U.S. patent application number 13/132583 was filed with the patent office on 2011-11-10 for microrna-based methods and compositions for the diagnosis, prognosis and treatment of ovarian cancer using a real-time pcr platform.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY. Invention is credited to Hansjuerg Alder, David E. Cohn, Carlo M. Croce, Kimberly Resnick.
Application Number | 20110275534 13/132583 |
Document ID | / |
Family ID | 42233538 |
Filed Date | 2011-11-10 |
United States Patent
Application |
20110275534 |
Kind Code |
A1 |
Cohn; David E. ; et
al. |
November 10, 2011 |
MicroRNA-Based Methods and Compositions for the Diagnosis,
Prognosis and Treatment of Ovarian Cancer Using a Real-Time PCR
Platform
Abstract
Methods and compositions for the diagnosis, prognosis and/or
treatment of ovarian cancer are disclosed.
Inventors: |
Cohn; David E.; (Bexley,
OH) ; Resnick; Kimberly; (Shaker Hts., OH) ;
Alder; Hansjuerg; (Columbus, OH) ; Croce; Carlo
M.; (Columbus, OH) |
Assignee: |
THE OHIO STATE UNIVERSITY
Columbus
OH
|
Family ID: |
42233538 |
Appl. No.: |
13/132583 |
Filed: |
March 25, 2009 |
PCT Filed: |
March 25, 2009 |
PCT NO: |
PCT/US09/38214 |
371 Date: |
June 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61120123 |
Dec 5, 2008 |
|
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|
Current U.S.
Class: |
506/9 ; 435/6.12;
435/6.14; 436/501; 536/23.1 |
Current CPC
Class: |
C12Q 2600/178 20130101;
C12N 15/113 20130101; C12N 15/1138 20130101; C12Q 1/6809 20130101;
C12N 2320/30 20130101; C12Q 1/6809 20130101; A61K 31/7105 20130101;
A61P 15/00 20180101; C12Q 2525/207 20130101; C12N 2310/113
20130101; C12N 2310/141 20130101; C12Q 2525/207 20130101; C12Q
1/6886 20130101; A61P 35/00 20180101; C12Q 2600/136 20130101 |
Class at
Publication: |
506/9 ; 435/6.12;
436/501; 435/6.14; 536/23.1 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/574 20060101 G01N033/574; C07H 21/02 20060101
C07H021/02; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A method of diagnosing whether a subject has, or is at risk for
developing, an epithelial ovarian cancer, comprising measuring the
level of at least one biomarker in a test sample of serum from the
subject, wherein the at least one biomarker differentially
expressed between tumor tissue and non-tumor tissue comprises
miR-21; and wherein an increase in the level of the miR-21
biomarker in the test sample, relative to the level of a
corresponding biomarker in a control sample, is indicative of the
subject either having, or being at risk for developing, epithelial
ovarian cancer.
2. The method of claim 1, wherein the at least one additional
biomarker is one or more of miR-92, miR-93, miR-126, miR-29a,
miR-155, miR-127 and miR-99b, or functional variants thereof.
3. (canceled)
4. The method of claim 2, wherein the at least one additional
biomarker is over-expressed, and wherein the at least one
additional over-expressed biomarker is selected from the group
consisting of: miR-92, miR-93, miR-126 and miR-29a, or functional
variants thereof.
5. (canceled)
6. The method of claim 2, wherein the level of the at least one
additional biomarker in the test sample is less than the level of
the corresponding biomarker in the control sample, wherein the at
least one biomarker is one or more of miR-155, miR-127 and miR-99b,
or functional variants thereof.
7. (canceled)
8. A method of screening for one or more biomarkers for ovarian
cancer in a subject, comprising: obtaining a sample of serum from
the subject, conducting quantitative real-time polymerase chain
reaction (RT-PCR), and quantifying one or more one biomarkers
differentially expressed between tumor tissue and non-tumor tissue,
wherein the biomarkers are selected from one or more of miR-21,
miR-92, miR-93, miR-126, miR-29a, miR-155, miR-127 and miR-99b, or
functional variants thereof.
9. (canceled)
10. (canceled)
11. A biomarker for ovarian cancer, comprising at least one
biomarker differentially expressed between tumor tissue and
non-tumor tissue, wherein the biomarkers are selected from one or
more of miR-21, miR-92, miR-93, miR-126, miR-29a, miR-155, miR-127
and miR-99b, or functional variants thereof.
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. A method for determining the prognosis of a subject with
ovarian cancer, comprising measuring the level of at least one
biomarker in a test sample of serum from the subject, wherein the
biomarker is selected from one or more of miR-21, miR92, miR-93,
miR-126, miR-29a, miR-155, miR-127 and miR-99b, or functional
variants thereof, and wherein: i) the biomarker is associated with
an adverse prognosis in ovarian cancer; and ii) an alteration in
the level of the at least one biomarker in the test sample,
relative to the level of a corresponding biomarker in a control
sample, is indicative of an adverse prognosis.
39. The method of claim 38, comprising: a) reverse transcribing RNA
from the test sample of serum obtained from the subject to provide
a set of target oligodeoxynucleotides; b) hybridizing the target
oligodeoxynucleotides to a microarray comprising miRNA-specific
probe oligonucleotides to provide a hybridization profile for the
test sample; and c) comparing the test sample hybridization profile
to a hybridization profile generated from a control sample, wherein
an alteration in the signal of at least one biomarker is indicative
of the subject either having, or being at risk for developing,
ovarian cancer.
40. The method of claim 38, wherein the signal of at least miR-155,
miR-127 and miR-99, relative to the signal generated from the
control sample, is down-regulated, and/or wherein the signal of at
least miR-21, miR92, miR-93, miR-126, miR-29a, relative to the
signal generated from the control sample, is up-regulated.
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. A kit for screening for epithelial ovarian cancer, wherein the
kit comprises: one or more reagents of at least one biomarker
selected from one or more of: miR-21, miR-92, miR-93, miR-126,
miR-29a, miR-155, miR-127 and miR-99b, or functional variants
thereof.
54. The kit of claim 53, wherein the presence of the biomarker is
detected using a reagent comprising an antibody or an antibody
fragment which specifically binds with at least one biomarker.
55. (canceled)
56. (canceled)
57. (canceled)
58. (canceled)
59. (canceled)
60. (canceled)
61. (canceled)
62. (canceled)
63. (canceled)
64. (canceled)
65. (canceled)
66. (canceled)
67. The method of claim 1, further comprising collecting a
plurality of test samples from the subject at different time points
and comparing the amount of the at least one biomarker in each test
sample to determine if the amount of the at least one biomarker is
increasing or decreasing in the subject over time.
68. (canceled)
69. (canceled)
70. (canceled)
71. (canceled)
72. (canceled)
73. (canceled)
74. (canceled)
75. (canceled)
76. (canceled)
77. A method for classifying an ovarian tissue from a subject
comprising: a) measuring the expression of one or more of: miR-21,
miR-92, miR-93, miR-126, miR-29a, miR-155, miR-127 and miR-99b, or
functional variants thereof, among the miR in a test cell
population, wherein at least one cell in the test cell population
is capable of expressing one or more of: miR-21, miR92, miR-93,
miR-126, miR-29a, miR-155, miR-127 and miR-99b, or functional
variants thereof among the miR; b) comparing the expression of the
miR(s) to the expression of the miR(s) in a reference cell
population comprising at least one cell for which an ovarian cancer
classification is known; and c) identifying a difference, if
present, in expression levels of one or more miR(s) in the test
cell population and reference cell population, thereby classifying
the ovarian cancer in the subject.
78. The method of claim 77, wherein a difference in the expression
in the test cell population as compared to the reference cell
population indicates that the test cell population has a different
classification as the cells from the reference cell population.
79. The method of claim 77, wherein a similar expression pattern in
the test cell population as compared to the reference cell
population indicates that the test cell population has the same
classification as the cells from the reference cell population.
80. The method of claim 77, wherein the reference cell population
is a plurality of cells or a database.
81. The method of claim 80, wherein the reference cell population
is selected from the group consisting of: a reference cell
population classified as a cell population from normal ovarian
tissue, a reference cell population classified as a cell population
from benign ovarian tissue and a reference cell population
classified as a cell population from malignant ovarian tissue.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/120,123, filed Dec. 28, 2008, the entire
disclosure of which is expressly incorporated herein by
reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was not made with government support and the
government has no rights in this invention.
TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION
[0003] This invention relates generally to the field of molecular
biology. Certain aspects of the invention include application in
diagnostics, therapeutics, and prognostics of ovarian cancer
related disorders.
BACKGROUND
[0004] There is no admission that the background art disclosed in
this section legally constitutes prior art.
[0005] In 2008, it is expected that 20,180 women will be diagnosed
with ovarian cancer and 15,310 will succumb to the disease [1].
Ovarian cancer is a devastating illness in which only 20% of
patients are diagnosed with stage I disease [2]. The poor prognosis
associated with ovarian cancer is multi-factorial; a lack of
minimally invasive, early detection tests, subtle symptom
development and tumor chemo-resistance. Even with the advent of
chemo-resistance assays it is still difficult to predict drug
resistance and only 10-15% of patients will remain in prolonged
remission after initial cytotoxic therapy.
[0006] While annual pelvic examination is widely practiced, it
lacks the sensitivity to be used a screening strategy for ovarian
cancer [3]. Women at high risk for ovarian cancer may typically
undergo screening with trans-vaginal ultrasound and serum CA-125.
CA-125, however, remains a poor marker for early stage disease with
a documented sensitivity of 40% [4,5]. Additionally, it has been
shown that even in a high-risk, screened population, incident cases
are still more likely to be advanced stage [6]. The identification
of biomarkers that may assist in treatment planning and prediction
of chemotherapy outcomes is highly desirable in this population of
patients.
[0007] There is emerging research about the role of microRNAs in a
variety of pathologic conditions; including both solid and
hematologic malignancies. MicroRNAs (miRNAs) are small, 22-25
nucleotide non-coding sequences of RNA. These sequences control
gene expression either by translational repression or degradation
of the messenger RNA transcript after targeting the 3'UTR. Early
studies with Caenorhabditis elegans showed that a great number of
these sequences are highly conserved across all species,
demonstrating the important roles that miRNAs play in cellular
differentiation, proliferation and cell cycle control [7]. It is
now recognized that miRNAs are frequently de-regulated in
malignancy. Under-expressed miRNAs such as let-7 in lung cancer and
mirs-15/16 in leukemia, are tumor suppressor genes, suppressing Ras
and BCL2 respectively [8,9]. Over-expressed miRNAs such as mir-21
and the cluster mir-17-92, are oncogenes (oncomirs), targeting
tumor suppressors PTEN and E2F1 in solid and hematologic
malignancies respectively [10,11]. While miRNA research in
gynecologic malignancies is in its infancy, the miRNA signature
profile of ovarian cancer has recently been published [12-14].
[0008] The diagnostic and prognostic utility of circulating RNAs in
both benign and malignant conditions has recently been revealed.
Placental-associated circulating miRNAs correlate with pregnancy
progression [15]. In malignant states, circulating mRNAs in renal
cell carcinoma patients [16], as well as miRNAs from the serum of
patients with diffuse large B cell lymphoma [17], have been shown
to be stable and highly predictive of malignancy as well as
survival. Recently, it has been demonstrated that the miRNA
signature of circulating tumor exosomes of ovarian cancer patients
demonstrates high correlation with miRNA expression of the primary
tumor [18]. Ovarian cancer remains a disease for which improved
non-invasive, serum screening tests are highly desirable.
[0009] In spite of considerable research into therapies to treat
these diseases, they remain difficult to diagnose and treat
effectively, and the mortality observed in patients indicates that
improvements are needed in the diagnosis, treatment and prevention
of ovarian cancer.
SUMMARY
[0010] In a first broad aspect, there is provided herein, a method
of diagnosing whether a subject has, or is at risk for developing a
ovarian-related disorder, determining a prognosis of a subject with
ovarian related disorder, and/or treating a ovarian related
disorder in a subject who has the ovarian related disorder,
comprising: measuring the level of at least one biomarker in a test
sample of serum from the subject, wherein an alteration in the
level of the biomarker in the test sample, relative to the level of
a corresponding biomarker in a control sample, is indicative of the
subject either having, or being at risk for developing, the
disorder.
[0011] In certain embodiments, the at least one biomarker
differentially expressed between tumor tissue and non-tumor tissue,
and is one or more of miR-21, miR92, miR-93, miR-126, miR-29a,
miR-155, miR-127 and miR-99b, or functional variants thereof.
[0012] In certain embodiments, the level of the biomarker in the
test sample is greater than the level of the corresponding
biomarker in the control sample.
[0013] In certain embodiments, the biomarker is differentially
expressed between tumor tissue and non-tumor tissue, and is one or
more of miR-21, miR92, miR-93, miR-126 and miR-29a, or functional
variants thereof.
[0014] In certain embodiments, the biomarker is differentially
expressed between tumor tissue and non-tumor tissue, and is one or
more of miR-21, miR92 and miR-93, or functional variants
thereof.
[0015] In certain embodiments, the level of the at least one
biomarker in the test sample is less than the level of the
corresponding biomarker in the control sample. In certain
embodiments, the biomarker is differentially expressed between
tumor tissue and non-tumor tissue, and is one or more of miR-155,
miR-127 and miR-99b, or functional variants thereof.
[0016] In another broad aspect, there is provided herein, a method
of screening for one or more biomarkers for ovarian cancer in a
subject, comprising: obtaining a sample of serum from the subject,
conducting quantitative real-time polymerase chain reaction
(RT-PCR), and quantifying one or more one biomarkers differentially
expressed between tumor tissue and non-tumor tissue, wherein the
biomarkers are selected from one or more of miR-21, miR92, miR-93,
miR-126, miR-29a, miR-155, miR-127 and miR-99b, or functional
variants thereof. In certain embodiments, the sample comprises a
blood sample.
[0017] In certain embodiments, the sample comprises one or more of
serum or plasma blood samples.
[0018] In another broad aspect, there is provided herein, a method
biomarker for ovarian cancer, comprising at least one biomarker
differentially expressed between tumor tissue and non-tumor tissue,
wherein the biomarkers are selected from one or more of miR-21,
miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and miR-99b, or
functional variants thereof.
[0019] In another broad aspect, there is provided herein, a method
distinct microRNA expression signature in ovarian tumors comprising
alterations in the expression of one or more biomarkers that
regulate tumor microRNA processing, wherein the biomarkers are
selected from one or more of miR-21, miR92, miR-93, miR-126,
miR-29a, miR-155, miR-127 and miR-99b, or functional variants
thereof.
[0020] In another broad aspect, there is provided herein, a method
for influencing transcript abundance and/or protein expression of
target mRNAs in the ovary of a subject in need thereof, comprising
deregulating one or more microRNAs selected from one or more of
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof.
[0021] In certain embodiments, the method further includes
inhibiting protein expression of cancer-related genes.
[0022] In certain embodiments, the method includes altering
expression of one or more of miR-21, miR-92, mir-93, miR-126 and
miR-129a to inhibit the protein expression of cancer-related
genes.
[0023] In another broad aspect, there is provided herein, use of a
large-scale gene expression profiling of microRNAs and/or
protein-encoding RNAs to identify alterations in microRNA function
that occur in human ovarian tumors.
[0024] In another broad aspect, there is provided herein, a method
for screening for ovarian in a subject in need thereof, comprising
the step of performing real-time polymerase chain reaction (RT-PCR)
on a serum sample from the subject.
[0025] In another broad aspect, there is provided herein, a tumor
gene signature for an ovarian related disorder comprising: one or
more of: miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127
and miR-99b, or functional variants thereof.
[0026] In another broad aspect, there is provided herein, a tumor
gene signature for an ovarian related disorder comprising: one or
more of: miR-21, miR92, miR-93, miR-126 and miR-29a, or functional
variants thereof that are up-regulated; and, miR-155, miR-127 and
miR-99b, or functional variants thereof, that are down
regulated.
[0027] In another broad aspect, there is provided herein, a tumor
gene signature for an ovarian related disorder comprising: one or
more of: miR-21, miR92 and miR-93, or functional variants
thereof.
[0028] In certain embodiments, the biomarker comprises host gene
expression in ovarian tumors that are increased in ovarian tumors.
In certain embodiments, the biomarkers include one or more of:
miR-21, miR92, miR-93, miR-126 and miR-29a, or functional variants
thereof.
[0029] In another broad aspect, there is provided herein, use of
miR-21, miR92 and/or miR-93, or functional variants thereof, as a
target for at least one gene in ovarian cancer cells and/or use in
inhibiting protein expression of such gene.
[0030] In another broad aspect, there is provided herein, a method
for regulating one or more of genes expressed by ovarian cancer
cells, comprising the step of altering expression of miR-21, miR92
and/or miR-93 in ovarian cancer cells.
[0031] In another broad aspect, there is provided herein, use of
binding of microRNAs to 3'UTR sequences to lead to degradation
and/or accumulation of targeted mRNA in mammalian ovarian cancer
cells.
[0032] In another broad aspect, there is provided herein, use of an
inverse and/or a positive correlation between a microRNA and a mRNA
in a human tissue predictive of a microRNA target gene for ovarian
cancer.
[0033] In another broad aspect, there is provided herein, a
miR-expression inhibitor comprising one or more of: miR-21, miR92,
miR-93, miR-126 and miR-29a, or functional variants thereof.
[0034] In another broad aspect, there is provided herein, a
miR-expression inhibitor comprising one or more of: miR-21, miR92
and/or miR-93, or functional variants thereof.
[0035] In another broad aspect, there is provided herein, a
miR-expression antisense inhibitor comprising one or more of:
miR-155, miR-127 and miR-99b, or functional variants thereof.
[0036] In another broad aspect, there is provided herein, an
oncomiR biomarker of an ovarian disorder or disease, comprising one
or more of: miR-21, miR92, miR-93, miR-126, miR-29a, miR-155,
miR-127 and miR-99b, or functional variants thereof.
[0037] In another broad aspect, there is provided herein, an
oncomiR biomarker of an ovarian disorder or disease, comprising one
or more of: miR-21, miR92 and miR-93, or functional variants
thereof.
[0038] In another broad aspect, there is provided herein, a method
for regulating protein expression in ovarian cancer cells,
comprising modulating the expression of one or more of: miR-21,
miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and miR-99b, or
functional variants thereof, in the ovarian cancer cells.
[0039] In another broad aspect, there is provided herein, a method
for regulating protein expression in ovarian cancer cells,
comprising modulating the expression of one or more of: miR-21,
miR92 and miR-93, or functional variants thereof, in the ovarian
cancer cells.
[0040] In another broad aspect, there is provided herein, a
composition for repressing expression of one or more of genes in
ovarian cancer cells, the composition comprising one or more of:
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof.
[0041] In another broad aspect, there is provided herein, a
composition for repressing expression of one or more of genes in
ovarian cancer cells, the composition comprising one or more of:
miR-21, miR92 and miR-93, or functional variants thereof.
[0042] In another broad aspect, there is provided herein, a method
for regulating one or more of protein levels in a subject with
ovarian cancer, comprising using one or more of: miR-21, miR92,
miR-93, miR-126, miR-29a, miR-155, miR-127 and miR-99b, or
functional variants thereof.
[0043] In another broad aspect, there is provided herein, a method
for regulating one or more of protein levels in a subject with
ovarian cancer, comprising using one or more of: miR-21, miR92 and
miR-93, or functional variants thereof.
[0044] In another broad aspect, there is provided herein, a method
for determining the prognosis of a subject with ovarian cancer,
comprising measuring the level of at least one biomarker in a test
sample of serum from the subject, wherein the biomarker is selected
from one or more of miR-21, miR92, miR-93, miR-126, miR-29a,
miR-155, miR-127 and miR-99b, or functional variants thereof, and
wherein: i) the biomarker is associated with an adverse prognosis
in ovarian cancer; and ii) an alteration in the level of the at
least one biomarker in the test sample, relative to the level of a
corresponding biomarker in a control sample, is indicative of an
adverse prognosis.
[0045] In another broad aspect, there is provided herein, a method
of diagnosing whether a subject has, or is at risk for developing,
ovarian cancer, comprising: reverse transcribing RNA from a test
sample of serum obtained from the subject to provide a set of
target oligodeoxynucleotides; hybridizing the target
oligodeoxynucleotides to a microarray comprising miRNA-specific
probe oligonucleotides to provide a hybridization profile for the
test sample; and comparing the test sample hybridization profile to
a hybridization profile generated from a control sample, wherein an
alteration in the signal of at least one miRNA is indicative of the
subject either having, or being at risk for developing, ovarian
cancer.
[0046] In certain embodiments, the signal of at least one miRNA,
relative to the signal generated from the control sample, is
down-regulated, and/or wherein the signal of at least one miRNA,
relative to the signal generated from the control sample, is
up-regulated.
[0047] In certain embodiments, an alteration in the signal of at
least one biomarker miR-21, miR92, miR-93, miR-126, miR-29a,
miR-155, miR-127 and miR-99b, or functional variants thereof are
indicative of the subject either having, or being at risk for
developing, an ovarian cancer with an adverse prognosis.
[0048] In another broad aspect, there is provided herein, a method
of treating ovarian cancer in a subject who has an ovarian cancer
in which at least one biomarker is down-regulated or up-regulated
in the cancer cells of the subject relative to control cells,
comprising: when the at least one biomarker is down-regulated in
the cancer cells, administering to the subject an effective amount
of at least one isolated biomarker, or an isolated variant or
biologically-active fragment thereof, such that proliferation of
cancer cells in the subject is inhibited; or when the at least one
biomarker is up-regulated in the cancer cells, administering to the
subject an effective amount of at least one compound for inhibiting
expression of the at least one biomarker, such that proliferation
of cancer cells in the subject is inhibited; wherein the biomarker
is selected from one or more of miR-21, miR92, miR-93, miR-126,
miR-29a, miR-155, miR-127 and miR-99b, or functional variants
thereof.
[0049] In another broad aspect, there is provided herein, a method
of treating ovarian cancer in a subject, comprising: determining
the amount of at least one biomarker in ovarian cancer cells,
relative to control cells; and altering the amount of biomarker
expressed in the ovarian cancer cells by: administering to the
subject an effective amount of at least one isolated biomarker, if
the amount of the biomarker expressed in the cancer cells is less
than the amount of the biomarker expressed in control cells; or
administering to the subject an effective amount of at least one
compound for inhibiting expression of the at least one biomarker,
if the amount of the biomarker expressed in the cancer cells is
greater than the amount of the biomarker expressed in control
cells; wherein the biomarker is selected from one or more of
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof.
[0050] In another broad aspect, there is provided herein, a
pharmaceutical composition for treating ovarian cancer, comprising
at least one isolated biomarker, and a pharmaceutically-acceptable
carrier, wherein the biomarker is selected from one or more of
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof. In certain embodiments,
the biomarker corresponds to a biomarker that is up-regulated in
ovarian cancer cells relative to control cells. In certain
embodiments, the pharmaceutical composition comprises at least one
miR expression-inhibitor compound and a pharmaceutically-acceptable
carrier.
[0051] In another broad aspect, there is provided herein, a method
of identifying an anti-ovarian cancer agent, comprising providing a
test agent to a cell and measuring the level of at least one
biomarker associated with increased expression levels in ovarian
cancer cells, wherein a decrease in the level of the biomarker in
the cell, relative to a control cell, is indicative of the test
agent being an anti-ovarian cancer agent; and wherein the biomarker
is selected from one or more of miR-21, miR92, miR-93, miR-126 and
miR-29a, or functional variants thereof.
[0052] In another broad aspect, there is provided herein, a method
of identifying an anti-ovarian cancer agent, comprising providing a
test agent to a cell and measuring the level of at least one
biomarker associated with decreased expression levels in ovarian
cancer cells, wherein an increase in the level of the biomarker in
the cell, relative to a control cell, is indicative of the test
agent being an anti-ovarian cancer agent; and wherein the biomarker
is selected from one or more of miR-155, miR-127 and miR-99b, or
functional variants thereof.
[0053] In another broad aspect, there is provided herein a method
of assessing the effectiveness of a therapy to prevent, diagnose
and/or treat an ovarian cancer associated disease, comprising:
subjecting an animal to a therapy whose effectiveness is being
assessed, and determining the level of effectiveness of the
treatment being tested in treating or preventing the disease, by
evaluating at least one biomarker selected from one or more of:
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof.
[0054] In certain embodiments, the candidate therapeutic agent
comprises one or more of: pharmaceutical compositions,
nutraceutical compositions, and homeopathic compositions.
[0055] In certain embodiments, the therapy being assessed is for
use in a human subject.
[0056] In another broad aspect, there is provided herein, an
article of manufacture comprising: at least one capture reagent
that binds to a marker for an ovarian cancer associated disease
comprising at least one biomarker selected from one or more of:
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof.
[0057] In another broad aspect, there is provided herein, a kit for
screening for a candidate compound for a therapeutic agent to treat
a ovarian cancer associated disease, wherein the kit comprises: one
or more reagents of at least one biomarker selected from one or
more of: miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127
and miR-99b, or functional variants thereof, and a cell expressing
at least one biomarker. In certain embodiments, the presence of the
biomarker is detected using a reagent comprising an antibody or an
antibody fragment which specifically binds with at least one
biomarker.
[0058] In another broad aspect, there is provided herein, use of an
agent that interferes with an ovarian cancer associated disease
response signaling pathway, for the manufacture of a medicament for
treating, preventing, reversing or limiting the severity of the
disease complication in an individual, wherein the agent comprises
at least one biomarker selected from one or more of: miR-21, miR92,
miR-93, miR-126, miR-29a, miR-155, miR-127 and miR-99b, or
functional variants thereof.
[0059] In another broad aspect, there is provided herein, a method
of treating, preventing, reversing or limiting the severity of an
ovarian cancer associated disease complication in an individual in
need thereof, comprising: administering to the individual an agent
that interferes with at least an ovarian cancer associated disease
response cascade, wherein the agent comprises at least one
biomarker selected from one or more of: miR-21, miR92, miR-93,
miR-126, miR-29a, miR-155, miR-127 and miR-99b, or functional
variants thereof.
[0060] In another broad aspect, there is provided herein, use of an
agent that interferes with at least an ovarian cancer associated
disease response cascade, for the manufacture of a medicament for
treating, preventing, reversing or limiting the severity of an
ovarian cancer-related disease complication in an individual,
wherein the agent comprises at least one biomarker selected from
one or more of: miR-21, miR92, miR-93, miR-126, miR-29a, miR-155,
miR-127 and miR-99b, or functional variants thereof.
[0061] In another broad aspect, there is provided herein, a
composition comprising an inhibitor of one or more of miR-21,
miR-92 and miR-93.
[0062] In another broad aspect, there is provided herein, a method
of treating an ovarian disorder in a subject in need thereof,
comprising administering to a subject a therapeutically effective
amount of the composition. In certain embodiments, the composition
is administered prophylactically. In certain embodiments,
administration of the composition delays the onset of one or more
symptoms of the disorder.
[0063] In certain embodiments, administration of the peptide
inhibits development of ovarian cancer.
[0064] In certain embodiments, administration of the peptide
inhibits tumor growth.
[0065] In another broad aspect, there is provided herein, a method
for detecting the presence of an ovarian cancer in a biological
sample, the method comprising: exposing the biological sample
suspected of containing ovarian cancer to a marker therefor; and
detecting the presence or absence of the marker, if any, in the
sample; wherein the biomarker is selected from one or more of
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof. In certain embodiments,
the marker includes a detectable label.
[0066] In certain embodiments, the method further comprising
comparing the amount of the marker in the biological sample from
the subject to an amount of the marker in a corresponding
biological sample from a normal subject. In certain embodiments,
the method further comprises collecting a plurality of biological
samples from a subject at different time points and comparing the
amount of the marker in each biological sample to determine if the
amount of the marker is increasing or decreasing in the subject
over time.
[0067] In another broad aspect, there is provided herein, a method
for treating an ovarian cancer in a subject, the method comprising:
administering to the subject in need thereof a therapeutically
effective amount of an ovarian receptor agonist comprising: an
inhibitor of one or more of: miR-21, miR92, miR-93, miR-126 and
miR-29a, or functional variants thereof.
[0068] In another broad aspect, there is provided herein, a method
for treating an ovarian cancer in a subject, the method comprising:
administering to the subject in need thereof a therapeutically
effective amount of an ovarian receptor agonist comprising: an
antisense inhibitor of one or more of: miR-155, miR-127 and
miR-99b, or functional variants thereof.
[0069] In another broad aspect, there is provided herein, a use, to
manufacture a drug for the treatment of an ovarian cancer,
comprised of a nucleic acid molecule chosen from one or more of:
miR-21, miR92, miR-93, miR-126, miR-29a, miR-155, miR-127 and
miR-99b, or functional variants thereof. In certain embodiments,
the drug comprises a nucleic acid molecule presenting a sequence
chosen from one or more of: miR-21, miR92, miR-93, miR-126,
miR-29a, miR-155, miR-127 and miR-99b, or functional variants
thereof.
[0070] In another broad aspect, there is provided herein, an in
vitro method to identify effective therapeutic agents or
combinations of therapeutic agents to induce the differentiation of
ovarian cancer cells, the method comprising the stages of: i)
culturing of cells derived from an ovarian tumor, ii) adding at
least one compound to the culture medium of the cell line, iii)
analyzing the evolution of the level of expression of at least one
miR between stages (i) and (ii), and iv) identifying compounds or
combinations of compounds inducing a change in the level of
expression of the miR between stages (i) and (ii). In certain
embodiments, stage (iii) includes the analysis of the level of
expression of at least one miR. In certain embodiments, stage (iv)
includes the identification of the compounds or combinations of
compounds modulating the level of expression of at least one miR.
In certain embodiments, stage (iv) includes the identification of
compounds or combinations of compounds reducing the level of
expression of at least one miR. In certain embodiments, the
compound is a therapeutic agent for the treatment of cancer.
[0071] In another broad aspect, there is provided herein, a method
for classifying an ovarian tissue from a subject comprising:
measuring the expression of one or more of: miR-21, miR92, miR-93,
miR-126, miR-29a, miR-155, miR-127 and miR-99b, or functional
variants thereof, among the miR in a test cell population, wherein
at least one cell in the test cell population is capable of
expressing one or more of: miR-21, miR92, miR-93, miR-126, miR-29a,
miR-155, miR-127 and miR-99b, or functional variants thereof among
the miR; comparing the expression of the miR(s) to the expression
of the miR(s) in a reference cell population comprising at least
one cell for which an ovarian cancer classification is known; and
identifying a difference, if present, in expression levels of one
or more miR(s) in the test cell population and reference cell
population, thereby classifying the ovarian cancer in the
subject.
[0072] In certain embodiments, a difference in the expression in
the test cell population as compared to the reference cell
population indicates that the test cell population has a different
classification as the cells from the reference cell population.
[0073] In certain embodiments, a similar expression pattern in the
test cell population as compared to the reference cell population
indicates that the test cell population has the same classification
as the cells from the reference cell population.
[0074] In certain embodiments, the reference cell population is a
plurality of cells or a database. In certain embodiments, the
reference cell population is selected from the group consisting of:
a reference cell population classified as a cell population from
normal ovarian tissue, a reference cell population classified as a
cell population from benign ovarian tissue and a reference cell
population classified as a cell population from malignant ovarian
tissue.
[0075] Various objects and advantages of this invention will become
apparent to those skilled in the art from the following detailed
description of the preferred embodiment, when read in light of the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] The patent or application file may contain one or more
drawings executed in color and/or one or more photographs. Copies
of this patent or patent application publication with color
drawing(s) and/or photograph(s) will be provided by the Patent
Office upon request and payment of the necessary fee.
[0077] FIG. 1: Comparison of published miRNA profile and
differentially expressed miRNAs from ovarian cancer patient
serum.
[0078] FIG. 2: Median fold-change differences in differentially
expressed miRNAs between patient and control serum.
DETAILED DESCRIPTION OF THE INVENTION
[0079] Throughout this disclosure, various publications, patents
and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure to more fully describe the
state of the art to which this invention pertains.
[0080] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0081] Although a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0082] MicroRNAs are small non-coding RNAs that regulate the
expression of protein-coding genes. MicroRNA expression becomes
altered with the development and progression of ovarian cancer.
Some of these microRNAs regulate the expression of cancer-related
genes in ovarian cancer cells. As used herein interchangeably, a
"miR gene product," "microRNA," "miR," or "miRNA" refers to the
unprocessed or processed RNA transcript from a miR gene.
[0083] As used herein, "biomarker" can include one or more of a
"miR gene product," "microRNA," "miR," or "miRNA," or a
protein-encoding RNA.
[0084] The active 19-25 nucleotide RNA molecule can be obtained
from the miR precursor through natural processing routes (e.g.,
using intact cells or cell lysates) or by synthetic processing
routes (e.g., using isolated processing enzymes, such as isolated
Dicer, Argonaut, or RNAse III). It is understood that the active
19-25 nucleotide RNA molecule can also be produced directly by
biological or chemical synthesis, without having to be processed
from the miR precursor. When a microRNA is referred to herein by
name, the name corresponds to both the precursor and mature forms,
unless otherwise indicated.
[0085] The present invention encompasses methods of diagnosing
whether a subject has, or is at risk for developing, a ovarian
related disorder. As used herein, a "subject" can be any mammal
that has, or is suspected of having, ovarian cancer.
[0086] We offer a description of miRNA extraction from the serum of
ovarian cancer patients, the differential expression of a number of
these miRNAs between patients and healthy controls as well as a
novel real-time PCR microarray detection method.
[0087] The present invention is further explained in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. All publications,
including patents and non-patent literature, referred to in this
specification are expressly incorporated by reference.
Example I
Methods
[0088] Following approval from the Institutional Review Board of
The Ohio State University College of Medicine we analyzed serum
samples from 28 patients with newly diagnosed ovarian cancer and 15
normal controls. These serum samples were collected at the time of
initial consultation, prior to definitive surgical management
and/or adjuvant therapy. The serum was obtained as part of a
prospective tissue and serum procurement study and was stored at
-80.degree. C. Fresh serum was obtained from 15 healthy women who
volunteered to serve as controls. The frozen serum was thawed and
RNA was extracted from the patient and control populations
simultaneously. None of the healthy controls had previously been
diagnosed with a malignancy.
[0089] RNA was extracted from 250 .mu.l of serum using the
Tri-Reagent BD (Molecular Research Center, Inc., Cincinnati, Ohio)
as described by the manufacturer. RNA quality was assessed with the
ThermoScientific Nanoprop1000 (Thermo Fisher Scientific, Inc.,
Waltham, Mass.). MicroRNA expression profiling was performed with
RNA from 4 controls and 9 cancer patients utilizing the TaqMan
Array Human MicroRNA Panel (v.1, Applied Biosystems, Foster City,
Calif.) using 50 ng of RNA per port for a total of 400 ng. This
array contains 365 miRNA targets as well as endogenous controls.
Normalization was performed with the small nuclear RNAs (snRNAs)
U44 and U48. These snRNAs are stably expressed reference genes
suitable for use as normalizers in TaqMan assays.
[0090] In addition to identifying differentially expressed miRNAs
on the microarray panel, a second goal was to identify miRNAs that
may serve as normalizers given the lack of published data on the
subject. Twenty-one miRNAs from the expression profile were
empirically chosen for further examination in control and patient
serum (11 controls and 19 patients). These were chosen based on
apparent Ct differences of 4 cycles or greater between controls and
patients. Two miRNAs (142-3p and 16) were identified as potential
normalizers given consistent expression across all patient and
control samples. For the miRNAs of interest the single tube TaqMan
MicroRNA Assays were used. All reagents, primers and probes were
obtained from Applied Biosystems (Applied Biosystems, Foster City,
Calif.). One nanogram of RNA per sample was used for the assays.
MiRNA-142-3p was used as a normalizer. All RT reactions, including
no-template (no cDNA) controls and minus controls (no reverse
transcriptase), were run in a GeneAmp PCR 9700 Thermocycler
(Applied Biosystems). Gene expression levels were quantified using
the ABI Prism 7900HT Sequence detection system (Applied
Biosystems). Comparative real-time PCR was performed in triplicate,
including no-template controls.
[0091] Expression of the microRNAs was calculated utilizing the
comparative Ct. method. Statistical analysis was performed with
STATA v. 10 (College Station, Tex.). Expression was compared using
the Mann-Whitney test. P-values >0.05 were considered
statistically significant.
[0092] Results
[0093] Twenty-eight patients with epithelial ovarian cancer were
included in this study. Stage breakdown was as follows: stage 18
(28.5%), stage 11-2 (7.1%), stage 111-8 (28.5%) stage 1V-10
(35.7%). Histologic breakdown was as follows: serous (60%), clear
cell (21.2%), endometrioid (12%), mucinous (6%). Median age was 57
years (age range 34-79). Similar to most groups with ovarian
cancer, the majority (66%) had stage III or IV disease, and was
predominately (60%) serous histology.
[0094] Primary miRNA expression profiling with microarray
identified 23 miRNAs (including 2 normalizers) of interest. We
created a Venn diagram in order to compare the 23 miRNAs of
interest from our initial test set with known miRNA signature
profiles. There were 10 miRNAs of interest in such group that were
in common with miRNAs that have been published in the literature as
part of the miRNA signatures of ovarian cancer (FIG. 1).
[0095] On follow up quantitative RT-PCR of the 21 miRNAs, 5 miRNAs
were over-expressed (miRNAs-21, 29a, 92, 93 and 126, p=0.0002,
p=0.003, p=0.0001, p=0.0003, p=0.007) and 3 miRNAs were
under-expressed (mir-127, 155 and 99b, p=0.0001, p=0.0003,
p=0.0001) in the serum of ovarian cancer patients compared to
controls. Fold-differences in median expression of the miRNAs in
patients versus controls are demonstrated in FIG. 2.
[0096] Three patients were identified with pre-operative
CA-125<35 U/ml. We then examined the three miRNAs with the
highest serum expression, miRs-21, 92 and 93 for expression
patterns in those patients with normal CA-125 in order to determine
if miRNA patterns mimicked CA-125 patterns. These three miRNAs were
found to be significantly over-expressed in these patients when
median expression in the patient population was compared to
controls (Table 1).
TABLE-US-00001 TABLE 1 miRNA over-expression in patients with
normal pre-operative CA-125 miR-21 miR-92 miR-93 FIGO CA-125
Patient .sup.a/ Patient/ Patient/ ID Stage (u/ml) Control .sup.b
Control .sup.c Control .sup.d 1050133 IIC 34 1.89/.79 27.5/4.3
2.37/.76 1050130 IV 13.4 1.54/.79 13.8/4.3 14/.76 1010026 IA 16.9
1.46/.79 16.1/4.3 3.5/.76 .sup.a Individual patient serum miRNA
expression is defined as the 2(.DELTA.Ct). .sup.b Inter-quartile
range for median expression of miR-21 in controls: (.68-.93).
.sup.c Inter-quartile range for median expression of miR-92 in
controls: (2.9-8.7). .sup.d Inter-quartile range for median
expression of miR-93 in controls: (.36-1.3).
[0097] There was no correlation between miRNA status and grade,
stage or histologic subtype. Due to the small sample size and
recent diagnosis of disease we did not attempt to correlate miRNA
status with progression-free interval or survival.
[0098] Discussion
[0099] We demonstrate that the extraction of RNA and identification
of miRNAs from the serum of individuals diagnosed with ovarian
cancer is practicable.
[0100] The inventors herein show herein the first description of
using a real-time PCR, microarray platform to screen large numbers
of miRNAs while minimizing the amount of RNA needed.
[0101] Additionally, the inventors herein show that miRNAs can as
early detection biomarkers in patients with normal CA-125.
[0102] A profile was created that was subsequently examined on a
set of 19 patients and 11 healthy controls. Out of the 21 miRNAs of
interest that we selected, 10 miRNAs were common to published
ovarian cancer profiles. Among the 5 over-expressed miRNAs that we
discovered are three potential oncomirs; miRs-21, 92 and 93.
Over-expression of miR-21 has been demonstrated in glioblastoma,
breast, colon, prostate, lung, pancreas and stomach cancers
[19,20]. It has been shown to modulate expression of PTEN in
hepatocellular carcinoma [10] as well as PDCD4 and maspin, two
genes involved in regulating invasion and metastasis [21,22].
[0103] The most consistently over-expressed miRNA in serum from
patients was miR-92. Mir-92a-1 is part of the mir-17-92
polycistron, located on chromosome 13q13. A known oncomir,
mir-17-92-enforced expression in a transgenic mouse model of
lymphoma unequivocally demonstrated accelerated lymphoma
progression [23]. Over-expression of miR-93 was associated with
decreased progression-free and overall survival in ovarian cancer
patients [13]. In gastric tumors, this cluster negatively regulated
TGF.beta. tumor suppressor activities [24]. The proposed oncogenic
activities of both miR-92 and miR-93 agree with our serum
findings.
[0104] Contrary to the published ovarian cancer profiles, the
inventors herein now have demonstrated significant over-expression
of miR-29a and miR-126 in the sera from ovarian cancer patients.
There have been a number of tumor suppressor activities proposed
for both miR-126 and 29a. Mir-126 has been implicated as a
"metastatic-suppressor" in breast cancer with loss associated with
poor outcome [25]. Mir-29a has been found to be under-expressed in
lung cancers; having been implicated in the modulation of
methylation patterns seen in lung cancer [26]. While over
expression of these miRNAs would tend to suggest they behave as
oncomirs, TargetScan (4.2) does predict PTEN as a potential target
of miR-29a.
[0105] Mir-127 has been identified as one of thirty-one
down-regulated miRNAs in ovarian cancer cell lines [14]. It has
recently been shown to be embedded in a CpG island and silenced
completely in most cancer cell lines. In this same study, it was
demonstrated that treatment of cell lines with 5-aza-2'
deoxycytidine not only restored miR-127 expression but also reduced
expression of the proto-oncogene BCL6 [27]. Taken together these
results identify miR-127 as a tumor suppressor gene, supporting our
findings of decreased expression in patient serum.
[0106] It is to be noted that there is a limited amount of
published data regarding the extraction of quality miRNA from
serum. Despite experiencing RNA degradation as well as genomic DNA
contamination (results not shown), the inventors found that only
400 ng of total RNA are required for the TaqMan Array Human
MicroRNA. Additionally, given that the amplicons of interest are
approximately 25-30 nucleotides, the inventors determined that some
degradation of the RNA is tolerable.
[0107] Also, the inventors herein have also determined that the
controls used in real-time PCR account for both cross contamination
by reagents (no template control) as well as genomic DNA
contamination (RT minus control).
[0108] Since the microarray chips typically only utilize up to
about 5 .mu.g of sample, the real-time based method described
herein does not require extracting large amounts of pure RNA from
serum. The inventors herein show for the first time that a
real-time PCT method can be to obtain a miRNA profile on serum
RNA.
EXAMPLES OF USES AND DEFINITIONS THEREOF
[0109] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of pharmacology,
chemistry, biochemistry, recombinant DNA techniques and immunology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Handbook of Experimental Immunology,
Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell
Scientific Publications); A. L. Lehninger, Biochemistry (Worth
Publishers, Inc., current addition); Sambrook, et al., Molecular
Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In
Enzymology (S. Colowick and N. Kaplan eds., Academic Press,
Inc.).
[0110] As such, the definitions herein are provided for further
explanation and are not to be construed as limiting.
[0111] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0112] A "marker" and "biomarker" is a gene and/or protein and/or
functional variants thereof o whose altered level of expression in
a tissue or cell from its expression level in normal or healthy
tissue or cell is associated with a disorder and/or disease
state.
[0113] The "normal" level of expression of a marker is the level of
expression of the marker in cells of a human subject or patient not
afflicted with a disorder and/or disease state.
[0114] An "over-expression" or "significantly higher level of
expression" of a marker refers to an expression level in a test
sample that is greater than the standard error of the assay
employed to assess expression, and in certain embodiments, at least
twice, and in other embodiments, three, four, five or ten times the
expression level of the marker in a control sample (e.g., sample
from a healthy subject not having the marker associated disorder
and/or disease state) and in certain embodiments, the average
expression level of the marker in several control samples.
[0115] A "significantly lower level of expression" of a marker
refers to an expression level in a test sample that is at least
twice, and in certain embodiments, three, four, five or ten times
lower than the expression level of the marker in a control sample
(e.g., sample from a healthy subject not having the marker
associated disorder and/or disease state) and in certain
embodiments, the average expression level of the marker in several
control samples.
[0116] A kit is any manufacture (e.g. a package or container)
comprising at least one reagent, e.g., a probe, for specifically
detecting the expression of a marker. The kit may be promoted,
distributed or sold as a unit for performing the methods of the
present invention.
[0117] "Proteins" encompass marker proteins and their fragments;
variant marker proteins and their fragments; peptides and
polypeptides comprising an at least 15 amino acid segment of a
marker or variant marker protein; and fusion proteins comprising a
marker or variant marker protein, or an at least 15 amino acid
segment of a marker or variant marker protein.
[0118] The compositions, kits and methods described herein have the
following non-limiting uses, among others:
[0119] 1) assessing whether a subject is afflicted with a disorder
and/or disease state;
[0120] 2) assessing the stage of a disorder and/or disease state in
a subject;
[0121] 3) assessing the grade of a disorder and/or disease state in
a subject;
[0122] 4) assessing the nature of a disorder and/or disease state
in a subject;
[0123] 5) assessing the potential to develop a disorder and/or
disease state in a subject;
[0124] 6) assessing the histological type of cells associated with
a disorder and/or disease state in a subject;
[0125] 7) making antibodies, antibody fragments or antibody
derivatives that are useful for treating a disorder and/or disease
state in a subject;
[0126] 8) assessing the presence of a disorder and/or disease state
in a subject's cells;
[0127] 9) assessing the efficacy of one or more test compounds for
inhibiting a disorder and/or disease state in a subject;
[0128] 10) assessing the efficacy of a therapy for inhibiting a
disorder and/or disease state in a subject;
[0129] 11) monitoring the progression of a disorder and/or disease
state in a subject;
[0130] 12) selecting a composition or therapy for inhibiting a
disorder and/or disease state in a subject;
[0131] 13) treating a subject afflicted with a disorder and/or
disease state;
[0132] 14) inhibiting a disorder and/or disease state in a
subject;
[0133] 15) assessing the harmful potential of a test compound;
and
[0134] 16) preventing the onset of a disorder and/or disease state
in a subject at risk therefor.
[0135] Screening Methods
[0136] Animal models can be created to enable screening of
therapeutic agents useful for treating or preventing a disorder
and/or disease state in a subject. Accordingly, the methods are
useful for identifying therapeutic agents for treating or
preventing a disorder and/or disease state in a subject. The
methods comprise administering a candidate agent to an animal model
made by the methods described herein, and assessing at least one
response in the animal model as compared to a control animal model
to which the candidate agent has not been administered. If at least
one response is reduced in symptoms or delayed in onset, the
candidate agent is an agent for treating or preventing the
disease.
[0137] The candidate agents may be pharmacologic agents already
known in the art or may be agents previously unknown to have any
pharmacological activity. The agents may be naturally arising or
designed in the laboratory. They may be isolated from
microorganisms, animals or plants, or may be produced
recombinantly, or synthesized by any suitable chemical method. They
may be small molecules, nucleic acids, proteins, peptides or
peptidomimetics. In certain embodiments, candidate agents are small
organic compounds having a molecular weight of more than 50 and
less than about 2,500 Daltons. Candidate agents comprise functional
groups necessary for structural interaction with proteins.
Candidate agents are also found among biomolecules including, but
not limited to: peptides, saccharides, fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0138] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. There are,
for example, numerous means available for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides and
oligopeptides. Alternatively, libraries of natural compounds in the
form of bacterial, fungal, plant and animal extracts are available
or readily produced. Additionally, natural or synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical and biochemical means, and may be
used to produce combinatorial libraries. In certain embodiments,
the candidate agents can be obtained using any of the numerous
approaches in combinatorial library methods art, including, by
non-limiting example: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection.
[0139] In certain further embodiments, certain pharmacological
agents may be subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification, etc. to produce structural analogs.
[0140] The same methods for identifying therapeutic agents for
treating a disorder and/or disease state in a subject can also be
used to validate lead compounds/agents generated from in vitro
studies.
[0141] The candidate agent may be an agent that up- or
down-regulates one or more of a disorder and/or disease state in a
subject response pathway. In certain embodiments, the candidate
agent may be an antagonist that affects such pathway.
[0142] Methods for Treating a Disorder and/or Disease State
[0143] There is provided herein methods for treating, inhibiting,
relieving or reversing a disorder and/or disease state response. In
the methods described herein, an agent that interferes with a
signaling cascade is administered to an individual in need thereof,
such as, but not limited to, subjects in whom such complications
are not yet evident and those who already have at least one such
response.
[0144] In the former instance, such treatment is useful to prevent
the occurrence of such response and/or reduce the extent to which
they occur. In the latter instance, such treatment is useful to
reduce the extent to which such response occurs, prevent their
further development or reverse the response.
[0145] In certain embodiments, the agent that interferes with the
response cascade may be an antibody specific for such response.
[0146] Expression of Biomarker(s)
[0147] Expression of a marker can be inhibited in a number of ways,
including, by way of a non-limiting example, an antisense
oligonucleotide can be provided to the disease cells in order to
inhibit transcription, translation, or both, of the marker(s).
Alternately, a polynucleotide encoding an antibody, an antibody
derivative, or an antibody fragment which specifically binds a
marker protein, and operably linked with an appropriate
promoter/regulator region, can be provided to the cell in order to
generate intracellular antibodies which will inhibit the function
or activity of the protein. The expression and/or function of a
marker may also be inhibited by treating the disease cell with an
antibody, antibody derivative or antibody fragment that
specifically binds a marker protein. Using the methods described
herein, a variety of molecules, particularly including molecules
sufficiently small that they are able to cross the cell membrane,
can be screened in order to identify molecules which inhibit
expression of a marker or inhibit the function of a marker protein.
The compound so identified can be provided to the subject in order
to inhibit disease cells of the subject.
[0148] Any marker or combination of markers, as well as any certain
markers in combination with the markers, may be used in the
compositions, kits and methods described herein. In general, it is
desirable to use markers for which the difference between the level
of expression of the marker in disease cells and the level of
expression of the same marker in normal system cells is as great as
possible. Although this difference can be as small as the limit of
detection of the method for assessing expression of the marker, it
is desirable that the difference be at least greater than the
standard error of the assessment method, and, in certain
embodiments, a difference of at least 2-, 3-, 4-, 5-, 6-, 7-, 8-,
9-, 10-, 15-, 20-, 100-, 500-, 1000-fold or greater than the level
of expression of the same marker in normal tissue.
[0149] It is recognized that certain marker proteins are secreted
to the extracellular space surrounding the cells. These markers are
used in certain embodiments of the compositions, kits and methods,
owing to the fact that such marker proteins can be detected in a
body fluid sample, which may be more easily collected from a human
subject than a tissue biopsy sample. In addition, in vivo
techniques for detection of a marker protein include introducing
into a subject a labeled antibody directed against the protein. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0150] In order to determine whether any particular marker protein
is a secreted protein, the marker protein is expressed in, for
example, a mammalian cell, such as a human cell line, extracellular
fluid is collected, and the presence or absence of the protein in
the extracellular fluid is assessed (e.g. using a labeled antibody
which binds specifically with the protein).
[0151] It will be appreciated that subject samples containing such
cells may be used in the methods described herein. In these
embodiments, the level of expression of the marker can be assessed
by assessing the amount (e.g., absolute amount or concentration) of
the marker in a sample. The cell sample can, of course, be
subjected to a variety of post-collection preparative and storage
techniques (e.g., nucleic acid and/or protein extraction, fixation,
storage, freezing, ultrafiltration, concentration, evaporation,
centrifugation, etc.) prior to assessing the amount of the marker
in the sample.
[0152] It will also be appreciated that the markers may be shed
from the cells into, for example, the respiratory system, digestive
system, the blood stream and/or interstitial spaces. The shed
markers can be tested, for example, by examining the sputum, BAL,
serum, plasma, urine, stool, etc.
[0153] The compositions, kits and methods can be used to detect
expression of marker proteins having at least one portion which is
displayed on the surface of cells which express it. For example,
immunological methods may be used to detect such proteins on whole
cells, or computer-based sequence analysis methods may be used to
predict the presence of at least one extracellular domain (i.e.,
including both secreted proteins and proteins having at least one
cell-surface domain). Expression of a marker protein having at
least one portion which is displayed on the surface of a cell which
expresses it may be detected without necessarily lysing the cell
(e.g., using a labeled antibody which binds specifically with a
cell-surface domain of the protein).
[0154] Expression of a marker may be assessed by any of a wide
variety of methods for detecting expression of a transcribed
nucleic acid or protein. Non-limiting examples of such methods
include immunological methods for detection of secreted,
cell-surface, cytoplasmic or nuclear proteins, protein purification
methods, protein function or activity assays, nucleic acid
hybridization methods, nucleic acid reverse transcription methods
and nucleic acid amplification methods.
[0155] In a particular embodiment, expression of a marker is
assessed using an antibody (e.g., a radio-labeled,
chromophore-labeled, fluorophore-labeled or enzyme-labeled
antibody), an antibody derivative (e.g., an antibody conjugated
with a substrate or with the protein or ligand of a protein-ligand
pair), or an antibody fragment (e.g., a single-chain antibody, an
isolated antibody hypervariable domain, etc.) which binds
specifically with a marker protein or fragment thereof, including a
marker protein which has undergone all or a portion of its normal
post-translational modification.
[0156] In another particular embodiment, expression of a marker is
assessed by preparing mRNA/cDNA (i.e., a transcribed
polynucleotide) from cells in a subject sample, and by hybridizing
the mRNA/cDNA with a reference polynucleotide which is a complement
of a marker nucleic acid, or a fragment thereof. cDNA can,
optionally, be amplified using any of a variety of polymerase chain
reaction methods prior to hybridization with the reference
polynucleotide; preferably, it is not amplified. Expression of one
or more markers can likewise be detected using quantitative PCR to
assess the level of expression of the marker(s). Alternatively, any
of the many methods of detecting mutations or variants (e.g.,
single nucleotide polymorphisms, deletions, etc.) of a marker may
be used to detect occurrence of a marker in a subject.
[0157] In a related embodiment, a mixture of transcribed
polynucleotides obtained from the sample is contacted with a
substrate having fixed thereto a polynucleotide complementary to or
homologous with at least a portion (e.g., at least 7, 10, 15, 20,
25, 30, 40, 50, 100, 500, or more nucleotide residues) of a marker
nucleic acid. If polynucleotides complementary to or homologous
with are differentially detectable on the substrate (e.g.,
detectable using different chromophores or fluorophores, or fixed
to different selected positions), then the levels of expression of
a plurality of markers can be assessed simultaneously using a
single substrate (e.g., a "gene chip" microarray of polynucleotides
fixed at selected positions). When a method of assessing marker
expression is used which involves hybridization of one nucleic acid
with another, it is desired that the hybridization be performed
under stringent hybridization conditions.
[0158] In certain embodiments, the biomarker assays can be
performed using mass spectrometry or surface plasmon resonance. In
various embodiments, the method of identifying an agent active
against a disorder and/or disease state in a subject can include
one or more of: a) providing a sample of cells containing one or
more markers or derivative thereof; b) preparing an extract from
such cells; c) mixing the extract with a labeled nucleic acid probe
containing a marker binding site; and, d) determining the formation
of a complex between the marker and the nucleic acid probe in the
presence or absence of the test agent. The determining step can
include subjecting said extract/nucleic acid probe mixture to an
electrophoretic mobility shift assay.
[0159] In certain embodiments, the determining step comprises an
assay selected from an enzyme linked immunoabsorption assay
(ELISA), fluorescence based assays and ultra high throughput
assays, for example surface plasmon resonance (SPR) or fluorescence
correlation spectroscopy (FCS) assays. In such embodiments, the SPR
sensor is useful for direct real-time observation of biomolecular
interactions since SPR is sensitive to minute refractive index
changes at a metal-dielectric surface. SPR is a surface technique
that is sensitive to changes of 10.sup.5 to 10.sup.-6 refractive
index (RI) units within approximately 200 nm of the SPR
sensor/sample interface. Thus, SPR spectroscopy is useful for
monitoring the growth of thin organic films deposited on the
sensing layer.
[0160] Because the compositions, kits, and methods rely on
detection of a difference in expression levels of one or more
markers, it is desired that the level of expression of the marker
is significantly greater than the minimum detection limit of the
method used to assess expression in at least one of normal cells
and cancer-affected cells.
[0161] It is understood that by routine screening of additional
subject samples using one or more of the markers, it will be
realized that certain of the markers are over-expressed in cells of
various types, including a specific disorder and/or disease state
in a subject.
[0162] In addition, as a greater number of subject samples are
assessed for expression of the markers and the outcomes of the
individual subjects from whom the samples were obtained are
correlated, it will also be confirmed that altered expression of
certain of the markers are strongly correlated with a disorder
and/or disease state in a subject and that altered expression of
other markers are strongly correlated with other diseases. The
compositions, kits, and methods are thus useful for characterizing
one or more of the stage, grade, histological type, and nature of a
disorder and/or disease state in a subject.
[0163] When the compositions, kits, and methods are used for
characterizing one or more of the stage, grade, histological type,
and nature of a disorder and/or disease state in a subject, it is
desired that the marker or panel of markers is selected such that a
positive result is obtained in at least about 20%, and in certain
embodiments, at least about 40%, 60%, or 80%, and in substantially
all subjects afflicted with a disorder and/or disease state of the
corresponding stage, grade, histological type, or nature. The
marker or panel of markers invention can be selected such that a
positive predictive value of greater than about 10% is obtained for
the general population (in a non-limiting example, coupled with an
assay specificity greater than 80%).
[0164] When a plurality of markers are used in the compositions,
kits, and methods, the level of expression of each marker in a
subject sample can be compared with the normal level of expression
of each of the plurality of markers in non-disorder and/or
non-disease samples of the same type, either in a single reaction
mixture (i.e. using reagents, such as different fluorescent probes,
for each marker) or in individual reaction mixtures corresponding
to one or more of the markers. In one embodiment, a significantly
increased level of expression of more than one of the plurality of
markers in the sample, relative to the corresponding normal levels,
is an indication that the subject is afflicted with a disorder
and/or disease state. When a plurality of markers is used, 2, 3, 4,
5, 8, 10, 12, 15, 20, 30, or 50 or more individual markers can be
used; in certain embodiments, the use of fewer markers may be
desired.
[0165] In order to maximize the sensitivity of the compositions,
kits, and methods (i.e. by interference attributable to cells of
system origin in a subject sample), it is desirable that the marker
used therein be a marker which has a restricted tissue
distribution, e.g., normally not expressed in a non-system
tissue.
[0166] It is recognized that the compositions, kits, and methods
will be of particular utility to subjects having an enhanced risk
of developing a disorder and/or disease state in a subject and
their medical advisors. Subjects recognized as having an enhanced
risk of developing a disorder and/or disease include, for example,
subjects having a familial history of such disorder or disease.
[0167] The level of expression of a marker in normal human system
tissue can be assessed in a variety of ways. In one embodiment,
this normal level of expression is assessed by assessing the level
of expression of the marker in a portion of system cells which
appear to be normal and by comparing this normal level of
expression with the level of expression in a portion of the system
cells which is suspected of being abnormal. Alternately, and
particularly as further information becomes available as a result
of routine performance of the methods described herein,
population-average values for normal expression of the markers may
be used. In other embodiments, the `normal` level of expression of
a marker may be determined by assessing expression of the marker in
a subject sample obtained from a non-afflicted subject, from a
subject sample obtained from a subject before the suspected onset
of a disorder and/or disease state in the subject, from archived
subject samples, and the like.
[0168] There is also provided herein compositions, kits, and
methods for assessing the presence of disorder and/or disease state
cells in a sample (e.g. an archived tissue sample or a sample
obtained from a subject). These compositions, kits, and methods are
substantially the same as those described above, except that, where
necessary, the compositions, kits, and methods are adapted for use
with samples other than subject samples. For example, when the
sample to be used is a parafinized, archived human tissue sample,
it can be necessary to adjust the ratio of compounds in the
compositions, in the kits, or the methods used to assess levels of
marker expression in the sample.
[0169] Kits and Reagents
[0170] The kits are useful for assessing the presence of disease
cells (e.g. in a sample such as a subject sample). The kit
comprises a plurality of reagents, each of which is capable of
binding specifically with a marker nucleic acid or protein.
Suitable reagents for binding with a marker protein include
antibodies, antibody derivatives, antibody fragments, and the like.
Suitable reagents for binding with a marker nucleic acid (e.g. a
genomic DNA, an MRNA, a spliced MRNA, a cDNA, or the like) include
complementary nucleic acids. For example, the nucleic acid reagents
may include oligonucleotides (labeled or non-labeled) fixed to a
substrate, labeled oligonucleotides not bound with a substrate,
pairs of PCR primers, molecular beacon probes, and the like.
[0171] The kits may optionally comprise additional components
useful for performing the methods described herein. By way of
example, the kit may comprise fluids (e.g. SSC buffer) suitable for
annealing complementary nucleic acids or for binding an antibody
with a protein with which it specifically binds, one or more sample
compartments, an instructional material which describes performance
of the method, a sample of normal system cells, a sample of
cancer-related disease cells, and the like.
[0172] Methods of Producing Antibodies
[0173] There is also provided herein a method of making an isolated
hybridoma which produces an antibody useful for assessing whether a
subject is afflicted with a disorder and/or disease state. In this
method, a protein or peptide comprising the entirety or a segment
of a marker protein is synthesized or isolated (e.g. by
purification from a cell in which it is expressed or by
transcription and translation of a nucleic acid encoding the
protein or peptide in vivo or in vitro). A vertebrate, for example,
a mammal such as a mouse, rat, rabbit, or sheep, is immunized using
the protein or peptide. The vertebrate may optionally (and
preferably) be immunized at least one additional time with the
protein or peptide, so that the vertebrate exhibits a robust immune
response to the protein or peptide. Splenocytes are isolated from
the immunized vertebrate and fused with an immortalized cell line
to form hybridomas, using any of a variety of methods. Hybridomas
formed in this manner are then screened using standard methods to
identify one or more hybridomas which produce an antibody which
specifically binds with the marker protein or a fragment thereof.
There is also provided herein hybridomas made by this method and
antibodies made using such hybridomas.
[0174] Methods of Assessing Efficacy
[0175] There is also provided herein a method of assessing the
efficacy of a test compound for inhibiting disease cells. As
described above, differences in the level of expression of the
markers correlate with the abnormal state of the subject's cells.
Although it is recognized that changes in the levels of expression
of certain of the markers likely result from the abnormal state of
such cells, it is likewise recognized that changes in the levels of
expression of other of the markers induce, maintain, and promote
the abnormal state of those cells. Thus, compounds which inhibit a
disorder and/or disease state in a subject will cause the level of
expression of one or more of the markers to change to a level
nearer the normal level of expression for that marker (i.e. the
level of expression for the marker in normal cells).
[0176] This method thus comprises comparing expression of a marker
in a first cell sample and maintained in the presence of the test
compound and expression of the marker in a second cell sample and
maintained in the absence of the test compound. A significantly
reduced expression of a marker in the presence of the test compound
is an indication that the test compound inhibits a related disease.
The cell samples may, for example, be aliquots of a single sample
of normal cells obtained from a subject, pooled samples of normal
cells obtained from a subject, cells of a normal cell line,
aliquots of a single sample of related disease cells obtained from
a subject, pooled samples of related disease cells obtained from a
subject, cells of a related disease cell line, or the like.
[0177] In one embodiment, the samples are cancer-related disease
cells obtained from a subject and a plurality of compounds believed
to be effective for inhibiting various cancer-related diseases are
tested in order to identify the compound which is likely to best
inhibit the cancer-related disease in the subject.
[0178] This method may likewise be used to assess the efficacy of a
therapy for inhibiting a related disease in a subject. In this
method, the level of expression of one or more markers in a pair of
samples (one subjected to the therapy, the other not subjected to
the therapy) is assessed. As with the method of assessing the
efficacy of test compounds, if the therapy induces a significantly
lower level of expression of a marker then the therapy is
efficacious for inhibiting a cancer-related disease. As above, if
samples from a selected subject are used in this method, then
alternative therapies can be assessed in vitro in order to select a
therapy most likely to be efficacious for inhibiting a
cancer-related disease in the subject.
[0179] As described herein, the abnormal state of human cells is
correlated with changes in the levels of expression of the markers.
There is also provided a method for assessing the harmful potential
of a test compound. This method comprises maintaining separate
aliquots of human cells in the presence and absence of the test
compound. Expression of a marker in each of the aliquots is
compared. A significantly higher level of expression of a marker in
the aliquot maintained in the presence of the test compound
(relative to the aliquot maintained in the absence of the test
compound) is an indication that the test compound possesses a
harmful potential. The relative harmful potential of various test
compounds can be assessed by comparing the degree of enhancement or
inhibition of the level of expression of the relevant markers, by
comparing the number of markers for which the level of expression
is enhanced or inhibited, or by comparing both. Various aspects are
described in further detail in the following subsections.
[0180] Isolated Proteins and Antibodies
[0181] One aspect pertains to isolated marker proteins and
biologically active portions thereof, as well as polypeptide
fragments suitable for use as immunogens to raise antibodies
directed against a marker protein or a fragment thereof. In one
embodiment, the native marker protein can be isolated from cells or
tissue sources by an appropriate purification scheme using standard
protein purification techniques. In another embodiment, a protein
or peptide comprising the whole or a segment of the marker protein
is produced by recombinant DNA techniques. Alternative to
recombinant expression, such protein or peptide can be synthesized
chemically using standard peptide synthesis techniques.
[0182] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein").
[0183] When the protein or biologically active portion thereof is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, 10%, or 5% of the volume of the protein preparation. When the
protein is produced by chemical synthesis, it is preferably
substantially free of chemical precursors or other chemicals, i.e.,
it is separated from chemical precursors or other chemicals which
are involved in the synthesis of the protein. Accordingly such
preparations of the protein have less than about 30%, 20%, 10%, 5%
(by dry weight) of chemical precursors or compounds other than the
polypeptide of interest.
[0184] Biologically active portions of a marker protein include
polypeptides comprising amino acid sequences sufficiently identical
to or derived from the amino acid sequence of the marker protein,
which include fewer amino acids than the full length protein, and
exhibit at least one activity of the corresponding full-length
protein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the corresponding
full-length protein. A biologically active portion of a marker
protein can be a polypeptide which is, for example, 10, 25, 50, 100
or more amino acids in length. Moreover, other biologically active
portions, in which other regions of the marker protein are deleted,
can be prepared by recombinant techniques and evaluated for one or
more of the functional activities of the native form of the marker
protein. In certain embodiments, useful proteins are substantially
identical (e.g., at least about 40%, and in certain embodiments,
50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and
retain the functional activity of the corresponding
naturally-occurring marker protein yet differ in amino acid
sequence due to natural allelic variation or mutagenesis.
[0185] In addition, libraries of segments of a marker protein can
be used to generate a variegated population of polypeptides for
screening and subsequent selection of variant marker proteins or
segments thereof.
[0186] Predictive Medicine
[0187] There is also provided herein uses of the animal models and
markers in the field of predictive medicine in which diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring
clinical trials are used for prognostic (predictive) purposes to
thereby treat an individual prophylactically. Accordingly, there is
also provided herein diagnostic assays for determining the level of
expression of one or more marker proteins or nucleic acids, in
order to determine whether an individual is at risk of developing a
particular disorder and/or disease. Such assays can be used for
prognostic or predictive purposes to thereby prophylactically treat
an individual prior to the onset of the disorder and/or
disease.
[0188] In another aspect, the methods are useful for at least
periodic screening of the same individual to see if that individual
has been exposed to chemicals or toxins that change his/her
expression patterns.
[0189] Yet another aspect pertains to monitoring the influence of
agents (e.g., drugs or other compounds) administered either to
inhibit a disorder and/or disease or to treat or prevent any other
disorder (e.g., in order to understand any system effects that such
treatment may have) on the expression or activity of a marker in
clinical trials.
[0190] Pharmaceutical Compositions
[0191] The compounds may be in a formulation for administration
topically, locally or systemically in a suitable pharmaceutical
carrier. Remington's Pharmaceutical Sciences, 15th Edition by E. W.
Martin (Mark Publishing Company, 1975), discloses typical carriers
and methods of preparation. The compound may also be encapsulated
in suitable biocompatible microcapsules, microparticles or
microspheres formed of biodegradable or non-biodegradable polymers
or proteins or liposomes for targeting to cells. Such systems are
well known to those skilled in the art and may be optimized for use
with the appropriate nucleic acid.
[0192] Various methods for nucleic acid delivery are described, for
example in Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York; and Ausubel et
al., 1994, Current Protocols in Molecular Biology, John Wiley &
Sons, New York. Such nucleic acid delivery systems comprise the
desired nucleic acid, by way of example and not by limitation, in
either "naked" form as a "naked" nucleic acid, or formulated in a
vehicle suitable for delivery, such as in a complex with a cationic
molecule or a liposome forming lipid, or as a component of a
vector, or a component of a pharmaceutical composition. The nucleic
acid delivery system can be provided to the cell either directly,
such as by contacting it with the cell, or indirectly, such as
through the action of any biological process.
[0193] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, or thickeners can be used as desired.
[0194] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions, solutions or emulsions that can include suspending
agents, solubilizers, thickening agents, dispersing agents,
stabilizers, and preservatives. Formulations for injection may be
presented in unit dosage form, e.g., in ampules or in multi-dose
containers, with an added preservative. Those of skill in the art
can readily determine the various parameters for preparing and
formulating the compositions without resort to undue
experimentation. The compound can be used alone or in combination
with other suitable components.
[0195] In general, methods of administering compounds, including
nucleic acids, are well known in the art. In particular, the routes
of administration already in use for nucleic acid therapeutics,
along with formulations in current use, provide preferred routes of
administration and formulation for the nucleic acids selected will
depend of course, upon factors such as the particular formulation,
the severity of the state of the subject being treated, and the
dosage required for therapeutic efficacy. As generally used herein,
an "effective amount" is that amount which is able to treat one or
more symptoms of the disorder, reverse the progression of one or
more symptoms of the disorder, halt the progression of one or more
symptoms of the disorder, or prevent the occurrence of one or more
symptoms of the disorder in a subject to whom the formulation is
administered, as compared to a matched subject not receiving the
compound. The actual effective amounts of compound can vary
according to the specific compound or combination thereof being
utilized, the particular composition formulated, the mode of
administration, and the age, weight, condition of the individual,
and severity of the symptoms or condition being treated.
[0196] Any acceptable method known to one of ordinary skill in the
art may be used to administer a formulation to the subject. The
administration may be localized (i.e., to a particular region,
physiological system, tissue, organ, or cell type) or systemic,
depending on the condition being treated.
[0197] Pharmacogenomics
[0198] The markers are also useful as pharmacogenomic markers. As
used herein, a "pharmacogenomic marker" is an objective biochemical
marker whose expression level correlates with a specific clinical
drug response or susceptibility in a subject. The presence or
quantity of the pharmacogenomic marker expression is related to the
predicted response of the subject and more particularly the
subject's tumor to therapy with a specific drug or class of drugs.
By assessing the presence or quantity of the expression of one or
more pharmacogenomic markers in a subject, a drug therapy which is
most appropriate for the subject, or which is predicted to have a
greater degree of success, may be selected.
[0199] Monitoring Clinical Trials
[0200] Monitoring the influence of agents (e.g., drug compounds) on
the level of expression of a marker can be applied not only in
basic drug screening, but also in clinical trials. For example, the
effectiveness of an agent to affect marker expression can be
monitored in clinical trials of subjects receiving treatment for a
cancer-related disease.
[0201] In one non-limiting embodiment, the present invention
provides a method for monitoring the effectiveness of treatment of
a subject with an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or
other drug candidate) comprising the steps of:
[0202] i) obtaining a pre-administration sample from a subject
prior to administration of the agent;
[0203] ii) detecting the level of expression of one or more
selected markers in the pre-administration sample;
[0204] iii) obtaining one or more post-administration samples from
the subject;
[0205] iv) detecting the level of expression of the marker(s) in
the post-administration samples;
[0206] v) comparing the level of expression of the marker(s) in the
pre-administration sample with the level of expression of the
marker(s) in the post-administration sample or samples; and
[0207] vi) altering the administration of the agent to the subject
accordingly.
[0208] For example, increased expression of the marker gene(s)
during the course of treatment may indicate ineffective dosage and
the desirability of increasing the dosage. Conversely, decreased
expression of the marker gene(s) may indicate efficacious treatment
and no need to change dosage.
[0209] Electronic Apparatus Readable Media, Systems, Arrays and
Methods of Using Same
[0210] As used herein, "electronic apparatus readable media" refers
to any suitable medium for storing, holding or containing data or
information that can be read and accessed directly by an electronic
apparatus. Such media can include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc storage medium, and
magnetic tape; optical storage media such as compact disc;
electronic storage media such as RAM, ROM, EPROM, EEPROM and the
like; and general hard disks and hybrids of these categories such
as magnetic/optical storage media. The medium is adapted or
configured for having recorded thereon a marker as described
herein.
[0211] As used herein, the term "electronic apparatus" is intended
to include any suitable computing or processing apparatus or other
device configured or adapted for storing data or information.
Examples of electronic apparatus suitable for use with the present
invention include stand-alone computing apparatus; networks,
including a local area network (LAN), a wide area network (WAN)
Internet, Intranet, and Extranet; electronic appliances such as
personal digital assistants (PDAs), cellular phone, pager and the
like; and local and distributed processing systems.
[0212] As used herein, "recorded" refers to a process for storing
or encoding information on the electronic apparatus readable
medium. Those skilled in the art can readily adopt any method for
recording information on media to generate materials comprising the
markers described herein.
[0213] A variety of software programs and formats can be used to
store the marker information of the present invention on the
electronic apparatus readable medium. Any number of data processor
structuring formats (e.g., text file or database) may be employed
in order to obtain or create a medium having recorded thereon the
markers. By providing the markers in readable form, one can
routinely access the marker sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences in readable form to compare a
target sequence or target structural motif with the sequence
information stored within the data storage means. Search means are
used to identify fragments or regions of the sequences which match
a particular target sequence or target motif.
[0214] Thus, there is also provided herein a medium for holding
instructions for performing a method for determining whether a
subject has a cancer-related disease or a pre-disposition to a
cancer-related disease, wherein the method comprises the steps of
determining the presence or absence of a marker and based on the
presence or absence of the marker, determining whether the subject
has a cancer-related disease or a pre-disposition to a
cancer-related disease and/or recommending a particular treatment
for a cancer-related disease or pre-cancer-related disease
condition.
[0215] There is also provided herein an electronic system and/or in
a network, a method for determining whether a subject has a
cancer-related disease or a pre-disposition to a cancer-related
disease associated with a marker wherein the method comprises the
steps of determining the presence or absence of the marker, and
based on the presence or absence of the marker, determining whether
the subject has a particular disorder and/or disease or a
pre-disposition to such disorder and/or disease, and/or
recommending a particular treatment for such disease or disease
and/or such pre-cancer-related disease condition. The method may
further comprise the step of receiving phenotypic information
associated with the subject and/or acquiring from a network
phenotypic information associated with the subject.
[0216] Also provided herein is a network, a method for determining
whether a subject has a disorder and/or disease or a
pre-disposition to a disorder and/or disease associated with a
marker, the method comprising the steps of receiving information
associated with the marker, receiving phenotypic information
associated with the subject, acquiring information from the network
corresponding to the marker and/or disorder and/or disease, and
based on one or more of the phenotypic information, the marker, and
the acquired information, determining whether the subject has a
disorder and/or disease or a pre-disposition thereto. The method
may further comprise the step of recommending a particular
treatment for the disorder and/or disease or pre-disposition
thereto.
[0217] There is also provided herein a business method for
determining whether a subject has a disorder and/or disease or a
pre-disposition thereto, the method comprising the steps of
receiving information associated with the marker, receiving
phenotypic information associated with the subject, acquiring
information from the network corresponding to the marker and/or a
disorder and/or disease, and based on one or more of the phenotypic
information, the marker, and the acquired information, determining
whether the subject has a disorder and/or disease or a
pre-disposition thereto. The method may further comprise the step
of recommending a particular treatment therefor.
[0218] There is also provided herein an array that can be used to
assay expression of one or more genes in the array. In one
embodiment, the array can be used to assay gene expression in a
tissue to ascertain tissue specificity of genes in the array. In
this manner, up to about 7000 or more genes can be simultaneously
assayed for expression. This allows a profile to be developed
showing a battery of genes specifically expressed in one or more
tissues.
[0219] In addition to such qualitative determination, there is
provided herein the quantitation of gene expression. Thus, not only
tissue specificity, but also the level of expression of a battery
of genes in the tissue is ascertainable. Thus, genes can be grouped
on the basis of their tissue expression per se and level of
expression in that tissue. This is useful, for example, in
ascertaining the relationship of gene expression between or among
tissues. Thus, one tissue can be perturbed and the effect on gene
expression in a second tissue can be determined. In this context,
the effect of one cell type on another cell type in response to a
biological stimulus can be determined.
[0220] Such a determination is useful, for example, to know the
effect of cell-cell interaction at the level of gene expression. If
an agent is administered therapeutically to treat one cell type but
has an undesirable effect on another cell type, the method provides
an assay to determine the molecular basis of the undesirable effect
and thus provides the opportunity to co-administer a counteracting
agent or otherwise treat the undesired effect. Similarly, even
within a single cell type, undesirable biological effects can be
determined at the molecular level. Thus, the effects of an agent on
expression of other than the target gene can be ascertained and
counteracted.
[0221] In another embodiment, the array can be used to monitor the
time course of expression of one or more genes in the array. This
can occur in various biological contexts, as disclosed herein, for
example development of a disorder and/or disease, progression
thereof, and processes, such as cellular transformation associated
therewith.
[0222] The array is also useful for ascertaining the effect of the
expression of a gene or the expression of other genes in the same
cell or in different cells. This provides, for example, for a
selection of alternate molecular targets for therapeutic
intervention if the ultimate or downstream target cannot be
regulated.
[0223] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes that could serve as a
molecular target for diagnosis or therapeutic intervention.
[0224] Surrogate Markers
[0225] The markers may serve as surrogate markers for one or more
disorders or disease states or for conditions leading up thereto.
As used herein, a "surrogate marker" is an objective biochemical
marker which correlates with the absence or presence of a disease
or disorder, or with the progression of a disease or disorder. The
presence or quantity of such markers is independent of the disease.
Therefore, these markers may serve to indicate whether a particular
course of treatment is effective in lessening a disease state or
disorder. Surrogate markers are of particular use when the presence
or extent of a disease state or disorder is difficult to assess
through standard methodologies, or when an assessment of disease
progression is desired before a potentially dangerous clinical
endpoint is reached.
[0226] The markers are also useful as pharmacodynamic markers. As
used herein, a "pharmacodynamic marker" is an objective biochemical
marker which correlates specifically with drug effects. The
presence or quantity of a pharmacodynamic marker is not related to
the disease state or disorder for which the drug is being
administered; therefore, the presence or quantity of the marker is
indicative of the presence or activity of the drug in a subject.
For example, a pharmacodynamic marker may be indicative of the
concentration of the drug in a biological tissue, in that the
marker is either expressed or transcribed or not expressed or
transcribed in that tissue in relationship to the level of the
drug. In this fashion, the distribution or uptake of the drug may
be monitored by the pharmacodynamic marker. Similarly, the presence
or quantity of the pharmacodynamic marker may be related to the
presence or quantity of the metabolic product of a drug, such that
the presence or quantity of the marker is indicative of the
relative breakdown rate of the drug in vivo.
[0227] Pharmacodynamic markers are of particular use in increasing
the sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug may be sufficient to activate multiple rounds of marker
transcription or expression, the amplified marker may be in a
quantity which is more readily detectable than the drug itself.
Also, the marker may be more easily detected due to the nature of
the marker itself; for example, using the methods described herein,
antibodies may be employed in an immune-based detection system for
a protein marker, or marker-specific radiolabeled probes may be
used to detect a mRNA marker. Furthermore, the use of a
pharmacodynamic marker may offer mechanism-based prediction of risk
due to drug treatment beyond the range of possible direct
observations.
[0228] Protocols for Testing
[0229] The method of testing for a disorder and/or disease may
comprise, for example measuring the expression level of each marker
gene in a biological sample from a subject over time and comparing
the level with that of the marker gene in a control biological
sample.
[0230] When the marker gene is one of the genes described herein
and the expression level is differentially expressed (for examples,
higher or lower than that in the control), the subject is judged to
be affected with a disorder and/or disease. When the expression
level of the marker gene falls within the permissible range, the
subject is unlikely to be affected therewith.
[0231] The standard value for the control may be pre-determined by
measuring the expression level of the marker gene in the control,
in order to compare the expression levels. For example, the
standard value can be determined based on the expression level of
the above-mentioned marker gene in the control. For example, in
certain embodiments, the permissible range is taken as .+-.2S.D.
based on the standard value. Once the standard value is determined,
the testing method may be performed by measuring only the
expression level in a biological sample from a subject and
comparing the value with the determined standard value for the
control.
[0232] Expression levels of marker genes include transcription of
the marker genes to mRNA, and translation into proteins. Therefore,
one method of testing for a disorder and/or disease is performed
based on a comparison of the intensity of expression of mRNA
corresponding to the marker genes, or the expression level of
proteins encoded by the marker genes.
[0233] The measurement of the expression levels of marker genes in
the testing for a disorder and/or disease can be carried out
according to various gene analysis methods. Specifically, one can
use, for example, a hybridization technique using nucleic acids
that hybridize to these genes as probes, or a gene amplification
technique using DNA that hybridize to the marker genes as
primers.
[0234] The probes or primers used for the testing can be designed
based on the nucleotide sequences of the marker genes. The
identification numbers for the nucleotide sequences of the
respective marker genes are described herein.
[0235] Further, it is to be understood that genes of higher animals
generally accompany polymorphism in a high frequency. There are
also many molecules that produce isoforms comprising mutually
different amino acid sequences during the splicing process. Any
gene associated with a cancer-related disease that has an activity
similar to that of a marker gene is included in the marker genes,
even if it has nucleotide sequence differences due to polymorphism
or being an isoform.
[0236] It is also to be understood that the marker genes can
include homologs of other species in addition to humans. Thus,
unless otherwise specified, the expression "marker gene" refers to
a homolog of the marker gene unique to the species or a foreign
marker gene which has been introduced into an individual.
[0237] Also, it is to be understood that a "homolog of a marker
gene" refers to a gene derived from a species other than a human,
which can hybridize to the human marker gene as a probe under
stringent conditions. Such stringent conditions are known to one
skilled in the art who can select an appropriate condition to
produce an equal stringency experimentally or empirically.
[0238] A polynucleotide comprising the nucleotide sequence of a
marker gene or a nucleotide sequence that is complementary to the
complementary strand of the nucleotide sequence of a marker gene
and has at least 15 nucleotides, can be used as a primer or probe.
Thus, a "complementary strand" means one strand of a double
stranded DNA with respect to the other strand and which is composed
of A:T (U for RNA) and G:C base pairs.
[0239] In addition, "complementary" means not only those that are
completely complementary to a region of at least 15 continuous
nucleotides, but also those that have a nucleotide sequence
homology of at least 40% in certain instances, 50% in certain
instances, 60% in certain instances, 70% in certain instances, 80%
in certain instances, 90% in certain instances, and 95% in certain
instances, or higher. The degree of homology between nucleotide
sequences can be determined by an algorithm, BLAST, etc.
[0240] Such polynucleotides are useful as a probe to detect a
marker gene, or as a primer to amplify a marker gene. When used as
a primer, the polynucleotide comprises usually 15 bp to 100 bp, and
in certain embodiments 15 bp to 35 bp of nucleotides. When used as
a probe, a DNA comprises the whole nucleotide sequence of the
marker gene (or the complementary strand thereof), or a partial
sequence thereof that has at least 15 bp nucleotides. When used as
a primer, the 3' region must be complementary to the marker gene,
while the 5' region can be linked to a restriction
enzyme-recognition sequence or a tag.
[0241] "Polynucleotides" may be either DNA or RNA. These
polynucleotides may be either synthetic or naturally-occurring.
Also, DNA used as a probe for hybridization is usually labeled.
Those skilled in the art readily understand such labeling methods.
Herein, the term "oligonucleotide" means a polynucleotide with a
relatively low degree of polymerization. Oligonucleotides are
included in polynucleotides.
[0242] Tests for a disorder and/or disease using hybridization
techniques can be performed using, for example, Northern
hybridization, dot blot hybridization, or the DNA microarray
technique. Furthermore, gene amplification techniques, such as the
RT-PCR method may be used. By using the PCR amplification
monitoring method during the gene amplification step in RT-PCR, one
can achieve a more quantitative analysis of the expression of a
marker gene.
[0243] In the PCR gene amplification monitoring method, the
detection target (DNA or reverse transcript of RNA) is hybridized
to probes that are labeled with a fluorescent dye and a quencher
which absorbs the fluorescence. When the PCR proceeds and Taq
polymerase degrades the probe with its 5'-3' exonuclease activity,
the fluorescent dye and the quencher draw away from each other and
the fluorescence is detected. The fluorescence is detected in real
time. By simultaneously measuring a standard sample in which the
copy number of a target is known, it is possible to determine the
copy number of the target in the subject sample with the cycle
number where PCR amplification is linear. Also, one skilled in the
art recognizes that the PCR amplification monitoring method can be
carried out using any suitable method.
[0244] The method of testing for a cancer-related disease can be
also carried out by detecting a protein encoded by a marker gene.
Hereinafter, a protein encoded by a marker gene is described as a
"marker protein." For such test methods, for example, the Western
blotting method, the immunoprecipitation method, and the ELISA
method may be employed using an antibody that binds to each marker
protein.
[0245] Antibodies used in the detection that bind to the marker
protein may be produced by any suitable technique. Also, in order
to detect a marker protein, such an antibody may be appropriately
labeled. Alternatively, instead of labeling the antibody, a
substance that specifically binds to the antibody, for example,
protein A or protein G, may be labeled to detect the marker protein
indirectly. More specifically, such a detection method can include
the ELISA method.
[0246] A protein or a partial peptide thereof used as an antigen
may be obtained, for example, by inserting a marker gene or a
portion thereof into an expression vector, introducing the
construct into an appropriate host cell to produce a transformant,
culturing the transformant to express the recombinant protein, and
purifying the expressed recombinant protein from the culture or the
culture supernatant. Alternatively, the amino acid sequence encoded
by a gene or an oligopeptide comprising a portion of the amino acid
sequence encoded by a full-length cDNA are chemically synthesized
to be used as an immunogen.
[0247] Furthermore, a test for a cancer-related disease can be
performed using as an index not only the expression level of a
marker gene but also the activity of a marker protein in a
biological sample. Activity of a marker protein means the
biological activity intrinsic to the protein. Various methods can
be used for measuring the activity of each protein.
[0248] Even if a subject is not diagnosed as being affected with a
disorder and/or disease in a routine test in spite of symptoms
suggesting these diseases, whether or not such a subject is
suffering from a disorder and/or disease can be easily determined
by performing a test according to the methods described herein.
[0249] More specifically, in certain embodiments, when the marker
gene is one of the genes described herein, an increase or decrease
in the expression level of the marker gene in a subject whose
symptoms suggest at least a susceptibility to a disorder and/or
disease indicates that the symptoms are primarily caused
thereby.
[0250] In addition, the tests are useful to determine whether a
disorder and/or disease are improving in a subject. In other words,
the methods described herein can be used to judge the therapeutic
effect of a treatment therefor. Furthermore, when the marker gene
is one of the genes described herein, an increase or decrease in
the expression level of the marker gene in a subject, who has been
diagnosed as being affected thereby, implies that the disease has
progressed more.
[0251] The severity and/or susceptibility to a disorder and/or
disease may also be determined based on the difference in
expression levels. For example, when the marker gene is one of the
genes described herein, the degree of increase in the expression
level of the marker gene is correlated with the presence and/or
severity of a disorder and/or disease.
[0252] Animal Models
[0253] Animal models for a disorder and/or disease where the
expression level of one or more marker genes or a gene functionally
equivalent to the marker gene has been elevated in the animal model
can also be made. A "functionally equivalent gene" as used herein
generally is a gene that encodes a protein having an activity
similar to a known activity of a protein encoded by the marker
gene. A representative example of a functionally equivalent gene
includes a counterpart of a marker gene of a subject animal, which
is intrinsic to the animal.
[0254] The animal model is useful for detecting physiological
changes due to a disorder and/or disease. In certain embodiments,
the animal model is useful to reveal additional functions of marker
genes and to evaluate drugs whose targets are the marker genes.
[0255] An animal model can be created by controlling the expression
level of a counterpart gene or administering a counterpart gene.
The method can include creating an animal model by controlling the
expression level of a gene selected from the group of genes
described herein. In another embodiment, the method can include
creating an animal model by administering the protein encoded by a
gene described herein, or administering an antibody against the
protein. It is to be also understood, that in certain other
embodiments, the marker can be over-expressed such that the marker
can then be measured using appropriate methods. In another
embodiment, an animal model can be created by introducing a gene
selected from such groups of genes, or by administering a protein
encoded by such a gene. In another embodiment, a disorder and/or
disease can be induced by suppressing the expression of a gene
selected from such groups of genes or the activity of a protein
encoded by such a gene. An antisense nucleic acid, a ribozyme, or
an RNAi can be used to suppress the expression. The activity of a
protein can be controlled effectively by administering a substance
that inhibits the activity, such as an antibody.
[0256] The animal model is useful to elucidate the mechanism
underlying a disorder and/or disease and also to test the safety of
compounds obtained by screening. For example, when an animal model
develops the symptoms of a particular disorder and/or disease, or
when a measured value involved in certain a disorder and/or disease
alters in the animal, a screening system can be constructed to
explore compounds having activity to alleviate the disease.
[0257] As used herein, the expression "an increase in the
expression level" refers to any one of the following: where a
marker gene introduced as a foreign gene is expressed artificially;
where the transcription of a marker gene intrinsic to the subject
animal and the translation thereof into the protein are enhanced;
or where the hydrolysis of the protein, which is the translation
product, is suppressed.
[0258] As used herein, the expression "a decrease in the expression
level" refers to either the state in which the transcription of a
marker gene of the subject animal and the translation thereof into
the protein are inhibited, or the state in which the hydrolysis of
the protein, which is the translation product, is enhanced. The
expression level of a gene can be determined, for example, by a
difference in signal intensity on a DNA chip. Furthermore, the
activity of the translation product--the protein--can be determined
by comparing with that in the normal state.
[0259] It is also within the contemplated scope that the animal
model can include transgenic animals, including, for example
animals where a marker gene has been introduced and expressed
artificially; marker gene knockout animals; and knock-in animals in
which another gene has been substituted for a marker gene. A
transgenic animal, into which an antisense nucleic acid of a marker
gene, a ribozyme, a polynucleotide having an RNAi effect, or a DNA
functioning as a decoy nucleic acid or such has been introduced,
can be used as the transgenic animal. Such transgenic animals also
include, for example, animals in which the activity of a marker
protein has been enhanced or suppressed by introducing a
mutation(s) into the coding region of the gene, or the amino acid
sequence has been modified to become resistant or susceptible to
hydrolysis. Mutations in an amino acid sequence include
substitutions, deletions, insertions, and additions.
[0260] Examples of Expression
[0261] In addition, the expression itself of a marker gene can be
controlled by introducing a mutation(s) into the transcriptional
regulatory region of the gene. Those skilled in the art understand
such amino acid substitutions. Also, the number of amino acids that
are mutated is not particularly restricted, as long as the activity
is maintained. Normally, it is within 50 amino acids, in certain
non-limiting embodiments, within 30 amino acids, within 10 amino
acids, or within 3 amino acids. The site of mutation may be any
site, as long as the activity is maintained.
[0262] In yet another aspect, there is provided herein screening
methods for candidate compounds for therapeutic agents to treat a
particular disorder and/or disease. One or more marker genes are
selected from the group of genes described herein. A therapeutic
agent for a cancer-related disease can be obtained by selecting a
compound capable of increasing or decreasing the expression level
of the marker gene(s).
[0263] It is to be understood that the expression "a compound that
increases the expression level of a gene" refers to a compound that
promotes any one of the steps of gene transcription, gene
translation, or expression of a protein activity. On the other
hand, the expression "a compound that decreases the expression
level of a gene", as used herein, refers to a compound that
inhibits any one of these steps.
[0264] In particular aspects, the method of screening for a
therapeutic agent for a disorder and/or disease can be carried out
either in vivo or in vitro. This screening method can be performed,
for example, by:
[0265] 1) administering a candidate compound to an animal
subject;
[0266] 2) measuring the expression level of a marker gene(s) in a
biological sample from the animal subject; or
[0267] 3) selecting a compound that increases or decreases the
expression level of a marker gene(s) as compared to that in a
control with which the candidate compound has not been
contacted.
[0268] In still another aspect, there is provided herein a method
to assess the efficacy of a candidate compound for a pharmaceutical
agent on the expression level of a marker gene(s) by contacting an
animal subject with the candidate compound and monitoring the
effect of the compound on the expression level of the marker
gene(s) in a biological sample derived from the animal subject. The
variation in the expression level of the marker gene(s) in a
biological sample derived from the animal subject can be monitored
using the same technique as used in the testing method described
above. Furthermore, based on the evaluation, a candidate compound
for a pharmaceutical agent can be selected by screening.
[0269] All patents, patent applications and references cited herein
are incorporated in their entirety by reference. While the
invention has been described and exemplified in sufficient detail
for those skilled in this art to make and use it, various
alternatives, modifications and improvements should be apparent
without departing from the spirit and scope of the invention. One
skilled in the art readily appreciates that the present invention
is well adapted to carry out the objects and obtain the ends and
advantages mentioned, as well as those inherent therein.
[0270] Certain Nucleobase Sequences
[0271] Nucleobase sequences of mature miRNAs and their
corresponding stem-loop sequences described herein are the
sequences found in miRBase, an online searchable database of miRNA
sequences and annotation, found at http://microrna.sanger.ac.uk/.
Entries in the miRBase Sequence database represent a predicted
hairpin portion of a miRNA transcript (the stem-loop), with
information on the location and sequence of the mature miRNA
sequence. The miRNA stem-loop sequences in the database are not
strictly precursor miRNAs (pre-miRNAs), and may in some instances
include the pre-miRNA and some flanking sequence from the presumed
primary transcript. The miRNA nucleobase sequences described herein
encompass any version of the miRNA, including the sequences
described in Release 10.0 of the miRBase sequence database and
sequences described in any earlier Release of the miRBase sequence
database. A sequence database release may result in the re-naming
of certain miRNAs. A sequence database release may result in a
variation of a mature miRNA sequence. The compounds that may
encompass such modified oligonucleotides may be complementary to
any nucleobase sequence version of the miRNAs described herein.
[0272] It is understood that any nucleobase sequence set forth
herein is independent of any modification to a sugar moiety, an
internucleoside linkage, or a nucleobase. It is further understood
that a nucleobase sequence comprising U's also encompasses the same
nucleobase sequence wherein `U` is replaced by `T` at one or more
positions having `U`. Conversely, it is understood that a
nucleobase sequence comprising T's also encompasses the same
nucleobase sequence wherein `T` is replaced by `U` at one or more
positions having `T`.
[0273] In certain embodiments, a modified oligonucleotide has a
nucleobase sequence that is complementary to a miRNA or a precursor
thereof, meaning that the nucleobase sequence of a modified
oligonucleotide is a least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 98% or 99% identical to the complement of a miRNA or precursor
thereof over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100 or more nucleobases, or that the two sequences
hybridize under stringent hybridization conditions. Accordingly, in
certain embodiments the nucleobase sequence of a modified
oligonucleotide may have one or more mismatched basepairs with
respect to its target miRNA or target miRNA precursor sequence, and
is capable of hybridizing to its target sequence. In certain
embodiments, a modified oligonucleotide has a nucleobase sequence
that is 100% complementary to a miRNA or a precursor thereof. In
certain embodiments, the nucleobase sequence of a modified
oligonucleotide has full-length complementary to a miRNA.
[0274] miRNA (miR) Therapies
[0275] In some embodiments, the present invention provides
microRNAs that inhibit the expression of one or more genes in a
subject. MicroRNA expression profiles can serve as a new class of
cancer biomarkers.
[0276] Included herein are methods of inhibiting gene expression
and/or activity using one or more MiRs. In some embodiments, the
miR(s) inhibit the expression of a protein. In other embodiments,
the miRNA(s) inhibits gene activity (e.g., cell invasion
activity).
[0277] The miRNA can be isolated from cells or tissues,
recombinantly produced, or synthesized in vitro by a variety of
techniques well known to one of ordinary skill in the art. In one
embodiment, miRNA is isolated from cells or tissues. Techniques for
isolating miRNA from cells or tissues are well known to one of
ordinary skill in the art. For example, miRNA can be isolated from
total RNA using the mirVana miRNA isolation kit from Ambion, Inc.
Another technique utilizes the flashIPAGE.TM. Fractionator System
(Ambion, Inc.) for PAGE purification of small nucleic acids.
[0278] For the use of miRNA therapeutics, it is understood by one
of ordinary skill in the art that nucleic acids administered in
vivo are taken up and distributed to cells and tissues.
[0279] The nucleic acid may be delivered in a suitable manner which
enables tissue-specific uptake of the agent and/or nucleic acid
delivery system. The formulations described herein can supplement
treatment conditions by any known conventional therapy, including,
but not limited to, antibody administration, vaccine
administration, administration of cytotoxic agents, natural amino
acid polypeptides, nucleic acids, nucleotide analogues, and
biologic response modifiers. Two or more combined compounds may be
used together or sequentially.
[0280] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more nucleic acid or small
molecule compounds and (b) one or more other chemotherapeutic
agents.
[0281] Additional Useful Definitions
[0282] "Subject" means a human or non-human animal selected for
treatment or therapy. "Subject suspected of having" means a subject
exhibiting one or more clinical indicators of a disorder, disease
or condition.
[0283] "Preventing" or "prevention" refers to delaying or
forestalling the onset, development or progression of a condition
or disease for a period of time, including weeks, months, or years.
"Treatment" or "treat" means the application of one or more
specific procedures used for the cure or amelioration of a disorder
and/or disease. In certain embodiments, the specific procedure is
the administration of one or more pharmaceutical agents.
[0284] "Amelioration" means a lessening of severity of at least one
indicator of a condition or disease. In certain embodiments,
amelioration includes a delay or slowing in the progression of one
or more indicators of a condition or disease. The severity of
indicators may be determined by subjective or objective measures
which are known to those skilled in the art.
[0285] "Subject in need thereof" means a subject identified as in
need of a therapy or treatment.
[0286] "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.
[0287] "Parenteral administration" means administration through
injection or infusion. Parenteral administration includes, but is
not limited to, subcutaneous administration, intravenous
administration, intramuscular administration, intra-arterial
administration, and intracranial administration. "Subcutaneous
administration" means administration just below the skin.
[0288] "Improves function" means the changes function toward normal
parameters. In certain embodiments, function is assessed by
measuring molecules found in a subject's bodily fluids.
Pharmaceutical composition" means a mixture of substances suitable
for administering to an individual that includes a pharmaceutical
agent. For example, a pharmaceutical composition may comprise a
modified oligonucleotide and a sterile aqueous solution.
[0289] "Target nucleic acid," "target RNA," "target RNA transcript"
and "nucleic acid target" all mean a nucleic acid capable of being
targeted by antisense compounds. Targeting" means the process of
design and selection of nucleobase sequence that will hybridize to
a target nucleic acid and induce a desired effect. "Targeted to"
means having a nucleobase sequence that will allow hybridization to
a target nucleic acid to induce a desired effect. In certain
embodiments, a desired effect is reduction of a target nucleic
acid.
[0290] "Modulation" means to a perturbation of function or
activity. In certain embodiments, modulation means an increase in
gene expression. In certain embodiments, modulation means a
decrease in gene expression.
[0291] "Expression" means any functions and steps by which a gene's
coded information is converted into structures present and
operating in a cell.
[0292] "Region" means a portion of linked nucleosides within a
nucleic acid. In certain embodiments, a modified oligonucleotide
has a nucleobase sequence that is complementary to a region of a
target nucleic acid. For example, in certain such embodiments a
modified oligonucleotide is complementary to a region of a miRNA
stem-loop sequence. In certain such embodiments, a modified
oligonucleotide is 100% identical to a region of a miRNA
sequence.
[0293] "Segment" means a smaller or sub-portion of a region.
[0294] "Nucleobase sequence" means the order of contiguous
nucleobases, in a 5' to 3' orientation, independent of any sugar,
linkage, and/or nucleobase modification.
[0295] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other in a nucleic acid.
[0296] "Nucleobase complementarity" means the ability of two
nucleobases to pair non-covalently via hydrogen bonding.
"Complementary" means a first nucleobase sequence is at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical, or is
100% identical, to the complement of a second nucleobase sequence
over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100 or more nucleobases, or that the two sequences
hybridize under stringent hybridization conditions. In certain
embodiments a modified oligonucleotide that has a nucleobase
sequence which is 100% complementary to a miRNA, or precursor
thereof, may not be 100% complementary to the miRNA, or precursor
thereof, over the entire length of the modified
oligonucleotide.
[0297] "Complementarity" means the nucleobase pairing ability
between a first nucleic acid and a second nucleic acid.
"Full-length complementarity" means each nucleobase of a first
nucleic acid is capable of pairing with each nucleobase at a
corresponding position in a second nucleic acid. For example, in
certain embodiments, a modified oligonucleotide wherein each
nucleobase has complementarity to a nucleobase in an miRNA has
full-length complementarity to the miRNA.
[0298] "Percent complementary" means the number of complementary
nucleobases in a nucleic acid divided by the length of the nucleic
acid. In certain embodiments, percent complementarity of a modified
oligonucleotide means the number of nucleobases that are
complementary to the target nucleic acid, divided by the number of
nucleobases of the modified oligonucleotide. In certain
embodiments, percent complementarity of a modified oligonucleotide
means the number of nucleobases that are complementary to a miRNA,
divided by the number of nucleobases of the modified
oligonucleotide.
[0299] "Percent region bound" means the percent of a region
complementary to an oligonucleotide region. Percent region bound is
calculated by dividing the number of nucleobases of the target
region that are complementary to the oligonucleotide by the length
of the target region. In certain embodiments, percent region bound
is at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%.
[0300] "Percent identity" means the number of nucleobases in first
nucleic acid that are identical to nucleobases at corresponding
positions in a second nucleic acid, divided by the total number of
nucleobases in the first nucleic acid.
[0301] "Substantially identical" used herein may mean that a first
and second nucleobase sequence are at least 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 97%, 98% or 99% identical, or 100% identical,
over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100 or more nucleobases.
[0302] "Hybridize" means the annealing of complementary nucleic
acids that occurs through nucleobase complementarity.
[0303] "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.
[0304] "Non-complementary nucleobase" means two nucleobases that
are not capable of pairing through hydrogen bonding.
[0305] "Identical" means having the same nucleobase sequence.
[0306] "miRNA" or "miR" means a non-coding RNA between 18 and 25
nucleobases in length which hybridizes to and regulates the
expression of a coding RNA. In certain embodiments, a miRNA is the
product of cleavage of a pre-miRNA by the enzyme Dicer. Examples of
miRNAs are found in the miRNA database known as miRBase
(http://microrna.sanger.ac.uk/).
[0307] "Pre-miRNA" or "pre-miR" means a non-coding RNA having a
hairpin structure, which contains a miRNA. In certain embodiments,
a pre-miRNA is the product of cleavage of a pri-miR by the
double-stranded RNA-specific ribonuclease known as Drosha.
[0308] "Stem-loop sequence" means an RNA having a hairpin structure
and containing a mature miRNA sequence. Pre-miRNA sequences and
stem-loop sequences may overlap. Examples of stem-loop sequences
are found in the miRNA database known as miRBase
(microrna.sanger.ac.uk/.
[0309] "miRNA precursor" means a transcript that originates from a
genomic DNA and that comprises a non-coding, structured RNA
comprising one or more miRNA sequences. For example, in certain
embodiments a miRNA precursor is a pre-miRNA. In certain
embodiments, a miRNA precursor is a pri-miRNA.
[0310] "Antisense compound" means a compound having a nucleobase
sequence that will allow hybridization to a target nucleic acid. In
certain embodiments, an antisense compound is an oligonucleotide
having a nucleobase sequence complementary to a target nucleic
acid.
[0311] "Oligonucleotide" means a polymer of linked nucleosides,
each of which can be modified or unmodified, independent from one
another. "Naturally occurring internucleoside linkage" means a 3'
to 5' phosphodiester linkage between nucleosides. "Natural
nucleobase" means a nucleobase that is unmodified relative to its
naturally occurring form. "miR antagonist"+means an agent designed
to interfere with or inhibit the activity of a miRNA. In certain
embodiments, a miR antagonist comprises an antisense compound
targeted to a miRNA. In certain embodiments, a miR antagonist
comprises a modified oligonucleotide having a nucleobase sequence
that is complementary to the nucleobase sequence of a miRNA, or a
precursor thereof. In certain embodiments, an miR antagonist
comprises a small molecule, or the like that interferes with or
inhibits the activity of an miRNA.
[0312] The methods and reagents described herein are representative
of preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Modifications therein
and other uses will occur to those skilled in the art. These
modifications are encompassed within the spirit of the invention
and are defined by the scope of the claims. It will also be readily
apparent to a person skilled in the art that varying substitutions
and modifications may be made to the invention disclosed herein
without departing from the scope and spirit of the invention.
[0313] It should be understood that although the present invention
has been specifically disclosed by preferred embodiments and
optional features, modifications and variations of the concepts
herein disclosed may be resorted to by those skilled in the art,
and that such modifications and variations are considered to be
within the scope of this invention as defined by the appended
claims.
[0314] While the invention has been described with reference to
various and preferred embodiments, it should be understood by those
skilled in the art that various changes may be made and equivalents
may be substituted for elements thereof without departing from the
essential scope of the invention. In addition, many modifications
may be made to adapt a particular situation or material to the
teachings of the invention without departing from the essential
scope thereof.
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[0316] Citation of the any of the documents recited herein is not
intended as an admission that any of the foregoing is pertinent
prior art. All statements as to the date or representation as to
the contents of these documents is based on the information
available to the applicant and does not constitute any admission as
to the correctness of the dates or contents of these documents.
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