is introduced.
[0067] In some embodiments, modified nucleosides include
pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,
2-thiomidine, 4-thio-pseudomidine, 2-thio-pseudowidine,
5-hydroxyuridine, 3-methylmidine, 5-carboxymethyl-uridine,
1-carboxymethyl-pseudoutidine, 5-propynyl-uridine,
1-propynyl-pseudomidine, 5-taurinomethyluridine,
1-taurinomethyl-pseudouridine, 5-taw.inomethyl-2-thio-utidine,
1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,
1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,
2-thio-1-methyl-pseudoutidine, 1-methyl-1-deaza-pseudomidine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydromidine,
2-thio-dihydropseudoulidine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudomidine, and
4-methoxy-2-thio-pseudouridine.
[0068] In some embodiments, modified nucleosides include
5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine,
N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine,
5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,
pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-meth yl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebulruine,
5-methyl-zebularine, 5-aza-2-thio-zebulru.ine, 2-thio-zebulaiine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine.
[0069] In other embodiments, modified nucleosides include
2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine,
7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,
7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,
7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine,
N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine.
[0070] In specific embodiments, a modified nucleoside is
5'-O-(1-Thiophosphate)-Adenosine, 5'-O-(1-Thiophosphate)-Cytidine,
5'-O-(1-thiophosphate)-Guanosine, 5'-O-(1-Thiophosphate)-Uridine or
5'-O-(1-Thiophosphate)-Pseudouridine.
##STR00001##
[0071] 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
nucleic acids are expected to also reduce the innate immune
response through weaker binding/activation of cellular innate
immune molecules.
[0072] In certain embodiments it is desirable to intracellularly
degrade a modified nucleic acid introduced into the cell, for
example if precise timing of protein production is desired. Thus,
the disclosure provides a modified nucleic acid containing a
degradation domain, which is capable of being acted on in a
directed manner within a cell.
[0073] In other embodiments, modified nucleosides include inosine,
1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,
7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-meth
yl-8-oxo-guanosine, J-methyl-6-thio-guanosine,
N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine.
[0074] Other components of nucleic acid are optional, and are
beneficial in some embodiments. For example, a 5' untranslated
region (UTR) and/or a 3'UTR are provided, wherein either or both
may independently contain one or more different nucleoside
modifications. In such embodiments, nucleoside modifications may
also be present in the translatable region. Also provided are
nucleic acids containing a Kozak sequence.
[0075] Additionally, provided are nucleic acids containing one or
more intronic nucleotide sequences capable of being excised from
the nucleic acid.
[0076] Further, provided are nucleic acids containing an internal
ribosome entry site (IRES). An IRES may act as the sole ribosome
binding site, or may serve as one of multiple ribosome binding
sites of an mRNA. An mRNA containing more than one functional
ribosome binding site may encode several peptides or polypeptides
that are translated independently by the ribosomes ("multicistronic
mRNA"). When nucleic acids are provided with an IRES, further
optionally provided is a second translatable region. Examples of
IRES sequences that can be used according to the disclosure 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 (STY) or cricket paralysis
viruses (CrPV).
Prevention or Reduction of Innate Cellular Immune Response
Activation Using Modified Nucleic Acids.
[0077] The modified nucleic acids described herein are capable of
evading an innate immune response of a cell into which the nucleic
acids are introduced, thus increasing the efficiency of protein
production in the cell. The term "innate immune response" includes
a cellular response to exogenous single stranded nucleic acids,
generally of viral or bacterial origin, which involves the
induction of cytokine expression and release, particularly the
interferons, and cell death. Protein synthesis is also reduced
during the innate cellular immune response. While it is
advantageous to eliminate the innate immune response in a cell, the
disclosure provides modified mRNAs that substantially reduce the
immune response, including interferon signaling, without entirely
eliminating such a response. In some embodiments, the immune
response is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 99%, 99.9%, or greater than 99.9%, as compared to
the immune response induced by a corresponding unmodified nucleic
acid. Such a reduction can be measured by expression or activity
level of Type 1 interferons or the expression of
interferon-regulated genes such as the toll-like receptors (e.g.,
TLR7 and TLR8). Reduction of innate immune response can also be
measured by decreased cell death following one or more
administration s of modified RNA s to a cell population; e.g., cell
death is about 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less
than the cell death frequency observed with a corresponding
unmodified nucleic acid. Moreover, cell death may affect fewer than
about 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, or fewer than
0.01% of cells contacted with the modified nucleic acids.
[0078] The disclosure provides for the repeated introduction (e.g.,
transfection) of modified nucleic acids into a target cell
population, e.g., in vitro, ex vivo, or in vivo. The step of
contacting the cell population may be repeated one or more times
(such as two, three, four, five, or more than five times). In some
embodiments, the step of contacting the cell population with the
modified nucleic acids is repeated a number of times sufficient
such that a predetermined efficiency of protein translation in the
cell population is achieved. Given the reduced cytotoxicity of the
target cell population provided by the nucleic acid modifications,
such repeated transfections are achievable in a diverse array of
cell types.
Modified Nucleic Acid Synthesis.
[0079] Nucleic acids for use in accordance with the disclosure may
be prepared according to any available technique including, but not
limited to chemical synthesis, enzymatic synthesis, which is
generally termed in vitro transcription, 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).
[0080] Modified nucleic acids need not be uniformly modified along
the entire length of the molecule. Different nucleotide
modifications and/or backbone structures may exist at various
positions in the nucleic acid. One of ordinary skill in the alt
will appreciate that the nucleotide analogs or other
modification(s) may be located at any position(s) of a nucleic acid
such that the function of the nucleic acid is not substantially
decreased. A modification may also be a 5' or 3' terminal
modification. The nucleic acids may contain at a minimum one and at
maximum 100% modified nucleotides, or any intervening percentage,
such as at least about 50% modified nucleotides, at least about 80%
modified nucleotides, or at least about 90% modified
nucleotides.
[0081] Generally, the length of a modified mRNA of the present
disclosure is suitable for protein, polypeptide, or peptide
production in a cell (e.g., a human cell). For example, the mRNA is
of a length sufficient to allow translation of at least a dipeptide
in a cell. In one embodiment, the length of the modified mRNA is
greater than 30 nucleotides. In another embodiment, the length is
greater than 35 nucleotides. In another embodiment, the length is
at least 40 nucleotides. In another embodiment, the length is at
least 45 nucleotides. In another embodiment, the length is at least
55 nucleotides. In another embodiment, the length is at least 60
nucleotides. In another embodiment, the length is at least 60
nucleotides. In another embodiment, the length is at least 80
nucleotides. In another embodiment, the length is at least 90
nucleotides. In another embodiment, the length is at least 100
nucleotides. In another embodiment, the length is at least 120
nucleotides. In another embodiment, the length is at least 140
nucleotides. In another embodiment, the length is at least 160
nucleotides. In another embodiment, the length is at least 180
nucleotides. In another embodiment, the length is at least 200
nucleotides. In another embodiment, the length is at least 250
nucleotides. In another embodiment, the length is at least 300
nucleotides. In another embodiment, the length is at least 350
nucleotides. In another embodiment, the length is at least 400
nucleotides. In another embodiment, the length is at least 450
nucleotides. In another embodiment, the length is at least 500
nucleotides. In another embodiment, the length is at least 600
nucleotides. In another embodiment, the length is at least 700
nucleotides. In another embodiment, the length is at least 800
nucleotides. In another embodiment, the length is at least 900
nucleotides. In another embodiment, the length is at least 1000
nucleotides. In another embodiment, the length is at least 1100
nucleotides. In another embodiment, the length is at least 1200
nucleotides. In another embodiment, the length is at least 1300
nucleotides. In another embodiment, the length is at least 1400
nucleotides. In another embodiment, the length is at least 1500
nucleotides. In another embodiment, the length is at least 1600
nucleotides. In another embodiment, the length is at least 1800
nucleotides. In another embodiment, the length is at least 2000
nucleotides. In another embodiment, the length is at least 2500
nucleotides. In another embodiment, the length is at least 3000
nucleotides. In another embodiment, the length is at least 4000
nucleotides. In another embodiment, the length is at least 5000
nucleotides, or greater than 5000 nucleotides.
Uses of Modified Nucleic Acids.
[0082] The proteins, polypeptides, or peptides produced by the
methods described herein can be used as therapeutic agents to treat
or prevent one or more diseases or conditions described herein.
[0083] Therapeutic Agents. Provided are compositions, methods,
kits, and reagents for treatment or prevention of disease or
conditions in humans and other animals (e.g., mammals). The active
therapeutic agents of the disclosure include polypeptides
translated from modified nucleic acids, cells containing modified
nucleic acids or polypeptides translated from the modified nucleic
acids, and cells contacted with cells containing modified nucleic
acids or polypeptides translated from the modified nucleic
acids.
[0084] Provided are methods of inducing translation of a
recombinant polypeptide in a cell population using the modified
nucleic acids 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 a nucleic acid
that has at least one nucleoside modification, and a translatable
region encoding the recombinant polypeptide. The population is
contacted under conditions such that the nucleic acid is localized
into one or more cells of the cell population and the recombinant
polypeptide is translated in the cell from the nucleic acid.
[0085] 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 protein translated
from the modified nucleic acid (e.g., size), and other
determinants.
[0086] Compositions containing modified nucleic acids are
formulated for administration intramuscularly, transarterially,
intraperitoneally, intravenously, intranasally, subcutaneously,
endoscopically, transdermally, or intrathecally. In some
embodiments, the composition is formulated for extended
release.
[0087] The subject to whom the therapeutic agent is administered
suffers from or is at risk of developing a disease, disorder, or
deleterious condition. Provided are methods of identifying,
diagnosing, and classifying subjects on these bases, which may
include clinical diagnosis, biomarker levels, genome-wide
association studies (GWAS), and other methods known in the art.
[0088] In certain embodiments, the administered recombinant
polypeptide translated from the modified nucleic acid described
herein provide a functional activity which is substantially absent
in the cell in which the recombinant polypeptide is administered.
For example, the missing functional activity may be enzymatic,
structural, or gene regulatory in nature.
[0089] In other embodiments, the administered recombinant
polypeptide replaces a polypeptide (or multiple polypeptides) that
is substantially absent in the cell in which the recombinant
polypeptide is administered. Such absence may be due to genetic
mutation of the encoding gene or regulatory pathway thereof.
Alternatively, the recombinant 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 protein is deleterious to the subject, for example, due
to mutation of the endogenous protein resulting in altered activity
or localization. Additionally, the recombinant 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, or a small molecule toxin.
[0090] The recombinant proteins described herein are 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.
[0091] As described herein, a useful feature of the modified
nucleic acids of the disclosure is the capacity to reduce the
innate immune response of a cell to an exogenous nucleic acid,
e.g., to increase protein production. Provided are methods for
performing the titration, reduction or elimination of the immune
response in a cell or a population of cells. In some embodiments,
the cell is contacted with a first composition that contains a
first dose of a first exogenous nucleic acid including a
translatable region and at least one nucleoside modification, and
the level of the innate immune response of the cell to the first
exogenous nucleic acid is determined. Subsequently, the cell is
contacted with a second composition, which includes a second dose
of the first exogenous nucleic acid, the second dose containing a
lesser amount of the first exogenous nucleic acid as compared to
the first dose. Alternatively, the cell is contacted with a first
dose of a second exogenous nucleic acid. The second exogenous
nucleic acid may contain one or more modified nucleosides, which
may be the same or different from the first exogenous nucleic acid
or, alternatively, the second exogenous nucleic acid may not
contain modified nucleosides. The steps of contacting the cell with
the first composition and/or the second composition may be repeated
one or more times. Additionally, efficiency of protein production
(e.g., protein translation) i n the cell is optionally determined,
and the cell may be re-transfected with the first and/or second
composition repeatedly until a target protein production efficiency
is achieved.
[0092] Therapeutics for diseases and conditions. Provided are
methods for treating or preventing a symptom of diseases
characterized by missing or aberrant protein activity, by replacing
the missing protein activity or overcoming the aberrant protein
activity.
[0093] Diseases characterized by dysfunctional or aberrant protein
activity include, but not limited to, cancer and proliferative
diseases, genetic diseases (e.g., cystic fibrosis), autoimmune
diseases, diabetes, neurodegenerative diseases, cardiovascular
diseases, and metabolic diseases. The present disclosure provides a
method for treating such conditions or diseases in a subject by
introducing protein or cell-based therapeutics produced by a method
using the modified nucleic acids provided herein, wherein the
modified nucleic acids encode for a protein that antagonizes or
otherwise overcomes the aberrant protein activity present in the
cell of the subject. Specific examples of a dysfunctional protein
are the missense mutation variants of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene, which produce a
dysfunctional protein variant of CFTR protein, which causes cystic
fibrosis.
[0094] Multiple diseases are characterized by missing (or
substantially diminished such that proper protein function does not
occur) protein activity. Such proteins may not be present, or are
essentially non-functional. The present disclosure provides a
method for treating such conditions or diseases in a subject by
introducing nucleic acid or cell-based therapeutics containing the
modified nucleic acids provided herein, wherein the modified
nucleic acids encode for a protein that replaces the protein
activity missing from the target cells of the subject. Specific
examples of a dysfunctional protein are the nonsense mutation
variants of the cystic fibrosis transmembrane conductance regulator
(CFTR) gene, which produce a nonfunctional protein valiant of CFTR
protein, which causes cystic fibrosis.
[0095] Thus, provided are methods of treating cystic fibrosis in a
mammalian subject by contacting a cell of the subject with a
modified nucleic acid having a translatable region that encodes a
functional CFTR polypeptide, under conditions such that an
effective amount of the CFTR polypeptide is present in the cell.
Typical target cells are epithelial cells, such as the lung, and
methods of administration are determined in view of the target
tissue; i.e., for lung delivery, the RNA molecules are formulated
for administration by inhalation.
[0096] In another embodiment, the present disclosure provides a
method for treating hyperlipidemia in a subject, by introducing
into a cell population of the subject with Sortilin (a protein
recently characterized by genomic studies) produced by a method
described herein using a modified mRNA molecule encoding Sortilin,
thereby ameliorating the hyperlipidemia in a subject. The SORT1
gene encodes a trans-Golgi network (TGN) transmembrane protein
called Sortilin. Genetic studies have shown that one of five
individuals has a single nucleotide polymorphism, rs12740374, in
the 1p13 locus of the SORT1 gene that predisposes them to having
low levels of low-density lipoprotein (LDL) and very-low-density
lipoprotein (VLDL). Each copy of the minor allele, present in about
30% of people, alters LDL cholesterol by 8 mg/dL, while two copies
of the minor allele, present in about 5% of the population, lowers
LDL cholesterol 16 mg/dL. Carriers of the minor allele have also
been shown to have a 40% decreased risk of myocardial
infarction.
[0097] Functional in vivo studies in mice describes that
overexpression of SORT1 in mouse liver tissue led to significantly
lower LDL-cholesterol levels, as much as 80% lower, and that
silencing SORT1 increased LDL cholesterol approximately 200%
(Musunuru K. et al. From noncoding variant to phenotype via SORT1
at the 1p13 cholesterol locus. Nature 2010; 466: 714-721).
Pharmaceutical Compositions
[0098] The present disclosure provides proteins generated from
modified mRNAs and proteins produced by the methods described
herein can be used in pharmaceutical compositions. Pharmaceutical
compositions may optionally comprise one or more additional
therapeutically active substances. In accordance with some
embodiments, a method of administering pharmaceutical compositions
comprising one or more proteins to be delivered to a subject in
need thereof is provided. In some embodiments, compositions are
administered to humans. For the purposes of the present disclosure,
the phrase "active ingredient" generally refers to a protein or
protein-containing complex as described herein.
[0099] 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 animals of all sorts.
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 chickens, ducks, geese, and/or turkeys.
[0100] 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, shaping and/or packaging
the product into a desired single- or multi-dose unit.
[0101] A pharmaceutical composition in accordance with the
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" 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.
[0102] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
disclosure will vary, depending upon the identity, size, and/or
condition of the subject heated 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% (w/w)
active ingredient.
[0103] Pharmaceutical compositions may be formulated to
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.51
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 disclosure.
[0104] 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.
[0105] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inlet 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. 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.
[0106] 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.
[0107] 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), sodium lauryl sulfate, quaternary ammonium compounds,
etc., and/or combinations thereof.
[0108] 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 [Tween.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.RTM.30]), poly(vinyl-pyrrolidone), diethylene
glycol monolaurate, triethanolamine oleate, sodium oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium
lauryl sulfate, Pluronic.RTM. F 68, Poloxamer.RTM.188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, etc. and/or combinations thereof.
[0109] 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.
[0110] 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,
ascorbyl 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, editic acid,
fumaric acid, malic acid, phosphoric acid, sodium edetate, trellis
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..
[0111] 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, o-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.
[0112] 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.
[0113] Exemplary oils include, but are not limited to, almond,
apricot kernel, avocado, babassu, bergamot, black current seed,
borage, cade, chamomile, 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 mylistate, 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.
[0114] 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.
[0115] 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.
[0116] Injectable compositions 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.
[0117] 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 compositions are formulated or prepared by entrapping
the drug in liposomes or microemulsions which are compatible with
body tissues.
[0118] 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.
[0119] 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.
[0120] 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. 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 rut. 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.
[0121] Dosage forms for topical and/or transdermal administration
of a composition may include ointments, pastes, creams, lotions,
gels, 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 disclosure 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 ruld/or gel.
[0122] 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.
[0123] Compositions formulated 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. Topically-administrable compositions may be
formulated, for example, to comprise from about 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. Compositions formulated for topical
administration may further comprise one or more of the additional
ingredients described herein.
[0124] A pharmaceutical composition may be formulated, prepared,
packaged, and/or sold for pulmonary administration via the buccal
cavity. Such a composition may be formulated to 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 conveniently 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.
[0125] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute about 50% to
about 99.9% (w/w) of the composition, and active ingredient may
constitute about 0.1% to about 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).
[0126] Pharmaceutical compositions formulated for pulmonary
delivery may provide an active ingredient in the form of droplets
of a solution and/or suspension. Such compositions may be
formulated, 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 an y nebulization and/or atomization device. Such
compositions 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.
[0127] Formulations described herein as being useful for pulmonary
delivery rule useful for intranasal delivery of a pharmaceutical
composition. Another composition formulated 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 composition is formulated for administration 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.
[0128] Compositions formulated 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 formulated, prepared, packaged, and/or sold for
buccal administration. Such compositions may, for example, be
formulated in the form of tablets and/or lozenges made using
conventional methods, and may contain, 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,
compositions formulated 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 compositions, 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.
[0129] A pharmaceutical composition may be formulated, prepared,
packaged, and/or sold for ophthalmic administration. Such
compositions may, for example, be formulated in the form of eye
drops including, for example, a 0.111.0% (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 opthalmically-administrable compositions 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
disclosure.
[0130] General considerations in the formulation and/or manufacture
of pharmaceutical agents may be found, for example, in Remington:
The Science and Practice of Pharmacy 21st ed., Lippincott Williams
& Wilkins, 2005 (incorporated herein by reference).
Administration.
[0131] The present disclosure provides methods comprising
administering proteins or compositions produced by the methods
described herein to a subject in need thereof. 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
disclosure are typically formulated in dosage unit from for ease of
administration and uniformity of dosage. It will be understood,
however, that the total daily usage of the compositions of the
present disclosure will 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.
[0132] Proteins to be delivered and/or pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof may be
administered to animals, such as mammals (e.g., humans,
domesticated animals, cats, dogs, mice, rats, etc.). In some
embodiments, pharmaceutical, prophylactic, diagnostic, or imaging
compositions thereof are administered to humans.
[0133] Proteins to be delivered and/or pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof in
accordance with the present disclosure may be administered by any
route. In some embodiments, proteins and/or pharmaceutical,
prophylactic, diagnostic, or imaging compositions thereof, are
administered by one or more of a variety of routes, including oral,
intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, subcutaneous, intraventricular, transdermal,
interdermal, rectal, intravaginal, intraperitoneal, topical (e.g.,
by powders, ointments, creams, gels, lotions, and/or drops),
mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual;
by intratracheal instillation, bronchial instillation, and/or
inhalation; as an oral spray, nasal spray, and/or aerosol, and/or
through a portal vein catheter. In some embodiments, proteins or
complexes, and/or pharmaceutical, prophylactic, diagnostic, or
imaging compositions thereof, are administered by systemic
intravenous injection. In specific embodiments, proteins or
complexes and/or pharmaceutical, prophylactic, diagnostic, or
imaging compositions thereof may be administered intravenously
and/or orally. In specific embodiments, proteins or complexes,
and/or pharmaceutical, prophylactic, diagnostic, or imaging
compositions thereof, may be administered in a way which allows the
protein or complex to cross the blood-brain barrier, vascular
barrier, or other epithelial barrier.
[0134] However, the disclosure encompasses the delivery of proteins
or complexes, and/or pharmaceutical, prophylactic, diagnostic, or
imaging compositions thereof, by any appropriate route taking into
consideration likely advances in the sciences of drug delivery.
[0135] In general the most appropriate route of administration will
depend upon a variety of factors including the nature of the
protein or complex comprising proteins associated with at least one
agent to be delivered (e.g., its stability in the environment of
the gastrointestinal tract, bloodstream, etc.), the condition of
the patient (e.g., whether the patient is able to tolerate
particular routes of administration), etc. The disclosure
encompasses the delivery of the pharmaceutical, prophylactic,
diagnostic, or imaging compositions by any appropriate route taking
into consideration likely advances in the sciences of drug
delivery.
[0136] In certain embodiments, compositions in accordance with the
disclosure may be administered at dosage levels sufficient to
deliver from about 0.0001 mg/kg to about 100 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).
[0137] Proteins or complexes 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 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 disclosure
encompasses the delivery of pharmaceutical, prophylactic,
diagnostic, or imaging compositions in combination with agents that
improve their bioavailability, reduce and/or modify their
metabolism, inhibit their excretion, and/or modify their
distribution within the body. In certain embodiments, provided are
combination therapeutics containing one or more modified nucleic
acids containing translatable regions that encode for a protein or
protein s that boost a mammalian subject's immunity along with a
protein that induces antibody-dependent cellular toxicity. For
example, provided are therapeutics containing one or more nucleic
acids that encode trastuzumab and granulocyte-colony stimulating
factor (G-CSF). In particular, such combination therapeutics are
useful in Her2+ breast cancer patients who develop induced
resistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy.
2(6):795-8 (2010)).
[0138] It will further be appreciated that therapeutically,
prophylactically, diagnostically, or imaging active agents utilized
in combination may be administered together in a single composition
or administered separately in different compositions. In general,
it is expected that agents utilized in combination with be utilized
at levels that do not exceed the levels at which they are utilized
individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually.
[0139] The particular combination of therapies (therapeutics or
procedures) to employ in a combination regimen will take into
account compatibility of the desired therapeutics and/or procedures
and the desired therapeutic effect to be achieved. It will also be
appreciated that the therapies employed may achieve a desired
effect for the same disorder (for example, a composition useful for
treating cancer in accordance with the disclosure may be
administered concurrently with a chemotherapeutic agent), or they
may achieve different effects (e.g., control of any adverse
effects).
Kits.
[0140] The disclosure provides a variety of kits for conveniently
and/or effectively carrying out methods of the present disclosure.
For example, described herein are kits for protein production using
a modified nucleic acid described herein. 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.
Definitions
[0141] 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.
[0142] 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.
[0143] 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).
[0144] 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.
[0145] 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, where a nucleic
acid is biologically active, a portion of that nucleic acid that
shares at least one biological activity of the whole nucleic acid
is typically referred to as a "biologically active" portion.
[0146] Conserved: As used herein, the term "conserved" refers to
nucleotides or amino acid residues of a polynucleotide sequence or
amino acid sequence, respectively, that are those that
occur-unaltered in the same position of two or more related
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. 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.
[0147] 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.
[0148] Ex vivo: As used herein, "ex vivo" refers to events that
which occur outside an organism, e.g., in or on tissue in an
artificial environment outside the organism, e.g., with the minimum
alteration of natural conditions.
[0149] 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.
[0150] 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%, 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%, or at least 99%
identical. In some embodiments, polymeric molecules are considered
to be "homologous" to one another if their sequences are 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%, or at least 99% similar. The term "homologous" necessarily
refers to a comparison between at least two sequences (nucleotides
sequences or amino acid sequences). In accordance with the
disclosure, two nucleotide sequences are considered to be
homologous if the polypeptides they encode are at least about 50%
identical, at least about 60% identical, at least about 70%
identical, at least about 80% identical, or at least about 90%
identical for at least one stretch of at least about 20 amino
acids. In some embodiments, homologous nucleotide sequences are
characterized by the ability to encode a stretch of at least 4-5
uniquely specified amino acids. Both the identity and the
approximate spacing of these amino acids relative to one another
must be considered for nucleotide sequences to be considered
homologous. For nucleotide 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 disclosure, two protein sequences are considered to be
homologous if the proteins are at least about 50% identical, at
least about 60% identical, at least about 70% identical, at least
about 80% identical, or at least about 90% identical for at least
one stretch of at least about 20 amino acids.
[0151] Identity: As used herein, the term "identity" 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. Calculation of the percent
identity of two nucleic acid 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,
alliteratively, 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
Atschul, S. F. et al., J MoZee. Bioi., 215, 403 (1990)).
[0152] 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.
[0153] 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).
[0154] In vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant, or
microbe).
[0155] Isolated: As used herein, the term "isolated" refers to a
substance or entity that has been (1) separated from at least some
of the components with which it was associated when initially
produced (whether in nature or in an experimental setting), and/or
(2) produced, prepared, and/or manufactured by the hand of man.
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.
[0156] Preventing: As used herein, the term "preventing" refers to
partially or completely delaying onset of a particular disease,
disorder, and/or condition; partially or completely delaying onset
of one or more symptoms, features, or clinical manifestations of a
particular disease, disorder, and/or condition (e.g., prior to an
identifiable disease, disorder, and/or condition); partially or
completely delaying progression from a latent disease, disorder,
and/or condition to an active disease, disorder, and/or condition;
and/or decreasing the risk of developing pathology associated with
a particular disease, disorder, and/or condition.
[0157] Similarity: As used herein, the term "similarity" 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. 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 rut.
[0158] Subject: As used herein, the term "subject" or "patient"
refers to any organ ism to which a composition in accordance with
the disclosure 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.
[0159] 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.
[0160] 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.
[0161] 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. 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.
[0162] 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 a
disease, disorder, and/or condition, to treat, improve symptoms of,
diagnose, prevent, and/or delay the onset of the disease, disorder,
and/or condition.
[0163] 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.
[0164] 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,
features, or clinical manifestations of a particular disease,
disorder, and/or condition. For example, "treating" cancer may
refer to inhibiting survival, growth, rule/or spread of a tumor.
Treatment may be administered to a subject who does not exhibit
signs of a disease, disorder, and/or condition (e.g., prior to an
identifiable 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. In some embodiments, treatment comprises delivery of a
protein associated with a therapeutically active nucleic acid to a
subject in need thereof.
[0165] Unmodified: As used herein, "unmodified" refers to a nucleic
acid prior to being modified.
EXAMPLES
Example 1: Synthesis of Modified mRNA
[0166] Modified mRNAs (modRNAs) according to the invention were
made using standard laboratory methods and materials. The open
reading frame (ORF) of the gene of interest is flanked by a 5'
untranslated region (UTR) containing a strong Kozak translational
initiation signal and an alpha-globin 3' UTR terminating with an
oligo(dT) sequence for templated addition of a polyA tail. The
modRNAs were modified with pseudouridine (.psi.) and
5-methyl-cytidine (5meC) to reduce the cellular innate immune
response. 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).
[0167] The cloning, gene synthesis and vector sequencing was
performed by DNA2.0 Inc. (Menlo Park, Calif.). Vector sequences and
insert sequences are set forth in SEQ ID NOs: 5-8. The ORFs were
restriction digested using XbaI or HindIII and used for cDNA
synthesis using tailed-PCR. This tailed-PCR cDNA product was used
as the template for the modified mRNA synthesis reaction using 25
mM each modified nucleotide mix (modified U/C was manufactured by
TriLink Biotech, San Diego, Calif., unmodified A/G was purchased
from Epicenter Biotechnologies, Madison, Wis.) and CellScript
MegaScript.TM. (Epicenter Biotechnologies, Madison, Wis.) complete
mRNA synthesis kit. The in vitro transcription reaction was run for
3-4 hours at 37.degree. C. PCR reaction used HiFi PCR 2.times.
Master Mix.TM. (Kapa Biosystems, Woburn, Mass.). The In vitro
transcribed mRNA product was run on an agarose gel and visualized.
mRNA was purified with Ambion/Applied Biosystems (Austin, Tex.)
MEGAClear RNA.TM. purification kit PCR used PureLink.TM. PCR
purification kit (Invitrogen, Carlsbad, Calif.) or PCR cleanup kit
(Qiagen, Valencia, Calif.). The product was quantified on
Nanodrop.TM. UV Absorbance (ThermoFisher, Waltham, Mass.). Quality,
UV absorbance quality and visualization of the product was
performed on an 1.2% agarose gel. The product was resuspended in TE
buffer.
[0168] Modified RNAs incorporating adenosine analogs were poly (A)
tailed using yeast Poly (A) Polymerase (Affymetrix, Santa Clara,
Calif.). PCR reaction used HiFi PCR 2.times. Master Mix.TM. (Kapa
Biosystems, Woburn, Mass.). Modified RNAs were
post-transcriptionally capped using recombinant Vaccinia Virus
Capping Enzyme (New England BioLabs, Ipswich, Mass.) and a
recombinant 2'-o-methyltransferase (Epicenter Biotechnologies,
Madison, Wis.) to generate the 5'-guanosine Cap1 structure. Cap 2
structure and Cap 3 structure may be generated using additional
2'-o-methyltransferases. The in vitro transcribed mRNA product was
run on an agarose gel and visualized. Modified RNA was purified
with Ambion/Applied Biosystems (Austin, Tex.) MEGAClear RNA.TM.
purification kit. PCR used PureLink.TM. PCR purification kit
(Invitrogen, Carlsbad, Calif.). The product was quantified on
Nanodrop.TM. UV Absorbance (ThermoFisher, Waltham, Mass.). Quality,
UV absorbance quality and visualization of the product was
performed on an 1.2% agarose gel. The product was resuspended in TE
buffer.
[0169] Exemplary capping structures. 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; 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 Vims 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 ViJ.us Capping Enzyme and a 2'-0 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'-0 methyl-transferase.
Enzymes are preferably derived from a recombinant source.
[0170] When transfected into mammalian cells, the modified mRNAs
may 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 2: De Novo Generation of a Mammalian Commercial Production
Cell Line Expressing Human G-CSF as a Therapeutic Agent in Model
Bioreactor
[0171] The nucleic acid sequence for the precursor of human
granulocyte colony stimulating factor (G-CSF) is set forth in SEQ
ID NO: 1:
TABLE-US-00001 (SEQ ID NO: 1)
agcttttggaccctcgtacagaagctaatacgactcactatagggaaat
aagagagaaaagaagagtaagaagaaatataagagccaccatggccggt
cccgcgacccaaagccccatgaaacttatggccctgcagttgctgcttt
ggcactcggccctctggacagtccaagaagcgactcctctcggacctgc
ctcatcgttgccgcagtcattccttttgaagtgtctggagcaggtgcga
aagattcagggcgatggagccgcactccaagagaagctctgcgcgacat
acaaactttgccatcccgaggagctcgtactgctcgggcacagcttggg
gattccctgggctcctctctcgtcctgtccgtcgcaggctttgcagttg
gcagggtgcctttcccagctccactccggtttgttcttgtatcagggac
tgctgcaagcccttgagggaatctcgccagaattgggcccgacgctgga
cacgttgcagctcgacgtggcggatttcgcaacaaccatctggcagcag
atggaggaactggggatggcacccgcgctgcagcccacgcagggggcaa
tgccggcctttgcgtccgcgtttcagcgcagggcgggtggagtcctcgt
agcgagccaccttcaatcatttttggaagtctcgtaccgggtgctgaga
catcttgcgcagccgtgaagcgctgccttctgcggggcttgccttctgg
ccatgcccttcttctctcccttgcacctgtacctcttggtattgaataa
agcctgagtaggaaggcggccgctcgagcatgcatctagagggcccaat
tcgccctattcgaagtcg
[0172] The nucleic acid sequence for G-CSF mRNA is set forth in SEQ
ID NO: 2:
TABLE-US-00002 (SEQ ID NO: 2)
agcuuuuggacccucguacagaagcuaauacgacucacuauagggaaau
aagagagaaaagaagaguaagaagaaauauaagagccaccauggccggu
cccgcgacccaaagccccaugaaacuuauggcccugcaguugcugcuuu
ggcacucggcccucuggacaguccaagaagcgacuccucucggaccugc
cucaucguugccgcagucauuccuuuugaagugucuggagcaggugcga
aagauucagggcgauggagccgcacuccaagagaagcucugcgcgacau
acaaacuuugccaucccgaggagcucguacugcucgggcacagcuuggg
gauucccugggcuccucucucguccuguccgucgcaggcuuugcaguug
gcagggugccuuucccagcuccacuccgguuuguucuuguaucagggac
ugcugcaagcccuugagggaaucucgccagaauugggcccgacgcugga
cacguugcagcucgacguggcggauuucgcaacaaccaucuggcagcag
auggaggaacuggggauggcacccgcgcugcagcccacgcagggggcaa
ugccggccuuugcguccgcguuucagcgcagggcggguggaguccucgu
agcgagccaccuucaaucauuuuuggaagucucguaccgggugcugaga
caucuugcgcagccgugaagcgcugccuucugcggggcuugccuucugg
ccaugcccuucuucucucccuugcaccuguaccucuuggucuuugaaua
aagccugaguaggaaggcggccgcucgagcaugcaucuagagggcccaa
uucgcccuauucgaagucg
[0173] The nucleic acid sequence for an exemplary G-CSF modified
mRNA (modRNA) is set forth in SEQ ID NO: 3:
TABLE-US-00003 (SEQ ID NO: 3)
ag5meC.psi..psi..psi..psi.gga5meC5meC5meC.psi.5meCg.psi.a5meCagaag5meC.psi-
.aa.psi.a5meCga5meC.psi.5meCa5me
C.psi.a.psi.agggaaa.psi.aagagagaaaagaagag.psi.aagaagaaa.psi.a.psi.aagag5me-
C5meCa5meC5meCa.psi.gg5meC5me
Cgg.psi.5meC5meC5meCg5meCga5meC5meC5meCaaag5meC5meC5meC5meCa.psi.gaaa5meC.-
psi..psi.a.psi.
gg5meC5meC5meC.psi.g5meCag.psi..psi.g5meC.psi.g5meC.psi..psi..psi.gg5meCa5-
meC.psi.5meCgg5meC5meC5meC
.psi.5meC.psi.gga5meCag.psi.5meC5meCaagaag5meCga5meC.psi.5meC5meC.psi.5meC-
.psi.5meCgga5meC5me
C.psi.g5meC5meC.psi.5meCa.psi.5meCg.psi..psi.g5meC5meCg5meCag.psi.5meCa.ps-
i..psi.5meC5meC.psi..psi..psi..psi.gaag.psi.g.psi.
5meC.psi.ggag5meCagg.psi.g5meCgaaaga.psi..psi.5meCaggg5meCga.psi.ggag5meC5-
meCg5meCa5meC.psi.5me
C5meCaagagaag5meC.psi.5meC.psi.g5meCg5meCga5meCa.psi.a5meCaaa5meC.psi..psi-
..psi.g5meC5meCa.psi.5me
C5meC5meCgaggag5meC.psi.5meCg.psi.a5meC.psi.g5meC.psi.5meCggg5meCa5meCag5m-
eC.psi..psi.gggga.psi..psi.
5meC5meC5meC.psi.ggg5meC.psi.5meC5meC.psi.5meC.psi.5meC.psi.5meCg.psi.5meC-
5meC.psi.g.psi.5meC5meCg
.psi.5meCg5meCagg5meC.psi..psi..psi.g5meCag.psi..psi.gg5meCaggg.psi.g5meC5-
meC.psi..psi..psi.5meC5meC5meCag5m
eC.psi.5meC5meCa5meC.psi.5meC5meCgg.psi..psi..psi.g.psi..psi.5meC.psi..psi-
.g.psi.a.psi.5meCaggga5meC.psi.g5meC.psi.g5meC
aag5meC5meC5meC.psi..psi.gagggaa.psi.5meC.psi.5meCg5meC5meCagaa.psi..psi.g-
gg5meC5meC5meCga5meC
g5meC.psi.gga5meCa5meCg.psi..psi.g5meCag5meC.psi.5meCga5meCg.psi.gg5meCgg
a.psi..psi..psi.5meCg5meCaa5meCaa5meC5meCa.psi.5meC.psi.gg5meCag5meCaga.ps-
i.ggaggaa5meC.psi.gggga.psi.g
g5meCa5meC5meC5meCg5meCg5meC.psi.g5meCag5meC5meC5meCa5meCg5meCaggggg5meCa
a.psi.g5meC5meCgg5meC5meC.psi..psi..psi.g5meCg.psi.5meC5meCg5meCg.psi..psi-
..psi.5meCag5meCg5meCaggg5
meCggg.psi.ggag.psi.5meC5meC.psi.5meCg.psi.ag5meCgag5meC5meCa5meC5meC.psi.-
.psi.5meCaa.psi.5meCa.psi..psi.
.psi..psi..psi.ggaag.psi.5meC.psi.5meCg.psi.a5meC5meCggg.psi.g5meC.psi.gag-
a5meCa.psi.5meC.psi..psi.g5meCg5meCag5m
eC5meCg.psi.gaag5meCg5meC.psi.g5meC5meC.psi..psi.5meC.psi.g5meCgggg5meC.ps-
i..psi.g5meC5meC.psi..psi.5meC
.psi.gg5meC5meCa.psi.g5meC5meC5meC.psi..psi.5meC.psi..psi.5meC.psi.5meC.ps-
i.5meC5meC5meC.psi..psi.g5meCa5me
C5meC.psi.g.psi.a5meC5meC.psi.5meC.psi..psi.gg.psi.5meC.psi..psi..psi.gaa.-
psi.aaag5meC5meC.psi.gag.psi.aggaagg5meCgg5m
eC5meCg5meC.psi.5meCgag5meCa.psi.g5meCa.psi.5meC.psi.agaggg5meC5meC5meCaa.-
psi..psi.5meCg5meC5
meC5meC.psi.a.psi..psi..psi.5meCgaag.psi.5meCg
[0174] FIG. 1 shows an Enzyme-linked immunosorbent assay (ELISA)
for Human Granulocyte-Colony Stimulating Factor (G-CSF) from
Chinese Hamster Ovary Cells (CHO) transfected with modRNA for
G-CSF. The CHO cells were grown in CD CHO Medium with Supplement of
L-Glutamine, Hypoxanthine and Thymidine. 2.times.10.sub.6 Cells
were transfected with 24 ug modRNA complexed with RNAiMax from
Invitrogen in a 75 cm2 culture flask from Corning with 7 ml of
medium. The RNA:RNAiMAX complex was formed by first incubating the
RNA with CD CHO Medium in a 5.times. volumetric dilution for 10
minutes at room temperature. In a second vial, RNAiMAX reagent was
incubated with CD CHO Medium in a 10.times. volumetric dilution for
10 minutes at room temperature. The RNA vial was then mixed with
the RNAiMAX vial and incubated for 20-30 at room temperature before
being added to the cells in a drop-wise fashion. The concentration
of secreted huG-CSF in the culture medium was measured at 12 and 24
hours post-transfection. Cell supernatants were stored at
-20.degree. C. Secretion of Human Granulocyte-Colony Stimulating
Factor (G-CSF) from transfected Human Embryonic Kidney cells was
quantified using an ELISA kit from Invitrogen following the
manufacturers recommended instructions. These data show that
huG-CSF modRNA (SEQ ID NO: 3) is capable of being translated in CHO
cells, and that huG-CSF is secreted out of the cells and released
into the extracellular environment. Furthermore these data
demonstrate that transfection of cells with modRNA huG-CSF for the
production of secreted protein can be scaled up to a bioreactor or
large cell culture conditions.
Example 3: De Novo Generation of a Mammalian Commercial Production
Cell Line Expressing Humanized IgG Antibodies (Trastuzumab and
Rituximab) as a Therapeutic Agent in Model Bioreactor
[0175] The nucleic acid sequence for the Heavy Chain of Rituximab
is set forth in SEQ ID NO: 4:
TABLE-US-00004 (SEQ ID NO: 4) CTCGTACAGAAGCTAATACGACTCACTATAGGGAA
ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG
AGCCACCATGGCCGTGATGGCGCCGAGGACCCTGG
TGCTCTTGCTCACGGGTGCCTTGGCCCTCACGCAA
ACATGGGCGGGACAGGCGTACTTGCAGCAGTCAGG
GGCAGAACTCGTAAGGCCCGGAGCGTCGGTGAAGA
TGTCGTGTAAAGCGTCGGGCTATACTTTCACATCG
TACAACATGCACTGGGTCAAACAGACGCCCCGACA
AGGGCTGGAGTGGATTGGAGCTATCTACCCCGGTA
ACGGGGATACGTCGTACAACCAGAAGTTTAAGGGG
AAGGCGACTCTTACTGTCGACAAGTCGTCCTCCAC
CGCCTATATGCAGCTGTCGAGCCTGACTTCGGAAG
ATTCAGCGGTGTACTTTTGTGCGCGCGTGGTCTAT
TACTCAAATTCGTATTGGTATTTCGATGTGTGGGG
TACGGGGACCACTGTGACCGTGTCAGGACCCTCGG
TATTCCCCCTCGCGCCTAGCTCAAAGTCCACCTCC
GGGGGAACAGCCGCCTTGGGTTGCTTGGTAAAGGA
CTATTTCCCCGAGCCCGTCACAGTGAGCTGGAACT
CCGGGGCACTGACATCGGGAGTGCACACGTTTCCC
GCGGTACTTCAGTCATCAGGACTCTACTCGCTGTC
AAGCGTGGTCACGGTGCCTTCATCCTCCCTTGGAA
CGCAGACTTACATCTGCAACGTGAATCATAAGCCT
AGCAATACCAAGGTCGACAAGAAAGCCGAACCCAA
ATCATGTGATAAAACACACACGTGTCCTCCCTGCC
CCGCACCGGAGCTTCTCGGGGGACCGAGCGTGTTC
TTGTTTCCACCTAAGCCGAAAGATACGCTTATGAT
CTCCCGGACCCCCGAAGTAACTTGCGTAGTAGTAG
ACGTAAGCCACGAGGACCCCGAAGTGAAATTCAAT
TGGTACGTCGACGGAGTGGAGGTCCATAATGCGAA
AACAAAGCCGAGAGAGGAACAGTACAATTCCACAT
ACCGCGTCGTAAGCGTCTTGACAGTATTGCATCAG
GATTGGCTGAACGGAAAGGAATACAAGTGCAAAGT
ATCAAACAAAGCACTTCCGGCACCGATTGAAAAGA
CGATCTCAAAAGCAAAAGGGCAACCTCGGGAGCCA
CAAGTCTATACTCTCCCGCCGTCGCGCGATGAATT
GACCAAAAACCAGGTGTCCCTTACATGTCTCGTAA
AGGGTTTTTACCCGTCAGACATCGCCGTCGAGTGG
GAGTCAAACGGTCAGCCGGAGAATAACTATAAGAC
GACCCCACCAGTCTTGGACAGCGATGGCTCCTTCT
TCTTGTATTCAAAGCTGACGGTGGACAAATCGAGA
TGGCAGCAGGGTAATGTGTTTTCGTGCAGCGTCAT
GCACGAGGCGCTTCATAATCATTACACTCAAAAGT
CCCTGTCGCTGTCGCCCGGAAAGCACCATCACCAC
CACCATTGAAGCGCTGCCTTCTGCGGGGCTTGCCT
TCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTC
TTGGTCTTTGAATAAAGCCTGAGTAGGAAG GCGGCCGCTCGAGCATGCATCTAGA
[0176] The nucleic acid sequence for the mRNA for the Heavy Chain
of Rituximab is set forth in SEQ ID NO: 5:
TABLE-US-00005 (SEQ ID NO: 5) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAA
AUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG
AGCCACCAUGGCCGUGAUGGCGCCGAGGACCCUGG
UGCUCUUGCUCACGGGUGCCUUGGCCCUCACGCAA
ACAUGGGCGGGACAGGCGUACUUGCAGCAGUCAGG
GGCAGAACUCGUAAGGCCCGGAGCGUCGGUGAAGA
UGUCGUGUAAAGCGUCGGGCUAUACUUUCACAUCG
UACAACAUGCACUGGGUCAAACAGACGCCCCGACA
AGGGCUGGAGUGGAUUGGAGCUAUCUACCCCGGUA
ACGGGGAUACGUCGUACAACCAGAAGUUUAAGGGG
AAGGCGACUCUUACUGUCGACAAGUCGUCCUCCAC
CGCCUAUAUGCAGCUGUCGAGCCUGACUUCGGAAG
AUUCAGCGGUGUACUUUUGUGCGCGCGUGGUCUAU
UACUCAAAUUCGUAUUGGUAUUUCGAUGUGUGGGG
UACGGGGACCACUGUGACCGUGUCAGGACCCUCGG
UAUUCCCCCUCGCGCCUAGCUCAAAGUCCACCUCC
GGGGGAACAGCCGCCUUGGGUUGCUUGGUAAAGGA
CUAUUUCCCCGAGCCCGUCACAGUGAGCUGGAACU
CCGGGGCACUGACAUCGGGAGUGCACACGUUUCCC
GCGGUACUUCAGUCAUCAGGACUCUACUCGCUGUC
AAGCGUGGUCACGGUGCCUUCAUCCUCCCUUGGAA
CGCAGACUUACAUCUGCAACGUGAAUCAUAAGCCU
AGCAAUACCAAGGUCGACAAGAAAGCCGAACCCAA
AUCAUGUGAUAAAACACACACGUGUCCUCCCUGCC
CCGCACCGGAGCUUCUCGGGGGACCGAGCGUGUUC
UUGUUUCCACCUAAGCCGAAAGAUACGCUUAUGAU
CUCCCGGACCCCCGAAGUAACUUGCGUAGUAGUAG
ACGUAAGCCACGAGGACCCCGAAGUGAAAUUCAAU
UGGUACGUCGACGGAGUGGAGGUCCAUAAUGCGAA
AACAAAGCCGAGAGAGGAACAGUACAAUUCCACAU
ACCGCGUCGUAAGCGUCUUGACAGUAUUGCAUCAG
GAUUGGCUGAACGGAAAGGAAUACAAGUGCAAAGU
AUCAAACAAAGCACUUCCGGCACCGAUUGAAAAGA
CGAUCUCAAAAGCAAAAGGGCAACCUCGGGAGCCA
CAAGUCUAUACUCUCCCGCCGUCGCGCGAUGAAUU
GACCAAAAACCAGGUGUCCCUUACAUGUCUCGUAA
AGGGUUUUUACCCGUCAGACAUCGCCGUCGAGUGG
GAGUCAAACGGUCAGCCGGAGAAUAACUAUAAGAC
GACCCCACCAGUCUUGGACAGCGAUGGCUCCUUCU
UCUUGUAUUCAAAGCUGACGGUGGACAAAUCGAGA
UGGCAGCAGGGUAAUGUGUUUUCGUGCAGCGUCAU
GCACGAGGCGCUUCAUAAUCAUUACACUCAAAAGU
CCCUGUCGCUGUCGCCCGGAAAGCACCAUCACCAC
CACCAUUGAAGCGCUGCCUUCUGCGGGGCUUGCCU
UCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGU
ACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG GCGGCCGCUCGAGCAUGCAUCUAGA
[0177] The nucleic acid sequence for the nucleic acid sequence for
the Light Chain of Rituximab is set forth in SEQ ID NO: 6:
TABLE-US-00006 (SEQ ID NO: 6) CTCGTACAGAAGCTAATACGACTCACTATAGGGAA
ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG
AGCCACCATGGCTGTCATGGCCCCGAGAACACTTG
TGCTGTTGTTGACAGGAGCGCTCGCACTCACACAG
ACTTGGGCCGGTCAGATTGTGCTCAGCCAGTCGCC
AGCGATCCTTTCGGCCTCCCCTGGTGAGAAAGTAA
CGATGACGTGCCGAGCCTCCTCAAGCGTGTCATAC
ATGCATTGGTATCAGCAGAAGCCTGGGTCGTCGCC
CAAGCCCTGGATCTACGCCCCGTCCAATCTTGCGT
CAGGGGTCCCGGCACGGTTCAGCGGATCGGGGTCG
GGTACATCGTATTCACTCACGATTAGCCGCGTAGA
GGCCGAGGACGCGGCGACTTACTACTGTCAGCAAT
GGTCCTTTAATCCACCCACGTTTGGAGCGGGCACC
AAGCTCGAACTTAAAAGAACGGTCGCCGCACCCTC
AGTGTTTATCTTCCCGCCCTCGGACGAACAACTTA
AGTCGGGGACCGCTTCCGTGGTGTGCTTGCTGAAC
AATTTCTATCCTCGGGAAGCTAAAGTGCAATGGAA
AGTCGATAACGCATTGCAGAGCGGAAACTCACAAG
AGTCGGTAACTGAGCAGGATAGCAAGGATTCGACA
TACTCGCTGAGCAGCACGCTGACGTTGTCCAAGGC
GGACTACGAGAAACACAAGGTATATGCGTGTGAAG
TCACCCACCAGGGATTGTCATCGCCGGTCACCAAA
TCATTCAACAGGTGATAAAGCGCTGCCTTCTGCGG
GGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCT
TGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTG
AGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA
[0178] The nucleic acid sequence for the mRNA of the Light Chain of
Rituximab is set forth in SEQ ID NO: 7:
TABLE-US-00007 (SEQ ID NO: 7) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAA
AUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG
AGCCACCAUGGCUGUCAUGGCCCCGAGAACACUUG
UGCUGUUGUUGACAGGAGCGCUCGCACUCACACAG
ACUUGGGCCGGUCAGAUUGUGCUCAGCCAGUCGCC
AGCGAUCCUUUCGGCCUCCCCUGGUGAGAAAGUAA
CGAUGACGUGCCGAGCCUCCUCAAGCGUGUCAUAC
AUGCAUUGGUAUCAGCAGAAGCCUGGGUCGUCGCC
CAAGCCCUGGAUCUACGCCCCGUCCAAUCUUGCGU
CAGGGGUCCCGGCACGGUUCAGCGGAUCGGGGUCG
GGUACAUCGUAUUCACUCACGAUUAGCCGCGUAGA
GGCCGAGGACGCGGCGACUUACUACUGUCAGCAAU
GGUCCUUUAAUCCACCCACGUUUGGAGCGGGCACC
AAGCUCGAACUUAAAAGAACGGUCGCCGCACCCUC
AGUGUUUAUCUUCCCGCCCUCGGACGAACAACUUA
AGUCGGGGACCGCUUCCGUGGUGUGCUUGCUGAAC
AAUUUCUAUCCUCGGGAAGCUAAAGUGCAAUGGAA
AGUCGAUAACGCAUUGCAGAGCGGAAACUCACAAG
AGUCGGUAACUGAGCAGGAUAGCAAGGAUUCGACA
UACUCGCUGAGCAGCACGCUGACGUUGUCCAAGGC
GGACUACGAGAAACACAAGGUAUAUGCGUGUGAAG
UCACCCACCAGGGAUUGUCAUCGCCGGUCACCAAA
UCAUUCAACAGGUGAUAAAGCGCUGCCUUCUGCGG
GGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCU
UGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUG
AGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAGA
[0179] The nucleic acid sequence for the nucleic acid sequence for
the Heavy Chain of Trastuzumab is set forth in SEQ ID NO: 8:
TABLE-US-00008 (SEQ ID NO: 8) CTCGTACAGAAGCTAATACGACTCACTATAGGGAA
ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG
AGCCACCATGGCCGTGATGGCGCCGCGGACCCTGG
TCCTCCTGCTGACCGGCGCCCTCGCCCTGACGCAG
ACCTGGGCCGGGGAGGTGCAGCTGGTCGAGAGCGG
CGGGGGCCTCGTGCAGCCGGGCGGGTCGCTGCGGC
TGAGCTGCGCCGCGAGCGGGTTCAACATCAAGGAC
ACCTACATCCACTGGGTGCGCCAGGCCCCCGGCAA
GGGCCTCGAGTGGGTCGCCCGGATCTACCCCACGA
ACGGGTACACCCGCTACGCCGACAGCGTGAAGGGC
CGGTTCACCATCAGCGCGGACACCTCGAAGAACAC
GGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGG
ACACCGCCGTGTACTACTGCAGCCGGTGGGGCGGC
GACGGGTTCTACGCCATGGACTACTGGGGGCAGGG
CACCCTCGTCACCGTGAGCAGCGCGTCGACGAAGG
GGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAG
AGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCT
CGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGT
CGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCAC
ACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTA
CTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCA
GCCTGGGGACCCAGACGTACATCTGCAACGTGAAC
CACAAGCCCTCGAACACCAAGGTCGACAAGAAGGT
GGAGCCCCCGAAGAGCTGCGACAAGACCCACACCT
GCCCGCCCTGCCCCGCCCCCGAGCTCCTGGGCGGG
CCCAGCGTGTTCCTGTTCCCGCCCAAGCCCAAGGA
CACGCTCATGATCAGCCGCACCCCCGAGGTCACCT
GCGTGGTGGTCGACGTGAGCCACGAGGACCCCGAG
GTGAAGTTCAACTGGTACGTCGACGGCGTGGAGGT
GCACAACGCCAAGACCAAGCCGCGGGAGGAGCAGT
ACAACTCGACGTACCGCGTCGTGAGCGTGCTGACC
GTCCTGCACCAGGACTGGCTCAACGGCAAGGAGTA
CAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCGC
CCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAG
CCCCGGGAGCCGCAGGTGTACACCCTGCCCCCCAG
CCGCGACGAGCTCACGAAGAACCAGGTCAGCCTGA
CCTGCCTGGTGAAGGGCTTCTACCCCTCGGACATC
GCCGTGGAGTGGGAGAGCAACGGGCAGCCGGAGAA
CAACTACAAGACCACCCCGCCCGTCCTCGACAGCG
ACGGCAGCTTCTTCCTGTACAGCAAGCTGACGGTG
GACAAGTCGCGGTGGCAGCAGGGCAACGTGTTCAG
CTGCAGCGTCATGCACGAGGCCCTCCACAACCACT
ACACCCAGAAGAGCCTGAGCCTGAGCCCCGGGAAG
CATCATCATCATCATCATTGAAGCGCTGCCTTCTG
CGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTC
CCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGC
CTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTA GA
[0180] The nucleic acid sequence of the mRNA for the Heavy Chain of
Trastuzumab is set forth in SEO ID NO: 9:
TABLE-US-00009 (SEQ ID NO: 9) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAA
AUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG
AGCCACCAUGGCCGUGAUGGCGCCGCGGACCCUGG
UCCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAG
ACCUGGGCCGGGGAGGUGCAGCUGGUCGAGAGCGG
CGGGGGCCUCGUGCAGCCGGGCGGGUCGCUGCGGC
UGAGCUGCGCCGCGAGCGGGUUCAACAUCAAGGAC
ACCUACAUCCACUGGGUGCGCCAGGCCCCCGGCAA
GGGCCUCGAGUGGGUCGCCCGGAUCUACCCCACGA
ACGGGUACACCCGCUACGCCGACAGCGUGAAGGGC
CGGUUCACCAUCAGCGCGGACACCUCGAAGAACAC
GGCCUACCUGCAGAUGAACAGCCUGCGCGCCGAGG
ACACCGCCGUGUACUACUGCAGCCGGUGGGGCGGC
GACGGGUUCUACGCCAUGGACUACUGGGGGCAGGG
CACCCUCGUCACCGUGAGCAGCGCGUCGACGAAGG
GGCCCAGCGUGUUCCCGCUGGCCCCCAGCAGCAAG
AGCACCAGCGGCGGGACCGCCGCCCUGGGCUGCCU
CGUCAAGGACUACUUCCCCGAGCCCGUGACCGUGU
CGUGGAACAGCGGCGCGCUGACGAGCGGGGUCCAC
ACCUUCCCGGCCGUGCUGCAGAGCAGCGGCCUCUA
CUCGCUGAGCAGCGUGGUCACCGUGCCCAGCAGCA
GCCUGGGGACCCAGACGUACAUCUGCAACGUGAAC
CACAAGCCCUCGAACACCAAGGUCGACAAGAAGGU
GGAGCCCCCGAAGAGCUGCGACAAGACCCACACCU
GCCCGCCCUGCCCCGCCCCCGAGCUCCUGGGCGGG
CCCAGCGUGUUCCUGUUCCCGCCCAAGCCCAAGGA
CACGCUCAUGAUCAGCCGCACCCCCGAGGUCACCU
GCGUGGUGGUCGACGUGAGCCACGAGGACCCCGAG
GUGAAGUUCAACUGGUACGUCGACGGCGUGGAGGU
GCACAACGCCAAGACCAAGCCGCGGGAGGAGCAGU
ACAACUCGACGUACCGCGUCGUGAGCGUGCUGACC
GUCCUGCACCAGGACUGGCUCAACGGCAAGGAGUA
CAAGUGCAAGGUGAGCAACAAGGCCCUGCCCGCGC
CCAUCGAGAAGACCAUCAGCAAGGCCAAGGGGCAG
CCCCGGGAGCCGCAGGUGUACACCCUGCCCCCCAG
CCGCGACGAGCUCACGAAGAACCAGGUCAGCCUGA
CCUGCCUGGUGAAGGGCUUCUACCCCUCGGACAUC
GCCGUGGAGUGGGAGAGCAACGGGCAGCCGGAGAA
CAACUACAAGACCACCCCGCCCGUCCUCGACAGCG
ACGGCAGCUUCUUCCUGUACAGCAAGCUGACGGUG
GACAAGUCGCGGUGGCAGCAGGGCAACGUGUUCAG
CUGCAGCGUCAUGCACGAGGCCCUCCACAACCACU
ACACCCAGAAGAGCCUGAGCCUGAGCCCCGGGAAG
CAUCAUCAUCAUCAUCAUUGAAGCGCUGCCUUCUG
CGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUC
CCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGC
CUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUA GA
[0181] The nucleic acid sequence for the nucleic acid sequence for
the Light Chain of Trastuzumab is set forth in SEQ ID NO: 10:
TABLE-US-00010 (SEQ ID NO: 10) CTCGTACAGAAGCTAATACGACTCACTATAGGGAA
ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG
AGCCACCATGGCCGTGATGGCGCCGCGGACCCTGG
TCCTCCTGCTGACCGGCGCCCTCGCCCTGACGCAG
ACCTGGGCCGGGGACATCCAGATGACCCAGAGCCC
GTCGAGCCTGAGCGCCAGCGTGGGCGACCGGGTCA
CGATCACCTGCCGCGCGAGCCAGGACGTGAACACC
GCCGTGGCCTGGTACCAGCAGAAGCCCGGGAAGGC
CCCCAAGCTCCTGATCTACTCGGCGAGCTTCCTGT
ACAGCGGCGTCCCCAGCCGGTTCAGCGGGTCGCGC
AGCGGCACCGACTTCACGCTCACCATCAGCAGCCT
GCAGCCGGAGGACTTCGCCACCTACTACTGCCAGC
AGCACTACACCACGCCCCCCACCTTCGGGCAGGGC
ACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCC
CAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGC
TGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTG
AACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTG
GAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCC
AGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA
GGCCGACTACGAGAAGCACAAGGTCTACGCCTGCG
AGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACC
AAGAGCTTCAACCGGGGCGAGTGCTGAAGCGCTGC
CTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCT
TCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAA
TAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATG CATCTAGA
[0182] The nucleic acid sequence for the mRNA of the Light Chain of
Trastuzumab is set forth in SEQ ID NO: 11:
TABLE-US-00011 (SEQ ID NO: 11)
CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAG
AAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGUGAUGGCG
CCGCGGACCCUGGUCCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAG
ACCUGGGCCGGGGACAUCCAGAUGACCCAGAGCCCGUCGAGCCUGAGC
GCCAGCGUGGGCGACCGGGUCACGAUCACCUGCCGCGCGAGCCAGGAC
GUGAACACCGCCGUGGCCUGGUACCAGCAGAAGCCCGGGAAGGCCCCC
AAGCUCCUGAUCUACUCGGCGAGCUUCCUGUACAGCGGCGUCCCCAGC
CGGUUCAGCGGGUCGCGCAGCGGCACCGACUUCACGCUCACCAAGCAG
CCUGCAGCCGGAGGACUUCGCCACCUACUACUGCCAGCAGCACUACAC
CACGCCCCCCACCUUCGGGCAGGGCACCAAGGUGGAGAUCAAGCGGAC
CGUGGCCGCCCCCAGCGUCUUCAUCUUCCCGCCCAGCGACGAGCAGCU
GAAGUCGGGCACGGCCAGCGUGGUGUGCCUCCUGAACAACUUCUACCC
CCGCGAGGCGAAGGUCCAGUGGAAGGUGGACAACGCCCUGCAGAGCGG
GAACAGCCAGGAGAGCGUGACCGAGCAGGACUCGAAGGACAGCACCUA
CAGCCUCAGCAGCACCCUGACGCUGAGCAAGGCCGACUACGAGAAGCA
CAAGGUCUACGCCUGCGAGGUGACCCACCAGGGGCUCUCGAGCC
CCGUGACCAAGAGCUUCAACCGGGGCGAGUGCUGAAGCGCUGCCUUCU
GCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGU
ACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAG CAUGCAUCUAGA
[0183] The nucleic acid sequence for nucleotide sequence of the
wild type CERT protein is set forth in SEQ ID NO: 12:
TABLE-US-00012 (SEQ ID NO: 12) atgtcggata atcagagctg gaactcgtcg
ggctcggagg aggatccaga gacggagtct gggccgcctg tggagcgctg cggggtcctc
agtaagtgga caaactacat tcatgggtgg caggatcgtt gggtagtttt gaaaaataat
gctctgagtt actacaaatc tgaagatgaa acagagtatg gctgcagagg atccatctgt
cttagcaagg ctgtcatcac acctcacgat tttgatgaat gtcgatttga tattagtgta
aatgatagtg tttggtatct tcgtgctcag gatccagatc atagacagca atggatagat
gccattgaac agcacaagac tgaatctgga tatggatctg aatccagctt gcgtcgacat
ggctcaatgg tgtccctggt gtctggagca agtggctact ctgcaacatc cacctcttca
ttcaagaaag gccacagttt acgtgagaag ttggctgaaa tggaaacatt tagagacatc
ttatgtagac aagttgacac gctacagaag tactttgatg cctgtgctga tgctgtctct
aaggatgaac ttcaaaggga taaagtggta gaagatgatg aagatgactt tcctacaacg
cgttctgatg gtgacttctt gcatagtacc aacggcaata aagaaaagtt atttccacat
gtgacaccaa aaggaattaa tggtatagac tttaaagggg aagcgataac ttttaaagca
actactgctg gaatccttgc aacactttct cattgtattg aactaatggt taaacgtgag
gacagctggc agaagagact ggataaggaa actgagaaga aaagaagaac agaggaagca
tataaaaatg caatgacaga acttaagaaa aaatcccact ttggaggacc agattatgaa
gaaggcccta acagtctgat taatgaagaa gagttctttg atgctgttga agctgctctt
gacagacaag ataaaataga agaacagtca cagagtgaaa aggtgagatt acattggcct
acatccttgc cctctggaga tgccttttct tctgtgggga cacatagatt tgtccaaaag
gttgaagaga tggtgcagaa ccacatgact tactcattac aggatgtagg cggagatgcc
aattggcagt tggttgtaga agaaggagaa atgaaggtat acagaagaga agtagaagaa
aatgggattg ttctggatcc tttaaaagct acccatgcag ttaaaggcgt cacaggacat
gaagtctgca attatttctg gaatgttgac gttcgcaatg actgggaaac aactatagaa
aactttcatg tggtggaaac attagctgat aatgcaatca tcatttatca aacacacaag
agggtgtggc ctgcttctca gcgagacgta ttatatcttt ctgtcattcg aaagatacca
gccttgactg aaaatgaccc tgaaacttgg atagtttgta atttttctgt ggatcatgac
agtgctcctc taaacaaccg atgtgtccgt gccaaaataa atgttgctat gatttgtcaa
accttggtaa gcccaccaga gggaaaccag gaaattagca gggacaacat tctatgcaag
attacatatg tagctaatgt gaaccctgga ggatgggcac cagcctcagt gttaagggca
gtggcaaagc gagagtatcc taaatttcta aaacgtttta cttcttacgt ccaagaaaaa
actgcaggaa agcctatttt gttctag
[0184] The protein sequence for the wild type CERT protein is set
forth in SEQ ID NO: 13:
TABLE-US-00013 (SEQ ID NO: 13) Met Ser Asp Asn Gin Ser Trp Asn Ser
Ser Gly Ser Glu Glu Asp Pro Glu Thr Glu Ser Gly Pro Pro Val Glu Arg
Cys Gly Val Leu Ser Lys Trp Thr Asn Tyr Ile His Gly Trp Gin Asp Arg
Trp Val Val Leu Lys Asn Asn Ala Leu Ser Tyr Tyr Lys Ser Glu Asp Glu
Thr Glu Tyr Gly Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala Val Ile Thr
Pro His Asp Phe Asp Glu Cys Arg Phe Asp Ile Ser Val Asn Asp Ser Val
Trp Tyr Leu Arg Ala Gin Asp Pro Asp His Arg Gin Gin Trp Ile Asp Ala
Ile Glu Gin His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg
Arg His Gly Ser Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser Ala
Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg Glu Lys Leu Ala
Glu Met Glu Thr Phe Arg Asp Ile Leu Cys Arg Gin Val A sp Thr Leu
Gin Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys Asp Glu Leu Gin
Arg Asp Lys Val Val Glu Asp Asp Glu Asp Asp Phe Pro Thr Thr Arg Ser
Asp Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys Glu Lys Leu Phe Pro
His Val Thr Pro Lys Gly Ile Asn Gly Ile Asp Phe Lys Gly Glu Ala Ile
Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu Ala Thr Leu Ser His Cys Ile
Glu Leu Met Val Lys Arg Glu Asp Ser Trp Gin Lys Arg Leu Asp Lys Glu
Thr Glu Lys Lys Arg Arg Thr Glu Glu Ala Tyr Lys Asn Ala Met Thr Glu
Leu Lys Lys Lys Ser His Phe Gly Gly Pro Asp Tyr Glu Glu Gly Pro Asn
Ser Leu Ile Asn Glu Glu Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp
Arg Gin Asp Lys Ile Glu Glu Gin Ser Gin Ser Glu Lys Val Arg Leu His
Trp Pro Thr Ser Leu Pro Ser Gly Asp Ala Phe Ser Ser Val Gly Thr His
Arg Phe Val Gin Lys Val Glu Glu Met Val Gin Asn His Met Thr Tyr Ser
Leu Gin Asp Val Gly Gly Asp Ala Asn Trp Gin Leu Val Val Glu Glu Gly
Glu Met Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu Asp
Pro Leu Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu Val Cys
Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp Trp Glu Thr Thr Ile Glu
Asn Phe His Val Val Glu Thr Leu Ala Asp Asn Ala Ile Ile Ile Tyr Gin
Thr His Lys Arg Val Trp Pro Ala Ser Gin Arg Asp Val Leu Tyr Leu Ser
Val Ile Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp Pro Glu Thr Trp Ile
Val Cys Asn Phe Ser Val Asp His Asp Ser Ala Pro Leu Asn Asn Arg Cys
Val Arg Ala Lys Ile Asn Val Ala Met Ile Cys Gin Thr Leu Val Ser Pro
Pro Glu Gly Asn Gin Glu Ile Ser Arg
[0185] The nucleic acid sequence for the nucleotide sequence of the
Ser132A Cert mutant is set forth as SEQ ID NO: 14:
TABLE-US-00014 (SEQ ID NO: 14) atgtcggata atcagagctg gaactcgtcg
ggctcggagg aggatccaga gacggagtct gggccgcctg tggagcgctg cggggtcctc
agtaagtgga caaactacat tcatgggtgg caggatcgtt gggtagtttt gaaaaataat
gctctgagtt actacaaatc tgaagatgaa acagagtatg gctgcagagg atccatctgt
cttagcaagg ctgtcatcac acctcacgat tttgatgaat gtcgatttga tattagtgta
aatgatagtg tttggtatct tcgtgctcag gatccagatc atagacagca atggatagat
gccattgaac agcacaagac tgaatctgga tatggatctg aatccagctt gcgtcgacat
ggcgcaatgg tgtccctggt gtctggagca agtggctact ctgcaacatc cacctcttca
ttcaagaaag gccacagttt acgtgagaag ttggctgaaa tggaaacatt tagagacatc
ttatgtagac aagttgacac gctacagaag tactttgatg cctgtgctga tgctgtctct
aaggatgaac ttcaaaggga taaagtggta gaagatgatg aagatgactt tcctacaacg
cgttctgatg gtgacttctt gcatagtacc aacggcaata aagaaaagtt atttccacat
gtgacaccaa aaggaattaa tggtatagac tttaaagggg aagcgataac ttttaaagca
actactgctg gaatccttgc aacactttct cattgtattg aactaatggt taaacgtgag
gacagctggc agaagagact ggataaggaa actgagaaga aaagaagaac agaggaagca
tataaaaatg caatgacaga acttaagaaa aaatcccact ttggaggacc agattatgaa
gaaggcccta acagtctgat taatgaagaa gagttctttg atgctgttga agctgctctt
gacagacaag ataaaataga agaacagtca cagagtgaaa aggtgagatt acattggcct
acatccttgc cctctggaga tgccttttct tctgtgggga cacatagatt tgtccaaaag
gttgaagaga tggtgcagaa ccacatgact tactcattac aggatgtagg cggagatgcc
aattggcagt tggttgtaga agaaggagaa atgaaggtat acagaagaga agtagaagaa
aatgggattg ttctggatcc tttaaaagct acccatgcag ttaaaggcgt cacaggacat
gaagtctgca attatttctg gaatgttgac gttcgcaatg actgggaaac aactatagaa
aactttcatg tggtggaaac attagctgat aatgcaatca tcatttatca aacacacaag
agggtgtggc ctgcttctca gcgagacgta ttatatcttt ctgtcattcg aaagatacca
gccttgactg aaaatgaccc tgaaacttgg atagtttgta atttttctgt ggatcatgac
agtgctcctc taaacaaccg atgtgtccgt gccaaaataa atgttgctat gatttgtcaa
accttggtaa gcccaccaga gggaaaccag gaaattagca gggacaacat tctatgcaag
attacatatg tagctaatgt gaaccctgga ggatgggcac cagcctcagt gttaagggca
gtggcaaagc gagagtatcc taaatttcta aaacgtttta cttcttacgt ccaagaaaaa
actgcaggaa agcctatttt gttctag
[0186] The protein sequence of the Ser132A Cert mutant is set forth
as SEQ ID NO. 15:
TABLE-US-00015 (SEQ ID NO: 15) Met Ser Asp Asn Gin Ser Trp Asn Ser
Ser Gly Ser Glu Glu Asp Pro Glu Thr Glu Ser Gly Pro Pro Val Glu Arg
Cys Gly Val Leu Ser Lys Trp Thr Asn Tyr Ile His Gly Trp Gin Asp Arg
Trp Val Val Leu Lys Asn Asn Ala Leu Ser Tyr Tyr Lys Ser Glu Asp Glu
Thr Glu Tyr Gly Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala Val Ile Thr
Pro His Asp Phe Asp Glu Cys Arg Phe Asp Ile Ser Val Asn Asp Ser Val
Trp Tyr Leu Arg Ala Gin Asp Pro Asp His Arg Gin Gin Trp Ile Asp Ala
Ile Glu Gin His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg
Arg His Gly Ala Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser Ala
Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg Glu Lys Leu Ala
Glu Met Glu Thr Phe Arg Asp Ile Leu Cys Arg Gin Val Asp Thr Leu Gin
Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys Asp Glu Leu Gin Arg
Asp Lys Val Val Glu Asp Asp Glu Asp Asp Phe Pro Thr Thr Arg Ser Asp
Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys Glu Lys Leu Phe Pro His
Val Thr Pro Lys Gly Ile Asn Gly Ile Asp Phe Lys Gly Glu Ala Ile Thr
Phe Lys Ala Thr Thr Ala Gly Ile Leu Ala Thr Leu Ser His Cys Ile Glu
Leu Met Val Lys Arg Glu Asp Ser Trp Gin Lys Arg Leu Asp Lys Glu Thr
Glu Lys Lys Arg Arg Thr Glu Glu Ala Tyr Lys Asn Ala Met Thr Glu Leu
Lys Lys Lys Ser His Phe Gly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Glu
Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gin Asp Lys Ile Glu Glu
Gin Ser Gin Ser Glu Lys Val Arg Leu His Trp Pro Thr Ser Leu Pro Ser
Gly Asp Ala Phe Ser Ser Val Gly Thr His Arg Phe Val Gin Lys Val Glu
Glu Met Val Gin Asn His Met Thr Tyr Ser Leu Gin Asp Val Gly Gly Asp
Ala Asn Trp Gin Leu Val Val Glu Glu Gly Glu Met Lys Val Tyr Arg Arg
Glu Val Glu Glu Asn Gly Ile Val Leu Asp Pro Leu Lys Ala Thr His Ala
Val Lys Gly Val Thr Gly His Glu Val Cys Asn Tyr Phe Trp Asn Val Asp
Val Arg Asn Asp Trp Glu Thr Thr Ile Glu Asn Phe His Val Val Glu Thr
Leu Ala Asp Asn Ala Ile Ile Ile Tyr Gin Thr His Lys Arg Val Trp Pro
Ala Ser Gin Arg A sp Val Leu Tyr Leu Ser Val Ile Arg Lys Ile Pro
Ala Leu Thr Glu Asn Asp Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val
Asp His Asp Ser Ala Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn
Val Ala Met Ile Cys Gin Thr Leu Val Ser Pro Pro Glu Gly Asn Gin Glu
Ile Ser Arg Asp Asn Ile Leu Cys Lys Ile Thr Tyr Val Ala Asn Val Asn
Pro Gly Gly Trp Ala Pro Ala Ser Val Leu Arg Ala Val Ala Lys Arg Glu
Tyr Pro Lys Phe Leu Lys Arg Phe Thr Ser Tyr Val Gin Glu Lys Thr Ala
Gly Lys Pro Ile Leu Phe
ELISA Detection of Human IgG Antibodies
[0187] FIG. 2 and FIG. 3 show an Enzyme-linked immunosorbent assay
(ELISA) for Human IgG from Chinese Hamster Ovary's (CHO) and Human
Embryonic Kidney (HEK, HER-2 Negative) 293 cells transfected with
human IgG modRNA, respectively. The Human Embryonic Kidney (HEK)
293 were grown in CD 293 Medium with Supplement of L-Glutamine from
Invitrogen until they reached a confluence of 80-90%. The CHO cells
were grown in CD CHO Medium with Supplement of L-Glutamine,
Hypoxanthine and Thymidine. In FIG. 2, 2.times.10.sub.6 cells were
transfected with 24 ug modRNA complexed with RNAiMax from
Invitrogen in a 75 cm2 culture flask from Corning in 7 ml of
medium. In FIG. 3, 80,000 cells were transfected with 1 ug modRNA
complexed with RNAiMax from Invitrogen in a 24-well plate. The
RNA:RNAiMAX complex was formed by first incubating the RNA with CD
293 or CD CHO Medium in a 5.times. volumetric dilution for 10
minutes at room temperature. In a second vial, RNAiMAX reagent was
incubated with CD 293 Medium or CD CHO Medium in a 10.times.
volumetric dilution for 10 minutes at room temperature. The RNA
vial was then mixed with the RNAiMAX vial and incubated for 20-30
at room temperature before being added to the cells in a drop-wise
fashion. In FIG. 2, the concentration of secreted human IgG in the
culture medium was measured at 12, 24, 36 hours post-transfection.
In FIG. 3, secreted human IgG was measured at 36 hours. The culture
supernatants were stored at 4 degrees. Secretion of Trastuzumab
from transfected Human Embryonic Kidney 293 cells was quantified
using an ELISA kit from Abcam following the manufacturers
recommended instructions. This data show that a Humanized IgG
antibody (Trastuzumab) modRNA (SEQ ID NOs: 6 and 7) is capable of
being translated in Human Embryonic Kidney Cells and that
Trastuzumab is secreted out of the cells and released into the
extracellular environment. Furthermore these data demonstrate that
transfection of cells with modRNA encoding Trastuzumab for the
production of secreted protein can be scaled up to a bioreactor or
large cell culture conditions.
Western Detection of modRNA Produced Human IgG Antibody.
[0188] FIG. 4 shows a Western Blot of CHO-K1 cells co-transfected
with 1 mg each of Heavy and Light Chain of Trastuzumab modRNA. In
order to detect translation of protein product, cells were grown
using standard protocols in 24-well plates, and cell supernatants
or cell lysates were collected at 24 hours post-transfection and
separated on a 12% SDS-Page gel and transferred onto a
nitrocellulose membrane using the iBlot by Invitrogen. After
incubation with a rabbit polyclonal antibody to Human IgG
conjugated to DyLight.RTM. 594 (ab96904, abcam, Cambridge, Mass.)
and a secondary goat polyclonal antibody to Rb IgG which was
conjugated to alkaline phosphatase, the antibody was detected using
Novex.RTM. alkaline phosphatase chromogenic substrate by
Invitrogen.
Cell Immuno Staining of modRNA Produced Trastuzumab and
Rituximab
[0189] FIG. 5 shows CHO-K1 cells co-transfected with 500 ng each of
Heavy and Light Chain of Trastuzumab or Rituximab. Cells were grown
in F-12K Medium from Gibco and 10% PBS. Cells were fixed with 4%
paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in
PBS for 5-10 minutes at room temperature. Cells were then washed
3.times. with room temperature PBS. Trastuzumab and Rituximab
staining was performed using rabbit polyclonal antibody to Human
IgG conjugated to DyLight.RTM. 594 (ab96904, abcam, Cambridge,
Mass.) according to the manufacturer's recommended dilutions.
Nuclear DNA staining was performed with DAPI dye from Invitrogen.
The protein for Trastuzumab and Rituximab is translated and
localized to the cytoplasm upon modRNA transfection. The pictures
were taken 13 hours post-transfection.
Binding Immunoblot Assay for modRNA Produced Trastuzumab and
Rituximab
[0190] FIG. 6 shows a Binding Immunoblot detection assay for
Trastuzumab and Rituximab. Varying concentrations of the ErB2
peptide (ab40048, abcam, Cambridge, Mass.),
[0191] antigen for Trastuzumab and the CD20 peptide (ab97360,
abcam, Cambridge, Mass.), antigen for Rituximab were run at varying
concentrations (100 ng/ul to 0 ng/.mu.l) on a 12% SDS-Page gel and
transferred onto a membrane using the iBlot from Invitrogen. The
membranes were incubated for 1 hour with their respective cell
supernatants from CHO-K1 cells co-transfected with 500 ng each of
Heavy and Light Chain of Trastuzumab or Rituximab. The membranes
were blocked with 1% BSA and a secondary anti-human IgG antibody
conjugated to alkaline phosphatase (abcam, Cambridge, Mass.) was
added. Antibody detection was conducted using the Novex.RTM.
alkaline phosphatase chromogenic substrate by Invitrogen. This data
show that a humanized IgG antibodies generated from modRNA are
capable of recognizing and binding to their respective
antigens.
Cell Proliferation Assay
[0192] The SK-BR-3 cell line, an adherent cell line derived from a
human breast adenocarcinoma, which overexpress the HER2/neu
receptor can be used to compare the antiproliferative properties of
modRNA generated Trastuzumab. Varying concentrations of purified
Trastuzumab generated from modRNA and trastuzumab can be added to
cell cultures, and their effects on cell growth can be assessed in
triplicate cytotoxicity and viability assays.
SKOV-3 Tumor Model
[0193] The anti-cancer effects of modRNA generated Trastuzumab can
be determined by consecutive injections of 1) modRNA Trastuzumab,
2) trastuzumab, and 3) modRNA Trastuzumab+modRNA GCSF over a period
of 28 days in SKOV-3 xenograft mice. The reduction in tumor growth
size can be monitored over time.
Example 4. Overexpression of Ceramide Transfer Protein to Increase
Therapeutic Antibody Protein Production in Established CHO Cell
Lines
a) Batch Culture
[0194] An antibody producing CHO cell line (CHO DG44) secreting a
humanized therapeutic IgG antibody is transfected a single time
with lipid cationic delivery agent alone (control) or a synthetic
mRNA transcript encoding wild type ceramide transfer protein (CERT)
or a non-phosphorylation competent Ser132A CERT mutant. CERT is an
essential cytosolic protein in mammalian cells that transfers the
sphingolipid ceramide from the endoplasmic reticulum to the Golgi
complex where it is converted to sphingomyelin (Hanada et al.,
2003). Overexpression of CERT significantly enhances the transport
of secreted proteins to the plasma membrane and improves the
production of proteins that are transported via the secretory
pathway from eukaryotic cells thereby enhancing secretion of
protein s in the culture medium. Synthetic mRNA transcripts are
pre-mixed with a lipid cationic delivery agent at a 2-5:1
carrier:RNA ratio. The initial seeding density is about
2.times.10.sub.5 viable cells/mL. The synthetic mRNA transcript is
delivered after initial culture seeding during the exponential
culture growth phase to achieve a final synthetic mRNA copy number
between 10.times.10.sub.2 and 10.times.10.sub.3 per cell. The basal
cell culture medium used for all phases of cell inoculum generation
and for growth of cultures in bioreactors is modified CD-CHO medium
containing glutamine, sodium bicarbonate, insulin and methotrexate.
The pH of the medium is adjusted to 7.0 with 1 N HCl or 1N NaOH
after addition of all components. Culture run times end on days 7,
14, 21 or 28+. Production-level 50 L scale reactors (stainless
steel reactor with two marine impellers) may be used and are
scalable to >10,000 L stainless steel reactors (described in
commonly-assigned patent application U.S. Ser. No. 60/436,050,
filed Dec. 23, 2002, and U.S. Ser. No. 10/740,645). A data
acquisition system (Intellution Fix 32) records temperature, pH,
and dissolved oxygen (DO) throughout runs. Gas flows are controlled
via rotameters. Air is sparged into the reactor via a submerged
frit (5 nm pore size) and through the reactor head space for
CO.sub.2 removal. Molecular oxygen is sparged through the same frit
for DO control CO.sub.2 is sparged through same flit as used for pH
control. Samples of cells are removed from the reactor on a daily
basis. A sample used for cell counting is stained with trypan blue
(Sigma, St. Louis, Mo.). Cell count and cell viability
determination are performed via hemocytometry using a microscope.
For analysis of metabolites, additional samples are centrifuged for
20 minutes at 2000 rpm (4.degree. C.) for cell separation.
Supernatant is analyzed for the following parameters: titer, sialic
acid, glucose, lactate, glutamine, glutamate, pH, pO.sub.2,
pCO.sub.2, ammonia, and, optionally, lactate dehydrogenase (LDH).
Additional back-up samples are frozen at -20.degree. C. To measure
secreted humanized IgG antibody titers, supernatant is taken from
seed-stock cultures of all stable cell pools, the IgG titer is
determined by ELISA and divided by the mean number of cells to
calculate the specific productivity. The highest values are the
cell pools with the Ser132A CERT mutant (SEQ ID No. 14), followed
by wild type CERT (SEQ ID No. 12. In both, IgG expression is
markedly enhanced compared to carrier-alone or untransfected
cells.
b) Continuous or Batch-Fed Culture
[0195] An antibody producing CHO cell line (CHO DG44) secreting
humanized IgG antibody is transfected with lipid cationic delivery
agent alone (control) or a synthetic mRNA transcript encoding wild
type ceramide transfer protein or a non-phosphorylation competent
Ser132A CERT mutant. Synthetic mRNA transcripts are pre-mixed with
a lipid cationic delivery agent at a 2-5:1 carrier:RNA ratio. The
initial seeding density was about 2.times.10.sub.5 viable cells/mL.
Synthetic mRNA transcript is delivered after initial culture
seeding during the exponential culture growth phase to achieve a
final synthetic mRNA copy number between 10.times.10.sub.2 and
10.times.10.sub.3 per cell. The basal cell culture medium used for
all phases of cell inoculum generation and for growth of cultures
in bioreactors was modified CD-CHO medium containing lutamide,
sodium bicarbonate, insulin and methotrexate. The pH of the medium
is adjusted to 7.0 with 1 N HCl or 1N NaOH after addition of all
components. Bioreactors of 5 L scale (glass reactor with one marine
impeller) are used to obtain maximum CERT protein production and
secreted humanized IgG antibody curves. For continuous or fed-batch
cultures, the culturing run time is increased by supplementing the
culture medium one or more times daily (or continuously) with fresh
medium during the run. In the a continuous and fed-batch feeding
regimens, the cultures receive feeding medium as a
continuously-supplied infusion, or other automated addition to the
culture, in a timed, regulated, and/or programmed fashion so as to
achieve and maintain the appropriate amount of synthetic
mRNA:carrier in the culture. The typical method is a feeding
regimen of a once per day bolus feed with feeding medium containing
synthetic mRNA: carrier on each day of the culture run, from the
beginning of the culture run to the day of harvesting the cells.
The daily feed amount is recorded on batch sheets. Production-level
50 L scale reactors (stainless steel reactor with two murine
impellers) were used and are scalable to >10,000 L stainless
steel reactors. A data acquisition system (Intellution Fix 32)
record temperature, pH, and dissolved oxygen (DO) throughout runs.
Gas flows are controlled via rotameters. Air is sparged into the
reactor via a submerged frit (5 .mu.m pore size) and through the
reactor head space for CO.sub.2 removal. Molecular oxygen was
sparged through the same frit for DO control. CO.sub.2 is sparged
through same frit as used for pH control. Samples of cells are
removed from the reactor on a daily basis. A sample used for cell
counting is typically stained with trypan blue (Sigma, St. Louis,
Mo.). Cell count and cell viability determination are performed via
hemocytometry using a microscope. For analysis of metabolites,
additional samples are centrifuged for 20 minutes at 2000 rpm
(4.degree. C.) for cell separation. Supernatant is analyzed for the
following parameters titer, sialic acid, glucose, lactate,
glutamine, glutamate, pH, pO.sub.2, pCO.sub.2, ammonia, and,
optionally, lactate dehydrogenase (LDH). Additional back-up samples
are frozen at -20.degree. C. To measure secreted humanized IgG
antibody titers, supernatant is taken from seed-stock cultures of
all stable cell pools, the IgG titer is determined by ELISA and
divided by the mean number of cells to calculate the specific
productivity. The highest values are the cell pools with the
Ser132A CERT mutant (SEQ ID NO: 14), followed by wild type CERT
(SEQ ID NO: 10 or 12). In both, IgG expression is markedly enhanced
compared to carrier--alone or untransfected cells.
EQUIVALENTS AND SCOPE
[0196] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments, described herein. The
scope of the present disclosure is not intended to be limited to
the above Description, but rather is as set forth in the appended
claims.
[0197] 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
disclosure described herein. The scope of the present disclosure is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0198] 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 disclosure 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 disclosure 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. Furthermore, it is to be understood that the disclosure
encompasses all variations, combinations, and permutations in which
one or more limitations, elements, clauses, descriptive terms,
etc., from one or more of the listed claims is introduced into
another claim. For example, any claim that is dependent on another
claim can be modified to include one or more limitations found in
any other claim that is dependent on the same base claim.
Furthermore, where the claims recite a composition, it is to be
understood that methods of using the composition for any of the
purposes disclosed herein are included, and methods of making the
composition according to any of the methods of making disclosed
herein or other methods known in the art are included, unless
otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise.
[0199] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that each subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It should it be understood that, in general, where the
disclosure, or aspects of the disclosure, is/are referred to as
comprising particular elements, features, etc., certain embodiments
of the disclosure or aspects of the disclosure consist, or consist
essentially of, such elements, features, etc. For purposes of
simplicity those embodiments have not been specifically set forth
in haec verba herein. It is also noted that the term "comprising"
is intended to be open and permits the inclusion of additional
elements or steps.
[0200] 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 disclosure, to the tenth of the unit of the
lower limit of the range, unless the context clearly dictates
otherwise.
[0201] In addition, it is to be understood that any particular
embodiment of the present disclosure 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 disclosure (e.g., any protein; any nucleic
acid; 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.
[0202] 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.
[0203] Other embodiments are in the claims.
Sequence CWU 1
1
151852DNAHomo sapiens 1agcttttgga ccctcgtaca gaagctaata cgactcacta
tagggaaata agagagaaaa 60gaagagtaag aagaaatata agagccacca tggccggtcc
cgcgacccaa agccccatga 120aacttatggc cctgcagttg ctgctttggc
actcggccct ctggacagtc caagaagcga 180ctcctctcgg acctgcctca
tcgttgccgc agtcattcct tttgaagtgt ctggagcagg 240tgcgaaagat
tcagggcgat ggagccgcac tccaagagaa gctctgcgcg acatacaaac
300tttgccatcc cgaggagctc gtactgctcg ggcacagctt ggggattccc
tgggctcctc 360tctcgtcctg tccgtcgcag gctttgcagt tggcagggtg
cctttcccag ctccactccg 420gtttgttctt gtatcaggga ctgctgcaag
cccttgaggg aatctcgcca gaattgggcc 480cgacgctgga cacgttgcag
ctcgacgtgg cggatttcgc aacaaccatc tggcagcaga 540tggaggaact
ggggatggca cccgcgctgc agcccacgca gggggcaatg ccggcctttg
600cgtccgcgtt tcagcgcagg gcgggtggag tcctcgtagc gagccacctt
caatcatttt 660tggaagtctc gtaccgggtg ctgagacatc ttgcgcagcc
gtgaagcgct gccttctgcg 720gggcttgcct tctggccatg cccttcttct
ctcccttgca cctgtacctc ttggtctttg 780aataaagcct gagtaggaag
gcggccgctc gagcatgcat ctagagggcc caattcgccc 840tattcgaagt cg
8522852RNAHomo sapiens 2agcuuuugga cccucguaca gaagcuaaua cgacucacua
uagggaaaua agagagaaaa 60gaagaguaag aagaaauaua agagccacca uggccggucc
cgcgacccaa agccccauga 120aacuuauggc ccugcaguug cugcuuuggc
acucggcccu cuggacaguc caagaagcga 180cuccucucgg accugccuca
ucguugccgc agucauuccu uuugaagugu cuggagcagg 240ugcgaaagau
ucagggcgau ggagccgcac uccaagagaa gcucugcgcg acauacaaac
300uuugccaucc cgaggagcuc guacugcucg ggcacagcuu ggggauuccc
ugggcuccuc 360ucucguccug uccgucgcag gcuuugcagu uggcagggug
ccuuucccag cuccacuccg 420guuuguucuu guaucaggga cugcugcaag
cccuugaggg aaucucgcca gaauugggcc 480cgacgcugga cacguugcag
cucgacgugg cggauuucgc aacaaccauc uggcagcaga 540uggaggaacu
ggggauggca cccgcgcugc agcccacgca gggggcaaug ccggccuuug
600cguccgcguu ucagcgcagg gcggguggag uccucguagc gagccaccuu
caaucauuuu 660uggaagucuc guaccgggug cugagacauc uugcgcagcc
gugaagcgcu gccuucugcg 720gggcuugccu ucuggccaug cccuucuucu
cucccuugca ccuguaccuc uuggucuuug 780aauaaagccu gaguaggaag
gcggccgcuc gagcaugcau cuagagggcc caauucgccc 840uauucgaagu cg
8523852RNAArtificial SequenceDescription of Artificial Sequence
Synthetic
polynucleotidemodified_base(3)..(3)5-methyl-cytidinemodified_base(4)..(7)-
Pseudouridinemodified_base(11)..(13)5-methyl-cytidinemodified_base(14)..(1-
4)Pseudouridinemodified_base(15)..(15)5-methyl-cytidinemodified_base(17)..-
(17)Pseudouridinemodified_base(19)..(19)5-methyl-cytidinemodified_base(25)-
..(25)5-methyl-cytidinemodified_base(26)..(26)Pseduouridinemodified_base(2-
9)..(29)Pseduouridinemodified_base(31)..(31)5-methyl-cytidinemodified_base-
(34)..(34)5-methyl-cytidinemodified_base(35)..(35)Pseudouridinemodified_ba-
se(36)..(36)5-methyl-cytidinemodified_base(38)..(38)5-methyl-cytidinemodif-
ied_base(39)..(39)Pseudouridinemodified_base(41)..(41)Pseudouridinemodifie-
d_base(49)..(49)Pseudouridinemodified_base(67)..(67)Pseudouridinemodified_-
base(77)..(77)Pseudouridinemodified_base(79)..(79)Pseudouridinemodified_ba-
se(85)..(86)5-methyl-cytidinemodified_base(88)..(89)5-methyl-cytidinemodif-
ied_base(91)..(91)Pseudouridinemodified_base(94)..(95)5-methyl-cytidinemod-
ified_base(98)..(98)Pseudouridinemodified_base(99)..(101)5-methyl-cytidine-
modified_base(103)..(103)5-methyl-cytidinemodified_base(106)..(108)5-methy-
l-cytidinemodified_base(113)..(116)5-methyl-cytidinemodified_base(118)..(1-
18)Pseudouridinemodified_base(123)..(123)5-methyl-cytidinemodified_base(12-
4)..(125)Pseudouridinemodified_base(127)..(127)Pseudouridinemodified_base(-
130)..(132)5-methyl-cytidinemodified_base(133)..(133)Pseudouridinemodified-
_base(135)..(135)5-methyl-cytidinemodified_base(138)..(139)Pseudouridinemo-
dified_base(141)..(141)5-methyl-cytidinemodified_base(144)..(144)5-methyl--
cytidinemodified_base(145)..(147)Pseudouridinemodified_base(150)..(150)5-m-
ethyl-cytidinemodified_base(152)..(152)5-methyl-cytidinemodified_base(153)-
..(153)Pseudouridinemodified_base(154)..(154)5-methyl-cytidinemodified_bas-
e(157)..(159)5-methyl-cytidinemodified_base(160)..(160)Pseudouridinemodifi-
ed_base(161)..(161)5-methyl-cytidinemodified_base(162)..(162)Pseudouridine-
modified_base(166)..(166)5-methyl-cytidinemodified_base(169)..(169)Pseudou-
ridinemodified_base(170)..(171)5-methyl-cytidinemodified_base(178)..(178)5-
-methyl-cytidinemodified_base(181)..(181)5-methyl-cytidinemodified_base(18-
2)..(182)Pseudouridinemodified_base(183)..(184)5-methyl-cytidinemodified_b-
ase(185)..(185)Pseudouridinemodified_base(186)..(186)5-methyl-cytidinemodi-
fied_base(187)..(187)Pseudouridinemodified_base(188)..(188)5-methyl-cytidi-
nemodified_base(192)..(193)5-methyl-cytidinemodified_base(194)..(194)Pseud-
ouridinemodified_base(196)..(197)5-methyl-cytidinemodified_base(198)..(198-
)Pseudouridinemodified_base(199)..(199)5-methyl-cytidinemodified_base(201)-
..(201)Pseudouridinemodified_base(202)..(202)5-methyl-cytidinemodified_bas-
e(204)..(205)Pseudouridinemodified_base(207)..(208)5-methyl-cytidinemodifi-
ed_base(210)..(210)5-methyl-cytidinemodified_base(213)..(213)Pseudouridine-
modified_base(214)..(214)5-methyl-cytidinemodified_base(216)..(217)Pseudou-
ridinemodified_base(218)..(219)5-methyl-cytidinemodified_base(220)..(223)P-
seudouridinemodified_base(228)..(228)Pseudouridinemodified_base(230)..(230-
)Pseudouridinemodified_base(231)..(231)5-methyl-cytidinemodified_base(232)-
..(232)Pseudouridinemodified_base(237)..(237)5-methyl-cytidinemodified_bas-
e(241)..(241)Pseudouridinemodified_base(243)..(243)5-methyl-cytidinemodifi-
ed_base(250)..(250)Pseudouridinemodified_base(252)..(252)5-methyl-cytidine-
modified_base(257)..(257)5-methyl-cytidinemodified_base(260)..(260)Pseudou-
ridinemodified_base(265)..(266)5-methyl-cytidinemodified_base(268)..(268)5-
-methyl-cytidinemodified_base(270)..(270)5-methyl-cytidinemodified_base(27-
1)..(271)Pseudouridinemodified_base(272)..(273)5-methyl-cytidinemodified_b-
ase(282)..(282)5-methyl-cytidinemodified_base(283)..(283)Pseudouridinemodi-
fied_base(284)..(284)5-methyl-cytidinemodified_base(285)..(285)Pseudouridi-
nemodified_base(287)..(287)5-methyl-cytidinemodified_base(289)..(289)5-met-
hyl-cytidinemodified_base(292)..(292)5-methyl-cytidinemodified_base(294)..-
(294)Pseudouridinemodified_base(296)..(296)5-methyl-cytidinemodified_base(-
300)..(300)5-methyl-cytidinemodified_base(301)..(303)Pseudouridinemodified-
_base(305)..(306)5-methyl-cytidinemodified_base(308)..(308)Pseudouridinemo-
dified_base(309)..(311)5-methyl-cytidinemodified_base(318)..(318)5-methyl--
cytidinemodified_base(319)..(319)Pseudouridinemodified_base(320)..(320)5-m-
ethyl-cytidinemodified_base(322)..(322)Pseudouridinemodified_base(325)..(3-
25)Pseudouridinemodified_base(327)..(327)5-methyl-cytidinemodified_base(32-
8)..(328)Pseudouridinemodified_base(329)..(329)5-methyl-cytidinemodified_b-
ase(333)..(333)5-methyl-cytidinemodified_base(335)..(335)5-methyl-cytidine-
modified_base(338)..(338)5-methyl-cytidinemodified_base(339)..(340)Pseudou-
ridinemodified_base(346)..(347)Pseudouridinemodified_base(348)..(350)5-met-
hyl-cytidinemodified_base(351)..(351)Pseudouridinemodified_base(355)..(355-
)5-methyl-cytidinemodified_base(356)..(356)Pseudouridinemodified_base(357)-
..(358)5-methyl-cytidinemodified_base(359)..(359)Pseudouridinemodified_bas-
e(360)..(360)5-methyl-cytidinemodified_base(361)..(361)Pseudouridinemodifi-
ed_base(362)..(362)5-methyl-cytidinemodified_base(363)..(363)Pseudouridine-
modified_base(364)..(364)5-methyl-cytidinemodified_base(366)..(366)Pseudou-
ridinemodified_base(367)..(368)5-methyl-cytidinemodified_base(369)..(369)P-
seudouridinemodified_base(371)..(371)Pseudouridinemodified_base(372)..(373-
)5-methyl-cytidinemodified_base(375)..(375)Pseudouridinemodified_base(376)-
..(376)5-methyl-cytidinemodified_base(378)..(378)5-methyl-cytidinemodified-
_base(382)..(382)5-methyl-cytidinemodified_base(383)..(385)Pseudouridinemo-
dified_base(387)..(387)5-methyl-cytidinemodified_base(390)..(391)Pseudouri-
dinemodified_base(394)..(394)5-methyl-cytidinemodified_base(399)..(399)Pse-
udouridinemodified_base(403)..(405)Pseudouridinemodified_base(406)..(408)5-
-methyl-cytidinemodified_base(411)..(411)5-methyl-cytidinemodified_base(41-
2)..(412)Pseudouridinemodified_base(413)..(414)5-methyl-cytidinemodified_b-
ase(416)..(416)5-methyl-cytidinemodified_base(417)..(417)Pseudouridinemodi-
fied_base(418)..(419)5-methyl-cytidinemodified_base(422)..(424)Pseudouridi-
nemodified_base(426)..(427)Pseudouridinemodified_base(428)..(428)5-methyl--
cytidinemodified_base(429)..(430)Pseudouridinemodified_base(432)..(432)Pse-
udouridinemodified_base(434)..(434)Pseudouridinemodified_base(435)..(435)5-
-methyl-cytidinemodified_base(441)..(441)5-methyl-cytidinemodified_base(44-
2)..(442)Pseudouridinemodified_base(444)..(444)5-methyl-cytidinemodified_b-
ase(445)..(445)Pseudouridinemodified_base(447)..(447)5-methyl-cytidinemodi-
fied_base(451)..(453)5-methyl-cytidinemodified_base(454)..(455)Pseudouridi-
nemodified_base(463)..(463)Pseudouridinemodified_base(464)..(464)5-methyl--
cytidinemodified_base(465)..(465)Pseudouridinemodified_base(466)..(466)5-m-
ethyl-cytidinemodified_base(468)..(469)5-methyl-cytidinemodified_base(474)-
..(475)Pseudouridinemodified_base(479)..(481)5-methyl-cytidinemodified_bas-
e(484)..(484)5-methyl-cytidinemodified_base(486)..(486)5-methyl-cytidinemo-
dified_base(487)..(487)Pseudouridinemodified_base(491)..(491)5-methyl-cyti-
dinemodified_base(493)..(493)5-methyl-cytidinemodified_base(495)..(496)Pse-
udouridinemodified_base(498)..(498)5-methyl-cytidinemodified_base(501)..(5-
01)5-methyl-cytidinemodified_base(502)..(502)Pseudouridinemodified_base(50-
3)..(503)5-methyl-cytidinemodified_base(506)..(506)5-methyl-cytidinemodifi-
ed_base(508)..(508)Pseudouridinemodified_base(511)..(511)5-methyl-cytidine-
modified_base(515)..(517)Pseudouridinemodified_base(518)..(518)5-methyl-cy-
tidinemodified_base(520)..(520)5-methyl-cytidinemodified_base(523)..(523)5-
-methyl-cytidinemodified_base(526)..(527)5-methyl-cytidinemodified_base(52-
9)..(529)Pseudouridinemodified_base(530)..(530)5-methyl-cytidinemodified_b-
ase(531)..(531)Pseudouridinemodified_base(534)..(534)5-methyl-cytidinemodi-
fied_base(537)..(537)5-methyl-cytidinemodified_base(541)..(541)Pseudouridi-
nemodified_base(549)..(549)5-methyl-cytidinemodified_base(550)..(550)Pseud-
ouridinemodified_base(556)..(556)Pseudouridinemodified_base(559)..(559)5-m-
ethyl-cytidinemodified_base(561)..(563)5-methyl-cytidinemodified_base(565)-
..(565)5-methyl-cytidinemodified_base(567)..(567)5-methyl-cytidinemodified-
_base(568)..(568)Pseudouridinemodified_base(570)..(570)5-methyl-cytidinemo-
dified_base(573)..(575)5-methyl-cytidinemodified_base(577)..(577)5-methyl--
cytidinemodified_base(579)..(579)5-methyl-cytidinemodified_base(586)..(586-
)5-methyl-cytidinemodified_base(589)..(589)Pseudouridinemodified_base(591)-
..(592)5-methyl-cytidinemodified_base(595)..(596)5-methyl-cytidinemodified-
_base(597)..(599)Pseudouridinemodified_base(601)..(601)5-methyl-cytidinemo-
dified_base(603)..(603)Pseudouridinemodified_base(604)..(604)5-methyl-cyti-
dinemodified_base(607)..(607)5-methyl-cytidinemodified_base(609)..(611)Pse-
udouridinemodified_base(612)..(612)5-methyl-cytidinemodified_base(615)..(6-
15)5-methyl-cytidinemodified_base(617)..(617)5-methyl-cytidinemodified_bas-
e(622)..(622)5-methyl-cytidinemodified_base(626)..(626)Pseudouridinemodifi-
ed_base(631)..(631)Pseudouridinemodified_base(632)..(633)5-methyl-cytidine-
modified_base(634)..(634)Pseudouridinemodified_base(635)..(635)5-methyl-cy-
tidinemodified_base(637)..(637)Pseudouridinemodified_base(640)..(640)5-met-
hyl-cytidinemodified_base(644)..(645)5-methyl-cytidinemodified_base(647)..-
(648)5-methyl-cytidinemodified_base(649)..(650)Pseudouridinemodified_base(-
651)..(651)5-methyl-cytidinemodified_base(654)..(654)Pseudouridinemodified-
_base(655)..(655)5-methyl-cytidinemodified_base(657)..(661)Pseudouridinemo-
dified_base(667)..(667)Pseudouridinemodified_base(668)..(668)5-methyl-cyti-
dinemodified_base(669)..(669)Pseudouridinemodified_base(670)..(670)5-methy-
l-cytidinemodified_base(672)..(672)Pseudouridinemodified_base(674)..(675)5-
-methyl-cytidinemodified_base(679)..(679)Pseudouridinemodified_base(681)..-
(681)5-methyl-cytidinemodified_base(682)..(682)Pseudouridinemodified_base(-
687)..(687)5-methyl-cytidinemodified_base(689)..(689)Pseudouridinemodified-
_base(690)..(690)5-methyl-cytidinemodified_base(691)..(692)Pseudouridinemo-
dified_base(694)..(694)5-methyl-cytidinemodified_base(696)..(696)5-methyl--
cytidinemodified_base(699)..(700)5-methyl-cytidinemodified_base(702)..(702-
)Pseudouridinemodified_base(707)..(707)5-methyl-cytidinemodified_base(709)-
..(709)5-methyl-cytidinemodified_base(710)..(710)Pseudouridinemodified_bas-
e(712)..(713)5-methyl-cytidinemodified_base(714)..(715)Pseudouridinemodifi-
ed_base(716)..(716)5-methyl-cytidinemodified_base(717)..(717)Pseudouridine-
modified_base(719)..(719)5-methyl-cytidinemodified_base(724)..(724)5-methy-
l-cytidinemodified_base(725)..(726)Pseudouridinemodified_base(728)..(729)5-
-methyl-cytidinemodified_base(730)..(731)Pseudouridinemodified_base(732)..-
(732)5-methyl-cytidinemodified_base(733)..(733)Pseudouridinemodified_base(-
736)..(737)5-methyl-cytidinemodified_base(739)..(739)Pseudouridinemodified-
_base(741)..(743)5-methyl-cytidinemodified_base(744)..(745)Pseudouridinemo-
dified_base(746)..(746)5-methyl-cytidinemodified_base(747)..(748)Pseudouri-
dinemodified_base(749)..(749)5-methyl-cytidinemodified_base(750)..(750)Pse-
udouridinemodified_base(751)..(751)5-methyl-cytidinemodified_base(752)..(7-
52)Pseudouridinemodified_base(753)..(755)5-methyl-cytidinemodified_base(75-
6)..(757)Pseudouridinemodified_base(759)..(759)5-methyl-cytidinemodified_b-
ase(761)..(762)5-methyl-cytidinemodified_base(763)..(763)Pseudouridinemodi-
fied_base(765)..(765)Pseudouridinemodified_base(767)..(768)5-methyl-cytidi-
nemodified_base(769)..(769)Pseudouridinemodified_base(770)..(770)5-methyl--
cytidinemodified_base(771)..(772)Pseudouridinemodified_base(775)..(775)Pse-
udouridinemodified_base(776)..(776)5-methyl-cytidinemodified_base(777)..(7-
79)Pseudouridinemodified_base(783)..(783)Pseudouridinemodified_base(788)..-
(789)5-methyl-cytidinemodified_base(790)..(790)Pseudouridinemodified_base(-
794)..(794)Pseudouridinemodified_base(802)..(802)5-methyl-cytidinemodified-
_base(805)..(806)5-methyl-cytidinemodified_base(808)..(808)5-methyl-cytidi-
nemodified_base(809)..(809)Pseudouridinemodified_base(810)..(810)5-methyl--
cytidinemodified_base(814)..(814)5-methyl-cytidinemodified_base(816)..(816-
)Pseudouridinemodified_base(818)..(818)5-methyl-cytidinemodified_base(820)-
..(820)Pseudouridinemodified_base(821)..(821)5-methyl-cytidinemodified_bas-
e(822)..(822)Pseudouridinemodified_base(829)..(831)5-methyl-cytidinemodifi-
ed_base(834)..(835)Pseudouridinemodified_base(836)..(836)5-methyl-cytidine-
modified_base(838)..(840)5-methyl-cytidinemodified_base(841)..(841)Pseudou-
ridinemodified_base(843)..(844)Pseudouridinemodified_base(845)..(845)5-met-
hyl-cytidinemodified_base(850)..(850)Pseudouridinemodified_base(851)..(851-
)5-methyl-cytidine 3agcuuuugga cccucguaca gaagcuaaua cgacucacua
uagggaaaua agagagaaaa 60gaagaguaag aagaaauaua agagccacca uggccggucc
cgcgacccaa agccccauga 120aacuuauggc ccugcaguug cugcuuuggc
acucggcccu cuggacaguc caagaagcga 180cuccucucgg accugccuca
ucguugccgc agucauuccu uuugaagugu cuggagcagg 240ugcgaaagau
ucagggcgau ggagccgcac uccaagagaa gcucugcgcg acauacaaac
300uuugccaucc cgaggagcuc guacugcucg ggcacagcuu ggggauuccc
ugggcuccuc 360ucucguccug uccgucgcag gcuuugcagu uggcagggug
ccuuucccag cuccacuccg 420guuuguucuu guaucaggga cugcugcaag
cccuugaggg aaucucgcca gaauugggcc 480cgacgcugga cacguugcag
cucgacgugg cggauuucgc aacaaccauc uggcagcaga 540uggaggaacu
ggggauggca cccgcgcugc agcccacgca gggggcaaug ccggccuuug
600cguccgcguu ucagcgcagg gcggguggag uccucguagc gagccaccuu
caaucauuuu 660uggaagucuc guaccgggug cugagacauc uugcgcagcc
gugaagcgcu gccuucugcg 720gggcuugccu ucuggccaug cccuucuucu
cucccuugca ccuguaccuc uuggucuuug 780aauaaagccu gaguaggaag
gcggccgcuc gagcaugcau cuagagggcc caauucgccc 840uauucgaagu cg
85241635DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 4ctcgtacaga agctaatacg actcactata
gggaaataag agagaaaaga agagtaagaa 60gaaatataag agccaccatg gccgtgatgg
cgccgaggac cctggtgctc ttgctcacgg 120gtgccttggc cctcacgcaa
acatgggcgg gacaggcgta cttgcagcag tcaggggcag 180aactcgtaag
gcccggagcg tcggtgaaga tgtcgtgtaa agcgtcgggc tatactttca
240catcgtacaa catgcactgg gtcaaacaga cgccccgaca agggctggag
tggattggag 300ctatctaccc cggtaacggg gatacgtcgt acaaccagaa
gtttaagggg aaggcgactc 360ttactgtcga caagtcgtcc tccaccgcct
atatgcagct gtcgagcctg acttcggaag 420attcagcggt gtacttttgt
gcgcgcgtgg tctattactc aaattcgtat tggtatttcg 480atgtgtgggg
tacggggacc actgtgaccg tgtcaggacc ctcggtattc cccctcgcgc
540ctagctcaaa gtccacctcc gggggaacag ccgccttggg ttgcttggta
aaggactatt 600tccccgagcc cgtcacagtg agctggaact ccggggcact
gacatcggga gtgcacacgt 660ttcccgcggt acttcagtca tcaggactct
actcgctgtc aagcgtggtc acggtgcctt 720catcctccct tggaacgcag
acttacatct gcaacgtgaa tcataagcct agcaatacca 780aggtcgacaa
gaaagccgaa cccaaatcat gtgataaaac acacacgtgt cctccctgcc
840ccgcaccgga gcttctcggg ggaccgagcg tgttcttgtt tccacctaag
ccgaaagata 900cgcttatgat ctcccggacc cccgaagtaa cttgcgtagt
agtagacgta agccacgagg 960accccgaagt gaaattcaat tggtacgtcg
acggagtgga ggtccataat gcgaaaacaa 1020agccgagaga ggaacagtac
aattccacat accgcgtcgt aagcgtcttg acagtattgc 1080atcaggattg
gctgaacgga aaggaataca agtgcaaagt atcaaacaaa gcacttccgg
1140caccgattga aaagacgatc tcaaaagcaa aagggcaacc tcgggagcca
caagtctata 1200ctctcccgcc gtcgcgcgat gaattgacca aaaaccaggt
gtcccttaca tgtctcgtaa 1260agggttttta cccgtcagac atcgccgtcg
agtgggagtc aaacggtcag ccggagaata 1320actataagac gaccccacca
gtcttggaca gcgatggctc cttcttcttg tattcaaagc 1380tgacggtgga
caaatcgaga tggcagcagg gtaatgtgtt ttcgtgcagc gtcatgcacg
1440aggcgcttca taatcattac actcaaaagt ccctgtcgct gtcgcccgga
aagcaccatc 1500accaccacca ttgaagcgct gccttctgcg gggcttgcct
tctggccatg cccttcttct 1560ctcccttgca cctgtacctc ttggtctttg
aataaagcct gagtaggaag gcggccgctc 1620gagcatgcat ctaga
163551635RNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 5cucguacaga agcuaauacg acucacuaua
gggaaauaag agagaaaaga agaguaagaa 60gaaauauaag agccaccaug gccgugaugg
cgccgaggac ccuggugcuc uugcucacgg 120gugccuuggc ccucacgcaa
acaugggcgg gacaggcgua cuugcagcag ucaggggcag
180aacucguaag gcccggagcg ucggugaaga ugucguguaa agcgucgggc
uauacuuuca 240caucguacaa caugcacugg gucaaacaga cgccccgaca
agggcuggag uggauuggag 300cuaucuaccc cgguaacggg gauacgucgu
acaaccagaa guuuaagggg aaggcgacuc 360uuacugucga caagucgucc
uccaccgccu auaugcagcu gucgagccug acuucggaag 420auucagcggu
guacuuuugu gcgcgcgugg ucuauuacuc aaauucguau ugguauuucg
480augugugggg uacggggacc acugugaccg ugucaggacc cucgguauuc
ccccucgcgc 540cuagcucaaa guccaccucc gggggaacag ccgccuuggg
uugcuuggua aaggacuauu 600uccccgagcc cgucacagug agcuggaacu
ccggggcacu gacaucggga gugcacacgu 660uucccgcggu acuucaguca
ucaggacucu acucgcuguc aagcgugguc acggugccuu 720cauccucccu
uggaacgcag acuuacaucu gcaacgugaa ucauaagccu agcaauacca
780aggucgacaa gaaagccgaa cccaaaucau gugauaaaac acacacgugu
ccucccugcc 840ccgcaccgga gcuucucggg ggaccgagcg uguucuuguu
uccaccuaag ccgaaagaua 900cgcuuaugau cucccggacc cccgaaguaa
cuugcguagu aguagacgua agccacgagg 960accccgaagu gaaauucaau
ugguacgucg acggagugga gguccauaau gcgaaaacaa 1020agccgagaga
ggaacaguac aauuccacau accgcgucgu aagcgucuug acaguauugc
1080aucaggauug gcugaacgga aaggaauaca agugcaaagu aucaaacaaa
gcacuuccgg 1140caccgauuga aaagacgauc ucaaaagcaa aagggcaacc
ucgggagcca caagucuaua 1200cucucccgcc gucgcgcgau gaauugacca
aaaaccaggu gucccuuaca ugucucguaa 1260aggguuuuua cccgucagac
aucgccgucg agugggaguc aaacggucag ccggagaaua 1320acuauaagac
gaccccacca gucuuggaca gcgauggcuc cuucuucuug uauucaaagc
1380ugacggugga caaaucgaga uggcagcagg guaauguguu uucgugcagc
gucaugcacg 1440aggcgcuuca uaaucauuac acucaaaagu cccugucgcu
gucgcccgga aagcaccauc 1500accaccacca uugaagcgcu gccuucugcg
gggcuugccu ucuggccaug cccuucuucu 1560cucccuugca ccuguaccuc
uuggucuuug aauaaagccu gaguaggaag gcggccgcuc 1620gagcaugcau cuaga
16356909DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 6ctcgtacaga agctaatacg actcactata
gggaaataag agagaaaaga agagtaagaa 60gaaatataag agccaccatg gctgtcatgg
ccccgagaac acttgtgctg ttgttgacag 120gagcgctcgc actcacacag
acttgggccg gtcagattgt gctcagccag tcgccagcga 180tcctttcggc
ctcccctggt gagaaagtaa cgatgacgtg ccgagcctcc tcaagcgtgt
240catacatgca ttggtatcag cagaagcctg ggtcgtcgcc caagccctgg
atctacgccc 300cgtccaatct tgcgtcaggg gtcccggcac ggttcagcgg
atcggggtcg ggtacatcgt 360attcactcac gattagccgc gtagaggccg
aggacgcggc gacttactac tgtcagcaat 420ggtcctttaa tccacccacg
tttggagcgg gcaccaagct cgaacttaaa agaacggtcg 480ccgcaccctc
agtgtttatc ttcccgccct cggacgaaca acttaagtcg gggaccgctt
540ccgtggtgtg cttgctgaac aatttctatc ctcgggaagc taaagtgcaa
tggaaagtcg 600ataacgcatt gcagagcgga aactcacaag agtcggtaac
tgagcaggat agcaaggatt 660cgacatactc gctgagcagc acgctgacgt
tgtccaaggc ggactacgag aaacacaagg 720tatatgcgtg tgaagtcacc
caccagggat tgtcatcgcc ggtcaccaaa tcattcaaca 780ggtgataaag
cgctgccttc tgcggggctt gccttctggc catgcccttc ttctctccct
840tgcacctgta cctcttggtc tttgaataaa gcctgagtag gaaggcggcc
gctcgagcat 900gcatctaga 9097909RNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 7cucguacaga agcuaauacg
acucacuaua gggaaauaag agagaaaaga agaguaagaa 60gaaauauaag agccaccaug
gcugucaugg ccccgagaac acuugugcug uuguugacag 120gagcgcucgc
acucacacag acuugggccg gucagauugu gcucagccag ucgccagcga
180uccuuucggc cuccccuggu gagaaaguaa cgaugacgug ccgagccucc
ucaagcgugu 240cauacaugca uugguaucag cagaagccug ggucgucgcc
caagcccugg aucuacgccc 300cguccaaucu ugcgucaggg gucccggcac
gguucagcgg aucggggucg gguacaucgu 360auucacucac gauuagccgc
guagaggccg aggacgcggc gacuuacuac ugucagcaau 420gguccuuuaa
uccacccacg uuuggagcgg gcaccaagcu cgaacuuaaa agaacggucg
480ccgcacccuc aguguuuauc uucccgcccu cggacgaaca acuuaagucg
gggaccgcuu 540ccguggugug cuugcugaac aauuucuauc cucgggaagc
uaaagugcaa uggaaagucg 600auaacgcauu gcagagcgga aacucacaag
agucgguaac ugagcaggau agcaaggauu 660cgacauacuc gcugagcagc
acgcugacgu uguccaaggc ggacuacgag aaacacaagg 720uauaugcgug
ugaagucacc caccagggau ugucaucgcc ggucaccaaa ucauucaaca
780ggugauaaag cgcugccuuc ugcggggcuu gccuucuggc caugcccuuc
uucucucccu 840ugcaccugua ccucuugguc uuugaauaaa gccugaguag
gaaggcggcc gcucgagcau 900gcaucuaga 90981647DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
8ctcgtacaga agctaatacg actcactata gggaaataag agagaaaaga agagtaagaa
60gaaatataag agccaccatg gccgtgatgg cgccgcggac cctggtcctc ctgctgaccg
120gcgccctcgc cctgacgcag acctgggccg gggaggtgca gctggtcgag
agcggcgggg 180gcctcgtgca gccgggcggg tcgctgcggc tgagctgcgc
cgcgagcggg ttcaacatca 240aggacaccta catccactgg gtgcgccagg
cccccggcaa gggcctcgag tgggtcgccc 300ggatctaccc cacgaacggg
tacacccgct acgccgacag cgtgaagggc cggttcacca 360tcagcgcgga
cacctcgaag aacacggcct acctgcagat gaacagcctg cgcgccgagg
420acaccgccgt gtactactgc agccggtggg gcggcgacgg gttctacgcc
atggactact 480gggggcaggg caccctcgtc accgtgagca gcgcgtcgac
gaaggggccc agcgtgttcc 540cgctggcccc cagcagcaag agcaccagcg
gcgggaccgc cgccctgggc tgcctcgtca 600aggactactt ccccgagccc
gtgaccgtgt cgtggaacag cggcgcgctg acgagcgggg 660tccacacctt
cccggccgtg ctgcagagca gcggcctcta ctcgctgagc agcgtggtca
720ccgtgcccag cagcagcctg gggacccaga cgtacatctg caacgtgaac
cacaagccct 780cgaacaccaa ggtcgacaag aaggtggagc ccccgaagag
ctgcgacaag acccacacct 840gcccgccctg ccccgccccc gagctcctgg
gcgggcccag cgtgttcctg ttcccgccca 900agcccaagga cacgctcatg
atcagccgca cccccgaggt cacctgcgtg gtggtcgacg 960tgagccacga
ggaccccgag gtgaagttca actggtacgt cgacggcgtg gaggtgcaca
1020acgccaagac caagccgcgg gaggagcagt acaactcgac gtaccgcgtc
gtgagcgtgc 1080tgaccgtcct gcaccaggac tggctcaacg gcaaggagta
caagtgcaag gtgagcaaca 1140aggccctgcc cgcgcccatc gagaagacca
tcagcaaggc caaggggcag ccccgggagc 1200cgcaggtgta caccctgccc
cccagccgcg acgagctcac gaagaaccag gtcagcctga 1260cctgcctggt
gaagggcttc tacccctcgg acatcgccgt ggagtgggag agcaacgggc
1320agccggagaa caactacaag accaccccgc ccgtcctcga cagcgacggc
agcttcttcc 1380tgtacagcaa gctgacggtg gacaagtcgc ggtggcagca
gggcaacgtg ttcagctgca 1440gcgtcatgca cgaggccctc cacaaccact
acacccagaa gagcctgagc ctgagccccg 1500ggaagcatca tcatcatcat
cattgaagcg ctgccttctg cggggcttgc cttctggcca 1560tgcccttctt
ctctcccttg cacctgtacc tcttggtctt tgaataaagc ctgagtagga
1620aggcggccgc tcgagcatgc atctaga 164791647RNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
9cucguacaga agcuaauacg acucacuaua gggaaauaag agagaaaaga agaguaagaa
60gaaauauaag agccaccaug gccgugaugg cgccgcggac ccugguccuc cugcugaccg
120gcgcccucgc ccugacgcag accugggccg gggaggugca gcuggucgag
agcggcgggg 180gccucgugca gccgggcggg ucgcugcggc ugagcugcgc
cgcgagcggg uucaacauca 240aggacaccua cauccacugg gugcgccagg
cccccggcaa gggccucgag ugggucgccc 300ggaucuaccc cacgaacggg
uacacccgcu acgccgacag cgugaagggc cgguucacca 360ucagcgcgga
caccucgaag aacacggccu accugcagau gaacagccug cgcgccgagg
420acaccgccgu guacuacugc agccgguggg gcggcgacgg guucuacgcc
auggacuacu 480gggggcaggg cacccucguc accgugagca gcgcgucgac
gaaggggccc agcguguucc 540cgcuggcccc cagcagcaag agcaccagcg
gcgggaccgc cgcccugggc ugccucguca 600aggacuacuu ccccgagccc
gugaccgugu cguggaacag cggcgcgcug acgagcgggg 660uccacaccuu
cccggccgug cugcagagca gcggccucua cucgcugagc agcgugguca
720ccgugcccag cagcagccug gggacccaga cguacaucug caacgugaac
cacaagcccu 780cgaacaccaa ggucgacaag aagguggagc ccccgaagag
cugcgacaag acccacaccu 840gcccgcccug ccccgccccc gagcuccugg
gcgggcccag cguguuccug uucccgccca 900agcccaagga cacgcucaug
aucagccgca cccccgaggu caccugcgug guggucgacg 960ugagccacga
ggaccccgag gugaaguuca acugguacgu cgacggcgug gaggugcaca
1020acgccaagac caagccgcgg gaggagcagu acaacucgac guaccgcguc
gugagcgugc 1080ugaccguccu gcaccaggac uggcucaacg gcaaggagua
caagugcaag gugagcaaca 1140aggcccugcc cgcgcccauc gagaagacca
ucagcaaggc caaggggcag ccccgggagc 1200cgcaggugua cacccugccc
cccagccgcg acgagcucac gaagaaccag gucagccuga 1260ccugccuggu
gaagggcuuc uaccccucgg acaucgccgu ggagugggag agcaacgggc
1320agccggagaa caacuacaag accaccccgc ccguccucga cagcgacggc
agcuucuucc 1380uguacagcaa gcugacggug gacaagucgc gguggcagca
gggcaacgug uucagcugca 1440gcgucaugca cgaggcccuc cacaaccacu
acacccagaa gagccugagc cugagccccg 1500ggaagcauca ucaucaucau
cauugaagcg cugccuucug cggggcuugc cuucuggcca 1560ugcccuucuu
cucucccuug caccuguacc ucuuggucuu ugaauaaagc cugaguagga
1620aggcggccgc ucgagcaugc aucuaga 164710918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
10ctcgtacaga agctaatacg actcactata gggaaataag agagaaaaga agagtaagaa
60gaaatataag agccaccatg gccgtgatgg cgccgcggac cctggtcctc ctgctgaccg
120gcgccctcgc cctgacgcag acctgggccg gggacatcca gatgacccag
agcccgtcga 180gcctgagcgc cagcgtgggc gaccgggtca cgatcacctg
ccgcgcgagc caggacgtga 240acaccgccgt ggcctggtac cagcagaagc
ccgggaaggc ccccaagctc ctgatctact 300cggcgagctt cctgtacagc
ggcgtcccca gccggttcag cgggtcgcgc agcggcaccg 360acttcacgct
caccatcagc agcctgcagc cggaggactt cgccacctac tactgccagc
420agcactacac cacgcccccc accttcgggc agggcaccaa ggtggagatc
aagcggaccg 480tggccgcccc cagcgtcttc atcttcccgc ccagcgacga
gcagctgaag tcgggcacgg 540ccagcgtggt gtgcctcctg aacaacttct
acccccgcga ggcgaaggtc cagtggaagg 600tggacaacgc cctgcagagc
gggaacagcc aggagagcgt gaccgagcag gactcgaagg 660acagcaccta
cagcctcagc agcaccctga cgctgagcaa ggccgactac gagaagcaca
720aggtctacgc ctgcgaggtg acccaccagg ggctctcgag ccccgtgacc
aagagcttca 780accggggcga gtgctgaagc gctgccttct gcggggcttg
ccttctggcc atgcccttct 840tctctccctt gcacctgtac ctcttggtct
ttgaataaag cctgagtagg aaggcggccg 900ctcgagcatg catctaga
91811918RNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 11cucguacaga agcuaauacg acucacuaua
gggaaauaag agagaaaaga agaguaagaa 60gaaauauaag agccaccaug gccgugaugg
cgccgcggac ccugguccuc cugcugaccg 120gcgcccucgc ccugacgcag
accugggccg gggacaucca gaugacccag agcccgucga 180gccugagcgc
cagcgugggc gaccggguca cgaucaccug ccgcgcgagc caggacguga
240acaccgccgu ggccugguac cagcagaagc ccgggaaggc ccccaagcuc
cugaucuacu 300cggcgagcuu ccuguacagc ggcgucccca gccgguucag
cgggucgcgc agcggcaccg 360acuucacgcu caccaucagc agccugcagc
cggaggacuu cgccaccuac uacugccagc 420agcacuacac cacgcccccc
accuucgggc agggcaccaa gguggagauc aagcggaccg 480uggccgcccc
cagcgucuuc aucuucccgc ccagcgacga gcagcugaag ucgggcacgg
540ccagcguggu gugccuccug aacaacuucu acccccgcga ggcgaagguc
caguggaagg 600uggacaacgc ccugcagagc gggaacagcc aggagagcgu
gaccgagcag gacucgaagg 660acagcaccua cagccucagc agcacccuga
cgcugagcaa ggccgacuac gagaagcaca 720aggucuacgc cugcgaggug
acccaccagg ggcucucgag ccccgugacc aagagcuuca 780accggggcga
gugcugaagc gcugccuucu gcggggcuug ccuucuggcc augcccuucu
840ucucucccuu gcaccuguac cucuuggucu uugaauaaag ccugaguagg
aaggcggccg 900cucgagcaug caucuaga 918121797DNAHomo sapiens
12atgtcggata atcagagctg gaactcgtcg ggctcggagg aggatccaga gacggagtct
60gggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacat tcatgggtgg
120caggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatc
tgaagatgaa 180acagagtatg gctgcagagg atccatctgt cttagcaagg
ctgtcatcac acctcacgat 240tttgatgaat gtcgatttga tattagtgta
aatgatagtg tttggtatct tcgtgctcag 300gatccagatc atagacagca
atggatagat gccattgaac agcacaagac tgaatctgga 360tatggatctg
aatccagctt gcgtcgacat ggctcaatgg tgtccctggt gtctggagca
420agtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagttt
acgtgagaag 480ttggctgaaa tggaaacatt tagagacatc ttatgtagac
aagttgacac gctacagaag 540tactttgatg cctgtgctga tgctgtctct
aaggatgaac ttcaaaggga taaagtggta 600gaagatgatg aagatgactt
tcctacaacg cgttctgatg gtgacttctt gcatagtacc 660aacggcaata
aagaaaagtt atttccacat gtgacaccaa aaggaattaa tggtatagac
720tttaaagggg aagcgataac ttttaaagca actactgctg gaatccttgc
aacactttct 780cattgtattg aactaatggt taaacgtgag gacagctggc
agaagagact ggataaggaa 840actgagaaga aaagaagaac agaggaagca
tataaaaatg caatgacaga acttaagaaa 900aaatcccact ttggaggacc
agattatgaa gaaggcccta acagtctgat taatgaagaa 960gagttctttg
atgctgttga agctgctctt gacagacaag ataaaataga agaacagtca
1020cagagtgaaa aggtgagatt acattggcct acatccttgc cctctggaga
tgccttttct 1080tctgtgggga cacatagatt tgtccaaaag gttgaagaga
tggtgcagaa ccacatgact 1140tactcattac aggatgtagg cggagatgcc
aattggcagt tggttgtaga agaaggagaa 1200atgaaggtat acagaagaga
agtagaagaa aatgggattg ttctggatcc tttaaaagct 1260acccatgcag
ttaaaggcgt cacaggacat gaagtctgca attatttctg gaatgttgac
1320gttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaac
attagctgat 1380aatgcaatca tcatttatca aacacacaag agggtgtggc
ctgcttctca gcgagacgta 1440ttatatcttt ctgtcattcg aaagatacca
gccttgactg aaaatgaccc tgaaacttgg 1500atagtttgta atttttctgt
ggatcatgac agtgctcctc taaacaaccg atgtgtccgt 1560gccaaaataa
atgttgctat gatttgtcaa accttggtaa gcccaccaga gggaaaccag
1620gaaattagca gggacaacat tctatgcaag attacatatg tagctaatgt
gaaccctgga 1680ggatgggcac cagcctcagt gttaagggca gtggcaaagc
gagagtatcc taaatttcta 1740aaacgtttta cttcttacgt ccaagaaaaa
actgcaggaa agcctatttt gttctag 179713544PRTHomo sapiens 13Met Ser
Asp Asn Gln Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp Pro1 5 10 15Glu
Thr Glu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser Lys 20 25
30Trp Thr Asn Tyr Ile His Gly Trp Gln Asp Arg Trp Val Val Leu Lys
35 40 45Asn Asn Ala Leu Ser Tyr Tyr Lys Ser Glu Asp Glu Thr Glu Tyr
Gly 50 55 60Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala Val Ile Thr Pro
His Asp65 70 75 80Phe Asp Glu Cys Arg Phe Asp Ile Ser Val Asn Asp
Ser Val Trp Tyr 85 90 95Leu Arg Ala Gln Asp Pro Asp His Arg Gln Gln
Trp Ile Asp Ala Ile 100 105 110Glu Gln His Lys Thr Glu Ser Gly Tyr
Gly Ser Glu Ser Ser Leu Arg 115 120 125Arg His Gly Ser Met Val Ser
Leu Val Ser Gly Ala Ser Gly Tyr Ser 130 135 140Ala Thr Ser Thr Ser
Ser Phe Lys Lys Gly His Ser Leu Arg Glu Lys145 150 155 160Leu Ala
Glu Met Glu Thr Phe Arg Asp Ile Leu Cys Arg Gln Val Asp 165 170
175Thr Leu Gln Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys Asp
180 185 190Glu Leu Gln Arg Asp Lys Val Val Glu Asp Asp Glu Asp Asp
Phe Pro 195 200 205Thr Thr Arg Ser Asp Gly Asp Phe Leu His Ser Thr
Asn Gly Asn Lys 210 215 220Glu Lys Leu Phe Pro His Val Thr Pro Lys
Gly Ile Asn Gly Ile Asp225 230 235 240Phe Lys Gly Glu Ala Ile Thr
Phe Lys Ala Thr Thr Ala Gly Ile Leu 245 250 255Ala Thr Leu Ser His
Cys Ile Glu Leu Met Val Lys Arg Glu Asp Ser 260 265 270Trp Gln Lys
Arg Leu Asp Lys Glu Thr Glu Lys Lys Arg Arg Thr Glu 275 280 285Glu
Ala Tyr Lys Asn Ala Met Thr Glu Leu Lys Lys Lys Ser His Phe 290 295
300Gly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Ser Leu Ile Asn Glu
Glu305 310 315 320Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg
Gln Asp Lys Ile 325 330 335Glu Glu Gln Ser Gln Ser Glu Lys Val Arg
Leu His Trp Pro Thr Ser 340 345 350Leu Pro Ser Gly Asp Ala Phe Ser
Ser Val Gly Thr His Arg Phe Val 355 360 365Gln Lys Val Glu Glu Met
Val Gln Asn His Met Thr Tyr Ser Leu Gln 370 375 380Asp Val Gly Gly
Asp Ala Asn Trp Gln Leu Val Val Glu Glu Gly Glu385 390 395 400Met
Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu Asp 405 410
415Pro Leu Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu Val
420 425 430Cys Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp Trp Glu
Thr Thr 435 440 445Ile Glu Asn Phe His Val Val Glu Thr Leu Ala Asp
Asn Ala Ile Ile 450 455 460Ile Tyr Gln Thr His Lys Arg Val Trp Pro
Ala Ser Gln Arg Asp Val465 470 475 480Leu Tyr Leu Ser Val Ile Arg
Lys Ile Pro Ala Leu Thr Glu Asn Asp 485 490 495Pro Glu Thr Trp Ile
Val Cys Asn Phe Ser Val Asp His Asp Ser Ala 500 505 510Pro Leu Asn
Asn Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile 515 520 525Cys
Gln Thr Leu Val Ser Pro Pro Glu Gly Asn Gln Glu Ile Ser Arg 530 535
540141797DNAHomo sapiens 14atgtcggata atcagagctg gaactcgtcg
ggctcggagg aggatccaga gacggagtct 60gggccgcctg tggagcgctg cggggtcctc
agtaagtgga caaactacat tcatgggtgg 120caggatcgtt gggtagtttt
gaaaaataat gctctgagtt actacaaatc tgaagatgaa 180acagagtatg
gctgcagagg atccatctgt cttagcaagg ctgtcatcac acctcacgat
240tttgatgaat gtcgatttga tattagtgta aatgatagtg tttggtatct
tcgtgctcag 300gatccagatc atagacagca atggatagat gccattgaac
agcacaagac tgaatctgga 360tatggatctg aatccagctt gcgtcgacat
ggcgcaatgg tgtccctggt gtctggagca 420agtggctact ctgcaacatc
cacctcttca ttcaagaaag gccacagttt acgtgagaag 480ttggctgaaa
tggaaacatt tagagacatc ttatgtagac aagttgacac gctacagaag
540tactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaaggga
taaagtggta 600gaagatgatg aagatgactt tcctacaacg cgttctgatg
gtgacttctt
gcatagtacc 660aacggcaata aagaaaagtt atttccacat gtgacaccaa
aaggaattaa tggtatagac 720tttaaagggg aagcgataac ttttaaagca
actactgctg gaatccttgc aacactttct 780cattgtattg aactaatggt
taaacgtgag gacagctggc agaagagact ggataaggaa 840actgagaaga
aaagaagaac agaggaagca tataaaaatg caatgacaga acttaagaaa
900aaatcccact ttggaggacc agattatgaa gaaggcccta acagtctgat
taatgaagaa 960gagttctttg atgctgttga agctgctctt gacagacaag
ataaaataga agaacagtca 1020cagagtgaaa aggtgagatt acattggcct
acatccttgc cctctggaga tgccttttct 1080tctgtgggga cacatagatt
tgtccaaaag gttgaagaga tggtgcagaa ccacatgact 1140tactcattac
aggatgtagg cggagatgcc aattggcagt tggttgtaga agaaggagaa
1200atgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcc
tttaaaagct 1260acccatgcag ttaaaggcgt cacaggacat gaagtctgca
attatttctg gaatgttgac 1320gttcgcaatg actgggaaac aactatagaa
aactttcatg tggtggaaac attagctgat 1380aatgcaatca tcatttatca
aacacacaag agggtgtggc ctgcttctca gcgagacgta 1440ttatatcttt
ctgtcattcg aaagatacca gccttgactg aaaatgaccc tgaaacttgg
1500atagtttgta atttttctgt ggatcatgac agtgctcctc taaacaaccg
atgtgtccgt 1560gccaaaataa atgttgctat gatttgtcaa accttggtaa
gcccaccaga gggaaaccag 1620gaaattagca gggacaacat tctatgcaag
attacatatg tagctaatgt gaaccctgga 1680ggatgggcac cagcctcagt
gttaagggca gtggcaaagc gagagtatcc taaatttcta 1740aaacgtttta
cttcttacgt ccaagaaaaa actgcaggaa agcctatttt gttctag
179715598PRTHomo sapiens 15Met Ser Asp Asn Gln Ser Trp Asn Ser Ser
Gly Ser Glu Glu Asp Pro1 5 10 15Glu Thr Glu Ser Gly Pro Pro Val Glu
Arg Cys Gly Val Leu Ser Lys 20 25 30Trp Thr Asn Tyr Ile His Gly Trp
Gln Asp Arg Trp Val Val Leu Lys 35 40 45Asn Asn Ala Leu Ser Tyr Tyr
Lys Ser Glu Asp Glu Thr Glu Tyr Gly 50 55 60Cys Arg Gly Ser Ile Cys
Leu Ser Lys Ala Val Ile Thr Pro His Asp65 70 75 80Phe Asp Glu Cys
Arg Phe Asp Ile Ser Val Asn Asp Ser Val Trp Tyr 85 90 95Leu Arg Ala
Gln Asp Pro Asp His Arg Gln Gln Trp Ile Asp Ala Ile 100 105 110Glu
Gln His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg 115 120
125Arg His Gly Ala Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser
130 135 140Ala Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg
Glu Lys145 150 155 160Leu Ala Glu Met Glu Thr Phe Arg Asp Ile Leu
Cys Arg Gln Val Asp 165 170 175Thr Leu Gln Lys Tyr Phe Asp Ala Cys
Ala Asp Ala Val Ser Lys Asp 180 185 190Glu Leu Gln Arg Asp Lys Val
Val Glu Asp Asp Glu Asp Asp Phe Pro 195 200 205Thr Thr Arg Ser Asp
Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys 210 215 220Glu Lys Leu
Phe Pro His Val Thr Pro Lys Gly Ile Asn Gly Ile Asp225 230 235
240Phe Lys Gly Glu Ala Ile Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu
245 250 255Ala Thr Leu Ser His Cys Ile Glu Leu Met Val Lys Arg Glu
Asp Ser 260 265 270Trp Gln Lys Arg Leu Asp Lys Glu Thr Glu Lys Lys
Arg Arg Thr Glu 275 280 285Glu Ala Tyr Lys Asn Ala Met Thr Glu Leu
Lys Lys Lys Ser His Phe 290 295 300Gly Gly Pro Asp Tyr Glu Glu Gly
Pro Asn Ser Leu Ile Asn Glu Glu305 310 315 320Glu Phe Phe Asp Ala
Val Glu Ala Ala Leu Asp Arg Gln Asp Lys Ile 325 330 335Glu Glu Gln
Ser Gln Ser Glu Lys Val Arg Leu His Trp Pro Thr Ser 340 345 350Leu
Pro Ser Gly Asp Ala Phe Ser Ser Val Gly Thr His Arg Phe Val 355 360
365Gln Lys Val Glu Glu Met Val Gln Asn His Met Thr Tyr Ser Leu Gln
370 375 380Asp Val Gly Gly Asp Ala Asn Trp Gln Leu Val Val Glu Glu
Gly Glu385 390 395 400Met Lys Val Tyr Arg Arg Glu Val Glu Glu Asn
Gly Ile Val Leu Asp 405 410 415Pro Leu Lys Ala Thr His Ala Val Lys
Gly Val Thr Gly His Glu Val 420 425 430Cys Asn Tyr Phe Trp Asn Val
Asp Val Arg Asn Asp Trp Glu Thr Thr 435 440 445Ile Glu Asn Phe His
Val Val Glu Thr Leu Ala Asp Asn Ala Ile Ile 450 455 460Ile Tyr Gln
Thr His Lys Arg Val Trp Pro Ala Ser Gln Arg Asp Val465 470 475
480Leu Tyr Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp
485 490 495Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val Asp His Asp
Ser Ala 500 505 510Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn
Val Ala Met Ile 515 520 525Cys Gln Thr Leu Val Ser Pro Pro Glu Gly
Asn Gln Glu Ile Ser Arg 530 535 540Asp Asn Ile Leu Cys Lys Ile Thr
Tyr Val Ala Asn Val Asn Pro Gly545 550 555 560Gly Trp Ala Pro Ala
Ser Val Leu Arg Ala Val Ala Lys Arg Glu Tyr 565 570 575Pro Lys Phe
Leu Lys Arg Phe Thr Ser Tyr Val Gln Glu Lys Thr Ala 580 585 590Gly
Lys Pro Ile Leu Phe 595
* * * * *