U.S. patent application number 15/975533 was filed with the patent office on 2019-04-18 for compositions and methods for prognosis of ovarian cancer.
This patent application is currently assigned to ROSETTA GENOMICS LTD.. The applicant listed for this patent is MOR RESEARCH APPLICATIONS, ROSETTA GENOMICS LTD.. Invention is credited to Ram Eitan, Moshe Hoshen, Gila Lithwick Yanai.
Application Number | 20190112665 15/975533 |
Document ID | / |
Family ID | 41171356 |
Filed Date | 2019-04-18 |
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United States Patent
Application |
20190112665 |
Kind Code |
A1 |
Hoshen; Moshe ; et
al. |
April 18, 2019 |
COMPOSITIONS AND METHODS FOR PROGNOSIS OF OVARIAN CANCER
Abstract
Described herein are compositions and methods for the prediction
of the prognosis of ovarian cancer subjects. The present invention
further provides methods for distinguishing between histological
subtypes of ovarian cancer tumors, and also methods and
compositions for the treatment or prevention of ovarian cancer.
Specifically the invention relates to microRNA molecules associated
with said methods and compositions, as well as various nucleic acid
molecules relating thereto or derived therefrom.
Inventors: |
Hoshen; Moshe; (Jerusalem,
IL) ; Yanai; Gila Lithwick; (Jerusalem, IL) ;
Eitan; Ram; (Einat, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ROSETTA GENOMICS LTD.
MOR RESEARCH APPLICATIONS |
Rehovot
Tel Aviv |
|
IL
IL |
|
|
Assignee: |
ROSETTA GENOMICS LTD.
Rehovot
IL
MOR RESEARCH APPLICATIONS
Tel Aviv
IL
|
Family ID: |
41171356 |
Appl. No.: |
15/975533 |
Filed: |
May 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15363822 |
Nov 29, 2016 |
9988690 |
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15975533 |
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12999201 |
Dec 15, 2010 |
9540695 |
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PCT/IL2009/000497 |
May 19, 2009 |
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15363822 |
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61108556 |
Oct 27, 2008 |
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61073036 |
Jun 17, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/178 20130101; A61K 31/713 20130101; C12Q 2600/106
20130101; C12Q 2600/112 20130101; C12Q 2600/118 20130101; A61K
45/00 20130101; C12Q 2600/16 20130101; A61P 35/00 20180101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; A61K 31/713 20060101 A61K031/713; A61K 45/00 20060101
A61K045/00 |
Claims
1. A method of determining the prognosis of ovarian cancer in a
subject comprising: (a) obtaining a biological sample from the
subject; (b) determining the expression level in said sample of a
nucleic acid sequence selected from the group consisting of SEQ ID
NOS: 1-71 and sequences at least about 80% identical thereto; and
(c) comparing said expression level to a threshold expression
level, wherein comparison of said expression level of said nucleic
acids to said threshold expression level is predictive of the
prognosis of said ovarian-cancer subject.
2.-44. (canceled)
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. 61/073,036, filed
Jun. 17, 2008 and U.S. Provisional Application No. 61/108,556,
filed Oct. 27, 2008 which are herein incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods for the
prediction of survival, time to progression, and response to
therapy in ovarian cancer subjects. Specifically the invention
relates to microRNA molecules associated with the prognosis 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. Patients who have
a prolonged disease-free-interval after first line platinum based
chemotherapy, are usually rechallenged with platinum and are more
likely to respond well to second line therapy. This group of
patients has an improved prognosis with a prolonged disease free
interval and longer overall survival. Patients who have progressive
disease during platinum treatment or who suffer first recurrent
disease within a short period of time are termed
platinum-resistant. These patients are given alternative
chemotherapy regimens who offer relatively small total response
rates reaching 20-30% at most. They will usually have a poorer
prognosis.
[0006] Comparison of the patterns of gene expressions in ovarian
cancer and normal ovarian tissue using cDNA micro-arrays revealed
several genes that are under- or over-expressed in ovarian cancer
(Collins Y, et al. Int J Mol Med 2004; 14:43-53). Patterns of gene
expression that predict response to chemotherapeutic agents and
prognosis have also been identified.
[0007] 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). In some cases miRNA overexpression results
in reduced expression of tumor suppressor genes, while loss of
miRNA expression often leads to oncogene activation.
[0008] Thus, there exists a need to identify biomarkers that will
make it possible to detect and predict which patients with ovarian
cancer will respond to platinum based chemotherapy and which
patients will remain refractory to this treatment. Specific data
may assist in tailoring treatment to each patient's specific
clinical situation during initial management of their disease and
also offer the opportunity for better counseling regarding
prognosis.
SUMMARY OF THE INVENTION
[0009] According to some aspects of the present invention, the
expression levels of any of SEQ ID NOS: 1-71 or combination thereof
is indicative of survival, time to progression, and response to
therapy in ovarian cancer subjects.
[0010] The present invention provides a method of determining the
prognosis of ovarian cancer in a subject comprising: [0011] (a)
obtaining a biological sample from the subject; [0012] (b)
determining the expression level in said sample of a nucleic acid
sequence selected from the group consisting of SEQ ID NOS: 1-71 and
sequences at least about 80% identical thereto; and [0013] (c)
comparing said expression level to a threshold expression level,
[0014] wherein comparison of said expression level of said nucleic
acids to said threshold expression level is predictive of the
prognosis of said ovarian-cancer subject.
[0015] In one aspect of the invention the prognosis is the
prediction of the clinical response of said subject to treatment
with a chemotherapeutic agent, and the nucleic acid sequence is
selected from the group consisting of SEQ ID NOS: 1-21 and
sequences at least about 80% identical thereto. According to one
embodiment, an expression level of a nucleic acid sequence selected
from the group consisting of SEQ ID NOS: 1-12, 19-21 and sequences
at least about 80% identical thereto above said threshold
expression level is predictive of resistance to said
chemotherapeutic agent. According to another embodiment, an
expression level of a nucleic acid sequence selected from the group
consisting of SEQ ID NOS: 13-18 and sequences at least about 80%
identical thereto above said threshold expression level is
predictive of sensitivity to said chemotherapeutic agent. In some
embodiments the chemotherapeutic agent is a platinum based agent.
In some embodiments the platinum based agent is an agent selected
from the group consisting of cisplatin and carboplatin.
[0016] In another aspect of the invention, the prognosis is time to
progression of the disease in a subject, and the nucleic acids are
selected from the group consisting of SEQ ID NOS: 4-7, 19-23, 55
and 71 and sequences at least about 80% identical thereto.
According to some embodiments, an expression level of a nucleic
acid sequence selected from the group consisting of SEQ ID NOS:
4-7, 19-21, 55 and 71 and sequences at least about 80% identical
thereto above said threshold expression level is predictive of
short time to progression. According to other embodiments,
expression levels of a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 22-23 and sequences at least about
80% identical thereto below said threshold expression level is
predictive of short time to progression.
[0017] According to another aspect of the invention, the prognosis
is the prediction of the survival of a subject, and the nucleic
acids are selected from the group consisting of SEQ ID NOS: 4-7,
19, 22-23, 55 and 71, and sequences at least about 80% identical
thereto. According to some embodiments, an expression level of a
nucleic acid sequence selected from the group consisting of SEQ ID
NOS: 4-7, 19 and 55 and 71 and sequences at least about 80%
identical thereto above said threshold expression level is
predictive of short survival. According to other embodiments,
expression levels of a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 22-23 and sequences at least about
80% identical thereto below said threshold expression level is
predictive of short survival.
[0018] According to one embodiment of the invention, the biological
sample is a tumor tissue at a specific stage. In some embodiments
the tumor tissue is at stage III.
[0019] According to one embodiment, the biological sample is a
tumor tissue of a specific histological subtype. In some
embodiments histological subtype is selected from the group
consisting of serous papillary cystadenocarcinoma and endometrioid
carcinoma.
[0020] In another aspect of the invention the prognosis is
distinguishing between stage I and stage III of ovarian cancer, and
the nucleic acids are selected from the group consisting of SEQ ID
NOS: 22, 23, 30, 31, 36, 38, 40-54, 56-70 and sequences at least
about 80% identical thereto. According to some embodiments,
expression levels of nucleic acids selected from the group
consisting of SEQ ID NOS: 22, 23, 30, 31, 36, 38, 40-44, 56-60 and
sequences at least about 80% identical thereto above a threshold
expression level is indicative of ovarian cancer stage I. According
to other embodiments, expression levels of a nucleic acid sequence
selected from the group consisting of SEQ ID NOS: 45-54, 61-70 and
sequences at least about 80% identical thereto above a threshold is
indicative of ovarian cancer stage II.
[0021] The invention further provides a method of distinguishing
between papillary serous cystadenocarcinoma and endometrioid
carcinoma subtypes of ovarian cancer tumors in a subject
comprising: [0022] (a) obtaining a biological sample from said
subject; [0023] (b) determining an expression profile in said
sample of a nucleic acid sequence selected from the group
consisting of SEQ ID NOS: 11-12 and 32-35 and sequences at least
about 80% identical thereto; and [0024] (c) comparing said
expression profile to a reference expression profile, wherein said
comparison is indicative of papillary serous cystadenocarcinoma or
endometrioid carcinoma subtype tumor.
[0025] According to one embodiment, a relative high expression
profile of SEQ ID NOS: 34-35 and sequences at least about 80%
identical thereto in said biological sample is indicative of an
endometrioid carcinoma subtype tumor. According to another
embodiment a relative high expression profile of a nucleic acid
selected from the group of 11-12 and 32-33 and sequences at least
about 80% identical thereto in said biological sample is indicative
of papillary serous cystadenocarcinoma carcinoma subtype tumor.
[0026] According to some embodiments, the subject is a human. In
some aspects the method is used to determine a course of treatment
for the subject.
[0027] In some embodiments of the invention the biological sample
is selected from the group consisting of bodily fluid, a cell line
and a tissue sample. In some embodiments the tissue is a fresh,
frozen, fixed, wax-embedded or formalin fixed paraffin-embedded
(FFPE) tissue.
[0028] In additional embodiments the expression levels of the
invention are determined by a method selected from the group
consisting of nucleic acid hybridization, nucleic acid
amplification, and a combination thereof. In some embodiments the
nucleic acid hybridization is performed using a solid-phase nucleic
acid biochip array or in situ hybridization. In some embodiments
the real-time PCR method comprises forward and reverse primers, and
may further comprise hybridization with a probe comprising a
nucleic acid sequence that is complementary to a sequence selected
from SEQ ID NOS: 1-71, to a fragment thereof, or to a sequence at
least about 80% identical thereto.
[0029] The invention further provides a kit for predicting a
clinical response of an ovarian cancer subject to treatment with a
chemotherapeutic agent, said kit comprising a probe comprising a
nucleic acid sequence that is complementary to a sequence selected
from SEQ ID NOS: 1-21, to a fragment thereof, or to a sequence at
least about 80% identical thereto.
[0030] The invention further provides a kit for predicting the time
to progression of disease in an ovarian cancer subject, said kit
comprising a probe comprising a nucleic acid sequence that is
complementary to a sequence selected from SEQ ID NOS: 4-7, 19-23,
55 and 71, to a fragment thereof, or to a sequence at least about
80% identical thereto.
[0031] The invention further provides a kit for predicting the
survival of an ovarian cancer subject, said kit comprising a probe
comprising a nucleic acid sequence that is complementary to a
sequence selected from SEQ ID NOS: 4-7, 19-23, 55 and 71, to a
fragment thereof; or to a sequence at least about 80% identical
thereto.
[0032] Further provided is a kit for distinguishing between stage I
and stage I of ovarian cancer in a subject, the kit comprising a
probe comprising a nucleic acid sequence that is complementary to a
sequence selected from SEQ ID NOS: 22, 23, 30, 31, 36, 38, 40-54,
56-70, to a fragment thereof, or to a sequence at least about 80%
identical thereto.
[0033] Also provided is a kit for distinguishing between papillary
serous cystadenocarcinoma and endometrioid subtypes of ovarian
cancer tumors in a subject, the kit comprising a probe comprising a
nucleic acid sequence that is complementary to a sequence selected
from SEQ ID NOS: 11-12 and 32-35, to a fragment thereof; or to a
sequence at least about 80% identical thereto.
[0034] According to some embodiments the kit of the invention
further comprises forward and reverse primers. According to other
embodiments the kit comprises reagents for performing in situ
hybridization analysis.
[0035] Further provided in accordance with the invention is a
method of treating or preventing ovarian cancer in a subject in
need thereof comprising administering to the subject an effective
amount of a composition comprising a nucleic acid sequence selected
from the group consisting of: [0036] (a) SEQ ID NOS: 22-25 and
30-31, [0037] (b) sequences at least about 80% identical to (a),
[0038] (c) sequences that are complementary to a sequence selected
from the group consisting of SEQ ID NOS: 4-7, 11, 12, 20 and 21;
and [0039] (d) sequences at least about 80% identical to (c).
[0040] An additional aspect of the invention is a use of an
effective amount of a composition comprising a nucleic acid
sequence selected from the group consisting of: [0041] (a) SEQ ID
NOS: 22-25 and 30-31, [0042] (b) sequences at least about 80%
identical to (a), [0043] (c) sequences that are complementary to a
sequence selected from the group consisting of SEQ ID NOS: 4-7, 11,
12, 20 and 21; and [0044] (d) sequences at least about 80%
identical to (c). in the preparation of a medicament suitable for
administration to a subject for the treatment or prevention of
ovarian cancer in said subject.
[0045] 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 and
cloposide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIGS. 1a-1b show differential expression of microRNAs in
ovarian cancers of different stages. Expression scale (Y-axis)
shows the logarithm (base 2) of the normalized fluorescence signal
by microarray. Boxplots show the median (horizontal line), 25 to 75
percentile (box) and extent of data ("whiskers") for stage I (left
box, n=19) and stage III (right box, n=38) patients. FIG. 1a
depicts the differential expression for hsa-miR-449b (SEQ ID NO:
22, p-value=0.048, median expression 4.6-fold higher in stage I)
and FIG. 1b depicts the differential expression for hsa-miR-200a
(SEQ ID NO: 30, p-value-0.00047, median expression 2.1-fold higher
in stage I).
[0047] FIGS. 2a-2b show differential expression of microRNAs in
stage I ovarian cancers that are resistant (left box) or sensitive
(right box) to platinum-based treatment. Expression scale (y-axis)
shows the logarithm (base 2) of the normalized fluorescence signal
by microarray. Boxplots show the median (horizontal line), 25 to 75
percentile (box) and extent of data ("whiskers") for resistant
(n=12) and sensitive (n=25) patients. FIG. 2a presents the
differential expression of hsa-miR-27a (SEQ ID NO: 4), with
p-value=0.0019 and median expression 1.7-fold higher in resistant
patients; FIG. 2b presents the differential expression of
hsa-miR-378 (SEQ ID NO: 15), with p-value=0.0055 and median
expression 1.8-fold higher in sensitive patients.
[0048] FIGS. 3a-3d show Kaplan Meier curves, which correct for
patients who were censored (subjects that may have dropped out of
the study and/or were lost to follow-up, or deliberately
withdrawn). Censoring events are marked by gray vertical lines.
[0049] In FIGS. 3a-3d each plot presents a curve for each of high
(dashed-dotted line, n=13), intermediate (dashed line, n=12) and
low (solid line, n=13) tertiles of expression; in FIGS. 3a and 3b
for the expression of hsa-miR-23a (SEQ ID NO: 6), and in FIGS. 3c
and 3d for the expression of hsa-miR-27a (SEQ ID NO: 4). The X-axis
in each of FIGS. 3a-3d depicts survival time, in months; in FIGS.
3a and 3c the Y-axis depicts the fraction of recurrence-free
surviving patients, and in FIGS. 3b and 3d the Y-axis depicts the
fraction of surviving patients.
[0050] FIGS. 4a-4b show boxplot presentations of microRNAs
comparing the distributions between the expressions of hsa-miR-93
(SEQ ID NO: 34, FIG. 4a) and hsa-miR-let-7i (SEQ ID NO: 32, FIG.
4b) in stage m of ovarian cancer patients with endometrioid
carcinoma tumors (n=13) and papillary serous cystadenocarcinoma
tumors (n=25) histological subtypes. The "box" part contains 50% of
the data, the line in the box indicates the median value, and the
ends of the vertical lines indicate the minimum and maximum data
values. The Y-axis represents the log(signal).
[0051] FIG. 5 shows Kaplan-Meier curves of recurrence-free survival
for groups of patients of stage 3 disease, stratified by expression
levels of hsa-miR-27a (SEQ ID NO: 4). Exceptionally high expression
level of hsa-miR-27a identifies a subgroup of patients (n=5) with
very poor prognosis. The samples with the highest expression level
of hsa-miR-27a (normalized fluorescence signal>9500) had a very
low time to progression and were resistant to platinum-based
treatment, and most (4 out of 5) had an incomplete response.
Samples with lower expression of hsa-miR-27a had a median survival
time of 20.6 months.
DETAILED DESCRIPTION
[0052] According to some aspects of the present invention miRNA
expression can serve as a novel tool for predicting survival, time
to progression, and response to therapy in ovarian cancer
subjects.
[0053] Several miRNAs were significantly differentially expressed
between the stage I and stage III ovarian cancers (Table 2). Of
particular interest are hsa-miR-200a (SEQ ID NO: 30), hsa-miR-34a
(SEQ ID NO: 36), and hsa-miR-449b (SEQ ID NO: 22), which were
down-regulated in the advanced (stage II) tumors.
[0054] The relation of miRNA expression to the prognosis of ovarian
cancer patients was studied. To avoid confounding effects of stage,
this analysis was performed in the group of 38 stage m ovarian
cancer patients, and two types of analyses were performed which
identified several miRNAs. Hsa-miR-378 (SEQ ID NO: 15) was found to
have significantly higher expression levels in the groups of
patients that were sensitive as compared to resistant to treatment
by platinum-based chemotherapy (FIG. 2). Expression of hsa-miR-449b
(SEQ ID NO: 22) divided the patients into groups with significantly
different disease-specific survival times. Patients with higher
expression of hsa-miR-449b were found to have an improved overall
survival (Table 4). Hsa-miR-23a (SEQ ID NO: 6) and hsa-miR-27a (SEQ
ID NO: 4) were found to be significantly associated with outcome by
both methods of analysis. High levels of these miRNAs were
associated in both cases with a poorer prognosis.
[0055] Many of the ovarian cancer patients who respond completely
to first line chemotherapy and are with no evidence of disease at
the end of treatment are unfortunately diagnosed with recurrent
disease during follow-up. In this study, an array of microRNA
markers has been found that are associated with response to
platinum-based first line chemotherapy. This approach can
potentially be used to tailor chemotherapy to specific patient
needs, to help in the selection of the most suitable treatment for
those at high risk for recurrence and to better counsel patients on
prognosis and the strategies planned to better their outcome. These
microRNAs present potential candidates for the development of
future therapeutic agents.
[0056] Methods and compositions are provided for predicting
survival, time to progression, and response to therapy in ovarian
cancer subjects. Other aspects of the invention will become
apparent to the skilled artisan by the following description of the
invention.
[0057] 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.
[0058] 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
a. Administering
[0059] "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.
[0060] "Parenteral administration," means administration through
injection or infusion. Parenteral administration includes, but is
not limited to, subcutaneous administration, intravenous
administration, or intramuscular administration.
[0061] "Subcutaneous administration" means administration just
below the skin.
[0062] "Intravenous administration" means administration into a
vein.
[0063] "Intratumoral administration" means administration within a
tumor.
[0064] "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.
b. Attached
[0065] "Attached" or "immobilized" as used herein to refer to a
probe and a solid support may mean that the binding between the
probe and the solid support is sufficient to be stable under
conditions of binding, washing, analysis, and removal. The binding
may be covalent or non-covalent. Covalent bonds may be formed
directly between the probe and the solid support or may be formed
by a cross linker or by inclusion of a specific reactive group on
either the solid support or the probe or both molecules.
Non-covalent binding may be one or more of electrostatic,
hydrophilic, and hydrophobic interactions. Included in non-covalent
binding is the covalent attachment of a molecule, such as
streptavidin, to the support and the non-covalent binding of a
biotinylated probe to the streptavidin. Immobilization may also
involve a combination of covalent and non-covalent
interactions.
c. Biological Sample
[0066] "Biological sample" as used herein means a sample of
biological tissue or fluid that comprises nucleic acids. Such
samples include, but are not limited to, tissue or fluid isolated
from subjects. Biological samples may also include sections of
tissues such as biopsy and autopsy samples, FFPE samples, frozen
sections taken for histological purposes, blood, plasma, serum,
sputum, stool, tears, mucus, hair, and skin. Biological samples
also include explants and primary and/or transformed cell cultures
derived from animal or patient tissues.
[0067] Biological samples may also be blood, a blood fraction,
urine, effusions, ascitic fluid, saliva, cerebrospinal fluid,
cervical secretions, vaginal secretions, endometrial secretions,
gastrointestinal secretions, bronchial secretions, sputum, cell
line, tissue sample, cellular content of fine needle aspiration
(FNA) or secretions from the breast. A biological sample may be
provided by removing a sample of cells from an animal, but can also
be accomplished by using previously isolated cells (e.g., isolated
by another person, at another time, and/or for another purpose), or
by performing the methods described herein in vivo. Archival
tissues, such as those having treatment or outcome history, may
also be used.
d. Cancer Prognosis
[0068] A forecast or prediction of the probable course or outcome
of the cancer and response to its treatment. As used herein, cancer
prognosis includes distinguishing between cancer stages and
subtypes, and the forecast or prediction of any one or more of the
following: duration of survival of a patient susceptible to or
diagnosed with a cancer, duration of recurrence-free survival,
duration of progression free survival of a patient susceptible to
or diagnosed with a cancer, response rate in a group of patients
susceptible to or diagnosed with a cancer, duration of response in
a patient or a group of patients susceptible to or diagnosed with a
cancer, and/or likelihood of metastasis in a patient susceptible to
or diagnosed with a cancer. As used herein, "prognostic for cancer"
means providing a forecast or prediction of the probable course or
outcome of the cancer. In some embodiments, "prognostic for cancer"
comprises providing the forecast or prediction of (prognostic for)
any one or more of the following: duration of survival of a patient
susceptible to or diagnosed with a cancer, duration of
recurrence-free survival, duration of progression free survival of
a patient susceptible to or diagnosed with a cancer, response rate
in a group of patients susceptible to or diagnosed with a cancer,
duration of response in a patient or a group of patients
susceptible to or diagnosed with a cancer, and/or likelihood of
metastasis in a patient susceptible to or diagnosed with a
cancer.
e. Chemotherapeutic Agent
[0069] 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.
f. Complement
[0070] "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.
g. Detection
[0071] "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.
h. Differential Expression
[0072] "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.
i. Dose
[0073] "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.
j. Dosage Unit
[0074] "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.
k. Expression Profile
[0075] "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, 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.
l. FDR
[0076] When performing multiple statistical tests, for example in
comparing the signal between two groups in multiple data features,
there is an increasingly high probability of obtaining false
positive results, by random differences between the groups that can
reach levels that would otherwise be considered as statistically
significant. In order to limit the proportion of such false
discoveries, statistical significance is defined only for data
features in which the differences reached a p-value (by two-sided
t-test) below a threshold, which is dependent on the number of
tests performed and the distribution of p-values obtained in these
tests.
m. Gene
[0077] "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 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.
n. Identity
[0078] "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.
o. Inhibit
[0079] "Inhibit" as used herein may mean prevent, suppress,
repress, reduce or eliminate.
p. Label
[0080] "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.
q. Metastasis
[0081] "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.
r. Mismatch
[0082] "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.
s. Nucleic Acid
[0083] "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.
[0084] 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.
[0085] 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.
t. Overall Survival Time
[0086] "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.
u. Progression-Free Survival
[0087] "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.
v. Probe
[0088] "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.
w. Promoter
[0089] "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 IB
promoter.
x. Reference Expression Profile
[0090] As used herein, the phrase "reference expression profile"
refers to a criterion expression value to which measured values are
compared in order to determine the detection of a subject with a
specific ovarian cancer sub-type. The reference expression profile
may be based on the expression of the nucleic acids, or may be
based on a combined metric score thereof.
y. Selectable Marker
[0091] "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.T), tetracycline-resistance gene (Tc.sup.T),
bacterial kanamycin-resistance gene (Kan), 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.
z. Stringent Hybridization Conditions
[0092] "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.
aa. Substantially Complementary
[0093] "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.
bb. Substantially Identical
[0094] "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.
cc. Subject
[0095] 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.
dd. Threshold Expression Level
[0096] As used herein, the phrase "threshold expression level"
refers to a reference expression value. Measured values are
compared to a corresponding threshold expression level to determine
the prognosis of a subject.
ee. Therapeutically Effective Amount
[0097] "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.
ff. 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.
gg. Treat
[0099] "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.
hh. Unit Dosage Form
[0100] "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.
ii. Variant
[0101] "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.
jj. Vector
[0102] "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
[0103] 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:
[0104] 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.
[0105] 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.
[0106] 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%
[0107] 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
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
2. Nucleic Acids
[0118] Nucleic acids are provided herein. The nucleic acid may
comprise the sequence of SEQ ID NOS: 1-71 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.
[0119] 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 name* miR SEQ ID NO: Hairpin SEQ ID NO:
hsa-miR-199a-3p 1 2, 3 hsa-miR-27a 4 5 hsa-miR-23a 6 7 hsa-miR-30c
8 9, 10 hsa-let-7g 11 12 MID-00689 13 14 hsa-miR-378 15 16
hsa-miR-625 17 18 hsa-miR-23a* 19 7 hsa-miR-21 20 21 hsa- miR-449b
22 23 hsa-miR-449a 24 25 hsa-miR-34c-5p 28 29 hsa-miR-200a 30 31
hsa -let-7i 32 33 hsa-miR-93 34 35 hsa-miR-34a 36 38 hsa-miR-34b*
37 39 hsa-miR-200b 40 56 hsa-miR-513a-5p 41 57 hsa-miR-509-3p 42 58
hsa-miR-509-3-5p 43 59 hsa-miR-574-5p 44 60 hsa-miR-423-3p 45 61
hsa-miR-130a 46 62 hsa-miR-146b-5p 47 63 hsa-miR-193a-3p 48 64
hsa-miR-193a-5p 49 65 hsa-miR-491-5p 50 66 hsa-miR-23b 51 67
hsa-miR-125a-3p 52 68 hsa-miR-125a-5p 53 69 hsa-miR-451 54 70
hsa-miR-24-2* 55 71 *MID-00689 was cloned at Rosetta Genomics. For
all the other sequences the miR name is the miRBase registry name
(release 10).
a. Nucleic Acid Complex
[0120] 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 al (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.
b. Pri-miRNA
[0121] 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: 1-71 or variants thereof.
[0122] 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.
c. Pre-miRNA
[0123] 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:
1-71 or variants thereof.
d. MiRNA
[0124] 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, 4, 6, 8, 11, 13, 15, 17, 19, 20, 22, 24, 28, 30 and
34, 36, 37 and 40-55, or variants thereof.
e. Anti-miRNA
[0125] 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, 4, 6, 8,
11, 13, 15, 17, 19, 20, 22, 24, 28, 30 and 34, 36, 37 and 40-55, or
variants thereof.
5. Probes
[0126] 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.
[0127] 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
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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
[0133] 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 miRNA molecules. Further, miRNA molecules may be
modified, in order that they are processed and then generated as
double-stranded siRNAs which are again directed against
therapeutically relevant targets.
[0134] As previously discussed the methods, compositions and
articles of manufacture of the present invention are particularly
useful in the treatment of cancer.
The compositions of the present invention may be combined with a
chemotherapeutic agent, a combination of chemotherapeutic agents
and/or radiotherapy.
[0135] 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.
[0136] 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
5-fluorouracil, gemcitabine, doxorubicine, mitomycin c, sorafenib,
etoposide, carboplatin, epirubicin, irinotecan and oxaliplatin. An
additional therapy may be surgical resection of tumor(s), or
chemoembolization.
8. Diagnostic
[0137] A method of diagnosis is also provided. The method comprises
detecting a differential expression level of a disease-associated
nucleic acid in a biological sample. The sample may be derived from
a patient. Diagnosis of a disease state in a patient may allow for
prognosis and selection of therapeutic strategy. Further, the
developmental stage of cells may be classified by determining
temporarily expressed disease-associated nucleic acids.
[0138] In situ hybridization of labeled probes to tissue arrays may
be performed. When comparing the fingerprints between an individual
and a standard, the skilled artisan can make a diagnosis, a
prognosis, or a prediction based on the findings. It is further
understood that the nucleic acids which indicate the diagnosis may
differ from those which indicate the prognosis and molecular
profiling of the condition of the cells may lead to distinctions
between responsive or refractory conditions or may be predictive of
outcomes.
9. Kits
[0139] A kit is also provided and may comprise a nucleic acid
described herein together with any or all of the following: assay
reagents, buffers, probes and/or primers, and sterile saline or
another pharmaceutically acceptable emulsion and suspension base.
In addition, the kits may include instructional materials
containing directions (e.g., protocols) for the practice of the
methods described herein.
[0140] For example, the kit may be a kit for the amplification,
detection, identification or quantification of a target nucleic
acid sequence. The kit may comprise a poly(T) primer, a forward
primer, a reverse primer, and a probe.
10. Compositions
[0141] 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.
[0142] 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.
[0143] 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.
[0144] The nucleic acids can be introduced into tissues or host
cells by any number of mutes, 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] In certain embodiments, pharmaceutical compositions are
prepared for buccal administration. Certain of such pharmaceutical
compositions are tablets or lozenges formulated in conventional
manner.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] In certain embodiments, one or more modified
oligonucleotides of the present invention are formulated as a
prodrug. In certain embodiments, upon in vive 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.
[0169] 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 pharnmacodynamic
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).
[0170] 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
a. Patients and Samples
[0171] 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.
Patients with progressive disease during first line platinum based
chemotherapy or those who suffered recurrent disease within 6
months of completing first line therapy were termed platinum
resistant. Patients with no recurrence or with recurrences beyond 6
months were termed platinum sensitive. Survival time was calculated
as the time from the end of treatment to the last follow-up date or
death. Recurrence time was calculated as the time from the end of
treatment to the time of detected recurrence/progression. The study
was approved by the institutional review board of the Rabin Medical
Center.
[0172] Fifty-seven patients were identified to fit study criteria.
Nineteen patients had stage I disease at diagnosis and 38 patients
had stage III at diagnosis. Due to small numbers, stage II and
stage IV patients were excluded from the study. One patient was
censored after 161 days (due to death of other causes). Median age
of the study cohort was 58 years. Of the stage III patients, 18 had
optimal surgical cytoreduction and 15 were left with sub-optimal
residual disease at the end of surgery. Thirty five patients were
diagnosed with serous adenocarcinoma and 22 with endometrioid
histology.
[0173] Most of the 19 patients with stage I disease were staged
according to FIGO guidelines. All of them had bilateral
salpingo-oophorectomy (BSO), cytology washings and omentectomy
performed; 12 (63%) had lymph-node sampling (LNS); 10 (52%)
appendectomy; and 14 (73%) total abdominal hysterectomy (TAH).
[0174] All patients received platinum based chemotherapy as
first-line treatment. 21 patients received platinum as a single
agent, 34 received paclitaxel with carboplatin, and 2 patients
received cyclophosphamide with cisplatin.
b. RNA Extraction
[0175] 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 570 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).
[0176] From frozen tissues, total RNA was extracted using the
miRvana microRNA isolation kit (Ambion).
c. microRNA Microarray Platform
[0177] 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 min at 50.degree. C., then
thoroughly rinsed with water and spun dry.
d. Cy-Dye Labeling of microRNA for Microarray
[0178] Five pig 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 CyS. The labeling
reaction contained total RNA, spikes (0.1-20 fmoles), 400 ng
RNA-linker-dye, 15% DMSO, 1.times. 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.
[0179] 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).
e. Data Analysis
[0180] Expression levels between groups of samples were compared
using the Mann-Whitney non-parametric test. Only microRNAs which
had a median signal higher than signal background levels
(normalized fluorescence signal of .about.300) in at least one of
the two groups were tested. Corrections for multiple pairwise
comparisons were performed using the Benjamini-Hochberg "False
Discovery Rate" (FDR) method. Survival time course was studied
using the Kaplan Meier method, and groups were compared using
logrank test. Stability of microRNAs in survival analysis was
assessed by repeated (100 times) random resampling (bootstrap) from
the original dataset (maintaining group sizes). Multivariate
analysis of microRNA expression (for hsa-miR-27a), grade, age,
optimal cytoreduction, and histological type was performed using
Cox regression. The values of these features were combined in order
to predict progression times in stage III patients. Histological
type was encoded such that endometrioid carcinoma samples were
given a value of one, while serous carcinomas were given a value of
zero. Similarly, a value of one or zero was assigned for samples
with or without optimal cytoreduction, respectively.
f. MicroRNA Target Prediction
[0181] Targets were selected from the intersection of the target
prediction results by Targetscan and Miranda. Only targets with a
Targetscan score lower than 0, a Miranda score>150 were used. In
order to retrieve only the most relevant targets, we listed only
genes targeted by at least three microRNAs that we found to be
associated with poor prognosis. This list included the microRNAs
that were over-expressed in platinum-resistant stage III patients
compared to platinum-sensitive stage II patients (including
hsa-miR-27a (SEQ ID NO: 4), hsa-miR-23a (SEQ ID NO: 6), hsa-miR-30c
(SEQ ID NO: 8), hsa-let-7g (SEQ ID NO: 11), hsa-miR-199a-3p (SEQ ID
NO: 1)) and the microRNAs that were associated with significantly
poorer recurrence-free survival (including hsa-miR-27a,
hsa-miR-23a, hsa-miR-21 (SEQ ID NO: 20)). In addition, since
hsa-miR-27a and hsa-miR-23a were significant in both the
differential expression analysis and in the progression-free
survival analysis, only genes that were targeted by at least one of
these two microRNAs were listed.
Example 2: miR Expression Patterns Correlate with Stage of
Disease
[0182] Time to progression and survival were clearly linked to
stage in the study cohort of patients. The microRNA expression were
compared between stage I (n=19) and stage III (n=38) cases. 18
microRNAs (Table 2) were differentially expressed with p<0.05
(Mann-Whitney test), including for example hsa-miR-449b (SEQ ID NO:
22) (p=0.048). hsa-miR-200a (SEQ ID NO: 30) (p=0.00047) was also
significant when allowing a False Discovery Rate (FDR) of 10%. Both
of these microRNAs were more highly expressed in stage I ovarian
cancers compared to stage III cases. Fold-change is the ratio of
the median signals in the two groups.
TABLE-US-00002 TABLE 2 miR SEQ ID NO: p-value fold-change higher in
hsa-miR-200a 30 0.00047 2.10 Stage I hsa-miR-200b 40 0.0043 1.63
Stage I hsa-miR-34a 36 0.0066 1.69 Stage I hsa-miR-513a-5p 41
0.0068 5.32 Stage I hsa-miR-509-3p 42 0.0074 10.3 Stage I
hsa-miR-509-3-5p 43 0.017 4.01 Stage I hsa-miR-574-5p 44 0.045 1.24
Stage I hsa-miR-449b 22 0.048 4.61 Stage I hsa-miR-423-3p 45 0.0024
1.33 Stage III hsa-miR-130a 46 0.0033 1.86 Stage III
hsa-miR-146b-5p 47 0.0037 2.27 Stage III hsa-miR-193a-3p 48 0.0056
1.42 Stage III hsa-miR-193a-5p 49 0.013 1.60 Stage III
hsa-miR-491-5p 50 0.028 1.40 Stage III hsa-miR-23b 51 0.028 1.10
Stage III hsa-miR-125a-3p 52 0.030 1.27 Stage III hsa-miR-125a-5p
53 0.034 1.25 Stage III hsa-miR-451 54 0.035 1.93 Stage III
[0183] The boxplots in FIGS. 1a-1b exemplify the differential
expression for hsa-miR-449b (SEQ ID NO: 22, FIG. 1a) and
hsa-miR-200a (SEQ ID NO: 30, FIG. 1b) such that the expression
levels of both these miRs are higher in stage I tumors than in
stage III tumors of ovarian cancer.
[0184] Accordingly, relatively high expressions of any of
hsa-miR-200a (SEQ ID NO: 30), hsa-miR-200b (SEQ ID NO: 40),
hsa-miR-34a (SEQ ID NO: 36), hsa-miR-513a-5p (SEQ ID NO: 41),
hsa-miR-509-3p (SEQ ID NO: 42), hsa-miR-509-3-5p (SEQ ID NO: 43),
hsa-miR-574-5p (SEQ ID NO: 44) and hsa-miR-449b (SEQ ID NO: 22) are
indicative of stage I tumors of ovarian cancer, and relatively high
expressions of any of hsa-miR-423-3p (SEQ ID NO: 45), hsa-miR-130a
(SEQ ID NO: 46), hsa-miR-146b-5p (SEQ ID NO: 47), hsa-miR-193a-3p
(SEQ ID NO: 48), hsa-miR-193a-5p (SEQ ID NO: 49), hsa-miR-491-5p
(SEQ ID NO: 50), hsa-miR-23b (SEQ ID NO: 51), hsa-miR-125a-3p (SEQ
ID NO: 52), hsa-miR-125a-5p (SEQ ID NO: 53) and hsa-miR-451 (SEQ ID
NO: 54) are indicative of stage 1 tumors of ovarian cancer.
Example 3: miR Expression Patterns in Patients with Stage III
Disease Correlate with Response to Platinum Therapy
[0185] The relation between miR expression and disease progression
was studied. Since patient prognosis and disease characteristics
vary for different stages of the disease, the inventors focused on
the larger, higher risk group of patients in stage II. 25 patients
achieved a complete response with no recurrence within 6 months of
the end of treatment, and were termed platinum-sensitive. Twelve
patients had rapid progression of the disease (partial response or
recurrence within 6 months of the end of treatment) and were termed
platinum-resistant. The patient censored before 6 months was not
included in this analysis.
[0186] miR expression patterns were examined in tumors from
platinum-resistant stage III patients (n=12) and in tumors from
platinum-sensitive stage II patients (n=25). As shown in table 3,
hsa-miR-199a-3p (SEQ ID NO: 1), hsa-miR-27a (SEQ ID NO: 4),
hsa-miR-23a (SEQ ID NO: 6), hsa-miR-30c (SEQ ID NO: 8), hsa-let-7g
(SEQ ID NO: 11), MID-00689 (SEQ ID NO: 13), hsa-miR-23a* (SEQ ID
NO: 19), hsa-miR-21 (SEQ ID NO: 20), hsa-miR-378 (SEQ ID NO: 15),
and hsa-miR-625 (SEQ ID NO: 17) were found to be significantly
differentially expressed in tumors from platinum sensitive vs.
platinum resistant patients (p-value<0.05).
TABLE-US-00003 TABLE 3 miR SEQ ID NO: p-value fold-change higher in
hsa-miR-199a-3p 1 0.046 2.32 resistant hsa-miR-27a 4 0.0019 1.67
resistant hsa-miR-23a 6 0.011 1.44 resistant hsa-miR-30c 8 0.029
1.41 resistant hsa-let-7g 11 0.043 1.41 resistant
.sup.1hsa-miR-23a* 19 0.0106 1.54 resistant .sup.2hsa-miR-21 20
0.0621 1.75 resistant MID-00689 13 0.005 2.1 sensitive hsa-miR-378
15 0.0055 1.84 sensitive hsa-miR-625 17 0.029 1.81 sensitive
.sup.1hsa-miR-23a* did not pass the signal threshold of >300
.sup.2hsa-miR-21 did not pass the p-value cutoff (Mann-Whitney
pValue < 0.05)
[0187] The differential expression of hsa-miR-27a (SEQ ID NO: 4),
hsa-miR-378 (SEQ ID NO: 15) and hsa-miR-23a (SEQ ID NO: 6) between
sensitive and resistant tumors was also observed in a subset of
stage III patients (n=30) treated by the combined paclitaxel with
carboplatin treatment, and in the subset of stage III serous tumors
(n=25). With the exception of hsa-let-7g (SEQ ID NO: 11), which was
over-expressed in serous papillary tumors (p=0.013), no difference
was found between tumors of serous and endometrioid histologies
with similar response to chemotherapy in the expression of relevant
microRNAs.
[0188] The distributions of two of the significantly differentially
expressed miRs, hsa-miR-27a (SEQ ID NO: 4) and hsa-miR-378 (SEQ ID
NO: 15), are presented in the boxplots of FIGS. 2a and 2b,
respectively.
[0189] Accordingly, relatively high expressions of any of
hsa-miR-199a-3p (SEQ ID NO: 1), hsa-miR-27a (SEQ ID NO: 4),
hsa-miR-23a (SEQ ID NO: 6), hsa-miR-30c (SEQ ID NO: 8), hsa-let-7g
(SEQ ID NO: 11), hsa-miR-23a* (SEQ ID NO: 19) and hsa-miR-21 (SEQ
ID NO: 20) are predictive of platinum-resistance in tumors from
patients with stage I ovarian cancer, and relatively high
expression levels of any of MID-00689 (SEQ ID NO: 13), hsa-miR-378
(SEQ ID NO: 15) and hsa-miR-625 (SEQ ID NO: 17) are predictive of
platinum-sensitivity in tumors from patients with stage III ovarian
cancer.
Example 4: miR Expression Patterns in Patients with Stage II
Ovarian Carcinoma Correlate with Prognosis
[0190] The prognosis of groups of patients, stratified according
the expression levels of individual microRNAs, was compared. For
each microRNA the samples were divided into tertiles according to
high (n=13), intermediate (n=12) or low (n=13) expression level of
the microRNA.
[0191] Survival and time to progression were compared between the
two groups with high and low microRNA expression levels. The
microRNAs associated with significant differences (logrank
p-value<0.05) in survival or time to progression are presented
in Table 4.
TABLE-US-00004 TABLE 4 p value time to higher expression miR SEQ ID
NO. progression survival associated with- hsa-miR-27a 4 0.0176
0.0215 poorer prognosis hsa-miR-23a 6 0.0049 0.0025 poorer
prognosis .sup.1hsa-miR-23a* 19 0.0053 0.0007 poorer prognosis
hsa-miR-21 20 0.0493 0.222 poorer prognosis hsa-miR-24-2* 55 0.225
0.0493 poorer prognosis hsa-miR-449b 22 0.1 0.0379 better prognosis
.sup.1hsa-miR-23a* did not pass the signal threshold of >300
[0192] The correlation between miR expression and survival time and
time to progression in stage II patients is further indicated in
FIGS. 3a-3d, which show Kaplan Meier plots of survival time and
recurrence-free survival curves plotted for each of the three
expression-level groups, for hsa-miR-23a (SEQ ID NO: 6) and
hsa-miR-27a (SEQ ID NO: 4), which were associated with significant
differences in both survival and recurrence-free survival.
[0193] Accordingly, in patients with stage II ovarian cancer,
relatively high expressions of any of hsa-miR-27a (SEQ ID NO: 4),
hsa-miR-23a (SEQ ID NO: 6), hsa-miR-23a* (SEQ ID NO: 19),
hsa-miR-21 (SEQ ID NO: 20) and hsa-miR-24-2* (SEQ ID NO: 55) is
predictive of a poor prognosis, whereas high expression of
hsa-miR-449b (SEQ ID NO: 22) is indicative of better prognosis.
Exceptionally high expression level of hsa-miR-27a (SEQ ID NO: 4)
was further found to identify a subgroup of patients with very poor
prognosis (FIG. 5) that had progressive disease during first line
chemotherapy and extremely short progression-free survival.
[0194] In order to assess the relative contribution of various
parameters on progression times in stage III, the Cox proportional
hazards model was used. The parameters used were hsa-miR-27a (SEQ
ID NO: 4) expression (b=0.99, p=0.02), grade (b=-0.15, p=0.76), age
(b=0.03, p=0.13), optimal cytoreduction status (b=-1.1, p=0.05) and
histological type (b=0.37, p=0.51). The results thus indicated that
grade, age and histological type do not contribute to progression
times within stage II beyond the effect of hsa-miR-27a expression
and optimal debulking status. In order to further examine the
connection between hsa-miR-27a expression and optimal cytoreduction
status, the association of this microRNA with disease progression
was analyzed separately for stage III patients with or without
optimal cytoreduction. For patients with optimal cytoreduction,
hsa-miR-27a was not a good predictor of progression times (logrank
p-value of 0.62, comparing the upper and lower tertiles). However,
interestingly, for patients without optimal debulking, hsa-miR-27a
was a significant predictor of progression times (logrank p-value
of 0.046, comparing the upper and lower tertiles).
Example 5: miR Expression Patterns in Patients with Stage III
Ovarian Carcinoma Correlate with Histological Subtype
[0195] The miR expression patterns of stage m ovarian cancer were
examined in tumors of various histological subtypes. As indicated
in FIG. 4, the expression of hsa-miR-93 (SEQ ID NO: 34) in
endometrioid carcinoma tumors (n=13) was two fold higher than its
expression in papillary serous cystadenocarcinoma tumors (n=25),
with a p-value of 0.004216. Contrastingly, the expression of
hsa-let-7i (SEQ ID NO. 32) in cystadenocarcinoma tumors was
2.2-fold higher than in endometrioid carcinoma tumors, with a
p-value of 0.000637. These two miRs were significant when limiting
the False Detection Rate (FDR) to 0.1. Several other miRs were
significant using this threshold, although with a lower fold
change. These include hsa-let-7g (SEQ ID NO: 11) (P=0.0005, 1.6
fold higher in serous cystadenocarcinoma). Accordingly, the
expression pattern of endometrioid carcinoma tumors differs
significantly from the expression pattern of cystadenocarcinoma
tumors.
Example 6: Therapeutic Uses of HSA-miR-449A (Seq Id No: 24),
HSA-miR-449B (Seq Id No: 22) and HSA-miR-200A (Seq Id No: 30)
[0196] Hsa-miR-449b (SEQ ID NO: 22) and hsa-miR-449a (SEQ ID NO:
24) bear a high similarity in sequence to the hsa-miR-34 family. In
particular, residues 2-8 (5' end) of hsa-miR-449 are identical to
those of hsa-miR-34a (SEQ ID NO: 36). Residues 2-8 of microRNAs,
also referred to as the "seed" sequence, are the most strongly
conserved sequences in microRNAs and microRNA families and are
considered the most important for determination of mRNA targets of
microRNAs. Thus, similarity in the seed sequence may suggest
similar activity in a cancer cell.
[0197] The common seed of hsa-miR-449a (SEQ ID NO: 24) and
hsa-miR-449b (SEQ ID NO: 22) is identical to the seeds of most of
the members of the hsa-miR-34 family. The sequences of the
hsa-miR-34 and the hsa-miR-449 families are presented in table 5,
with the sequences of the seeds underlined.
TABLE-US-00005 TABLE 5 hsa-miR-34a SEQ ID T GGCAGTGTC TT AGCTGGTTGT
NO: 36 hsa-miR-34b* SEQ ID TAGGCAGTGTCATT AGCTGATTG NO: 37
hsa-miR-34c-5p SEQ ID AGGCAGTGTAGTT AGCTGATTGC NO: 28 hsa-miR-449a
SEQ ID TGGCAGTGT ATTGTTAGCTGGT NO: 24 hsa-miR-449b SEQ ID AGGCAGTGT
ATTGTTAGCTGGC NO: 22
[0198] According to the present invention, hsa-miR-34a (SEQ ID NO:
36) was down-regulated in advanced (stage III) tumors. Hsa-miR-449b
(SEQ ID NO: 22) was similarly down-regulated in advanced tumors
while its high expression was associated with a better response to
platinum-based chemotherapy among stage III cases.
[0199] He et al. (Nature 2007; 447:1130-4) have shown that genes
encoding miRNAs in the miR-34 family are direct transcriptional
targets of p53, and that ectopic expression of miR-34 induces cell
cycle arrest in both primary and tumor-derived cell lines. p53 is
an important tumor suppressor gene in many human cancers including
ovarian carcinoma. Mutations in p53 are known to be associated with
tumor aggressiveness and prognosis. miRNA components of tumor
suppressor pathways have been described by comparing miRNA
expression profiles of wild-type and p53-deficient cells.
[0200] In addition to regulating the expression of hundreds of
protein-coding genes, p53 also modulates the levels of miRNAs. p53
can induce expression of miR-34a (SEQ ID NO: 36) in cultured cells
as well as in irradiated mice, by binding to a perfect p53 binding
site located within the gene that gives rise to miR-34a (SEQ ID NO:
36). Inactivation of miR-34a (SEQ ID NO: 36) strongly attenuates
p53-mediated apoptosis in cells exposed to genotoxic stress,
whereas overexpression of miR-34a (SEQ ID NO: 36) mildly increases
apoptosis. Hence, miR-34a (SEQ ID NO: 36) is a direct pro-apoptotic
transcriptional target of p53 that can mediate some of p53's
biological effects. It has been postulated that decreased
expression of miR-34a (SEQ ID NO: 36) may contribute to
tumorigenesis by attenuating p53-dependent apoptosis (Nina
Raver-Shapira, et. al, Mol Cell, 2007; 26:731-743).
[0201] The similarity between the seed sequence of the hsa-miR-34
and the hsa-miR-449 families may suggest similar activity. The
level of hsa-miR-449b (SEQ ID NO: 22) was also found to differ
significantly between stages (4.6-fold higher in stage I vs. stage
III, P=0.048). Taken together with its correlation to stage, and to
better prognosis as described in Example 4, this makes hsa-miR-449b
(SEQ ID NO: 22) a promising therapeutic target.
[0202] A significant difference between stage I and stage I was
also found for hsa-miR-200a (SEQ ID NO: 30) (p=0.00047, 2.1 fold
higher in Stage I). Yang and coworkers recently studied expression
of microRNA in ovarian cancers of different stages (Yang H. et al.
Cancer Res. 2008 68: 425-433). Using a set of ovarian tumors of
mixed histologies, they found high expression of hsa-miR-200a (SEQ
ID NO: 30) associated with higher stage ovarian cancers. Nam and
coworkers recently described a correlation between tumor expression
of microRNAs and cumulative survival in ovarian carcinoma tumor
samples (Nam E J. Clin Cancer Res 2008; 14(9):2690-5). In their
data, high expression of hsa-miR-200a (SEQ ID NO: 30) was
associated with tumors from patients with poorer survival. In
contrast to their findings, in the data set of the present
invention, significantly higher expression of hsa-miR-200a (SEQ ID
NO: 30) was found in early stage disease, correlating with improved
survival. In the study by Nam and coworkers, no data is provided
regarding stage at diagnosis and its correlation to miR expression.
The study by Yang and coworkers included significant numbers of
mucinous and clear cell cystadenocarcinomas, histologies not
represented in the present study. This might explain the
discrepancy between the studies. Accordingly, hsa-miR-200a (SEQ ID
NO: 30) is also an interesting candidate for therapeutics.
Example 7: Genes Targeted by miRs Upregulated in Poor Prognosis
[0203] MicroRNA targets were selected from the intersection of the
target prediction results by Targetscan and Miranda. Only targets
with a Targetscan score lower than -0.2 were used. In order to
retrieve only the most relevant targets, only genes targeted by at
least two microRNAs were analyzed. The selected target genes
include EIF4EBP2, which is a target of hsa-miR-21 (SEQ ID NO: 20),
hsa-miR-23a (SEQ ID NO: 6) and less significantly of hsa-let-7g
(SEQ ID NO: 11), and EBIF4E3, a target of hsa-miR-23a (SEQ ID NO:
6) and less significantly hsa-miR-27a (SEQ ID NO: 4). These genes,
together with EIF4G2 (a predicted target of hsa-let-7g, SEQ ID NO:
11), take part in the eIF4F complex (Sonenberg N. Biochem Cell
Biol. 2008; 86(2):178-83) which was found to be associated with
good prognosis in ovarian cancer (Armengol G, et al. Cancer Res.
2007; 67(16):7551-5). Thus, high levels of hsa-miR-21 (SEQ ID NO:
20), hsa-miR-23a (SEQ ID NO: 6), hsa-miR-27a (SEQ ID NO: 4) and
hsa-let-7g (SEQ ID NO: 11) may contribute to poor prognosis or
chemotherapy resistance through their effect on this complex.
[0204] 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.
[0205] 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
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2gccaacccag uguucagacu accuguucag gaggcucuca auguguacag uagucugcac
60auugguuagg c 713110RNAHomo sapiens 3aggaagcuuc uggagauccu
gcuccgucgc cccaguguuc agacuaccug uucaggacaa 60ugccguugua caguagucug
cacauugguu agacugggca agggagagca 110421RNAHomo sapiens 4uucacagugg
cuaaguuccg c 21578RNAHomo sapiens 5cugaggagca gggcuuagcu gcuugugagc
aggguccaca ccaagucgug uucacagugg 60cuaaguuccg ccccccag 78621RNAHomo
sapiens 6aucacauugc cagggauuuc c 21773RNAHomo sapiens 7ggccggcugg
gguuccuggg gaugggauuu gcuuccuguc acaaaucaca uugccaggga 60uuuccaaccg
acc 73823RNAHomo sapiens 8uguaaacauc cuacacucuc agc 23989RNAHomo
sapiens 9accaugcugu agugugugua aacauccuac acucucagcu gugagcucaa
gguggcuggg 60agaggguugu uuacuccuuc ugccaugga 891072RNAHomo sapiens
10agauacugua aacauccuac acucucagcu guggaaagua agaaagcugg gagaaggcug
60uuuacucuuu cu 721122RNAHomo sapiens 11ugagguagua guuuguacag uu
221284RNAHomo sapiens 12aggcugaggu aguaguuugu acaguuugag ggucuaugau
accacccggu acaggagaua 60acuguacagg ccacugccuu gcca 841322RNAHomo
sapiens 13uggacuugga gucaggaggc cu 221478RNAHomo sapiens
14gagucacagu ggacuuggag ucaggaggcc ugagguccuu gaagaccucc cugaccugcu
60cugguccacu gugugcuc 781521RNAHomo sapiens 15acuggacuug gagucagaag
g 211666RNAHomo sapiens 16agggcuccug acuccagguc cuguguguua
ccuagaaaua gcacuggacu uggagucaga 60aggccu 661721RNAHomo sapiens
17agggggaaag uucuauaguc c 211885RNAHomo sapiens 18aggguagagg
gaugaggggg aaaguucuau aguccuguaa uuagaucuca ggacuauaga 60acuuuccccc
ucaucccucu gcccu 851922RNAHomo sapiens 19gggguuccug gggaugggau uu
222022RNAHomo sapiens 20uagcuuauca gacugauguu ga 222172RNAHomo
sapiens 21ugucggguag cuuaucagac ugauguugac uguugaaucu cauggcaaca
ccagucgaug 60ggcugucuga ca 722222RNAHomo sapiens 22aggcagugua
uuguuagcug gc 222397RNAHomo sapiens 23ugaccugaau cagguaggca
guguauuguu agcuggcugc uugggucaag ucagcagcca 60caacuacccu gccacuugcu
ucuggauaaa uucuucu 972422RNAHomo sapiens 24uggcagugua uuguuagcug gu
222591RNAHomo sapiens 25cuguguguga ugagcuggca guguauuguu agcugguuga
auaugugaau ggcaucggcu 60aacaugcaac ugcugucuua uugcauauac a
912622RNAHomo sapiens 26uuaucagaau cuccaggggu ac 222772RNAHomo
sapiens 27ggagcuuauc agaaucucca gggguacuuu auaauuucaa aaaguccccc
aggugugauu 60cugauuugcu uc 722823RNAHomo sapiens 28aggcagugua
guuagcugau ugc 232977RNAHomo sapiens 29agucuaguua cuaggcagug
uaguuagcug auugcuaaua guaccaauca cuaaccacac 60ggccagguaa aaagauu
773022RNAHomo sapiens 30uaacacuguc ugguaacgau gu 223190RNAHomo
sapiens 31ccgggccccu gugagcaucu uaccggacag ugcuggauuu cccagcuuga
cucuaacacu 60gucugguaac gauguucaaa ggugacccgc 903222RNAHomo sapiens
32ugagguagua guuugugcug uu 223384RNAHomo sapiens 33cuggcugagg
uaguaguuug ugcuguuggu cggguuguga cauugcccgc uguggagaua 60acugcgcaag
cuacugccuu gcua 843423RNAHomo sapiens 34caaagugcug uucgugcagg uag
233580RNAHomo sapiens 35cugggggcuc caaagugcug uucgugcagg uagugugauu
acccaaccua cugcugagcu 60agcacuuccc gagcccccgg 803622RNAHomo sapiens
36uggcaguguc uuagcugguu gu 223723RNAHomo sapiens 37uaggcagugu
cauuagcuga uug 2338110RNAHomo Sapiens 38ggccagcugu gaguguuucu
uuggcagugu cuuagcuggu uguugugagc aauaguaagg 60aagcaaucag caaguauacu
gcccuagaag ugcugcacgu uguggggccc 1103984RNAHomo Sapiens
39gugcucgguu uguaggcagu gucauuagcu gauuguacug uggugguuac aaucacuaac
60uccacugcca ucaaaacaag gcac 844022RNAHomo Sapiens 40uaauacugcc
ugguaaugau ga 224118RNAHomo Sapiens 41uucacaggga ggugucau
184222RNAHomo Sapiens 42ugauugguac gucugugggu ag 224322RNAHomo
Sapiens 43uacugcagac guggcaauca ug 224423RNAHomo Sapiens
44ugagugugug ugugugagug ugu 234523RNAHomo Sapiens 45agcucggucu
gaggccccuc agu 234622RNAHomo Sapiens 46cagugcaaug uuaaaagggc au
224722RNAHomo Sapiens 47ugagaacuga auuccauagg cu 224822RNAHomo
Sapiens 48aacuggccua caaaguccca gu 224922RNAHomo Sapiens
49ugggucuuug cgggcgagau ga 225022RNAHomo Sapiens 50aguggggaac
ccuuccauga gg 225121RNAHomo Sapiens 51aucacauugc cagggauuac c
215222RNAHomo Sapiens 52acaggugagg uucuugggag cc 225324RNAHomo
Sapiens 53ucccugagac ccuuuaaccu guga 245422RNAHomo Sapiens
54aaaccguuac cauuacugag uu 225522RNAHomo Sapiens 55ugccuacuga
gcugaaacac ag 225695RNAHomo Sapiens 56ccagcucggg cagccguggc
caucuuacug ggcagcauug gauggaguca ggucucuaau 60acugccuggu aaugaugacg
gcggagcccu gcacg 9557127RNAHomo Sapiens 57ggaugccaca uucagccauu
cagugugcag ugccuuucac agggaggugu cauuuaugug 60aacuaaaaua uaaauuucac
cuuucugaga aggguaaugu acagcaugca cugcauaugu 120ggugucc
1275894RNAHomo Sapiens 58caugcugugu gugguacccu acugcagaca
guggcaauca uguauaauua aaaaugauug 60guacgucugu ggguagagua cugcaugaca
caug 945975RNAHomo Sapiens 59gugguacccu acugcagacg uggcaaucau
guauaauuaa aaaugauugg uacgucugug 60gguagaguac ugcau 756096RNAHomo
Sapiens 60gggaccugcg ugggugcggg cgugugagug ugugugugug aguguguguc
gcuccggguc 60cacgcucaug cacacaccca cacgcccaca cucagg 966194RNAHomo
Sapiens 61auaaaggaag uuaggcugag gggcagagag cgagacuuuu cuauuuucca
aaagcucggu 60cugaggcccc ucagucuugc uuccuaaccc gcgc 946289RNAHomo
Sapiens 62ugcugcuggc cagagcucuu uucacauugu gcuacugucu gcaccuguca
cuagcagugc 60aauguuaaaa gggcauuggc cguguagug 896373RNAHomo Sapiens
63ccuggcacug agaacugaau uccauaggcu gugagcucua gcaaugcccu guggacucag
60uucuggugcc cgg 736488RNAHomo Sapiens 64cgaggauggg agcugagggc
ugggucuuug cgggcgagau gagggugucg gaucaacugg 60ccuacaaagu cccaguucuc
ggcccccg 886588RNAHomo Sapiens 65cgaggauggg agcugagggc ugggucuuug
cgggcgagau gagggugucg gaucaacugg 60ccuacaaagu cccaguucuc ggcccccg
886684RNAHomo Sapiens 66uugacuuagc uggguagugg ggaacccuuc caugaggagu
agaacacucc uuaugcaaga 60uucccuucua ccuggcuggg uugg 846797RNAHomo
Sapiens 67cucaggugcu cuggcugcuu ggguuccugg caugcugauu ugugacuuaa
gauuaaaauc 60acauugccag ggauuaccac gcaaccacga ccuuggc 976886RNAHomo
Sapiens 68ugccagucuc uaggucccug agacccuuua accugugagg acauccaggg
ucacagguga 60gguucuuggg agccuggcgu cuggcc 866986RNAHomo Sapiens
69ugccagucuc uaggucccug agacccuuua accugugagg acauccaggg ucacagguga
60gguucuuggg agccuggcgu cuggcc 867072RNAHomo Sapiens 70cuugggaaug
gcaaggaaac cguuaccauu acugaguuua guaaugguaa ugguucucuu 60gcuauaccca
ga 727173RNAHomo Sapiens 71cucugccucc cgugccuacu gagcugaaac
acaguugguu uguguacacu ggcucaguuc 60agcaggaaca ggg 73
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