U.S. patent application number 15/284693 was filed with the patent office on 2017-01-19 for compositions and methods for treatment of ovarian cancer.
The applicant listed for this patent is Rosetta Genomics Ltd.. Invention is credited to Noga Yerushalmi.
Application Number | 20170015999 15/284693 |
Document ID | / |
Family ID | 46457283 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170015999 |
Kind Code |
A1 |
Yerushalmi; Noga |
January 19, 2017 |
COMPOSITIONS AND METHODS FOR TREATMENT OF OVARIAN CANCER
Abstract
The disclosure provides compositions and methods for treating an
ovarian cancer in a subject. More specifically, the disclosure
provides microRNA (miRNA) inhibitor molecules that target to
different miRNAs for treating different types of ovarian cancers in
a subject. Furthermore, different modifications of miRNA inhibitor
molecules as well as different derivatives of miRNA inhibitor
molecules are also described.
Inventors: |
Yerushalmi; Noga;
(Nes-Ziona, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rosetta Genomics Ltd. |
Rehovot |
|
IL |
|
|
Family ID: |
46457283 |
Appl. No.: |
15/284693 |
Filed: |
October 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14505548 |
Oct 3, 2014 |
9487781 |
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15284693 |
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13977797 |
Jul 1, 2013 |
8883757 |
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PCT/IL2011/000892 |
Nov 20, 2011 |
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14505548 |
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61429194 |
Jan 3, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/141 20130101;
C12N 2310/11 20130101; C12N 15/113 20130101; C12N 2310/113
20130101; A61K 31/7088 20130101; C12N 2320/31 20130101; A61K 45/06
20130101; C12N 2310/315 20130101; C12N 2320/30 20130101; C12N
2310/321 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 45/06 20060101 A61K045/06; A61K 31/7088 20060101
A61K031/7088 |
Claims
1. A method of inhibiting proliferation of ovarian cancer cells,
the method comprising introducing into the cells a compound which
inhibit expression or activity of a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1-40 and sequences at
least about 80% identical thereto.
2. The method of claim 1, wherein the compound comprising a
modified oligonucleotide consisting of 15 to 30 linked nucleosides,
wherein the modified oligonucleotide has a nucleobase sequence that
is complementary to a nucleobase sequence selected from SEQ ID NOs:
1-40; or to a sequence at least 80% identical thereto.
3. The method of claim 2, wherein said modified oligonucleotide has
a nucleobase sequence selected from SEQ ID NOs: 41-58; or to a
sequence at least 80% identical thereto.
4. The method of claim 1, wherein the ovarian cancer cell is
selected from the group consisting of serous and endometrioid
ovarian cancer.
5. A method for treating ovarian cancer, comprising administering
to a subject in need thereof a pharmaceutical composition
comprising a modified oligonucleotide consisting of 15 to 30 linked
nucleosides, wherein the modified oligonucleotide has a nucleobase
sequence that is complementary to a nucleobase sequence selected
from SEQ ID NOs: 1-40; or to a sequence at least 80% identical
thereto.
6. The method of claim 5, wherein the modified oligonucleotide has
a nucleobase sequence comprising at least 15 contiguous nucleobases
of a nucleobase sequence selected from among the nucleobase
sequences recited in SEQ ID NOs: 41-58; or a sequence at least 80%
identical thereto.
7. The method of claim 5, wherein the subject is a human.
8. The method of claim 5, wherein the ovarian cancer is selected
from the group consisting of serous and endometrioid ovarian
cancer.
9. The method of any of claims 1-8, wherein the nucleobase sequence
of the modified oligonucleotide has no more than two mismatches to
a nucleobase sequence selected from SEQ ID NOs: 41-58.
10. The method of any of claims 1-8, wherein the nucleobase
sequence of the modified oligonucleotide has no more than one
mismatch to a nucleobase sequence selected from SEQ ID NO:
41-58.
11. The method of any of claims 1-8, wherein the nucleobase
sequence of the modified oligonucleotide has one mismatch to a
nucleobase sequence selected from SEQ ID NO: 41-58.
12. The method of any of claims 1-8, wherein the nucleobase
sequence of the modified oligonucleotide has no mismatch to a
nucleobase sequence selected from SEQ ID NO: 41-58.
13. The method of any of claims 1-12, wherein at least one
internucleoside linkage is a modified internucleoside linkage.
14. The method of any of claims 1-13, wherein each internucleoside
linkage is a modified internucleoside linkage.
15. The method of any of claims 1-15, wherein at least one
internucleoside linkage is a phosphorothioate internucleoside
linkage.
16. The method of any of claims 1-16, wherein each internucleoside
linkage is a phosphorothioate internucleoside linkage.
17. The method of any of claims 1-17, wherein at least one
nucleoside comprises a modified sugar.
18. The method of any of claims 1-13, wherein each of a plurality
of nucleosides comprises a modified sugar.
19. The method of any of claims 1-14, wherein each nucleoside
comprises a modified sugar.
20. The method of any of claims 1-15, wherein each nucleoside
comprises a 2'-O-methoxyethyl sugar.
21. The method of any of claims 5-20, wherein the administering
comprises intravenous administration, subcutaneous administration,
intratumoral administration, or chemoembolization.
22. The method of any of claims 5-21, further comprising
administering at least one additional therapy.
23. The method of claim 22, wherein the at least one additional
therapy is a chemotherapeutic agent.
24. The method of claim 23, wherein the chemotherapeutic agent may
be selected from cisplatin, carboplatin, camptothecins,
doxorubicin, cyclophosphamide, paclitaxel, etoposide, vinblastine,
Actinomycin D and cloposide.
25. The method of claim 22, wherein the at least one additional
therapy is administered at the same time as the modified
oligonucleotide.
26. The method of claim 22, wherein the at least one additional
therapy is administered less frequently than the modified
oligonucleotide.
27. The method of claim 22, wherein the at least one additional
therapy is administered more frequently than the modified
oligonucleotide.
28. The method of any of claims 5-27, wherein the modified
oligonucleotide is administered at a dose selected from 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,
425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725,
750, 775, and 800 mg.
29. The method of any of claims 5-28, wherein the modified
oligonucleotide is administered one per day, once per week, once
per two weeks, once per three weeks, or once per four weeks.
30. The method of any of claims 5-29, wherein the administering
results in reduction of tumor size.
31. The method of any of claims 5-30, wherein the administering
results in reduction of tumor number.
32. The method of any of claims 5-31, wherein the administering
prevents an increase in tumor size.
33. The method of any of claims 5-32, wherein the administering
prevents an increase in tumor number.
34. The method of any of claims 5-33, wherein the administering
prevents metastatic progression.
35. The method of any of claims 5-34, wherein the administering
slows or stops metastatic progression.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 61/429,194 filed
Jan. 3, 2011, which is herein incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods for the
treatment of ovarian cancer subjects. Specifically the invention
relates to microRNA inhibitor molecules associated with the
treatment of ovarian cancer subjects, as well as various nucleic
acid molecules relating thereto or derived therefrom.
BACKGROUND OF THE INVENTION
[0003] Epithelial ovarian cancer (EOC) is the fifth leading cause
of cancer-related deaths in women in the United States and the
leading cause of gynecologic cancer related deaths (Jemal A, Siegel
et. al, Cancer statistics, 2007, CA Cancer J Clin 2007; 57:43-66).
Annually, there are more than 22,000 new cases of ovarian cancer in
the United States and over 16,000 deaths. Despite efforts to
develop an effective ovarian cancer screening method, most patients
still present with advanced (Stages III-IV) disease. Survival of
patients diagnosed with ovarian cancer is known to closely
correlate with stage at diagnosis.
[0004] Treatment for advanced ovarian carcinoma is based on the
combination of surgery and chemotherapy. The objective of surgical
intervention in patients suffering from advanced disease is to
perform cytoreduction to minimal residual disease in the abdominal
cavity. Surgery is followed by adjuvant platinum based
chemotherapy. The two most important prognostic factors for
patients with advanced ovarian carcinoma are the amount of residual
disease left after surgery and the response to platinum based
chemotherapy.
[0005] Platinum-based cytotoxic chemotherapy in conjunction with
debulking surgery is currently the gold standard treatment for
patients with ovarian cancer. Although 80-90% of patients initially
respond to first line treatment, most will either later progress
during therapy or recur after complete remission.
[0006] microRNAs (miRNAs, miRs) are endogenous non-coding small
RNAs that interfere with the translation of coding messenger RNAs
(mRNAs) in a sequence specific manner, playing a critical role in
the control of gene expression during development and tissue
homeostasis (Yi et al., 2006, Nat Genet 38, 356-362). Certain
miRNAs have been shown to be deregulated in human cancer, and their
specific over- or under-expression has been shown to correlate with
particular tumor types (Calin and Croce, 2006, Nat Rev Cancer 6,
857-866), as well as to predict patient outcome (Yu et al., 2008,
Cancer Cell 13, 48-57).
[0007] In spite of considerable research into therapies for ovarian
cancer, ovarian cancer remains difficult to diagnose and treat
effectively, and the mortality observed in patients indicates that
improvements are needed in the treatment and prevention of the
disease.
SUMMARY OF THE INVENTION
[0008] The present invention is based in part on the development of
microRNA inhibitors, to be used for the treatment of ovarian cancer
patients. The microRNA inhibitors were shown to inhibit specific
microRNAs that are involved in proliferation of ovarian cells, and
therefore reduce cell proliferation. According to some embodiments,
the target cells are residual ovarian cancer cells in the abdominal
cavity where most ovarian cancer metastasis occur.
[0009] In order to develop a microRNA-based treatment for ovarian
cancer, candidate microRNA targets were identified for inhibition.
microRNAs that were over-expressed in ovarian tissue (both tumor,
metastases and normal) as compared to other normal tissues were
chosen, as well as highly expressed microRNAs. Candidates having a
greater potential of being a drug target were further selected,
using a proliferation assay for cells treated with a specific
microRNA inhibitor.
[0010] According to some aspects, the present invention provides a
method of inhibiting proliferation of ovarian cancer cells, the
method comprising introducing into the cells a compound which
inhibit expression or activity of a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1-40 and sequences at
least about 80% identical thereto.
[0011] According to some embodiments, said compound comprising a
modified oligonucleotide consisting of 15 to 30 linked nucleosides,
wherein the modified oligonucleotide has a nucleobase sequence that
is complementary to a nucleobase sequence selected from SEQ ID NOs:
1-40; or to a sequence at least 80% identical thereto.
[0012] According to some embodiments, said modified oligonucleotide
has a nucleobase sequence selected from SEQ ID NOs: 41-58; or to a
sequence at least 80% identical thereto.
[0013] According to one embodiment, the ovarian cancer cell is
selected from the group consisting of serous and endometrioid
ovarian cancer.
[0014] According to other aspects, the present invention provides a
method for treating ovarian cancer, comprising administering to a
subject in need thereof a pharmaceutical composition comprising a
modified oligonucleotide consisting of 15 to 30 linked nucleosides,
wherein the modified oligonucleotide has a nucleobase sequence that
is complementary to a nucleobase sequence selected from SEQ ID NOs:
1-40; or to a sequence at least 80% identical thereto.
[0015] In certain embodiments, the modified oligonucleotide has a
nucleobase sequence comprising at least 15 contiguous nucleobases
of a nucleobase sequence selected from among the nucleobase
sequences recited in SEQ ID NOs: 41-58; or a sequence at least 80%
identical thereto.
[0016] In certain embodiments, the subject is a human.
[0017] In certain embodiments, the present invention provides a
pharmaceutical composition comprising a modified oligonucleotide of
the invention or a salt thereof and a pharmaceutically acceptable
carrier or diluent.
[0018] In certain embodiments, the compound consists of a modified
oligonucleotide.
[0019] In certain embodiments, the modified oligonucleotide
consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 linked
nucleosides.
[0020] In certain embodiments, the nucleobase sequence of the
modified oligonucleotide has no more than two mismatches to a
nucleobase sequence selected from SEQ ID NO: 41-58. In certain
embodiments, the nucleobase sequence of the modified
oligonucleotide has no more than one mismatch to a nucleobase
sequence selected from SEQ ID NO: 41-58. In certain embodiments,
the nucleobase sequence of the modified oligonucleotide has one
mismatch to a nucleobase sequence selected from SEQ ID NO: 41-58.
In certain embodiments, the nucleobase sequence of the modified
oligonucleotide has no mismatches to a nucleobase sequence selected
from SEQ ID NO: 41-58.
[0021] In certain embodiments, the modified oligonucleotide
comprises one or more modified sugars, intemucleoside linkages, or
nucleobases. In certain embodiments, at least one internucleoside
linkage is a modified intemucleoside linkage. For example, at least
one internucleoside linkage may be a phosphorothioate
intemucleoside linkage. In certain embodiments, each
internucleoside linkage is a modified internucleoside linkage. For
example, each internucleoside linkage may be a phosphorothioate
intemucleoside linkage.
[0022] In certain embodiments, at least one nucleoside of the
modified oligonucleotide comprises a modified sugar. In certain
embodiments, each of a plurality of nucleosides comprises a
modified sugar. In certain embodiments, each nucleoside of the
modified oligonucleotide comprises a modified sugar. In each of
these embodiments, the modified sugar may be a 2'-O-methoxyethyl
sugar, a 2'-fluoro sugar, a 2'-O-methyl sugar, or a bicyclic sugar
moiety. In certain embodiments, each of a plurality of nucleosides
comprises a 2'-O-methoxyethyl sugar and each of a plurality of
nucleosides comprises a 2'-fluoro sugar.
[0023] In certain embodiments, the modified oligonucleotide
comprises at least one modified nucleobase. In certain such
embodiments, the modified nucleobase is a 5-methyleytosine. In
certain embodiments, at least one nucleoside comprises a cytosine,
wherein the cytosine is a 5-methylcytosine. In certain such
embodiments, each cytosine is a 5-methylcytosine.
[0024] In certain embodiments, administration of a compound of the
invention comprises intravenous administration, subcutaneous
administration, intratumoral administration, or
chemoembolization.
[0025] In certain embodiments, the methods of the present invention
further comprise administering at least one additional therapy. The
additional therapy may be a chemotherapeutic agent. The
chemotherapeutic agent may be selected from cisplatin, carboplatin,
camptothecins, doxorubicin, cyclophosphamide, paclitaxel,
etoposide, vinblastine, Actinomycin D and cloposide. The additional
therapy may be administered at the same time, less frequently, or
more frequently than a compound or pharmaceutical composition of
the invention.
[0026] In certain embodiments, the modified oligonucleotide is
administered at a dose selected from 50, 75, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg.
The modified oligonucleotide may be administered one per day, once
per week, once per two weeks, once per three weeks, or once per
four weeks.
[0027] In certain embodiments, the administration of a compound of
the invention results in reduction of ovarian tumor size. In
certain embodiments, the administration of a compound of the
invention prevents an increase in tumor size and/or tumor number.
In certain embodiments, the administration of a compound of the
invention prevents, slows, and/or stops metastatic progression. In
certain embodiments, the administration of a compound of the
invention extends the overall survival time of the subject. In
certain embodiments, the administration of a compound of the
invention extends the progression-free survival of the subject. In
certain embodiments, administration of a compound of the invention
prevents the recurrence of ovarian tumors. In certain embodiments,
administration of a compound of the invention prevents recurrence
of ovarian tumor metastasis.
[0028] According to some embodiments the composition is suitable
for administration in combination with at least one other
anticancer agent in unit dosage form. According to some embodiments
the anticancer agent is selected from the group consisting of
cisplatin, carboplatin, camptothecins, doxorubicin,
cyclophosphamide, etoposide, vinblastine, Actinomycin D, paclitaxel
and cloposide.
[0029] These and other embodiments of the present invention will
become apparent in conjunction with the figures, description and
claims that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIGS. 1A-1D demonstrate the effect of anti-miR (nM)
(squares) on proliferation (percentage) of OVCAR-3 cells, 72 hours
after transfection as compared to negative control anti-miR
(triangles). 1A--anti-miR-103 (SEQ ID NO: 42), 1B--anti-miR-100
(SEQ ID NO: 41), 1C--anti-miR-125b (SEQ ID NO: 43), 1D-anti-miR-191
(SEQ ID NO: 46).
[0031] FIGS. 2A-2D demonstrate the effect of anti-miR (nM)
(squares) on proliferation (percentage) of OVCAR-3 cells, 72 hours
after transfection as compared to negative control anti-miR
(triangles). 2A--anti-miR-21 (SEQ ID NO: 48), 2B--anti-miR-210 (SEQ
ID NO: 49), 2C--anti-miR-221 (SEQ ID NO: 51), 2D--anti-miR-99a (SEQ
ID NO: 58).
[0032] FIGS. 3A-3D demonstrate the effect of anti-miR (nM)
(squares) on proliferation (percentage) of OVCAR-3 cells, 72 hours
after transfection as compared to negative control anti-miR
(triangles). 3A--anti-miR-24 (SEQ ID NO: 53), 3B--anti-miR-25 (SEQ
ID NO: 54), 3C--anti-miR-26a (SEQ ID NO: 55), 3D--anti-miR-27a (SEQ
ID NO: 56).
[0033] FIGS. 4A-4D demonstrate the effect of anti-miR (nM)
(squares) on proliferation (percentage) of OVCAR-3 cells, 72 hours
after transfection as compared to negative control anti-miR
(triangles). 4A--anti-miR-18a (SEQ ID NO: 45), 4B--anti-miR-20b
(SEQ ID NO: 47), 4C--anti-miR-31 (SEQ ID NO: 57), 4D--anti-miR-23a
(SEQ ID NO: 52).
[0034] FIGS. 5A-5B demonstrate the effect of anti-miR (nM)
(squares) on proliferate (percentage) of OVCAR-3 cells, 72 hours
after transfection as compared to negative con anti-miR
(triangles). 5A--anti-miR-22 (SEQ ID NO: 50), 5B--anti-miR-17 (SEQ
ID NO: 44
DETAILED DESCRIPTION
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the arts to which the invention belongs. Unless
specific definitions are provided, the nomenclature utilized in
connection with, and the procedures and techniques of, analytical
chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and
commonly used in the art. In the event that there is a plurality of
definitions for terms herein, those in this section prevail.
Standard techniques may be used for chemical synthesis, chemical
analysis, pharmaceutical preparation, formulation and delivery, and
treatment of subjects. Certain such techniques and procedures may
be found for example in "Carbohydrate Modifications in Antisense
Research" Edited by Sangvi and Cook, American Chemical Society,
Washington D.C., 1994; and "Remington's Pharmaceutical Sciences,"
Mack Publishing Co., Easton, Pa., 18th edition, 1990; and which is
hereby incorporated by reference for any purpose. Where permitted,
all patents, patent applications, published applications and
publications, GENBANK sequences, websites and other published
materials referred to throughout the entire disclosure herein,
unless noted otherwise, are incorporated by reference in their
entirety. Where reference is made to a URL or other such identifier
or address, it is understood that such identifiers can change and
particular information on the internet can command go, but
equivalent information can be found by searching the internet.
Reference thereto evidences the availability and public
dissemination of such information.
[0036] Before the present compositions and methods are disclosed
and described, it is to be understood that the terminology used
herein is for the purpose of describing particular embodiments only
and is not intended to be limiting. It must be noted that, as used
in the specification and the appended claims, the singular forms
"a," "an" and "the" include plural referents unless the context
clearly dictates otherwise.
[0037] For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
1. DEFINITIONS
[0038] a. Administering
[0039] "Administering" means providing a pharmaceutical agent or
composition to a subject, and includes, but is not limited to,
administering by a medical professional and self-administering.
[0040] "Parenteral administration," means administration through
injection or infusion. Parenteral administration includes, but is
not limited to, subcutaneous administration, intravenous
administration, or intramuscular administration.
[0041] "Subcutaneous administration" means administration just
below the skin.
[0042] "Intravenous administration" means administration into a
vein.
[0043] "Intratumoral administration" means administration within a
tumor.
[0044] "Chemoembolization" means a procedure in which the blood
supply to a tumor is blocked surgically or mechanically and
chemotherapeutic agents are administered directly into the
tumor.
[0045] b. Chemotherapeutic Agent
[0046] A drug used to treat a disease, especially cancer. In
relation to cancer the drugs typically target rapidly dividing
cells, such as cancer cells. Non-limiting examples of
chemotherapeutic agents include cisplatin, carboplatin,
camptothecins, doxorubicin, cyclophosphamide, paclitaxel,
etoposide, vinblastine, Actinomycin D and cloposide.
[0047] c. Complement
[0048] "Complement" or "complementary" as used herein to refer to a
nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or
Hoogsteen base pairing between nucleotides or nucleotide analogs of
nucleic acid molecules. A full complement or fully complementary
may mean 100% complementary base pairing between nucleotides or
nucleotide analogs of nucleic acid molecules.
[0049] d. Detection
[0050] "Detection" means detecting the presence of a component in a
sample. Detection also means detecting the absence of a component.
Detection also means measuring the level of a component, either
quantitatively or qualitatively.
[0051] e. Differential Expression
[0052] "Differential expression" may mean qualitative or
quantitative differences in the temporal and/or cellular gene
expression patterns within and among cells and tissue. Thus, a
differentially expressed gene can qualitatively have its expression
altered, including an activation or inactivation, in, e.g., normal
versus disease tissue. Genes may be turned on or turned off in a
particular state, relative to another state thus permitting
comparison of two or more states. A qualitatively regulated gene
will exhibit an expression pattern within a state or cell type that
may be detectable by standard techniques. Some genes will be
expressed in one state or cell type, but not in both.
Alternatively, the difference in expression may be quantitative,
e.g., in that expression is modulated, up-regulated, resulting in
an increased amount of transcript, or down-regulated, resulting in
a decreased amount of transcript. The degree to which expression
differs need only be large enough to quantify via standard
characterization techniques such as expression arrays, quantitative
reverse transcriptase PCR, northern analysis, and RNase
protection.
[0053] f. Dose
[0054] "Dose" as used herein means a specified quantity of a
pharmaceutical agent provided in a single administration. In
certain embodiments, a dose may be administered in two or more
boluses, tablets, or injections. For example, in certain
embodiments, where subcutaneous administration is desired, the
desired dose requires a volume not easily accommodated by a single
injection. In such embodiments, two or more injections may be used
to achieve the desired dose. In certain embodiments, a dose may be
administered in two or more injections to minimize injection site
reaction in an individual.
[0055] g. Dosage Unit
[0056] "Dosage unit" as used herein means a form in which a
pharmaceutical agent is provided. In certain embodiments, a dosage
unit is a vial containing lyophilized oligonucleotide. In certain
embodiments, a dosage unit is a vial containing reconstituted
oligonucleotide.
[0057] h. Expression Profile
[0058] "Expression profile" as used herein may mean a genomic
expression profile, e.g., an expression profile of microRNAs.
Profiles may be generated by any convenient means for determining a
level of a nucleic acid sequence e.g. quantitative hybridization of
microRNA, labeled microRNA, amplified microRNA, cRNA, etc.,
quantitative PCR,
[0059] ELISA for quantitation, and the like, and allow the analysis
of differential gene expression between two samples. A subject or
patient tumor sample, e.g., cells or collections thereof, e.g.,
tissues, is assayed. Samples are collected by any convenient
method, as known in the art. Nucleic acid sequences of interest are
nucleic acid sequences that are found to be predictive, including
the nucleic acid sequences provided above, where the expression
profile may include expression data for 5, 10, 20, 25, 50, 100 or
more of, including all of the listed nucleic acid sequences. The
term "expression profile" may also mean measuring the abundance of
the nucleic acid sequences in the measured samples.
[0060] i. Gene
[0061] "Gene" used herein may be a natural (e.g., genomic) or
synthetic gene comprising transcriptional and/or translational
regulatory sequences and/or a coding region and/or non-translated
sequences (e.g., introns, 5'- and 3'-untranslated sequences). The
coding region of a gene may be a nucleotide sequence coding for an
amino acid sequence or a functional RNA, such as tRNA, rRNA,
catalytic RNA, siRNA, miRNA or antisense
[0062] RNA. A gene may also be an mRNA or cDNA corresponding to the
coding regions (e.g., exons and miRNA) optionally comprising 5'- or
3'-untranslated sequences linked thereto. A gene may also be an
amplified nucleic acid molecule produced in vitro comprising all or
a part of the coding region and/or 5'- or 3'-untranslated sequences
linked thereto.
[0063] j. Identity
[0064] "Identical" or "identity" as used herein in the context of
two or more nucleic acids or polypeptide sequences may mean that
the sequences have a specified percentage of residues that are the
same over a specified region. The percentage may be calculated by
optimally aligning the two sequences, comparing the two sequences
over the specified region, determining the number of positions at
which the identical residue occurs in both sequences to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the specified region,
and multiplying the result by 100 to yield the percentage of
sequence identity. In cases where the two sequences are of
different lengths or the alignment produces one or more staggered
ends and the specified region of comparison includes only a single
sequence, the residues of single sequence are included in the
denominator but not the numerator of the calculation. When
comparing DNA and RNA, thymine (T) and uracil (U) may be considered
equivalent. Identity may be performed manually or by using a
computer sequence algorithm such as BLAST or BLAST 2.0.
[0065] k. Inhibit
[0066] "Inhibit" as used herein may mean prevent, suppress,
repress, reduce or eliminate.
[0067] l. Label
[0068] "Label" as used herein may mean a composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
chemical, or other physical means. For example, useful labels
include .sup.32P, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and other entities which can be made detectable. A label
may be incorporated into nucleic acids and proteins at any
position.
[0069] m. Metastasis
[0070] "Metastasis" as used herein means the process by which
cancer spreads from the place at which it first arose as a primary
tumor to other locations in the body. The metastatic progression of
a primary tumor reflects multiple stages, including dissociation
from neighboring primary tumor cells, survival in the circulation,
and growth in a secondary location.
[0071] n. Mismatch
[0072] "Mismatch" means a nucleobase of a first nucleic acid that
is not capable of pairing with a nucleobase at a corresponding
position of a second nucleic acid.
[0073] o. Nucleic Acid
[0074] "Nucleic acid" or "oligonucleotide" or "polynucleotide" used
herein may mean at least two nucleotides covalently linked
together. The depiction of a single strand also defines the
sequence of the complementary strand. Thus, a nucleic acid also
encompasses the complementary strand of a depicted single strand.
Many variants of a nucleic acid may be used for the same purpose as
a given nucleic acid. Thus, a nucleic acid also encompasses
substantially identical nucleic acids and complements thereof. A
single strand provides a probe that may hybridize to a target
sequence under stringent hybridization conditions. Thus, a nucleic
acid also encompasses a probe that hybridizes under stringent
hybridization conditions.
[0075] Nucleic acids may be single stranded or double stranded, or
may contain portions of both double stranded and single stranded
sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA,
or a hybrid, where the nucleic acid may contain combinations of
deoxyribo- and ribo-nucleotides, and combinations of bases
including uracil, adenine, thymine, cytosine, guanine, inosine,
xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids
may be obtained by chemical synthesis methods or by recombinant
methods.
[0076] A nucleic acid will generally contain phosphodiester bonds,
although nucleic acid analogs may be included that may have at
least one different linkage, e.g., phosphoramidate,
phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite
linkages and peptide nucleic acid backbones and linkages. Other
analog nucleic acids include those with positive backbones;
non-ionic backbones, and non-ribose backbones, including those
described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are
incorporated by reference. Nucleic acids containing one or more
non-naturally occurring or modified nucleotides are also included
within one definition of nucleic acids. The modified nucleotide
analog may be located for example at the 5'-end and/or the 3'-end
of the nucleic acid molecule. Representative examples of nucleotide
analogs may be selected from sugar- or backbone-modified
ribonucleotides. It should be noted, however, that also
nucleobase-modified ribonucleotides, i.e. ribonucleotides,
containing a non-naturally occurring nucleobase instead of a
naturally occurring nucleobase such as uridines or cytidines
modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo
uridine; adenosines and guanosines modified at the 8-position, e.g.
8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O-
and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable.
The 2'-OH-group may be replaced by a group selected from H, OR, R,
halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or CN, wherein R is
C.sub.1-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or
I. Modified nucleotides also include nucleotides conjugated with
cholesterol through, e.g., a hydroxyprolinol linkage as described
in Krutzfeldt et al., Nature 438:685-689 (2005), Soutschek et al.,
Nature 432:173-178 (2004), and U.S. Patent Publication No.
20050107325, which are incorporated herein by reference. Additional
modified nucleotides and nucleic acids are described in U.S. Patent
Publication No. 20050182005, which is incorporated herein by
reference. Modifications of the ribose-phosphate backbone may be
done for a variety of reasons, e.g., to increase the stability and
half-life of such molecules in physiological environments, to
enhance diffusion across cell membranes, or as probes on a biochip.
The backbone modification may also enhance resistance to
degradation, such as in the harsh endocytic environment of cells.
The backbone modification may also reduce nucleic acid clearance by
hepatocytes, such as in the liver and kidney. Mixtures of naturally
occurring nucleic acids and analogs may be made; alternatively,
mixtures of different nucleic acid analogs, and mixtures of
naturally occurring nucleic acids and analogs may be made.
[0077] p. Overall Survival Time
[0078] "Overall survival time" or "survival time", as used herein
means the time period for which a subject survives after diagnosis
of or treatment for a disease. In certain embodiments, the disease
is cancer.
[0079] q. Progression-Free Survival
[0080] "Progression-free survival" means the time period for which
a subject having a disease survives, without the disease getting
worse. In certain embodiments, progression-free survival is
assessed by staging or scoring the disease. In certain embodiments,
progression-free survival of a subject having cancer is assessed by
evaluating tumor size, tumor number, and/or metastasis.
[0081] r. Probe
[0082] "Probe" as used herein may mean an oligonucleotide capable
of binding to a target nucleic acid of complementary sequence
through one or more types of chemical bonds, usually through
complementary base pairing, usually through hydrogen bond
formation. Probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the
stringency of the hybridization conditions. There may be any number
of base pair mismatches which will interfere with hybridization
between the target sequence and the single stranded nucleic acids
described herein. However, if the number of mutations is so great
that no hybridization can occur under even the least stringent of
hybridization conditions, the sequence is not a complementary
target sequence. A probe may be single stranded or partially single
and partially double stranded. The strandedness of the probe is
dictated by the structure, composition, and properties of the
target sequence. Probes may be directly labeled or indirectly
labeled such as with biotin to which a streptavidin complex may
later bind.
[0083] s. Promoter
[0084] "Promoter" as used herein may mean a synthetic or
naturally-derived molecule which is capable of conferring,
activating or enhancing expression of a nucleic acid in a cell. A
promoter may comprise one or more specific transcriptional
regulatory sequences to further enhance expression and/or to alter
the spatial expression and/or temporal expression of same. A
promoter may also comprise distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from
the start site of transcription. A promoter may be derived from
sources including viral, bacterial, fungal, plants, insects, and
animals. A promoter may regulate the expression of a gene component
constitutively or differentially with respect to cell, the tissue
or organ in which expression occurs or, with respect to the
developmental stage at which expression occurs, or in response to
external stimuli such as physiological stresses, pathogens, metal
ions, or inducing agents. Representative examples of promoters
include the bacteriophage T7 promoter, bacteriophage T3 promoter,
SP6 promoter, lac operator-promoter, tac promoter, SV40 late
promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter,
SV40 early promoter or SV40 late promoter and the CMV IE
promoter.
[0085] t. Selectable Marker
[0086] "Selectable marker" as used herein means any gene which
confers a phenotype on a host cell in which it is expressed to
facilitate the identification and/or selection of cells which are
transfected or transformed with a genetic construct. Representative
examples of selectable markers include the ampicillin-resistance
gene (Amp.sup.r), tetracycline-resistance gene (Tc.sup.r),
bacterial kanamycin-resistance gene (Kan.sup.r), zeocin resistance
gene, the AURI-C gene which confers resistance to the antibiotic
aureobasidin A, phosphinothricin-resistance gene, neomycin
phosphotransferase gene (nptII), hygromycin-resistance gene,
beta-glucuronidase (GUS) gene, chloramphenicol acetyltransferase
(CAT) gene, green fluorescent protein (GFP)-encoding gene and
luciferase gene.
[0087] u. Stringent Hybridization Conditions
[0088] "Stringent hybridization conditions" used herein may mean
conditions under which a first nucleic acid sequence (e.g., probe)
will hybridize to a second nucleic acid sequence (e.g., target),
such as in a complex mixture of nucleic acids. Stringent conditions
are sequence-dependent and will be different in different
circumstances. Stringent conditions may be selected to be about
5-10.degree. C. lower than the thermal melting point (T.sub.m) for
the specific sequence at a defined ionic strength pH. The T.sub.m
may be the temperature (under defined ionic strength, pH, and
nucleic concentration) at which 50% of the probes complementary to
the target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Stringent conditions may be
those in which the salt concentration is less than about 1.0 M
sodium ion, such as about 0.01-1.0 M sodium ion concentration (or
other salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g., about 10-50 nucleotides) and
at least about 60.degree. C. for long probes (e.g., greater than
about 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide. For
selective or specific hybridization, a positive signal may be at
least 2 to 10 times background hybridization. Exemplary stringent
hybridization conditions include the following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0089] v. Substantially Complementary
[0090] "Substantially complementary" used herein may mean that a
first sequence is at least 60%-99% identical to the complement of a
second sequence over a region of 8-50 or more nucleotides, or that
the two sequences hybridize under stringent hybridization
conditions.
[0091] w. Substantially Identical
[0092] "Substantially identical" used herein may mean that a first
and second sequence are at least 60%-99% identical over a region of
8-50 or more nucleotides or amino acids, or with respect to nucleic
acids, if the first sequence is substantially complementary to the
complement of the second sequence.
[0093] x. Subject
[0094] As used herein, the term "subject" refers to a mammal,
including both human and other mammals. The methods of the present
invention are preferably applied to human subjects.
[0095] y. Therapeutically Effective Amount
[0096] "Therapeutically effective amount" or "therapeutically
efficient" used herein as to a drug dosage, refer to dosage that
provides the specific pharmacological response for which the drug
is administered in a significant number of subjects in need of such
treatment. The "therapeutically effective amount" may vary
according, for example, the physical condition of the patient, the
age of the patient and the severity of the disease.
[0097] z. Therapy
[0098] "Therapy" as used herein means a disease treatment method.
In certain embodiments, therapy includes, but is not limited to,
chemotherapy, surgical resection, transplant, and/or
chemoembolization.
[0099] Aa. Treat
[0100] "Treat" or "treating" used herein when referring to
protection of a subject from a condition may mean preventing,
suppressing, repressing, or eliminating the condition. Preventing
the condition involves administering a composition described herein
to a subject prior to onset of the condition. Suppressing the
condition involves administering the composition to a subject after
induction of the condition but before its clinical appearance.
Repressing the condition involves administering the composition to
a subject after clinical appearance of the condition such that the
condition is reduced or prevented from worsening. Elimination of
the condition involves administering the composition to a subject
after clinical appearance of the condition such that the subject no
longer suffers from the condition.
[0101] Bb. Unit Dosage Form
[0102] "Unit dosage form," used herein may refer to a physically
discrete unit suitable as a unitary dosage for a human or animal
subject. Each unit may contain a predetermined quantity of a
composition described herein, calculated in an amount sufficient to
produce a desired effect in association with a pharmaceutically
acceptable diluent, carrier or vehicle. The specifications for a
unit dosage form may depend on the particular composition employed
and the effect to be achieved, and the pharmacodynamics associated
with the composition in the host.
[0103] cc. Variant
[0104] "Variant" used herein to refer to a nucleic acid may mean
(i) a portion of a referenced nucleotide sequence; (ii) the
complement of a referenced nucleotide sequence or portion thereof;
(iii) a nucleic acid that is substantially identical to a
referenced nucleic acid or the complement thereof; or (iv) a
nucleic acid that hybridizes under stringent conditions to the
referenced nucleic acid, complement thereof, or a sequences
substantially identical thereto.
[0105] dd. Vector
[0106] "Vector" used herein may mean a nucleic acid sequence
containing an origin of replication. A vector may be a plasmid,
bacteriophage, bacterial artificial chromosome or yeast artificial
chromosome. A vector may be a DNA or RNA vector. A vector may be
either a self-replicating extrachromosomal vector or a vector which
integrates into a host genome.
2. TREATMENT OF OVARIAN CANCER, ITS STAGES, AND HISTOLOGICAL
SUBTYPES
[0107] The treatment of ovarian cancer is based on the stage of the
disease which is a reflection of the extent or spread of the cancer
to other parts of the body. Staging is performed when the ovarian
cancer is removed. During the surgical procedure biopsies are
obtained from various sites in the abdominal cavity. During this
procedure, depending on the stage of the disease, the surgeon will
either remove just the ovary and fallopian tube or will remove
ovaries, fallopian tubes and uterus. In addition, the surgeon will
attempt to remove as much of the cancer as possible. Ovarian cancer
is staged as follows:
[0108] Stage I cancer is confined to one or both ovaries. The
cancer is Stage II if either one or both of the ovaries is involved
and has spread to the uterus and/or the fallopian tubes or other
sites in the pelvis. The cancer is Stage III cancer if one or both
of the ovaries is involved and has spread to lymph nodes or other
sites outside of the pelvis but is still within the abdominal
cavity, such as the surface of the intestine or liver. The cancer
is Stage IV cancer if one or both ovaries are involved and the
cancer has spread outside the abdomen or to the inside of the
liver.
[0109] The primary treatment of ovarian cancer is surgery at which
time the cancer is removed from the ovary and from as many other
sites as is possible. Chemotherapy is the second treatment
modality. Another treatment modality is radiation, which is used in
only certain instances. The treatment of ovarian cancer depends on
the stage of the disease, the histological cell type, and the
patient's age and overall condition. The histological cell type and
the extent of disease based on the biopsies performed during
surgery.
[0110] Over 75% of ovarian cancers cases are diagnosed at an
advanced stage. Overall 5-year survival in ovarian epithelial
carcinoma is low because of the preponderance of late-stage disease
at diagnosis. The overall 5-year survival rate, according to
stages, is:
a. Stage I and II: 80-100% b. Stage III: 15-20% c. Stage IV: 5%
[0111] Ovarian cancer is classified according to the histology of
the tumor. Histology dictates many aspects of clinical treatment,
management, and prognosis. Surface epithelial-stromal tumor, also
known as ovarian epithelial carcinoma, is the most common type of
ovarian cancer. It includes serous tumor (including serous
papillary cystadenocarcinoma), endometrioid tumor and mucinous
cystadenocarcinoma.
3. MICRORNAS AND THEIR PROCESSING
[0112] A gene coding for a miRNA may be transcribed leading to
production of a miRNA precursor known as the pri-miRNA. The
pri-miRNA may be part of a polycistronic RNA comprising multiple
pri-miRNAs. The pri-miRNA may form a hairpin with a stem and loop.
The stem may comprise mismatched bases.
[0113] The hairpin structure of the pri-miRNA may be recognized by
Drosha, which is an RNase III endonuclease. Drosha may recognize
terminal loops in the pri-miRNA and cleave approximately two
helical turns into the stem to produce a 30-200 nt precursor known
as the pre-miRNA. Drosha may cleave the pri-miRNA with a staggered
cut typical of Rnase III endonucleases yielding a pre-miRNA stem
loop with a 5' phosphate and .about.2 nucleotide 3' overhang.
Approximately one helical turn of stem (.about.10 nucleotides)
extending beyond the Drosha cleavage site may be essential for
efficient processing. The pre-miRNA may then be actively
transported from the nucleus to the cytoplasm by Ran-GTP and the
export receptor Ex-portin-5.
[0114] The pre-miRNA may be recognized by Dicer, which is also an
Rnase III endonuclease. Dicer may recognize the double-stranded
stem of the pre-miRNA. Dicer may also recognize the 5' phosphate
and 3' overhang at the base of the stem loop. Dicer may cleave off
the terminal loop two helical turns away from the base of the stem
loop leaving an additional 5' phosphate and .about.2 nucleotide 3'
overhang. The resulting siRNA-like duplex, which may comprise
mismatches, comprises the mature miRNA and a similar-sized fragment
known as the miRNA*. The miRNA and miRNA* may be derived from
opposing arms of the pri-miRNA and pre-miRNA. MiRNA* sequences may
be found in libraries of cloned miRNAs but typically at lower
frequency than the miRNAs.
[0115] Although initially present as a double-stranded species with
miRNA*, the miRNA may eventually become incorporated as a
single-stranded RNA into a ribonucleoprotein complex known as the
RNA-induced silencing complex (RISC). Various proteins can form the
RISC, which can lead to variability in specifity for miRNA/miRNA*
duplexes, binding site of the target gene, activity of miRNA
(repress or activate), and which strand of the miRNA/miRNA* duplex
is loaded in to the RISC.
[0116] When the miRNA strand of the miRNA:miRNA* duplex is loaded
into the RISC, the miRNA* may be removed and degraded. The strand
of the miRNA:miRNA* duplex that is loaded into the RISC may be the
strand whose 5' end is less tightly paired. In cases where both
ends of the miRNA:miRNA* have roughly equivalent 5' pairing, both
miRNA and miRNA* may have gene silencing activity.
[0117] The RISC may identify target nucleic acids based on high
levels of complementarity between the miRNA and the mRNA,
especially by nucleotides 2-8 of the miRNA. Only one case has been
reported in animals where the interaction between the miRNA and its
target was along the entire length of the miRNA. This was shown for
miR-196 and Hox B8 and it was further shown that miR-196 mediates
the cleavage of the Hox B8 mRNA (Yekta et al 2004, Science
304-594). Otherwise, such interactions are known only in plants
(Bartel & Bartel 2003, Plant Physiol 132-709).
[0118] A number of studies have looked at the base-pairing
requirement between miRNA and its mRNA target for achieving
efficient inhibition of translation (reviewed by Bartel 2004, Cell
116-281). In mammalian cells, the first 8 nucleotides of the miRNA
may be important (Doench & Sharp 2004 GenesDev 2004-504).
However, other parts of the microRNA may also participate in mRNA
binding. Moreover, sufficient base pairing at the 3' can compensate
for insufficient pairing at the 5' (Brennecke et al, 2005 PloS
3-e85). Computation studies, analyzing miRNA binding on whole
genomes have suggested a specific role for bases 2-7 at the 5' of
the miRNA in target binding but the role of the first nucleotide,
found usually to be "A" was also recognized (Lewis et at 2005 Cell
120-15). Similarly, nucleotides 1-7 or 2-8, the "seed", were used
to identify and validate targets. MiRNAs differ in their basic
structure and sequence of nucleotides; however similarity in seed
sequence may suggest similar activity.
[0119] The target sites in the mRNA may be in the 5' UTR, the 3'
UTR or in the coding region. Interestingly, multiple miRNAs may
regulate the same mRNA target by recognizing the same or multiple
sites. The presence of multiple miRNA binding sites in most
genetically identified targets may indicate that the cooperative
action of multiple RISCs provides the most efficient translational
inhibition.
[0120] miRNAs may direct the RISC to downregulate gene expression
by either of two mechanisms: mRNA cleavage or translational
repression. The miRNA may specify cleavage of the mRNA if the mRNA
has a certain degree of complementarity to the miRNA. When a miRNA
guides cleavage, the cut may be between the nucleotides pairing to
residues 10 and 11 of the miRNA. Alternatively, the miRNA may
repress translation if the miRNA does not have the requisite degree
of complementarity to the miRNA. Translational repression may be
more prevalent in animals since animals may have a lower degree of
complementarity between the miRNA and binding site.
[0121] It should be noted that there may be variability in the 5'
and 3' ends of any pair of miRNA and miRNA*. This variability may
be due to variability in the enzymatic processing of Drosha and
Dicer with respect to the site of cleavage. Variability at the 5'
and 3' ends of miRNA and miRNA* may also be due to mismatches in
the stem structures of the pri-miRNA and pre-miRNA. The mismatches
of the stem strands may lead to a population of different hairpin
structures. Variability in the stem structures may also lead to
variability in the products of cleavage by Drosha and Dicer.
4. NUCLEIC ACIDS
[0122] Nucleic acids are provided herein. The nucleic acid may
comprise the sequence of SEQ ID NOS: 1-58 presented in table 1 or
variants thereof. The variant may be a complement of the referenced
nucleotide sequence. The variant may also be a nucleotide sequence
that is substantially identical to the referenced nucleotide
sequence or the complement thereof. The variant may also be a
nucleotide sequence which hybridizes under stringent conditions to
the referenced nucleotide sequence, complements thereof, or
nucleotide sequences substantially identical thereto.
[0123] The nucleic acid may have a length of from 10 to 250
nucleotides. The nucleic acid may have a length of at least 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or
250 nucleotides. The nucleic acid may be synthesized or expressed
in a cell (in vitro or in vivo) using a synthetic gene described
herein. The nucleic acid may be synthesized as a single strand
molecule and hybridized to a substantially complementary nucleic
acid to form a duplex. The nucleic acid may be introduced to a
cell, tissue or organ in a single- or double-stranded form or
capable of being expressed by a synthetic gene using methods well
known to those skilled in the art, including as described in U.S.
Pat. No. 6,506,559 which is incorporated by reference.
TABLE-US-00001 TABLE 1 MiR Hairpin Antisense SEQ SEQ ID External
Antisense oligo SEQ ID Target miR ID NO: NO: ID Sequence NO:
hsa-miR-100 1 19 36858359 CACAAGUUCGGA 41 UCUACGGGUU hsa-miR-103 2
20, 21 36858360 UCAUAGCCCUGU 42 ACAAUGCUGCU hsa-miR-125b 3 22, 23
36858363 UCACAAGUUAGG 43 GUCUCAGGGA hsa-miR-17 4 24 60432524
CUACCUGCACUG 44 UAAGCACUUUG hsa-miR-18a 5 25 60432525 CUAUCUGCACUA
45 GAUGCACCUUA hsa-miR-191 6 26 36858368 CAGCUGCUUUUG 46
GGAUUCCGUUG hsa-miR-20b 7 27 36641194 CUACCUGCACUA 47 UGAGCACUUUG
hsa-miR-21 8 28 36858370 UCAACAUCAGUC 48 UGAUAAGCUA hsa-miR-210 9
29 36858371 UCAGCCGCUGUC 49 ACACGCACAG hsa-miR-22 10 30 36858373
ACAGUUCUUCAA 50 CUGGCAGCUU hsa-miR-221 11 31 36858374 GAAACCCAGCAG
51 ACAAUGUAGCU hsa-miR-23a 12 32 36858376 GGAAAUCCCUGG 52 CAAUGUGAU
hsa-miR-24 13 33, 34 36858377 CUGUUCCUGCUG 53 AACUGAGCCA hsa-miR-25
14 35 60432527 UCAGACCGAGAC 54 AAGUGCAAUG hsa-miR-26a 15 36, 37
36858378 AGCCUAUCCUGG 55 AUUACUUGAA hsa-miR-27a 16 38 38548308
GCGGAACUUAGC 56 CACUGUGAA hsa-miR-31 17 39 36858379 AGCUAUGCCAGC 57
AUCUUGCCU hsa-miR-99a 18 40 36858385 CACAAGAUCGGA 58 UCUACGGGUU
[0124] Nucleic Acid Complex
[0125] The nucleic acid may further comprise one or more of the
following: a peptide, a protein, a RNA-DNA hybrid, an antibody, an
antibody fragment, a Fab fragment, and an aptamer. The nucleic acid
may also comprise a protamine-antibody fusion protein as described
in Song et at (Nature Biotechnology 2005; 23:709-17) and Rossi
(Nature Biotechnology 2005: 23; 682-4), the contents of which are
incorporated herein by reference. The protamine-fusion protein may
comprise the abundant and highly basic cellular protein protamine.
The protamine may readily interact with the nucleic acid. The
protamine may comprise the entire 51 amino acid protamine peptide
or a fragment thereof. The protamine may be covalently attached to
another protein, which may be a Fab. The Fab may bind to a receptor
expressed on a cell surface.
[0126] Pri-miRNA
[0127] The nucleic acid may comprise a sequence of a pri-miRNA or a
variant thereof. The pri-miRNA sequence may comprise from
45-30,000, 50-25,000, 100-20,000, 1,000-1,500 or 80-100
nucleotides. The sequence of the pri-miRNA may comprise a
pre-miRNA, miRNA and miRNA*, as set forth herein, and variants
thereof. The sequence of the pri-miRNA may comprise the sequence of
SEQ ID NOS: 1-40 or variants thereof
[0128] The pri-miRNA may form a hairpin structure. The hairpin may
comprise first and second nucleic acid sequence that are
substantially complimentary. The first and second nucleic acid
sequence may be from 37-50 nucleotides. The first and second
nucleic acid sequence may be separated by a third sequence of from
8-12 nucleotides. The hairpin structure may have a free energy less
than -25 Kcal/mole as calculated by the Vienna algorithm with
default parameters, as described in Hofacker et al., Monatshefte f.
Chemie 125: 167-188 (1994), the contents of which are incorporated
herein. The hairpin may comprise a terminal loop of 4-20, 8-12 or
10 nucleotides. The pri-miRNA may comprise at least 19% adenosine
nucleotides, at least 16% cytosine nucleotides, at least 23%
thymine nucleotides and at least 19% guanine nucleotides.
[0129] Pre-miRNA
[0130] The nucleic acid may also comprise a sequence of a pre-miRNA
or a variant thereof. The pre-miRNA sequence may comprise from
45-200, 60-80 or 60-70 nucleotides. The sequence of the pre-miRNA
may comprise a miRNA and a miRNA* as set forth herein. The sequence
of the pre-miRNA may also be that of a pri-miRNA excluding from
0-160 nucleotides from the 5' and 3' ends of the pri-miRNA. The
sequence of the pre-miRNA may comprise the sequence of SEQ ID NOS:
1-40 or variants thereof.
[0131] MiRNA
[0132] The nucleic acid may also comprise a sequence of a miRNA
(including miRNA*) or a variant thereof. The miRNA sequence may
comprise from 13-33, 18-24 or 21-23 nucleotides. The miRNA may also
comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. The sequence of the
miRNA may be the first 13-33 nucleotides of the pre-miRNA. The
sequence of the miRNA may also be the last 13-33 nucleotides of the
pre-miRNA. The sequence of the miRNA may comprise the sequence of
SEQ ID NOS: 1-18, or variants thereof
[0133] Anti-miRNA
[0134] The nucleic acid may also comprise a sequence of an
anti-miRNA that is capable of blocking the activity of a miRNA or
miRNA*, such as by binding to the pri-miRNA, pre-miRNA, miRNA or
miRNA* (e.g. antisense or RNA silencing), or by binding to the
target binding site. The anti-miRNA may comprise a total of 5-100
or 10-60 nucleotides. The anti-miRNA may also comprise a total of
at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39 or 40 nucleotides. The sequence of the anti-miRNA may
comprise (a) at least 5 nucleotides that are substantially
identical or complimentary to the 5' of a miRNA and at least 5-12
nucleotides that are substantially complimentary to the flanking
regions of the target site from the 5' end of the miRNA, or (b) at
least 5-12 nucleotides that are substantially identical or
complimentary to the 3' of a miRNA and at least 5 nucleotide that
are substantially complimentary to the flanking region of the
target site from the 3' end of the miRNA. The sequence of the
anti-miRNA may comprise the compliment of SEQ ID NOS: 1-18, or
variants thereof.
5. PROBES
[0135] A probe is also provided comprising a nucleic acid described
herein. Probes may be used for screening and diagnostic methods.
The probe may be attached or immobilized to a solid substrate, such
as a biochip.
[0136] The probe may have a length of from 8 to 500, 10 to 100 or
20 to 60 nucleotides. The probe may also have a length of at least
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120,
140, 160, 180, 200, 220, 240, 260, 280 or 300 nucleotides. The
probe may further comprise a linker sequence of from 10-60
nucleotides.
6. BIOCHIP
[0137] A biochip is also provided. The biochip may comprise a solid
substrate comprising an attached probe or plurality of probes
described herein. The probes may be capable of hybridizing to a
target sequence under stringent hybridization conditions. The
probes may be attached at spatially defined address on the
substrate. More than one probe per target sequence may be used,
with either overlapping probes or probes to different sections of a
particular target sequence. The probes may be capable of
hybridizing to target sequences associated with a single disorder
appreciated by those in the art. The probes may either be
synthesized first, with subsequent attachment to the biochip, or
may be directly synthesized on the biochip.
[0138] The solid substrate may be a material that may be modified
to contain discrete individual sites appropriate for the attachment
or association of the probes and is amenable to at least one
detection method. Representative examples of substrates include
glass and modified or functionalized glass, plastics (including
acrylics, polystyrene and copolymers of styrene and other
materials, polypropylene, polyethylene, polybutylene,
polyurethanes, TeflonJ, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses and
plastics. The substrates may allow optical detection without
appreciably fluorescing.
[0139] The substrate may be planar, although other configurations
of substrates may be used as well. For example, probes may be
placed on the inside surface of a tube, for flow-through sample
analysis to minimize sample volume. Similarly, the substrate may be
flexible, such as a flexible foam, including closed cell foams made
of particular plastics.
[0140] The biochip and the probe may be derivatized with chemical
functional groups for subsequent attachment of the two. For
example, the biochip may be derivatized with a chemical functional
group including, but not limited to, amino groups, carboxyl groups,
oxo groups or thiol groups. Using these functional groups, the
probes may be attached using functional groups on the probes either
directly or indirectly using a linker. The probes may be attached
to the solid support by either the 5' terminus, 3' terminus, or via
an internal nucleotide.
[0141] The probe may also be attached to the solid support
non-covalently. For example, biotinylated oligonucleotides can be
made, which may bind to surfaces covalently coated with
streptavidin, resulting in attachment. Alternatively, probes may be
synthesized on the surface using techniques such as
photopolymerization and photolithography.
7. THERAPEUTIC
[0142] A method for treating a disease or disorder associated with
ovarian cancer is also provided. Furthermore, existing miRNA
molecules may be used as starting materials for the manufacture of
sequence-modified anti-miRNA molecules. As previously discussed the
methods, compositions and articles of manufacture of the present
invention are particularly useful in the treatment of cancer.
[0143] The compositions of the present invention may be combined
with a chemotherapeutic agent, a combination of chemotherapeutic
agents and/or radiotherapy.
[0144] Cancer treatments often comprise more than one therapy. As
such, in certain embodiments the present invention provides methods
for treating cancer comprising administering to a subject in need
thereof the composition of the present invention, and further
comprising administering at least one additional therapy.
[0145] In certain embodiments, an additional therapy may also be
designed to treat cancer. An additional therapy may be a
chemotherapeutic agent. Suitable chemotherapeutic agents include
cisplatin, carboplatin, camptothecins, doxorubiein,
cyclophosphamide, paclitaxel, etoposide, vinblastine, Actinomycin D
and cloposide An additional therapy may be surgical resection of
tumor(s), or chemoembolization.
8. COMPOSITIONS
[0146] A pharmaceutical composition is also provided. The
composition may comprise a nucleic acid described herein and
optionally a pharmaceutically acceptable carrier. The composition
may encompass modified oligonucleotides that are identical,
substantially identical, substantially complementary or
complementary to any nucleobase sequence version of the miRNAs or
nucleic acids described herein or a precursor thereof.
[0147] The compositions may be used for therapeutic applications.
The pharmaceutical composition may be administered by known
methods, including wherein a nucleic acid is introduced into a
desired target cell in vitro or in vivo.
[0148] Methods for the delivery of nucleic acid molecules are
described in Akhtar et al., (Trends Cell Bio. 2, 139, 1992). WO
94/02595 describes general methods for delivery of RNA molecules.
These protocols can be utilized for the delivery of virtually any
nucleic acid molecule. Nucleic acid molecules can be administered
to cells by a variety of methods known to those familiar to the
art, including, but not restricted to, encapsulation in liposomes,
by iontophoresis, or by incorporation into other vehicles, such as
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres. Alternatively, the nucleic acid/vehicle
combination is locally delivered by direct injection or by use of
an infusion pump. Other routes of delivery include, but are not
limited to oral (tablet or pill form) and/or intrathecal delivery
(Gold, 1997, Neuroscience, 76, 1153-1158). Other approaches include
the use of various transport and carrier systems, for example,
through the use of conjugates and biodegradable polymers. More
detailed descriptions of nucleic acid delivery and administration
are provided for example in WO93/23569, WO99/05094, and
WO99/04819.
[0149] The nucleic acids can be introduced into tissues or host
cells by any number of routes, including viral infection,
microinjection, or fusion of vesicles. Jet injection may also be
used for intra-muscular administration, as described by Furth et
al. (Anal Biochem 115 205:365-368, 1992). The nucleic acids can be
coated onto gold microparticles, and delivered intradermally by a
particle bombardment device, or "gene gun" as described in the
literature (see, for example, Tang et al. Nature 356:152-154,
1992), where gold microprojectiles are coated with the DNA, then
bombarded into skin cells.
[0150] The compositions of the present invention can be formulated
into pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents, and can be
formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants and
aerosols. As such, administration of the agents can be achieved in
various ways, including oral, buccal, rectal, parenteral,
intraperitoneal, intradermal, transdermal, intracheal, etc.
[0151] In certain embodiments, a pharmaceutical composition of the
present invention is administered in the form of a dosage unit
(e.g., tablet, capsule, bolus, etc.). In certain embodiments, such
pharmaceutical compositions comprise a modified oligonucleotide in
a dose selected from 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55
mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg,
105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145
mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg,
190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230
mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg,
270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315
mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg,
360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400
mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg,
445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485
mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg,
530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570
mg, 575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg,
615 mg, 620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655
mg, 660 mg, 665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg,
700 mg, 705 mg, 710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740
mg, 745 mg, 750 mg, 755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg,
785 mg, 790 mg, 795 mg, and 800 mg. In certain such embodiments, a
pharmaceutical composition of the present invention comprises a
dose of modified oligonucleotide selected from 25 mg, 50 mg, 75 mg,
100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600
mg, 700 mg, and 800 mg.
[0152] In certain embodiments, a pharmaceutical agent is sterile
lyophilized modified oligonucleotide that is reconstituted with a
suitable diluent, e.g., sterile water for injection or sterile
saline for injection. The reconstituted product is administered as
a subcutaneous injection or as an intravenous infusion after
dilution into saline. The lyophilized drug product consists of a
modified oligonucleotide which has been prepared in water for
injection, or in saline for injection, adjusted to pH 7.0-9.0 with
acid or base during preparation, and then lyophilized. The
lyophilized modified oligonucleotide may be 25-800 mg of a modified
oligonucleotide. It is understood that this encompasses 25, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425,
450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750,
775, and 800 mg of modified lyophilized oligonucleotide.
[0153] In certain embodiments, the compositions of the present
invention may additionally contain other adjunct components
conventionally found in pharmaceutical compositions, at their
art-established usage levels. Thus, for example, the compositions
may contain additional, compatible, pharmaceutically-active
materials such as, for example, antipruritics, astringents, local
anesthetics or anti-inflammatory agents, or may contain additional
materials useful in physically formulating various dosage forms of
the compositions of the present invention, such as dyes, flavoring
agents, preservatives, antioxidants, opacifiers, thickening agents
and stabilizers. However, such materials, when added, should not
unduly interfere with the biological activities of the components
of the compositions of the present invention. The formulations can
be sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the oligonucleotide(s) of the
formulation.
[0154] In certain embodiments, pharmaceutical compositions of the
present invention comprise one or more modified oligonucleotides
and one or more excipients. In certain such embodiments, excipients
are selected from water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylase, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose and
polyvinylpyrrolidone.
[0155] In certain embodiments, a pharmaceutical composition of the
present invention is prepared using known techniques, including,
but not limited to mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or tabletting
processes.
[0156] In certain embodiments, a pharmaceutical composition of the
present invention is a liquid (e.g., a suspension, elixir and/or
solution). In certain of such embodiments, a liquid pharmaceutical
composition is prepared using ingredients known in the art,
including, but not limited to, water, glycols, oils, alcohols,
flavoring agents, preservatives, and coloring agents.
[0157] In certain embodiments, a pharmaceutical composition of the
present invention is a solid (e.g., a powder, tablet, and/or
capsule). In certain of such embodiments, a solid pharmaceutical
composition comprising one or more oligonucleotides is prepared
using ingredients known in the art, including, but not limited to,
starches, sugars, diluents, granulating agents, lubricants,
binders, and disintegrating agents.
[0158] In certain embodiments, a pharmaceutical composition of the
present invention is formulated as a depot preparation. Certain
such depot preparations are typically longer acting than non-depot
preparations. In certain embodiments, such preparations are
administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. In certain
embodiments, depot preparations are prepared using suitable
polymeric or hydrophobic materials (for example an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
[0159] In certain embodiments, a pharmaceutical composition of the
present invention comprises a delivery system. Examples of delivery
systems include, but are not limited to, liposomes and emulsions.
Certain delivery systems are useful for preparing certain
pharmaceutical compositions including those comprising hydrophobic
compounds. In certain embodiments, certain organic solvents such as
dimethylsulfoxide are used.
[0160] In certain embodiments, a pharmaceutical composition of the
present invention comprises one or more tissue-specific delivery
molecules designed to deliver the one or more pharmaceutical agents
of the present invention to specific tissues or cell types. For
example, in certain embodiments, pharmaceutical compositions
include liposomes coated with a tissue-specific antibody.
[0161] In certain embodiments, a pharmaceutical composition of the
present invention comprises a co-solvent system. Certain of such
co-solvent systems comprise, for example, benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. In certain embodiments, such co-solvent systems are
used for hydrophobic compounds. A non-limiting example of such a
co-solvent system is the VPD co-solvent system, which is a solution
of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant Polysorbate 80.TM. and 65% w/v polyethylene
glycol 300. The proportions of such co-solvent systems may be
varied considerably without significantly altering their solubility
and toxicity characteristics. Furthermore, the identity of
co-solvent components may be varied: for example, other surfactants
may be used instead of Polysorbate 80.TM.; the fraction size of
polyethylene glycol may be varied; other biocompatible polymers may
replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
[0162] In certain embodiments, a pharmaceutical composition of the
present invention comprises a sustained-release system. A
non-limiting example of such a sustained-release system is a
semi-permeable matrix of solid hydrophobic polymers. In certain
embodiments, sustained-release systems may, depending on their
chemical nature, release pharmaceutical agents over a period of
hours, days, weeks or months.
[0163] In certain embodiments, a pharmaceutical composition of the
present invention is prepared for oral administration. In certain
of such embodiments, a pharmaceutical composition is formulated by
combining one or more compounds comprising modified
oligonucleotides with one or more pharmaceutically acceptable
carriers. Certain of such carriers enable pharmaceutical
compositions to be formulated as tablets, pills, dragees, capsules,
liquids, gels, syrups, slurries, suspensions and the like, for oral
ingestion by a subject. In certain embodiments, pharmaceutical
compositions for oral use are obtained by mixing oligonucleotide
and one or more solid excipient. Suitable excipients include, but
are not limited to, fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). In certain embodiments, such a
mixture is optionally ground and auxiliaries are optionally added.
In certain embodiments, pharmaceutical compositions are formed to
obtain tablets or dragee cores. In certain embodiments,
disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone,
agar, or alginic acid or a salt thereof, such as sodium alginate)
are added.
[0164] In certain embodiments, dragee cores are provided with
coatings. In certain such embodiments, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to tablets
or dragee coatings.
[0165] In certain embodiments, pharmaceutical compositions for oral
administration are push-fit capsules made of gelatin. Certain of
such push-fit capsules comprise one or more pharmaceutical agents
of the present invention in admixture with one or more filler such
as lactose, binders such as starches, and/or lubricants such as
talc or magnesium stearate and, optionally, stabilizers. In certain
embodiments, pharmaceutical compositions for oral administration
are soft, sealed capsules made of gelatin and a plasticizer, such
as glycerol or sorbitol. In certain soft capsules, one or more
pharmaceutical agents of the present invention are be dissolved or
suspended in suitable liquids, such as fatty oils, liquid paraffin,
or liquid polyethylene glycols. In addition, stabilizers may be
added.
[0166] In certain embodiments, pharmaceutical compositions are
prepared for buccal administration. Certain of such pharmaceutical
compositions are tablets or lozenges formulated in conventional
manner.
[0167] In certain embodiments, a pharmaceutical composition is
prepared for administration by injection (e.g., intravenous,
subcutaneous, intramuscular, etc.). In certain of such embodiments,
a pharmaceutical composition comprises a carrier and is formulated
in aqueous solution, such as water or physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. In certain embodiments, other
ingredients are included (e.g., ingredients that aid in solubility
or serve as preservatives). In certain embodiments, injectable
suspensions are prepared using appropriate liquid carriers,
suspending agents and the like. Certain pharmaceutical compositions
for injection are presented in unit dosage form, e.g., in ampoules
or in multi-dose containers. Certain pharmaceutical compositions
for injection are suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Certain solvents
suitable for use in pharmaceutical compositions for injection
include, but are not limited to, lipophilic solvents and fatty
oils, such as sesame oil, synthetic fatty acid esters, such as
ethyl oleate or triglycerides, and liposomes. Aqueous injection
suspensions may contain substances that increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, such suspensions may also contain suitable
stabilizers or agents that increase the solubility of the
pharmaceutical agents to allow for the preparation of highly
concentrated solutions.
[0168] In certain embodiments, a pharmaceutical composition is
prepared for transmucosal administration. In certain of such
embodiments penetrants appropriate to the barrier to be permeated
are used in the formulation. Such penetrants are generally known in
the art.
[0169] In certain embodiments, a pharmaceutical composition is
prepared for administration by inhalation. Certain of such
pharmaceutical compositions for inhalation are prepared in the form
of an aerosol spray in a pressurized pack or a nebulizer. Certain
of such pharmaceutical compositions comprise a propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
certain embodiments using a pressurized aerosol, the dosage unit
may be determined with a valve that delivers a metered amount. In
certain embodiments, capsules and cartridges for use in an inhaler
or insufflator may be formulated. Certain of such formulations
comprise a powder mixture of a pharmaceutical agent of the
invention and a suitable powder base such as lactose or starch.
[0170] In certain embodiments, a pharmaceutical composition is
prepared for rectal administration, such as a suppositories or
retention enema. Certain of such pharmaceutical compositions
comprise known ingredients, such as cocoa butter and/or other
glycerides.
[0171] In certain embodiments, a pharmaceutical composition is
prepared for topical administration. Certain of such pharmaceutical
compositions comprise bland moisturizing bases, such as ointments
or creams. Exemplary suitable ointment bases include, but are not
limited to, petrolatum, petrolatum plus volatile silicones, and
lanolin and water in oil emulsions. Exemplary suitable cream bases
include, but are not limited to, cold cream and hydrophilic
ointment.
[0172] In certain embodiments, a pharmaceutical composition of the
present invention comprises a modified oligonucleotide in a
therapeutically effective amount. In certain embodiments, the
therapeutically effective amount is sufficient to prevent,
alleviate or ameliorate symptoms of a disease or to prolong the
survival of the subject being treated. Determination of a
therapeutically effective amount is well within the capability of
those skilled in the art.
[0173] In certain embodiments, one or more modified
oligonucleotides of the present invention are formulated as a
prodrug. In certain embodiments, upon in vivo administration, a
prodrug is chemically converted to the biologically,
pharmaceutically or therapeutically more active form of a modified
oligonucleotide. In certain embodiments, prodrugs are useful
because they are easier to administer than the corresponding active
form. For example, in certain instances, a prodrug may be more
bioavailable (e.g., through oral administration) than is the
corresponding active form. In certain instances, a prodrug may have
improved solubility compared to the corresponding active form. In
certain embodiments, prodrugs are less water soluble than the
corresponding active form. In certain instances, such prodrugs
possess superior transmittal across cell membranes, where water
solubility is detrimental to mobility. In certain embodiments, a
prodrug is an ester. In certain such embodiments, the ester is
metabolically hydrolyzed to carboxylic acid upon administration. In
certain instances the carboxylic acid containing compound is the
corresponding active form. In certain embodiments, a prodrug
comprises a short peptide (polyaminoacid) bound to an acid group.
In certain of such embodiments, the peptide is cleaved upon
administration to form the corresponding active form.
[0174] In certain embodiments, a prodrug is produced by modifying a
pharmaceutically active compound such that the active compound will
be regenerated upon in vivo administration. The prodrug can be
designed to alter the metabolic stability or the transport
characteristics of a drug, to mask side effects or toxicity, to
improve the flavor of a drug or to alter other characteristics or
properties of a drug. By virtue of knowledge of pharmacodynamic
processes and drug metabolism in vivo, those of skill in this art,
once a pharmaceutically active compound is known, can design
prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal
Chemistry A Biochemical Approach, Oxford University Press, New
York, pages 388-392).
[0175] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples, which are provided by way of illustration and are not
intended to be limiting of the present invention.
EXAMPLES
Example 1
Materials and Methods
[0176] a. Patients and Samples
[0177] Patients, who were surgically treated for ovarian cancer at
the Rabin Medical Center between January, 2000 and December, 2004
were identified. All pathology slides were re-evaluated by an
expert pathologist. Tumor histology was established and the
diagnosis of EOC was confirmed. Only serous papillary and
endometrioid histology were included in the study. Patients found
to have a synchronous endometrial malignancy were excluded. For
each patient, a formalin-fixed paraffin embedded (FFPE) tumor
sample was obtained and tumor cell content was evaluated by a
pathologist. Only tumor samples with a minimum of 50% tumor tissue
content were included. Patient charts were reviewed for
clinicopathologic information--demographics, surgical procedure and
findings, pathology, chemotherapy regimens and response, follow-up
and survival. Optimal surgical cytoreduction was defined during the
study period as the largest residual tumor diameter of 1 cm.
[0178] 21 samples from ovarian tumor and 19 samples from ovarian
metastases were compared to 53 different normal tissues of which 18
were normal tissue of the abdominal area (including: colon, liver,
small intestine, fallopian tubes, endometrium, Small
intestine-Deudenum, Small intestine-Jejunum, Stomach, and Spleen)
for their miR expression. Comparison was also made to normal
ovarian tissues. Normal samples were purchased from Ambion.
[0179] b. RNA Extraction
[0180] For FFPE samples, total RNA was isolated from seven to ten
10-.mu.m-thick tissue sections using the microRNA extraction
protocol developed at Rosetta Genomics. Briefly, the sample is
incubated a few times in Xylene at 57.degree. to remove paraffin
excess, followed by Ethanol washes. Proteins are degraded by
proteinase K solution at 45.degree. C. for few hours. The RNA is
extracted with acid phenol:chloroform followed by ethanol
precipitation and DNAse digestion. Total RNA quantity and quality
is checked by spectrophotometer (Nanodrop ND-1000).
[0181] c. microRNA Microarray Platform
[0182] Custom microarrays were produced by printing DNA
oligonucleotide probes representing 903 human microRNAs. Each
probe, printed in triplicate, carries up to 22-nt linker at the 3'
end of the microRNA's complement sequence in addition to an amine
group used to couple the probes to coated glass slides. 20 .mu.M of
each probe were dissolved in 2.times.SSC+0.0035% SDS and spotted in
triplicate on Schott Nexterion.RTM. Slide E coated microarray
slides (Mainz, Germany) using a Genomic Solutions.RTM. BioRobotics
MicroGrid II according the MicroGrid manufacturer's directions. 22
negative control probes were designed using the sense sequences of
different microRNAs. Two groups of positive control probes were
designed to hybridize to the microarray (i) synthetic small RNA
were spiked to the RNA before labeling to verify the labeling
efficiency and (ii) probes for abundant small RNA (e.g. small
nuclear RNAs (U43, U49, U24, Z30, U6, U48, U44), 5.8 s and 5 s
ribosomal RNA) are spotted on the array to verify RNA quality. The
slides were blocked in a solution containing 50 mM ethanolamine, 1M
Tris (pH9.0) and 0.1% SDS for 20 mM at 50.degree. C., then
thoroughly rinsed with water and spun dry.
[0183] d. Cy-Dye Labeling of microRNA for Microarray
[0184] Five .mu.g of total RNA were labeled by ligation (Thomson et
al., Nature Methods 2004, 1:47-53) of an RNA-linker, p-rCrU-Cy/dye
(Dharmacon, Lafayette), to the 3'-end with Cy3 or Cy5. The labeling
reaction contained total RNA, spikes (0.1-20 fmoles), 400 ng
RNA-linker-dye, 15% DMSO, lx ligase buffer and 20 units of T4 RNA
ligase (NEB) and proceeded at 4.degree. C. for 1 hr followed by 1
hr at 37.degree. C. The labeled RNA was mixed with 3.times.
hybridization buffer (Ambion), heated to 95.degree. C. for 3 min
and then added on top of the microarray. Slides were hybridized
12-16 hr in 42.degree. C., followed by two washes in room
temperature with 1.times.SSC and 0.2% SDS and a final wash with
0.1.times.SSC.
[0185] Arrays were scanned using an Agilent Microarray Scanner
Bundle G2565BA (resolution of 10 .mu.m at 100% and 10% power).
Array images were analyzed using SpotReader software (Niles
Scientific).
[0186] e. Data Analysis
[0187] Triplicate spots were combined to produce one signal for
each probe by taking the logarithmic mean of reliable spots. All
data was log-transformed (natural base) and the analysis was
performed in log-space. A reference data vector for normalization R
was calculated by taking the median expression level for each probe
across all samples. For each sample data vector S, a 2nd degree
polynomial F was found so as to provide the best fit between the
sample data and the reference data, such that R.apprxeq.F(S).
Remote data points ("outliers") were not used for fitting the
polynomial F. For each probe in the sample (element S.sub.i in the
vector 5), the normalized value (in log-space) M.sub.i was
calculated from the initial value S.sub.i by transforming it with
the polynomial function F, so that M.sub.i=F(S.sub.i).
[0188] f. Cell Culture Maintenance and Transfections
[0189] OVCAR-3 and SKOV-3 cell lines were purchased from NCI. Cells
were grown under standard growth conditions. Transfection of cells
with anti-miRs was conducted using Oligofectamine (Invitrogen,
Cat#12252011), according to manufacturer instructions. Briefly,
cells were exposed to anti-miR and transfection reagent complex in
OptiMEM for 4 h. After that transfection media was removed and full
supplemented media was applied.
[0190] g. Proliferation Assays
[0191] 72 h after transfection, cells were tested for proliferation
using cell Proliferation Assay kit-cellTiter 96 AQueous One
solution (Promega, Cat# G3581), according to manufacture
instructions. Absorbance was measured at 490 nm, using ELx808 ultra
microplate reader (BIO-TEK instruments, INC). Absorbance was
blanked using medium and calculated as % of untreated cells. At day
of transfection another plate with non-treated cells were also
tested for proliferation. This result was subtracted from the 72 h
read, so proliferation is calculated from the time of
transfection.
[0192] h. Anti-miR Molecules
[0193] 2'-O-Me modified Antisense oligonucleotides having the
sequence of the reverse complement of the relevant miRs were
ordered from IDT.
Example 2
Expression Analysis
[0194] Comparison of ovarian (tumors and metastases) samples to
normal abdominal tissues and to normal ovarian tissues resulted
with a list of upregulated or highly expressed miRs. Table 2
discloses the list of miRs that were later shown to be important in
cell proliferation inhibition by anti-miRs.
TABLE-US-00002 TABLE 2 Signal Signal in in Normal miRname Tumor
tissue fold-change p-value Reason for election hsa-miR-100 9800
3400 2.91(+) 0.0000061 differentially expressed-- upregulated in
cancer hsa-miR-17 4600 2100 2.16(+) 4.4E-09 differentially
expressed-- upregulated in cancer hsa-miR-18a 760 160 4.61(+)
3.9E-09 differentially expressed-- upregulated in cancer hsa-miR-21
70000 32000 2.17(+) 5.1E-11 differentially expressed-- upregulated
in cancer hsa-miR-210 2300 340 6.70(+) 9.1E-13 differentially
expressed-- upregulated in cancer hsa-miR-24 31000 15000 2.09(+)
2.4E-13 differentially expressed-- upregulated in cancer hsa-miR-25
2000 880 2.29(+) 0.00000078 differentially expressed-- upregulated
in cancer hsa-miR-27a 17000 6400 2.66(+) 1.3E-10 differentially
expressed-- upregulated in cancer hsa-miR-99a 16000 3800 4.18(+)
0.000016 differentially expressed-- upregulated in cancer
hsa-mir-103 14000 8600 1.62(+) 0.0011 differentially expressed--
upregulated in cancer hsa-mir-22 5500 2500 2.18(+) 0.0034
differentially expressed-- upregulated in cancer hsa-mir-221 12000
7700 1.59(+) 0.00041 differentially expressed-- upregulated in
cancer hsa-mir-23a 30000 16000 1.84(+) 0.00025 differentially
expressed-- upregulated in cancer hsa-mir-26a 31000 22000 1.43(+)
0.013 differentially expressed-- upregulated in cancer hsa-mir-31
2700 720 3.73(+) 0.043 differentially expressed-- upregulated in
cancer hsa-mir-125b 30000 50000 1.69(-) 0.038 Highly expressed in
30000 25000 1.17(+) 0.76 ovarian tissue hsa-mir-20b 650 160 NA NA
differentially expressed-- upregulated in cancer hsa-mir-191 7100
6200 1.15(+) 0.45 Highly expressed in 7100 7000 1.02(+) 0.85
ovarian tissue
Example 3
Proliferation Assays
[0195] OVCAR-3 cells (ovarian adenocarcinoma), were used to study
the effect of miR inhibition on proliferation of cells. Cells were
transfected with increasing amounts of anti-miR in the range of
10-300 nM. Proliferation was assayed 72 hours after transfection.
FIGS. 1-5 demonstrate the inhibition of proliferation for some of
the upregulated and highly expressed miRs. Assay was repeated in
SKOV-3 ovarian adenocarcinoma cells with similar trends. Table 3
shows the results of the concentration needed for 50% proliferation
inhibition for the two cell lines. Negative control anti-miR was an
oligonucleotide with similar chemical modifications, but an
irrelevant sequence (Antisense for miR-122 harboring 6
missmatches).
TABLE-US-00003 TABLE 3 IC-50(nM) Candidate OVCAR-3 SK-OV-3
hsa-mir-100 60 60 hsa-mir-103 32 38 hsa-mir-125b 48 80 hsa-mir-17
82 280 hsa-mir-18a 100 300 hsa-mir-191 40 30 hsa-mir-20b 45 90
hsa-mir-21 70 50 hsa-mir-210 65 58 hsa-mir-22 50 55 hsa-mir-221 85
65 hsa-mir-23a 82 60 hsa-mir-24 38 68 hsa-mir-25 85 65 hsa-mir-26a
28 58 hsa-mir-27a 68 150 hsa-mir-31 75 100 hsa-mir-99a 42 60
Example 4
In Vivo Study
[0196] In order to demonstrate the efficacy of the anti-miR
molecules of the invention, and the potential of those molecules in
ovarian cancer therapy, an in vivo study is performed using a mouse
model for peritoneal ovarian metastases.
female BALB/c nude mice are injected IP with 5.times.10.sup.6
SKOV-3 cells in 200 ul PBS for tumor induction. 24 hours after
tumor inoculation, anti-miR-oligonucleotides are injected IP every
other day at a dose of 50 mg/Kg, for a period of two weeks into 15
animals. A second group of animals are injected with a control
anti-miR with an irrelevant sequence.
[0197] The body weight and the survival of all animals are measured
and vital parameters are monitored.
[0198] At termination of the study the following parameters are
measured: tumors in the peritoneal cavity, number of tumor nodules,
total tumor volume and weight, invasion into other organs and
ascites volume.
[0199] Serum is sampled from the animals and the tumors are fresh
frozen, and fixed in formalin for FFPE.
[0200] The following parameters are compared between the study and
control groups: number of tumor nodules, total tumor volume, total
tumor weight, ascites volume, CA-125 in serum, miR-concentration in
the tumor using qRT-PCR, and in situ hybridization analysis of the
specific miR in the tumor tissue.
[0201] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. Although the invention
has been described in conjunction with specific embodiments
thereof, it is evident that many alternatives, modifications and
variations will be apparent to those skilled in the art.
Accordingly, it is intended to embrace all such alternatives,
modifications and variations that fall within the spirit and broad
scope of the appended claims.
[0202] It should be understood that the detailed description and
specific examples, while indicating preferred embodiments of the
invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
Sequence CWU 1
1
58122RNAHomo sapiens 1aacccguaga uccgaacuug ug 22223RNAHomo sapiens
2agcagcauug uacagggcua uga 23322RNAHomo sapiens 3ucccugagac
ccuaacuugu ga 22423RNAHomo sapiens 4caaagugcuu acagugcagg uag
23523RNAHomo sapiens 5uaaggugcau cuagugcaga uag 23623RNAHomo
sapiens 6caacggaauc ccaaaagcag cug 23723RNAHomo sapiens 7caaagugcuc
auagugcagg uag 23822RNAHomo sapiens 8uagcuuauca gacugauguu ga
22922RNAHomo sapiens 9cugugcgugu gacagcggcu ga 221022RNAHomo
sapiens 10aagcugccag uugaagaacu gu 221123RNAHomo sapiens
11agcuacauug ucugcugggu uuc 231221RNAHomo sapiens 12aucacauugc
cagggauuuc c 211322RNAHomo sapiens 13uggcucaguu cagcaggaac ag
221422RNAHomo sapiens 14cauugcacuu gucucggucu ga 221522RNAHomo
sapiens 15uucaaguaau ccaggauagg cu 221621RNAHomo sapiens
16uucacagugg cuaaguuccg c 211721RNAHomo sapiens 17aggcaagaug
cuggcauagc u 211822RNAHomo sapiens 18aacccguaga uccgaucuug ug
221980RNAHomo sapiens 19ccuguugcca caaacccgua gauccgaacu ugugguauua
guccgcacaa gcuuguaucu 60auagguaugu gucuguuagg 802078RNAHomo sapiens
20uacugcccuc ggcuucuuua cagugcugcc uuguugcaua uggaucaagc agcauuguac
60agggcuauga aggcauug 782178RNAHomo sapiens 21uugugcuuuc agcuucuuua
cagugcugcc uuguagcauu caggucaagc agcauuguac 60agggcuauga aagaacca
782288RNAHomo sapiens 22ugcgcuccuc ucagucccug agacccuaac uugugauguu
uaccguuuaa auccacgggu 60uaggcucuug ggagcugcga gucgugcu
882389RNAHomo sapiens 23accagacuuu uccuaguccc ugagacccua acuugugagg
uauuuuagua acaucacaag 60ucaggcucuu gggaccuagg cggagggga
892484RNAHomo sapiens 24gucagaauaa ugucaaagug cuuacagugc agguagugau
augugcaucu acugcaguga 60aggcacuugu agcauuaugg ugac 842571RNAHomo
sapiens 25uguucuaagg ugcaucuagu gcagauagug aaguagauua gcaucuacug
cccuaagugc 60uccuucuggc a 712692RNAHomo sapiens 26cggcuggaca
gcgggcaacg gaaucccaaa agcagcuguu gucuccagag cauuccagcu 60gcgcuuggau
uucguccccu gcucuccugc cu 922769RNAHomo sapiens 27aguaccaaag
ugcucauagu gcagguaguu uuggcaugac ucuacuguag uaugggcacu 60uccaguacu
692872RNAHomo sapiens 28ugucggguag cuuaucagac ugauguugac uguugaaucu
cauggcaaca ccagucgaug 60ggcugucuga ca 7229110RNAHomo sapiens
29acccggcagu gccuccaggc gcagggcagc cccugcccac cgcacacugc gcugccccag
60acccacugug cgugugacag cggcugaucu gugccugggc agcgcgaccc
1103085RNAHomo sapiens 30ggcugagccg caguaguucu ucaguggcaa
gcuuuauguc cugacccagc uaaagcugcc 60aguugaagaa cuguugcccu cugcc
8531110RNAHomo sapiens 31ugaacaucca ggucuggggc augaaccugg
cauacaaugu agauuucugu guucguuagg 60caacagcuac auugucugcu ggguuucagg
cuaccuggaa acauguucuc 1103273RNAHomo sapiens 32ggccggcugg
gguuccuggg gaugggauuu gcuuccuguc acaaaucaca uugccaggga 60uuuccaaccg
acc 733368RNAHomo sapiens 33cuccggugcc uacugagcug auaucaguuc
ucauuuuaca cacuggcuca guucagcagg 60aacaggag 683473RNAHomo sapiens
34cucugccucc cgugccuacu gagcugaaac acaguugguu uguguacacu ggcucaguuc
60agcaggaaca ggg 733584RNAHomo sapiens 35ggccaguguu gagaggcgga
gacuugggca auugcuggac gcugcccugg gcauugcacu 60ugucucgguc ugacagugcc
ggcc 843677RNAHomo sapiens 36guggccucgu ucaaguaauc caggauaggc
ugugcagguc ccaaugggcc uauucuuggu 60uacuugcacg gggacgc 773784RNAHomo
sapiens 37ggcuguggcu ggauucaagu aauccaggau aggcuguuuc caucugugag
gccuauucuu 60gauuacuugu uucuggaggc agcu 843878RNAHomo sapiens
38cugaggagca gggcuuagcu gcuugugagc aggguccaca ccaagucgug uucacagugg
60cuaaguuccg ccccccag 783971RNAHomo sapiens 39ggagaggagg caagaugcug
gcauagcugu ugaacuggga accugcuaug ccaacauauu 60gccaucuuuc c
714081RNAHomo sapiens 40cccauuggca uaaacccgua gauccgaucu uguggugaag
uggaccgcac aagcucgcuu 60cuaugggucu gugucagugu g 814122RNAArtificial
sequenceSynthetic 41cacaaguucg gaucuacggg uu 224223RNAArtificial
sequenceSynthetic 42ucauagcccu guacaaugcu gcu 234322RNAArtificial
sequenceSynthetic 43ucacaaguua gggucucagg ga 224423RNAArtificial
sequenceSynthetic 44cuaccugcac uguaagcacu uug 234523RNAArtificial
sequenceSynthetic 45cuaucugcac uagaugcacc uua 234623RNAArtificial
sequenceSynthetic 46cagcugcuuu ugggauuccg uug 234723RNAArtificial
sequenceSynthetic 47cuaccugcac uaugagcacu uug 234822RNAArtificial
sequenceSynthetic 48ucaacaucag ucugauaagc ua 224922RNAArtificial
sequenceSynthetic 49ucagccgcug ucacacgcac ag 225022RNAArtificial
sequenceSynthetic 50acaguucuuc aacuggcagc uu 225123RNAArtificial
sequenceSynthetic 51gaaacccagc agacaaugua gcu 235221RNAArtificial
sequenceSynthetic 52ggaaaucccu ggcaauguga u 215322RNAArtificial
sequenceSynthetic 53cuguuccugc ugaacugagc ca 225422RNAArtificial
sequenceSynthetic 54ucagaccgag acaagugcaa ug 225522RNAArtificial
sequenceSynthetic 55agccuauccu ggauuacuug aa 225621RNAArtificial
sequenceSynthetic 56gcggaacuua gccacuguga a 215721RNAArtificial
sequenceSynthetic 57agcuaugcca gcaucuugcc u 215822RNAArtificial
sequenceSynthetic 58cacaagaucg gaucuacggg uu 22
* * * * *