U.S. patent application number 14/104886 was filed with the patent office on 2014-06-26 for dual targeting of mir-208 and mir-499 in the treatment of cardiac disorders.
This patent application is currently assigned to THE BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. The applicant listed for this patent is THE BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Eric N. OLSON, Eva van Rooij.
Application Number | 20140179764 14/104886 |
Document ID | / |
Family ID | 42542383 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140179764 |
Kind Code |
A1 |
OLSON; Eric N. ; et
al. |
June 26, 2014 |
DUAL TARGETING OF MIR-208 AND MIR-499 IN THE TREATMENT OF CARDIAC
DISORDERS
Abstract
The present invention provides a method of treating or
preventing cardiac disorders in a subject in need thereof by
inhibiting the expression or function of both miR-499 and miR-208
in the heart cells of the subject. In particular, specific
protocols for administering inhibitors of the two miRNAs that
achieve efficient, long-term suppression are disclosed. In
addition, the invention provides a method for treating or
preventing musculoskeletal disorders in a subject in need thereof
by increasing the expression or activity of both miR-208 and
miR-499 in skeletal muscle cells of the subject.
Inventors: |
OLSON; Eric N.; (Dallas,
TX) ; van Rooij; Eva; (Utrecht, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM |
Austin |
TX |
US |
|
|
Assignee: |
THE BOARD OF REGENTS, THE
UNIVERSITY OF TEXAS SYSTEM
Austin
TX
|
Family ID: |
42542383 |
Appl. No.: |
14/104886 |
Filed: |
December 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13147784 |
Oct 18, 2011 |
8629119 |
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PCT/US10/23234 |
Feb 4, 2010 |
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14104886 |
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61149915 |
Feb 4, 2009 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61P 9/04 20180101; C12N
15/113 20130101; A61P 21/00 20180101; C12N 2310/141 20130101; C12N
2320/31 20130101; C12N 2320/50 20130101; C12N 2330/51 20130101;
A61P 9/00 20180101; A61P 9/10 20180101; C12N 2310/113 20130101 |
Class at
Publication: |
514/44.A |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Number HL53351-06 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-25. (canceled)
26. A method for treating pathologic cardiac hypertrophy,
myocardial infarction, or heart failure in a subject in need
thereof comprising administering to the subject a nucleic acid
molecule comprising a first miRNA targeting sequence and a second
miRNA targeting sequence, wherein the first miRNA targeting
sequence is at least partially complementary to a mature miR-208a
or miR-208b sequence and the second miRNA targeting sequence is at
least partially complementary to a mature miR-499 sequence, and
wherein the expression or activity of miR-208a or miR-208b and
miR-499 is reduced in the heart cells of the subject following
administration of the nucleic acid.
27. The method of claim 26, wherein the nucleic acid is expressed
from a vector delivered to the heart cells of the subject.
28. The method of claim 26, wherein the first and second miRNA
targeting sequences are separated by one or more spacer
nucleotides.
29. The method of claim 28, wherein the first and second miRNA
targeting sequences are separated by about 10 to about 50 spacer
nucleotides.
30. The method of claim 28, wherein the first and second miRNA
targeting sequences are separated by about 5 spacer
nucleotides.
31. The method of claim 26, wherein the mature miR-208a sequence is
SEQ ID NO: 5.
32. The method of claim 31, wherein the first miRNA targeting
sequence is at least 80% complementary to SEQ ID NO: 5.
33. The method of claim 31, wherein the first miRNA targeting
sequence is at least 90% complementary to SEQ ID NO: 5.
34. The method of claim 31, wherein the first miRNA targeting
sequence is fully complementary to SEQ ID NO: 5.
35. The method of claim 26, wherein the mature miR-208b sequence is
SEQ ID NO: 19.
36. The method of claim 35, wherein the first miRNA targeting
sequence is at least 80% complementary to SEQ ID NO: 19.
37. The method of claim 35, wherein the first miRNA targeting
sequence is at least 90% complementary to SEQ ID NO: 19.
38. The method of claim 35, wherein the first miRNA targeting
sequence is fully complementary to SEQ ID NO: 19.
39. The method of claim 26, wherein the mature miR-499 sequence is
SEQ ID NO: 14.
40. The method of claim 39, wherein the second miRNA targeting
sequence is at least 80% complementary to SEQ ID NO: 14.
41. The method of claim 39, wherein the second miRNA targeting
sequence is at least 90% complementary to SEQ ID NO: 14.
42. The method of claim 39, wherein the second miRNA targeting
sequence is fully complementary to SEQ ID NO: 14.
43. The method of claim 26, wherein the nucleic acid comprises at
least one sugar and/or backbone modification.
44. The method of claim 43, wherein said at least one sugar
modification is a bicyclic sugar nucleoside modification,
2'-O-alkyl modification, or 2'-fluoro modification.
45. The method of claim 44, wherein the bicyclic sugar nucleoside
modification is a locked nucleic acid.
46. The method of claim 43, wherein said at least one backbone
modification is a phosphorothioate linkage.
47. The method of claim 26, wherein the first miRNA targeting
sequence and/or the second miRNA targeting sequence are about 8 to
about 18 nucleotides in length.
48. The method of claim 26, wherein the first miRNA targeting
sequence and/or the second miRNA targeting sequence are about 12 to
about 16 nucleotides in length.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/149,915, filed Feb. 4, 2009, which is herein
incorporated by reference in its entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0003] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
MIRG.sub.--013.sub.--01WO_SeqList_ST25.txt, date recorded: Feb. 1,
2010, file size 5 kilobytes).
FIELD OF THE INVENTION
[0004] The present invention relates to the treatment of cardiac
and musculoskeletal disorders by administering agents that modulate
the activity or expression of microRNAs (miRNAs). In particular,
the invention provides a method for treating or preventing cardiac
disorders by inhibiting the expression or activity of both
miR-208a/miR-208b and miR-499 in the heart cells of a subject,
including humans. In addition, the invention provides a method for
treating or preventing musculoskeletal disorders by increasing the
expression or activity of both miR-208b and miR-499 in skeletal
muscle cells of a subject.
BACKGROUND OF THE INVENTION
[0005] Heart disease and its manifestations, including coronary
artery disease, myocardial infarction, congestive heart failure and
cardiac hypertrophy, clearly present a major health risk in the
United States today. The cost to diagnose, treat and support
patients suffering from these diseases is well into the billions of
dollars. Two particularly severe manifestations of heart disease
are myocardial infarction and cardiac hypertrophy.
[0006] Myocardial infarction, commonly known as a heart attack, is
caused by a sudden and sustained lack of blood flow to the heart
tissue, which is usually the result of a narrowing or occlusion of
a coronary artery. Without adequate blood supply, the tissue
becomes ischemic, leading to the death of cardiomyocytes (e.g.
heart muscle cells) and vascular structures. The necrotic tissue
resulting from the death of the cardiomyocytes is generally
replaced by scar tissue, which is not contractile, fails to
contribute to cardiac function, and often plays a detrimental role
in heart function by expanding during cardiac contraction, or by
increasing the size and effective radius of the ventricle, for
example, becoming hypertrophic.
[0007] Cardiac hypertrophy is an adaptive response of the heart to
virtually all forms of cardiac disease, including those arising
from hypertension, mechanical load, myocardial infarction, cardiac
arrhythmias, endocrine disorders, and genetic mutations in cardiac
contractile protein genes. While the hypertrophic response is
initially a compensatory mechanism that augments cardiac output,
sustained hypertrophy can lead to dilated cardiomyopathy (DCM),
heart failure, and sudden death. In the United States,
approximately half a million individuals are diagnosed with heart
failure each year, with a mortality rate approaching 50%.
[0008] Numerous signaling pathways, especially those involving
aberrant calcium signaling, drive cardiac hypertrophy and
pathological remodeling (Heineke & Molkentin, 2006).
Hypertrophic growth in response to stress involves different
signaling pathways and gene expression patterns than physiological
hypertrophy, which occurs in response to exercise. Stress-mediated
myocardial hypertrophy is a complex phenomenon associated with
numerous adverse consequences with distinct molecular and
histological characteristics causing the heart to fibrose, dilate
and decompensate which, through cardiomyocyte degeneration and
death, often culminates in heart failure. As such, there has been
intense interest in deciphering the underlying molecular mechanisms
and in discovering novel therapeutic targets for suppressing
adverse cardiac growth and ultimately failure. Understanding these
mechanisms is essential to the design of new therapies to treat
cardiac hypertrophy and heart failure.
[0009] MicroRNAs have recently been implicated in a number of
biological processes including regulation of developmental timing,
apoptosis, fat metabolism, and hematopoietic cell differentiation
among others. MicroRNAs (miRNAs) are small, non-protein coding RNAs
of about 18 to about 25 nucleotides in length that are derived from
individual miRNA genes, from introns of protein coding genes, or
from poly-cistronic transcripts that often encode multiple, closely
related miRNAs. See review by Carrington et al. (Science, Vol.
301(5631):336-338, 2003). MiRNAs act as repressors of target mRNAs
by promoting their degradation, when their sequences are perfectly
complementary, or by inhibiting translation, when their sequences
contain mismatches.
[0010] MiRNAs are transcribed by RNA polymerase II (pol II) or RNA
polymerase III (pol III; see Qi et al. (2006) Cellular &
Molecular Immunology, Vol. 3:411-419) and arise from initial
transcripts, termed primary miRNA transcripts (pri-miRNAs), that
are generally several thousand bases long. Pri-miRNAs are processed
in the nucleus by the RNase Drosha into about 70- to about
100-nucleotide hairpin-shaped precursors (pre-miRNAs). Following
transport to the cytoplasm, the hairpin pre-miRNA is further
processed by Dicer to produce a double-stranded miRNA. The mature
miRNA strand is then incorporated into the RNA-induced silencing
complex (RISC), where it associates with its target mRNAs by
base-pair complementarity. In the relatively rare cases in which a
miRNA base pairs perfectly with an mRNA target, it promotes mRNA
degradation. More commonly, miRNAs form imperfect heteroduplexes
with target mRNAs, affecting either mRNA stability or inhibiting
mRNA translation.
[0011] Recently, the inventors reported a cardiac-specific
microRNA, miR-208a, which is encoded by an intron of the
.alpha.-myosin heavy chain (MHC) gene, and is required for
up-regulation of .beta.-MHC expression in response to cardiac
stress and for repression of fast skeletal muscle genes in the
heart (van Rooij et al., (2007) Science, Vol. 316: 575-579). The
present invention expands on this discovery and provides a novel
therapeutic approach to the treatment of cardiac and
musculoskeletal disorders.
SUMMARY OF THE INVENTION
[0012] The present invention is based, in part, on the discovery
that systematic downregulation of both miR-208a and miR-499 in
heart cells produces a synergistic effect on the development of
cardiac hypertrophy, enhanced contractility, and pathological
cardiac remodeling in response to stress. The inventors have
surprisingly found that the time period for regulating expression
of stress-related genes, such as .beta.-MHC, is dramatically
decreased by downregulating both miR-208a and miR-499 by either
simultaneous or sequential administration of miR-208a and miR-499
inhibitors. Such dual targeting produces immediate effects on
stress-related gene expression as compared to the several month
delay required to observe similar effects with downregulation of
miR-208a alone. Accordingly, the present invention provides a novel
therapeutic approach for the treatment of pathologic cardiac
hypertrophy, heart failure, and myocardial infarction in a subject
in need thereof, including a human.
[0013] In one embodiment, the method comprises administering an
inhibitor of miR-208a or miR-208b and an inhibitor of miR-499 to a
subject, wherein the expression or activity of miR-208a or miR-208b
and miR-499 is reduced in the heart cells of the subject following
administration. In some embodiments, the expression or activity of
miR-208a or miR-208b and miR-499 is reduced by greater than 60
percent in the heart cells of the subject following administration
of the inhibitors. The miR-208 and miR-499 inhibitors include
antagomirs or antisense oligonucleotides. In one embodiment, the
miRNA inhibitors are encoded on a expression vector.
[0014] In another embodiment, the cardiac stress response is
reduced in the subject following administration of an inhibitor of
miR-208a or miR-208b and an inhibitor of miR-499. The cardiac
stress response includes hypertrophy of cardiomyocytes, fibrosis of
the heart, reduced expression of .alpha.-MHC, and/or increased
expression of .beta.-MHC in the heart cells of said subject. In
certain embodiments, the reduction of the cardiac stress response
occurs less than two months after administration of the
miR-208a/miR-208b and miR-499 inhibitors. In a preferred
embodiment, the reduction of the cardiac stress response occurs
less than one week after administration of the inhibitors.
[0015] In some embodiments, the miR-208a/miR-208b inhibitor and the
miR-499 inhibitor are administered sequentially. Administration of
the two inhibitors can be separated by an interval that can be on
the order of minutes to weeks. In one embodiment, the
miR-208a/miR-208b inhibitor and the miR-499 inhibitor are
administered at least 24 hours apart. In another embodiment, the
miR-208a/miR-208b inhibitor and the miR-499 inhibitor are
co-administered. The two inhibitors can be administered each at a
dosage of about 1 mg/kg to about 200 mg/kg.
[0016] The present invention also provides a method of treating or
preventing a musculoskeletal disorder in a subject in need thereof
comprising administering an agonist of miR-208 and an agonist of
miR-499 to the subject, wherein the expression or activity of
miR-208 and miR-499 is increased in the skeletal muscle cells of
the subject following administration. In one embodiment, the method
comprises administering an agonist of miR-208b and an agonist of
miR-499 to the subject. The miRNA agonists can be polynucleotides
encoding mature miR-208a, miR-208b, or miR-499 sequences. In some
embodiments, such polynucleotides are operably linked to a promoter
sequence and provided to the subject's cells in an expression
vector.
[0017] The miRNA agonists may be co-administered or administered
sequentially separated by a particular time interval. In some
embodiments, the expression of one or more fast skeletal muscle
genes in the skeletal muscle cells of a subject is reduced
following administration of the miR-499 and miR-208a or miR-208b
agonists to the subject. One or more fast skeletal muscle genes can
include troponin I2, troponin T3, fast skeletal myosin light chain,
and alpha skeletal actin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. MiR-208 Knockout Animals Exhibit Less Cardiac
Hypertrophy and Fibrosis in Response to Thoracic Aortic
Banding.
[0019] A. Schematic illustrating the thoracic aortic banding (TAB)
procedure (left). Histological sections of hearts of wild-type and
miR-208-/- mice stained for Masson trichrome after TAB procedure or
sham procedure (right). The absence of miR-208 diminishes
hypertrophy and fibrosis seen in wild-type mice subjected to TAB
for 21 days. B. Relative expression levels for beta-myosin heavy
chain (.beta.-MHC), atrial natriuretic factor (ANF), and brain
natriuretic peptide (BNP) in hearts of wild-type and miR-208-/-
animals after a sham or TAB procedure. C. Western analysis of
.alpha.-MHC and .beta.-MHC protein levels in hearts of wild-type
and miR-208-/- animals after a sham or TAB procedure. GAPDH was
detected as a loading control.
[0020] FIG. 2. Longterm Knockdown of miR-208 Phenocopies the
Inhibition of the Stress Response in the miR-208 Knockout
Animals.
[0021] A. Sequence of a synthetic oligonucleotide targeted to the
mature miR-208 sequence (SEQ ID NO: 16). The mismatch control
sequence contains four base mismatches compared to the anti miR-208
sequence (SEQ ID NO: 17). B. Realtime PCR analysis shows efficient
knockdown of miR-208 in hearts of animals treated with the
anti-miR-208 oligonucleotide. C. Relative expression levels for
beta-myosin heavy chain (.beta.-MHC), atrial natriuretic factor
(ANF), and brain natriuretic peptide (BNP) in hearts of animals
that received an anti-miR-208 oligonucleotide (anti 208) or a
mismatched control (mm) after a sham or thoracic aortic banding
(TAB) procedure. While the stress markers ANF and BNP are induced
in response to TAB, the animals that received anti-miR-208 showed a
decreased induction of .beta.MBC expression.
[0022] FIG. 3. Myh7b and miR-499 are Regulated by miR-208.
[0023] Northern blot showing expression of miR-499 in hearts of
wild-type (+/+), miR-208 heterozygotes (+/-) and miR-208 knockout
(-/-) mice. There is a direct correlation between the expression of
miR-208 and miR-499, as well as Myh7b in wild-type and mutant mice.
The expression of GAPDH was measured as a control.
[0024] FIG. 4. Myh7b and miR-499 are Expressed in Cardiac and Slow
Skeletal Muscle.
[0025] A. Northern analysis indicates that miR-499 is expressed in
heart and slow skeletal muscle (e.g. soleus). RT-PCR for Myh7b
shows that miR-499 is co-expressed with its hostgene. B. Real-time
PCR analysis for miR-499 on heart and four skeletal muscle types
(gastrocnemius/plantaris (GP), tibialis anterior (TA), extensor
digitorum longus (EDL), and soleus) confirms that miR-499 is
predominantly expressed in the heart and soleus. Only minor levels
of miR-499 expression can be detected in TA and EDL. C. In situ
hybridization indicates that during embryogenesis, Myh7b is
specifically expressed in the heart and somites.
[0026] FIG. 5. MiR-499 does not Affect Myosin Expression.
[0027] A. RT-PCR analysis for Myh7b shows that genetic deletion of
miR-499 does not affect the expression of its hostgene, Myh7b.
GP-gastrocnemius/plantaris; TA-tibialis anterior; EDL-extensor
digitorum longus. Expression of GAPDH was measured as a control. B.
Western blot analysis of hearts from wild-type (WT), heterozygote
(+/-), and miR-499 knockout (KO) animals for both .alpha.- and
.beta.-MHC shows that deletion of miR-499 does not affect the
expression of either gene at the protein level. C. Propylthiouracil
(PTU), which blocks thyroid hormone biogenesis and is a strong
inducer of .beta.-MHC, produces a decrease in .alpha.-MHC and an
increase in .beta.-MHC in both wildtype (WT) and miR-499 knockout
(KO) animals.
[0028] FIG. 6. The Regulation of miR-499 by In Vivo Knockdown of
miR-208.
[0029] A. Northern analysis of miR-208 and miR-499 expression three
days after tail vein injection of the indicated amount of
anti-miR-208 oligonucleotide or saline (Sal). B. Northern analysis
for miR-208 and miR-499 expression of cardiac tissue of animals
injected with either a single 80 mg/kg dose of anti-miR-208,
2.times.80 mg/kg doses of anti-miR-208 on two consecutive days, or
a mismatched control oligonucleotide (mm) two months after
treatment. C. Realtime PCR analysis for miR-208, miR-499, miR-208b,
.alpha.-MHC, Myh7b, and .beta.-MHC expression in cardiac tissue two
months after treatment with a single dose of anti-miR-208, two
doses of anti-miR-208 on two consecutive days, or two doses of a
mismatched oligonucleotide on two consecutive days. D. Western blot
analysis of .beta.-MHC expression in cardiac tissue two months
after anti-miR-208 (single 80 mg/kg dose or two consecutive 80
mg/kg doses) or mismatch (mm) control treatment.
[0030] FIG. 7. Dual Targeting of miR-208 and miR-499.
[0031] A. Northern analysis for miR-208, miR-208b and miR-499 in
cardiac tissue of wild-type and miR-499 knockout animals shows a
strong induction of miR-208b in response to PTU. MiR-208b is
co-expressed with .beta.-MHC and is indicative of its expression.
In the miR-499 knockout animals, the induction of miR-208b is
comparable to wild-type. However, knockdown of miR-208 in miR-499
knockout animals suppresses the induction of miR-208b expression by
PTU. B. Realtime PCR analysis for miR-208, .alpha.-MHC, and
.beta.-MHC in cardiac tissue of wild-type animals, miR-208 knockout
animals, miR-499 knockout animals, and miR-499 knockout animals
treated with anti-miR-208 in the presence and absence of PTU.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Cardiac and skeletal muscles respond to a variety of
pathophysiological stimuli such as workload, thyroid hormone
signaling and injury by modulating the expression of myosin
isoforms, which regulate the efficiency of contraction.
[0033] The ratio of .alpha.- to .beta.-MHC isoforms in the adult
heart is a major determinant of cardiac contractility. .beta.-MHC,
the major myosin isoform in the adult heart, displays relatively
low ATPase activity, whereas .alpha.-MHC has high ATPase activity.
In response to a variety of pathological stimuli such as myocardial
infarction, hypertension, and other disorders, .beta.-MHC
expression increases, while .alpha.-MHC expression decreases with a
consequent reduction in myofibrillar ATPase activity and reduced
shortening velocity of cardiac myofibers, leading to eventual
contractile dysfunction. Remarkably, minor changes in .alpha.-MHC
content of the heart can have a profound influence on cardiac
performance.
[0034] Recently, the inventors reported a cardiac-specific miRNA,
miR-208a, which is encoded by an intron of the .alpha.-myosin heavy
chain gene, and is required for up-regulation of .beta.-MHC
expression in response to cardiac stress and for repression of fast
skeletal muscle genes in the heart (see co-pending application WO
2008/016924, which is herein incorporated by reference in its
entirety).
[0035] The inventors have also recently discovered that miR-208a is
also required for cardiac expression of a closely related miRNA,
miR-499, which is encoded by an intron of the Myh7b gene (see
co-pending application PCT/US08/71837, which is herein incorporated
by reference in its entirety). Expression of Myh7b and miR-499 in
the heart, as well as in slow skeletal muscle, is controlled by the
MEF2 transcription factor, a signal-dependent regulator of striated
muscle gene expression. Forced expression of miR-499 or miR-208 is
sufficient to mediate a fast to slow myofiber conversion in vivo.
MiR-208 and miR-499 can negatively regulate the expression of
Thrap1, a thyroid hormone receptor coregulator, and members of the
PUR family of transcription factors, which in turn negatively
regulate .beta.-MHC expression in cardiac and skeletal muscle. Sox6
functions as a repressor of slow fiber type-specific genes.
Knockdown of Sox6 expression in wild-type myotubes results in a
significant increase in .beta.-MHC expression. Analysis of the
.beta.-MHC promoter revealed a Sox consensus sequence which
suggests that Sox6 plays a critical role in the fiber type
differentiation of fetal skeletal muscle and .beta.-MHC regulation
in the heart. These findings unveil a common regulatory mechanism
in which Myh genes regulate the gene expression patterns of
striated muscles by encoding regulatory miRNAs that govern
contractility and signal responsiveness (van Rooij et al. (2009)
Developmental Cell, Vol. 17: 662-673).
[0036] The present invention is based, in part, on the discovery
that downregulation of both miR-208 and miR-499 in heart cells
produces a synergistic effect in suppressing the cardiac stress
response. Inhibition of miR-208a expression in heart cells results
in a reduction of stress-induced expression of .beta.-MHC. However,
this effect is not observed until two months following
administration of the miR-208 inhibitor. The inventors have
surprisingly found that the inhibition of both miR-208a and miR-499
result in suppression of stress-induced .beta.-MHC expression
almost immediately after administration, thus accelerating the
effect on the cardiac stress response. Accordingly, strategies to
manipulate skeletal and cardiac muscle gene expression by
modulating miR-208 and miR-499 expression, either simultaneously or
sequentially, for the treatment and prevention of cardiac diseases
are described in light of these discoveries.
[0037] MiR-208a is located within an intron of the .alpha.-MHC
gene. The precise intron location is dependent on the particular
species and specific transcript. For example, in humans, miR-208a
is encoded within the 28.sup.th intron of the .alpha.-MHC gene,
while in mice, it is encoded within the 29.sup.th intron. The
pre-miRNA encoding sequences for miR-208a for human, mouse, rat,
and canine are shown below as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, and SEQ ID NO: 4, respectively. The mature miR-208a sequence
is provided in SEQ ID NO: 5. Like .alpha.-MHC, miR-208a is
expressed solely in the heart.
TABLE-US-00001 Human pre-miR-208a (SEQ ID NO: 1) ACGGGCGAGC
TTTTGGCCCG GGTTATACCT GATGCTCACG TATAAGACGA GCAAAAAGCT TGTTGGTCAG A
Mouse pre-miR-208a (SEQ ID NO: 2) ACGGGTGAGC TTTTGGCCCG GGTTATACCT
GACTCTCACG TATAAGACGA GCAAAAAGCT TGTTGGTCAG A Rat pre-miR-208a (SEQ
ID NO: 3) ACGGGTGAGC TTTTGGCCCG GGTTATACCT GACTCTCACG TATAAGACGA
GCAAAAAGCT TGTTGGTCAG A Canine pre-miR-208a (SEQ ID NO: 4)
ACGCATGAGC TTTTGGCTCG GGTTATACCT GATGCTCACG TATAAGACGA GCAAAAAGCT
TGTTGGTCAG A Mature miR-208a (SEQ ID NO: 5)
AUAAGACGAGCAAAAAGCUUGU
[0038] Analysis of the genomic location of the miR-499 gene showed
it to be contained within the 20.sup.th intron of the Myh7b gene, a
homolog of the .alpha.-MHC gene. The pre-miRNA encoding sequences
for miR-499 for mouse, rat, human, canine, opposum, chicken and X.
tropicalis are provided in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12,
respectively. SEQ ID NO: 13 is the stem-loop structure of the mouse
precursor sequence and SEQ ID NO: 14 is the mature miR-499
sequence. The Myh7b gene is conserved in vertebrates and is
expressed solely in the heart and slow skeletal muscle (e.g.
soleus).
TABLE-US-00002 Mouse pre-miR-499 (SEQ ID NO: 6) TCCCTGTGTC
TTGGGTGGGC AGCTGTTAAG ACTTGCAGTG ATGTTTAGCT CCTCTGCATG TGAACATCAC
AGCAAG Rat pre-miR-499 (SEQ ID NO: 7) TCCCTGTCTT GGGTGGGCAG
CTGTTAAGAC TTGCAGTGAT GTTTAGCTCC TCTCCATGTG AACATCACAG CAAG Human
pre-miR-499 (SEQ ID NO: 8) CCCCTGTGCC TTGGGCGGGC GGCTGTTAAG
ACTTGCAGTG ATGTTTAACT CCTCTCCACG TGAACATCAC AGCAAG Canine
pre-miR-499 (SEQ ID NO: 9) CCCTTGCACC CTGGGCGGGC GGCCGTTAAG
ACTTGCAGTG ATGTTTAACT CCTCTCCACG TGAACATCAC AGCAAG Opposum
pre-miR-499 (SEQ ID NO: 10) CCCCTGCCTC CCCGGCGGGC AGCTGTTAAG
ACTTGCAGTG ATGTTTAATT CTTCTCTATG TGAACATCAC AACAAG Chicken
pre-miR-499 (SEQ ID NO: 11) GGAGCGGCAG TTAAGACTTG TAGTGATGTT
TAGATAATGT ATTACATGGA CATCACTTTA AG X tropicalis pre-miR-499 (SEQ
ID NO: 12) GTCTTAGCGA GGCAGTTAAG ACTTGCAGTG ATGTTTAGTT AAAATCTTTT
CATGAACATC ACTTTAAG Mouse stem-loop of the pre-miR-499 sequence
(SEQ ID NO: 13) GGGUGGGCAG CUGUUAAGAC UUGCAGUGAU GUUUAGCUCC
UCUGCAUGUG AACAUCACAG CAAGUCUGUG CUGCUGCCU Mature miR-499 (SEQ ID
NO: 14) UUAAGACUUG CAGUGAUGUU U
[0039] The inventors have also discovered that the genome contains
a second version of miR208a, called miR-208b, which is located
within the .beta.-MHC gene at intron 31, and like .beta.-MHC, miRNA
208b is expressed solely in the heart and slow skeletal muscle
(e.g. soleus). Genes regulated by miR-208b include, for example,
Sp3, Myostatin, PURbeta, THRAP1, and fast skeletal muscle protein
genes. The sequence of this miRNA is largely overlapping with
miR-208a with a 100% homology in the "seed region," the region that
defines mRNA targets of a certain miRNA. Thus, miR-208b can have
profound effects on cardiac and skeletal muscle contractility in
humans. The pre-miR-208b sequence is conserved across several
mammalian species (e.g. human, mouse, rat, and canine). The
pre-miR-208b sequence as well as the mature miR-208b sequence is
shown below:
TABLE-US-00003 pre-miR-208b (SEQ ID NO: 18) TTTCTGATCC GAATATAAGA
CGAACAAAAG GTTTGTCTGA GGG Mature miR-208b (SEQ ID NO: 19)
AUAAGACGAA CAAAAGGUUU GU
[0040] It is understood that when the RNA sequences disclosed
herein are used in embodiments that require deoxyribonucleotides, a
thymidine residue is substituted for a uridine residue. Similarly,
in embodiments requiring ribonucleotides, a uridine residue is
substituted for a thymidine residue in the DNA sequences disclosed
herein.
[0041] In one embodiment, the present invention provides a method
of treating pathologic cardiac hypertrophy, myocardial infarction,
or heart failure in a subject in need thereof, including a human,
by targeting the expression and/or activity of either or both
miR-208 (e.g., miR-208a and/or miR-208b, or in other words,
miR208a/miR208b) and miR-499 in the heart cells of the subject. In
some embodiments, an inhibitor of miR-208a/miR-208b and an
inhibitor of miR-499 are administered to the subject to reduce the
expression or activity of miR-208a/miR-208b and miR-499 in the
heart cells of the subject.
[0042] In another embodiment, the subject in need thereof may be at
risk for developing pathologic cardiac hypertrophy, heart failure,
or myocardial infarction. Such a subject may exhibit one or more
risk factors including, but not limited to, long standing
uncontrolled hypertension, uncorrected valvular disease, chronic
angina, recent myocardial infarction, congenital predisposition to
heart disease or pathological hypertrophy. The subject at risk may
be diagnosed as having a genetic predisposition to cardiac
hypertrophy or may have a familial history of cardiac
hypertrophy.
[0043] Preferably, administration of both an inhibitor of
miR-208a/miR-208b and an inhibitor of miR-499 to the subject
results in the improvement of one or more symptoms of cardiac
hypertrophy, heart failure, or myocardial infarction in the
subject, or in the delay in the transition from cardiac hypertrophy
to heart failure. The one or more improved symptoms may be, for
example, increased exercise capacity, increased cardiac ejection
volume, decreased left ventricular end diastolic pressure,
decreased pulmonary capillary wedge pressure, increased cardiac
output, increased cardiac index, lowered pulmonary artery
pressures, decreased left ventricular end systolic and diastolic
dimensions, decreased cardiac fibrosis, decreased collagen
deposition in cardiac muscle, decreased left and right ventricular
wall stress, decreased wall tension, increased quality of life, and
decreased disease related morbidity or mortality.
[0044] In one embodiment of the invention, the cardiac stress
response is reduced in the subject following administration of the
miR-208 (e.g., miR-208a and/or miR-208b) and miR-499 inhibitors.
The cardiac stress response includes, inter alia, cardiomyocyte
hypertrophy, fibrosis of the heart, reduced expression of
.alpha.-MHC in the heart cells, and/or increased expression of
.beta.-MHC in the heart cells. Administration of both an inhibitor
of miR-208a/miR-208b and an inhibitor of miR-499 to the subject
results in a more rapid effect on the cardiac stress response as
compared to administration of either inhibitor alone. For instance,
the reduction of the cardiac stress response occurs less than eight
weeks, less than six weeks, less than four weeks, less than three
weeks, less than two weeks, less than one week, less than five
days, less than three days, or less than one day following
administration of the inhibitors. In another embodiment, the
reduction in the cardiac stress response occurs less than twelve
hours following administration of the inhibitors.
[0045] In some embodiments, miR-208 (e.g., miR-208a and/or
miR-208b) and miR-499 inhibitors may be antisense oligonucleotides
targeting the mature miR-499 and/or miR-208a or miR-208b sequences.
The antisense oligonucleotides may be ribonucleotides or
deoxyribonucleotides. Preferably, the antisense oligonucleotides
have at least one chemical modification. For instance, suitable
antisense oligonucleotides may be comprised of one or more
"conformationally constrained" or bicyclic sugar nucleoside
modifications, for example, "locked nucleic acids." "Locked nucleic
acids" (LNAs) are modified ribonucleotides that contain an extra
bridge between the 2' and 4' carbons of the ribose sugar moiety
resulting in a "locked" conformation that confers enhanced thermal
stability to oligonucleotides containing the LNAs. The antisense
oligonucleotides targeting miR-208a/miR-208b and miR-499 can
contain combinations of LNAs or other modified nucleotides and
ribonucleotides or deoxyribonucleotides. Alternatively, the
antisense oligonucleotides may comprise peptide nucleic acids
(PNAs), which contain a peptide-based backbone rather than a
sugar-phosphate backbone. Other chemical modifications that the
antisense oligonucleotides may contain include, but are not limited
to, sugar modifications, such as 2'-O-alkyl (e.g. 2'-O-methyl,
2'-.beta.-methoxyethyl), 2'-fluoro, and 4' thio modifications, and
backbone modifications, such as one or more phosphorothioate,
morpholino, or phosphonocarboxylate linkages (see, for example,
U.S. Pat. Nos. 6,693,187 and 7,067,641, which are herein
incorporated by reference in their entireties). For instance,
antisense oligonucleotides, particularly those of shorter lengths
(e.g., less than 15 nucleotides) can comprise one or more affinity
enhancing modifications, such as, but not limited to, LNAs,
bicyclic nucleosides, phosphonoformates, 2' O alkyl and the like.
In some embodiments, suitable antisense oligonucleotides are
2'-O-methoxyethyl "gapmers" which contain
2'-O-methoxyethyl-modified ribonucleotides on both 5' and 3' ends
with at least ten deoxyribonucleotides in the center. These
"gapmers" are capable of triggering RNase H-dependent degradation
mechanisms of RNA targets. Other modifications of antisense
oligonucleotides to enhance stability and improve efficacy, such as
those described in U.S. Pat. No. 6,838,283, which is herein
incorporated by reference in its entirety, are known in the art and
are suitable for use in the methods of the invention. Preferable
antisense oligonucleotides useful for inhibiting the activity of
miRNAs are about 5 to about 50 nucleotides in length, about 10 to
about 30 nucleotides in length, or about 20 to about 25 nucleotides
in length. In certain embodiments, antisense oligonucleotides
targeting miR-208a/miR-208b and miR-499 are about 8 to about 18
nucleotides in length, and in other embodiments about 12 to 16
nucleotides in length. In particular, any 8-mer or longer that is
complementary to miR208a or miR208b may be used, i.e., any antimir
sequence that is complementary to any consecutive sequence in
miR208a or miR208b, starting from the 5' end of the miR to the 3'
end of the mature sequence. Antisense oligonucleotides may in some
cases comprise a sequence that is at least partially complementary
to a mature miRNA sequence, e.g. at least about 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% complementary to a mature miRNA
sequence. In some embodiments, the antisense oligonucleotide may be
substantially complementary to a mature miRNA sequence, that is at
least about 95%, 96%, 97%, 98%, or 99% complementary to a target
polynucleotide sequence. In one embodiment, the antisense
oligonucleotide comprises a sequence that is 100% complementary to
a mature miRNA sequence.
[0046] In other embodiments, the antisense oligonucleotides are
antagomirs. "Antagomirs" are single-stranded, chemically-modified
ribonucleotides that are at least partially complementary to the
miRNA sequence. Antagomirs may comprise one or more modified
nucleotides, such as 2'-O-methyl-sugar modifications. In some
embodiments, antagomirs comprise only modified nucleotides.
Antagomirs may also comprise one or more phosphorothioate linkages
resulting in a partial or full phosphorothioate backbone. To
facilitate in vivo delivery and stability, the antagomir may be
linked to a steroid such as cholesterol, a fatty acid, a vitamin, a
carbohydrate, a peptide or another small molecule ligand at its 3'
end. Antagomirs suitable for inhibiting miRNAs may be about 15 to
about 50 nucleotides in length, more preferably about 18 to about
30 nucleotides in length, and most preferably about 20 to about 25
nucleotides in length. "Partially complementary" refers to a
sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% complementary to a target polynucleotide sequence. The
antagomirs may be at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% complementary to a mature miRNA sequence. In some
embodiments, the antagomir may be substantially complementary to a
mature miRNA sequence, that is at least about 95%, 96%, 97%, 98%,
or 99% complementary to a target polynucleotide sequence. In other
embodiments, the antagomirs are 100% complementary to the mature
miRNA sequence.
[0047] In some embodiments, inhibitors of miR-499 and
miR-208a/miR-208b are antagomirs comprising a sequence that is
perfectly complementary to the mature miR-499 and mature miR-208a
or miR-208b sequence. In one embodiment, an inhibitor of miR-499 is
an antagomir having a sequence that is partially or perfectly
complementary to 5'-UUAAGACUUGCAGUGAUGUUU-3' (SEQ ID NO: 14). In
another embodiment, an inhibitor of miR-208a is an antagomir having
a sequence that is partially or perfectly complementary to
5'-AUAAGACGAGCAAAAAGCUUGU-3' (SEQ ID NO: 5). In another embodiment,
an inhibitor of miR-208a is an antagomir having the sequence
5'-ACAAGCUUUUUGCUCGUCUTJAU-3' (SEQ ID NO: 15). In still another
embodiment, an inhibitor of miR-208a is an antagomir having the
sequence of SEQ ID NO: 16. In another embodiment, an inhibitor of
miR-208b is an antagomir having a sequence that is partially or
perfectly complementary to 5'-AUAAGACGAACAAAAGGUUUGU-3' (SEQ ID NO:
19).
[0048] In some embodiments, inhibitors of miR-499 and miR-208a or
miR-208b are chemically-modified antisense oligonucleotides. In one
embodiment, an inhibitor of miR-499 is a chemically-modified
antisense oligonucleotide comprising a sequence substantially
complementary to 5'-UUAAGACUUGCAGUGAUGUUU-3' (SEQ ID NO: 14). In
another embodiment, an inhibitor of miR-208a is a
chemically-modified antisense oligonucleotide comprising a sequence
substantially complementary to 5'-AUAAGACGAGCAAAAAGCUUGU-3' (SEQ ID
NO: 5). In yet another embodiment, an inhibitor of miR-208b is a
chemically-modified antisense oligonucleotide comprising a sequence
substantially complementary to 5'-AUAAGACGAACAAAAGGUUUGU-3' (SEQ ID
NO: 19). As used herein "substantially complementary" refers to a
sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100%
complementary to a target polynucleotide sequence (e.g. mature or
precursor miRNA sequence).
[0049] Antisense oligonucleotides may comprise a sequence that is
substantially complementary to a precursor miRNA sequence
(pre-miRNA) for miR-499 or miR-208a/miR-208b. In some embodiments,
the antisense oligonucleotide comprises a sequence that is
substantially complementary to a sequence located outside the
stem-loop region of the pre-miR-499 or pre-miR-208a/miR-208b
sequence. In one embodiment, an inhibitor of miR-499 function is an
antisense oligonucleotide having a sequence that is substantially
complementary to a pre-miR-499 sequence selected from the group
consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. In another
embodiment, an inhibitor of miR-208a function is an antisense
oligonucleotide having a sequence that is substantially
complementary to a pre-miR-208a sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID
NO: 4. In still another embodiment, an inhibitor of miR-208b
function is an antisense oligonucleotide having a sequence that is
substantially complementary to a pre-miR-208b sequence of SEQ ID
NO: 18.
[0050] In another embodiment of the invention, a single nucleic
acid molecule may be used to inhibit both miR-208 and miR-499
simultaneously. For instance, a single nucleic acid may contain a
sequence that is at least partially complementary to a mature
miR-208a sequence (e.g. SEQ ID NO: 5) and a sequence that is at
least partially complementary to a mature miR-499 sequence (e.g.
SEQ ID NO: 14). In another embodiment, a single nucleic acid may
contain a sequence that is at least partially complementary to a
mature miR-208b sequence (e.g. SEQ ID NO: 19) and a sequence that
is at least partially complementary to a mature miR-499 sequence
(e.g. SEQ ID NO: 14). In yet another embodiment, the single nucleic
acid molecule may contain a sequence that is at least partially
complementary to a pre-miR-208a sequence (e.g. SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4) and a sequence that is at
least partially complementary to a pre-miR-499 sequence (e.g. SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11 and SEQ ID NO: 12). In another embodiment, the single
nucleic acid molecule may contain a sequence that is at least
partially complementary to a pre-miR-208b sequence (e.g. SEQ ID NO:
18) and a sequence that is at least partially complementary to a
pre-miR-499 sequence (e.g. SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12).
The single nucleic acid molecule may further comprise one or more
spacer nucleotides between the miR-208 (e.g., miR-208a or miR-208b)
and miR-499 targeting sequences. For instance, the single nucleic
acid molecule may contain about 1 to about 200 spacer nucleotides,
more preferably about 5 to about 100 spacer nucleotides, most
preferably about 10 to about 50 spacer nucleotides between the
miR-208a/miR-208b and miR-499 targeting sequences.
[0051] Any of the inhibitors of miR-208a/miR-208b and miR-499
described herein can be delivered to the target cell (e.g. heart
cell, skeletal muscle cell) by delivering to the cell an expression
vector encoding the miR-208a/miR-208b and miR-499 inhibitors. The
inhibitor of miR-208a/miR-208b and the inhibitor of miR-499 can be
encoded by the same expression vector. Alternatively, the inhibitor
of miR-208 (e.g., miR-208a or miR-208b) and the inhibitor of
miR-499 are encoded on separate expression vectors. A "vector" is a
composition of matter which can be used to deliver a nucleic acid
of interest to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. Examples of viral
vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the like.
An expression construct can be replicated in a living cell, or it
can be made synthetically. For purposes of this application, the
terms "expression construct," "expression vector," and "vector,"
are used interchangeably to demonstrate the application of the
invention in a general, illustrative sense, and are not intended to
limit the invention.
[0052] In one embodiment, an expression vector for expressing an
inhibitor of miR-208a/miR-208b and/or miR-499 comprises a promoter
operably linked to a polynucleotide encoding an antisense
oligonucleotide, wherein the sequence of the expressed antisense
oligonucleotide is partially or perfectly complementary to a mature
sequence of miR-208 (e.g., miR-208a or miR-208b) and/or miR-499.
The phrase "operably linked" or "under transcriptional control" as
used herein means that the promoter is in the correct location and
orientation in relation to a polynucleotide to control the
initiation of transcription by RNA polymerase and expression of the
polynucleotide. In another embodiment, the expression vector may
encode a single nucleic acid that targets both miR-208 (e.g.,
miR-208a or miR-208b) and miR-499 as described herein, wherein the
single nucleic acid is operably linked to a promoter. In another
embodiment, a single expression vector may encode a
miR-208a/miR-208b inhibitor and a miR-499 inhibitor, wherein the
miR-208a/miR-208b inhibitor is driven by a different promoter than
the miR-499 inhibitor.
[0053] As used herein, a "promoter" refers to a DNA sequence
recognized by the synthetic machinery of the cell, or introduced
synthetic machinery, required to initiate the specific
transcription of a gene. Suitable promoters include, but are not
limited to RNA pol I, pol II, pol III, and viral promoters (e.g.
human cytomegalovirus (CMV) immediate early gene promoter, the SV40
early promoter, and the Rous sarcoma virus long terminal repeat).
In one embodiment, the promoter is a tissue specific promoter. Of
particular interest are muscle specific promoters, and more
particularly, cardiac specific promoters. These include the myosin
light chain-2 promoter (Franz et al. (1994) Cardioscience, Vol.
5(4):235-43; Kelly et al. (1995) J. Cell Biol., Vol.
129(2):383-396), the alpha actin promoter (Moss et al. (1996) Biol.
Chem., Vol. 271(49):31688-31694), the troponin 1 promoter (Bhaysar
et al. (1996) Genomics, Vol. 35(1):11-23); the Na+/Ca2+ exchanger
promoter, (Barnes et al. (1997) J. Biol. Chem., Vol.
272(17):11510-11517), the dystrophin promoter (Kimura et al. (1997)
Dev. Growth Differ., Vol. 39(3):257-265), the alpha7 integrin
promoter (Ziober and Kramer (1996) J. Bio. Chem., Vol.
271(37):22915-22), the brain natriuretic peptide promoter (LaPointe
et al. (1996) Hypertension, Vol. 27(3 Pt 2):715-22) and the alpha
B-crystallin/small heat shock protein promoter (Gopal-Srivastava
(1995) J. Mol. Cell. Biol., Vol. 15(12):7081-7090), alpha myosin
heavy chain promoter (Yamauchi-Takihara et al. (1989) Proc. Natl.
Acad. Sci. USA, Vol. 86(10):3504-3508) and the ANF promoter
(LaPointe et al. (1988) J. Biol. Chem., Vol.
263(19):9075-9078).
[0054] In certain embodiments, the promoter operably linked to a
polynucleotide encoding a miR-499 and/or a miR-208a/miR-208b
inhibitor may be an inducible promoter. Inducible promoters are
known in the art and include, but are not limited to, tetracycline
promoter, metallothionein IIA promoter, heat shock promoter,
steroid/thyroid hormone/retinoic acid response elements, the
adenovirus late promoter, and the inducible mouse mammary tumor
virus LTR. An expression vector may encode a single nucleic acid
that targets both miR-208 (e.g., miR-208a or miR-208b) and miR-499
as described herein, wherein the single nucleic acid is operably
linked to a inducible promoter. Alternatively, a single expression
vector may encode a miR-208a/miR-208b inhibitor and a miR-499
inhibitor, wherein the miR-208a/miR-208b inhibitor is driven by a
first inducible promoter and the miR-499 inhibitor is driven by a
second inducible promoter. In another embodiment, a first
expression vector may encode a miR-208a/miR-208b inhibitor, wherein
the miR-208a/miR-208b inhibitor is operably linked to a first
inducible promoter and a second expression vector may encode a
miR-499 inhibitor, wherein the miR-499 inhibitor is operably linked
to a second inducible promoter. Other combinations of inducible and
constitutive promoters for controlling the expression of the
miR-208 (e.g., miR-208a or miR-208b) and miR-499 inhibitors are
also contemplated. For instance, a miR-208a/miR-208b inhibitor may
be expressed from a vector using a constitutive promoter, while a
miR-499 inhibitor may be expressed from a vector using an inducible
promoter.
[0055] The present invention also includes methods for scavenging
or clearing miR-499 and miR-208a/miR-208b inhibitors following
treatment. The method may comprise overexpressing binding sites for
the miR-499 and miR-208a/miR-208b inhibitors in cardiac tissue. In
another embodiment, the present invention provides a method for
scavenging or clearing miR-499 and miR-208 (e.g., miR-208a or
miR-208b) following treatment. In one embodiment, the method
comprises overexpression of binding site regions for miR-499 and
miR-208a/miR-208b in skeletal muscle using a skeletal and heart
muscle specific promoter (muscle creatine kinase (MCK)). The
binding site regions preferably contain a sequence of the seed
region for miR-499 and miR-208a or miR-208b. The seed region is the
5' portion of a miRNA spanning bases 2-8, which is important for
target recognition. In some embodiments, the binding site may
contain a sequence from the 3'UTR of one or more targets of miR-499
or miR-208, such as THRAP1 or PURbeta. In another embodiment, a
miR-499 and miR-208 inhibitor may be administered after miR-499 and
miR-208 to attenuate or stop the function of the miRNA.
[0056] In another embodiment of the invention, the inhibitor of
miR-208 (e.g., miR-208a or miR-208b) and the inhibitor of miR-499
are co-administered. The miR-208 inhibitor and miR-208 may be
administered in a single formulation. For instance, a
pharmaceutical composition comprising a miR-208 inhibitor and a
miR-499 inhibitor can be used to co-administer the two inhibitors.
Alternatively, the miR-208 and miR-499 inhibitors may be encoded by
a single nucleic acid, such as an expression vector as described
herein. Multiple co-administrations of the two inhibitors can be
given over a sustained period of time, for instance, one week, two
weeks, three weeks, one month, two months, three months, four
months, five months, six months, nine months, one year, two years,
three years, four years, or five years.
[0057] In some embodiments, the inhibitor of miR-208 (e.g.,
miR-208a or miR-208b) and the inhibitor of miR-499 are administered
sequentially. In one embodiment, the inhibitor of miR-208 is
administered prior to the inhibitor of miR-499. In another
embodiment, the inhibitor of miR-499 is administered prior to the
inhibitor of miR-208. The interval separating the administration of
the miR-208 and miR-499 inhibitors may range from several minutes
to several days. For instance, the interval can be about one hour
to about 72 hours, six hours to about 48 hours, or about 12 hours
to about 24 hours. In a preferred embodiment, the interval between
the administration of the miR-208 inhibitor and the miR-499
inhibitor is at least 24 hours. The inventors have observed that
administering a miR-499 inhibitor at least about 24 hours before a
miR-208 inhibitor results in at least about a 50% reduction in
stress-induced .beta.-MHC expression at about three days after
administration of the miR-208 inhibitor. In the absence of a
miR-499 inhibitor, a comparable effect on stress-induced 13-MHC
expression is not observed until at least about two months after
administration of the miR-208 inhibitor.
[0058] In other embodiments of the invention, more than one
sequential administration of the miR-208 and miR-499 inhibitors may
be employed to produce a sustained effect. In this regard, various
combinations may be used. By way of illustration, where the
inhibitor of miR-499 is "A" and the inhibitor of miR-208 (e.g.,
miR-208a or miR-208b) is "B", the following permutations based on 3
and 4 total administrations are exemplary:
TABLE-US-00004 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B
B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
[0059] Other combinations are likewise contemplated.
[0060] Preferably, the expression or activity of miR-208 (e.g.,
miR-208a or miR-208b) and miR-499 is reduced in the heart cells of
a subject following administration of the miR-208 inhibitor and the
miR-499 inhibitor to the subject. In certain embodiments, the
expression or activity of miR-208a/miR-208b and/or miR-499 is
reduced by greater than 50%, greater than 60%, greater than 70%,
greater than 75%, greater than 80%, greater than 85%, greater than
90%, or greater than 95% following administration of a miR-208 and
miR-499 inhibitor. In one embodiment, the expression or activity of
miR-208a/miR-208b and miR-499 is reduced by greater than 60 percent
in the heart cells of the subject following administration of the
inhibitors. In another embodiment, the expression or activity of
miR-208a/miR-208b and miR-499 is reduced by greater than 80 percent
in the heart cells of the subject following administration of the
inhibitors. In still another embodiment, the expression or activity
of miR-208a/miR-208b and miR-499 is reduced by greater than 90
percent in the heart cells of the subject following administration
of the inhibitors.
[0061] The present invention also includes a method of regulating
cardiac and/or skeletal muscle contractility. Adult skeletal muscle
fibers can be categorized into fast and slow twitch subtypes based
on specialized contractile and metabolic properties. These
properties reflect the expression of specific sets of fast and slow
contractile protein isoforms of myosin heavy and light chains,
tropomyosin, and troponins, as well as myoglobin (Naya et al.
(2000) J Biol Chem, Vol. 275(7):4545-4548). Slow-twitch muscles are
primarily used in chronic activities such as posture maintenance
and sustained locomotor activity. Fast-twitch fibers are used
primarily for high-force burst activities. The adult skeletal
muscle phenotype is not static but instead retains the ability to
adjust to variations in load bearing and contractile usage
patterns, resulting in adaptations in morphology, phenotype, and
contractile properties.
[0062] The up-regulation of several fast skeletal muscle
contractile protein genes was observed in the hearts of mice
lacking both miR-208a alleles. This up-regulation of fast skeletal
muscle contractile protein genes in the hearts of miR-208a knockout
mice indicates that miR-208 normally functions to repress the fast
skeletal muscle gene program. A concomitant reduction of miR-499
expression was observed in miR-208a mutant mice (see Example 3),
suggesting that miR-499 may also negatively regulate the expression
of fast skeletal muscle contractile protein genes. As discussed
above, miR-208b is also expressed predominantly in slow skeletal
muscle (e.g., soleus). Thus, miR-208b may have profound effects on
cardiac and skeletal muscle contractility in humans, and may also
regulate the fast skeletal muscle gene program and determine fiber
identity. The inventors have recently shown that miR-208b and
miR-499 play important roles in the specification of muscle fiber
identity by activating slow and repressing fast myofiber gene
programs. The actions of these miRNAs are mediated in part by a
collection of transcriptional repressors of slow myofiber genes,
like Sox6, PUR.beta., Sp3 and HP1.beta.. Using the skeletal muscle
specific MCK-promoter miR-499 transgenic animals also revealed
conversion to a slower myofiber type. Even more remarkably, when
mice were subjected to a regimen of forced treadmill running, the
miR-499 transgenic animals ran more than 50% longer than wild-type
littermates, indicative of enhanced endurance resulting from the
reprogramming of fast myofibers to a slower fiber type. See van
Rooij et al. (2009) Developmental Cell, Vol. 17:662-673).
[0063] In one embodiment, the method of regulating cardiac and/or
skeletal muscle contractility comprises administering a modulator
of miR-499 and miR-208 (e.g., miR-208a or miR-208b) expression or
activity to heart and/or skeletal muscle cells. In another
embodiment, the method comprises administering a modulator of
miR-499 and miR-208b. In another embodiment, there is provided a
method of regulating cardiac contractile protein gene expression
comprising administering a modulator of miR-499 and miR-208 (e.g.,
miR-208a or miR-208b) expression or activity to heart cells. In
another embodiment, there is provided a method of regulating
skeletal muscle contractile protein gene expression comprising
administering to skeletal muscle cells a modulator of miR-499 and
miR-208 (e.g., miR-208a or miR-208b) expression or activity. In
another embodiment, there is provided a method of regulating
skeletal muscle contractile protein gene expression comprising
administering to skeletal muscle cells a modulator of miR-499 and
miR-208b expression or activity. In still another embodiment, the
present invention provides a method of inducing a fiber type switch
of a skeletal muscle cell comprising administering to skeletal
muscle cells a modulator of miR-499 and miR-208 expression or
activity to the skeletal muscle cell. In another embodiment, the
method of inducing a fiber type switch of a skeletal muscle cell
comprises administering to skeletal muscle cells a modulator of
miR-499 and miR-208b expression or activity. The modulator may be
an agonist or an inhibitor of miR-499, miR-208, and/or miR-208b
expression or activity. In some embodiments, the expression of
THRAP1, PURbeta, myostatin, Sp3, HP 1.beta., and Sox 6 are
increased in a cell by contacting the cell with a miR-499 and
miR-208a (or miR-208b) inhibitor. In other embodiments, expression
of THRAP1, PURbeta, myostatin, Sp3, HP1.beta., and Sox 6 are
decreased in a cell by contacting the cell with an agonist of
miR-499 and miR-208a (or miR-208b).
[0064] In certain embodiments of the invention, there is provided a
method of reducing (3-MHC expression in heart cells comprising
administering an inhibitor of miR-499 and miR-208 (e.g., miR-208a
or miR-208b) expression or activity to the heart cells. In one
embodiment, there is provided a method of reducing .beta.-MHC
expression in skeletal muscle cells comprising administering an
inhibitor of miR-499 and miR-208b expression or activity to the
skeletal muscle cells. In other embodiments of the invention, there
is provided a method of elevating .beta.-MHC expression in heart
cells and/or skeletal muscle cells comprising increasing endogenous
miR-499 and miR-208a (or miR-208b) expression or activity or
administering exogenous miR-499 and miR-208a (or miR-208b) to heart
cells and/or skeletal muscle cells.
[0065] In one embodiment of the invention, there is provided a
method of increasing the expression of a fast skeletal muscle
contractile protein gene in heart cells comprising administering to
the heart cells an inhibitor of miR-499 and miR-208 (e.g., miR-208a
or miR-208b) expression or activity. In another embodiment, there
is provided a method of increasing the expression of a fast
skeletal muscle contractile protein gene in skeletal muscle cells
comprising administering to the skeletal muscle cells an inhibitor
of miR-499 and miR-208b expression or activity. In another
embodiment of the invention, there is provided a method of
decreasing the expression of a fast skeletal muscle contractile
protein gene in heart cells and/or skeletal muscle cells comprising
increasing endogenous miR-499 and miR-208a (or miR-208b) expression
or activity or administering exogenous miR-499 and miR-208a (or
miR-208b) to the heart cells and/or skeletal muscle cells. Examples
of fast skeletal muscle contractile protein genes that may be
increased or decreased according to the methods of the present
invention include, but are not limited to, troponin I2; troponin
T3, fast skeletal myosin light chain, or alpha skeletal actin.
[0066] In skeletal muscle, the repression of slow fiber genes and
activation of fast fiber genes is associated with numerous
musculoskeletal disorders, including, but not limited to, disuse
atrophy, muscle wasting in response to anti-gravity, and
denervation. Thus, expression of miR-499 in combination with
miR-208a or miR-208b in skeletal muscle cells may be useful in
repressing fast fiber genes thereby activating the reciprocal
expression of slow fiber genes. Accordingly, the present invention
also encompasses a method for treating or preventing a
musculoskeletal disorder in a subject in need thereof. In one
embodiment, the method comprises administering an agonist of
miR-208 (e.g., miR-208a or miR-208b) and an agonist of miR-499 to
the subject, wherein the expression or activity of
miR-208a/miR-208b and miR-499 is increased in the skeletal muscle
cells of the subject following administration. In another
embodiment, the method comprises administering an agonist of
miR-208b and an agonist of miR-499 to the subject, wherein the
expression or activity of miR-208b and miR-499 is increased in the
skeletal muscle cells of the subject following administration.
Preferably, the expression of one or more fast skeletal muscle
genes in the skeletal muscle cells of the subject is reduced
following administration of the miR-499 and miR-208a (or miR-208b)
agonists. The one or more fast skeletal muscle genes can include,
but is not limited to, troponin I2, troponin T3, fast skeletal
myosin light chain, and alpha skeletal actin.
[0067] In another embodiment, the present invention provides a
method of treating or preventing muscle wasting in response to a
reduced gravity environment by administering an agonist of miR-499
and miR-208 (e.g., miR-208a or miR-208b) to the skeletal muscle. In
another embodiment, the method of treating or preventing muscle
wasting in response to a reduced gravity environment comprises
administering an agonist of miR-499 and miR-208b to the skeletal
muscle. In yet another embodiment, the present invention provides a
method of treating or preventing muscle atrophy by administering an
agonist of miR-499 and an agonist of miR-208 (e.g., miR-208a and/or
miR-208b) to the skeletal muscle. In another embodiment, the method
of treating or preventing muscle atrophy comprises administering an
agonist of miR-499 and an agonist of miR-208b to the skeletal
muscle.
[0068] In some embodiments, the agonist of miR-208 (miR208a or
miR-208b) and the agonist of miR-499 are polynucleotides encoding a
mature miR-208 (miR208a or miR-208b) and/or miR-499 sequence. In
one embodiment, the polynucleotide comprises a mature miR-208a
sequence (SEQ ID NO: 5) and a mature miR-499 sequence (SEQ ID NO:
14). In another embodiment, the polynucleotide comprises a mature
miR-208b sequence (SEQ ID NO: 19) and a mature miR-499 sequence
(SEQ ID NO: 14). In another embodiment, the agonist of miR-499 and
agonist of miR-208 (miR208a or miR-208b) may be a polynucleotide
comprising the pri-miRNA or pre-miRNA sequence for miR-499 and
miR-208 (miR208a or miR-208b). Alternatively, the agonist of
miR-208 (miR208a or miR-208b) and the agonist of miR-499 may be
separate polynucleotides each comprising a mature sequence or
pre-miRNA sequence of the miRNA. The polynucleotide comprising the
mature miR-499 and/or miR-208 (miR208a or miR-208b) sequence may be
single stranded or double stranded. The polynucleotides may contain
one or more chemical modifications, such as locked nucleic acids,
peptide nucleic acids, sugar modifications, such as 2'-O-alkyl
(e.g. 2'-O-methyl, 2'-O-methoxyethyl), 2'-fluoro, and 4' thio
modifications, and backbone modifications, such as one or more
phosphorothioate, morpholino, or phosphonocarboxylate linkages. In
one embodiment, the polynucleotide comprising a miR-499, miR-208,
and/or miR-208b sequence is conjugated to cholesterol.
[0069] In another embodiment, the agonist of miR-499 and miR-208
(miR208a or miR-208b) may be encoded on an expression vector. An
expression vector for expressing miR-499 and miR-208 (miR208a or
miR-208b) comprises at least one promoter operably linked to a
polynucleotide encoding miR-499 and/or miR-208 (miR208a or
miR-208b). The polynucleotide encoding miR-499 may encode the
primary-miRNA-499 sequence (pri-miR-499), the precursor-miRNA-499
sequence (pre-miR-499) or the mature miR-499 sequence. The
polynucleotide encoding miR-208a/miR-208b may encode the
primary-miRNA-208a/208b sequence (pri-miR-208/pri-miR-208b), the
precursor-miRNA-208/208b sequence (pre-miR-208a/pre-miR-208b) or
the mature miR-208a/208b sequence. In some embodiments, the
expression vector comprises a polynucleotide operably linked to a
promoter, wherein said polynucleotide comprises the sequence of SEQ
ID NO: 5 and SEQ ID NO: 14. In other embodiments, the expression
vector comprises a polynucleotide operably linked to a promoter,
wherein said polynucleotide comprises the sequence of SEQ ID NO: 19
and SEQ ID NO: 14. Such polynucleotides may be about 18 to about
2000 nucleotides in length, about 70 to about 200 nucleotides in
length, about 20 to about 50 nucleotides in length, or about 18 to
about 25 nucleotides in length. In another embodiment, the
expression vector may express a miR-499 agonist (e.g.
polynucleotide comprising a miR-499 sequence) and a miR-208 agonist
(e.g. polynucleotide comprising a miR-208a or miR-208b sequence)
from different promoters. Polynucleotides encoding miR-499,
miR-208a, and/or miR-208b may be located in a nucleic acid encoding
an intron or in a nucleic acid encoding an untranslated region of
an mRNA or in a non-coding RNA. In one embodiment, the expression
vector may contain sequences from the 20.sup.th intron from the
Myh7b gene or sequences from the 31.sup.st intron from the Myh7
(.beta.-MHC) gene.
[0070] The agonist of miR-208a or miR-208b may be co-administered
with the agonist of miR-499 to a subject. The two agonists may be
administered in a single formulation, e.g. a pharmaceutical
composition comprising a miR-208a or miR-208b agonist and a miR-499
agonist. Alternatively, the two agonists (e.g. miR-208a and miR-499
or miR-208b and miR-499) may be a single polynucleotide encoding
the mature or pre-miRNA sequence of the two miRNAs. Multiple
co-administrations of the two agonists can be given over a
sustained period of time, for instance, one week, two weeks, three
weeks, one month, two months, three months, four months, five
months, six months, nine months, one year, two years, three years,
four years, or five years.
[0071] In certain embodiments, the agonist of miR-208a or miR-208b
and the agonist of miR-499 are administered sequentially. In one
embodiment, the agonist of miR-208a or miR-208b is administered
prior to the agonist of miR-499. In another embodiment, the agonist
of miR-499 is administered prior to the agonist of miR-208a or
miR-208b. The interval separating the administration of the
agonists may range from several minutes to weeks, e.g. about one
hour to about 72 hours, six hours to about 48 hours, or about 12
hours to about 24 hours. In a preferred embodiment, the interval
between the administration of the miR-208a or miR-208b agonist and
the miR-499 agonist is at least 24 hours.
[0072] The present invention also includes pharmaceutical
compositions comprising an inhibitor or agonist of miR-499,
miR-208a, and/or miR-208b. Where clinical applications are
contemplated, pharmaceutical compositions will be prepared in a
form appropriate for the intended application. Generally, this will
entail preparing compositions that are essentially free of
pyrogens, as well as other impurities that could be harmful to
humans or animals.
[0073] In one embodiment, the pharmaceutical composition comprises
an effective dose of a miR-499 inhibitor and/or an effective dose
of a miR-208a or miR-208b inhibitor. In another embodiment, the
pharmaceutical composition comprises an effective dose of a miR-499
agonist and/or an effective dose of a miR-208a or miR-208b agonist.
An "effective dose" is an amount sufficient to effect a beneficial
or desired clinical result. An effective dose of an miRNA inhibitor
or miRNA agonist of the invention may be about 1 mg/kg to about 200
mg/kg, about 20 mg/kg to about 160 mg/kg, or about 40 mg/kg to
about 100 mg/kg. In one embodiment, the inhibitor of miR-208 or
miR-208b and the inhibitor of miR-499 are administered each at a
dosage of about 20 mg/kg to about 200 mg/kg. In another embodiment,
the inhibitor of miR-208 or miR-208b and the inhibitor of miR-499
are administered each at a dosage of about 80 mg/kg. In another
embodiment, the agonist of miR-208 or miR-208b and the agonist of
miR-499 are administered each at a dosage of about 20 mg/kg to
about 200 mg/kg. In still another embodiment, the agonist of
miR-208 or miR-208b and the agonist of miR-499 are administered
each at a dosage of about 80 mg/kg. The precise determination of
what would be considered an effective dose may be based on factors
individual to each patient, including their size, age, type of
disorder (e.g. myocardial infarction, heart failure, cardiac
hypertrophy, or musculoskeletal), and nature of inhibitor or
agonist (e.g. antagomir, expression construct, antisense
oligonucleotide, etc). Therefore, dosages can be readily
ascertained by those of ordinary skill in the art from this
disclosure and the knowledge in the art.
[0074] Colloidal dispersion systems, such as macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes, may be used as delivery vehicles for the
oligonucleotide inhibitors of miRNA function, polynucleotides
encoding miRNA agonists, or constructs expressing particular miRNA
inhibitors or agonists. Commercially available fat emulsions that
are suitable for delivering the nucleic acids of the invention to
cardiac and skeletal muscle tissues include Intralipid.RTM.,
Liposyn.RTM., Liposyn.RTM. II, Liposyn.RTM. III, Nutrilipid, and
other similar lipid emulsions. A preferred colloidal system for use
as a delivery vehicle in vivo is a liposome (i.e., an artificial
membrane vesicle). The preparation and use of such systems is well
known in the art. Exemplary formulations are also disclosed in U.S.
Pat. No. 5,981,505; U.S. Pat. No. 6,217,900; U.S. Pat. No.
6,383,512; U.S. Pat. No. 5,783,565; U.S. Pat. No. 7,202,227; U.S.
Pat. No. 6,379,965; U.S. Pat. No. 6,127,170; U.S. Pat. No.
5,837,533; U.S. Pat. No. 6,747,014; and WO03/093449, which are
herein incorporated by reference in their entireties.
[0075] One will generally desire to employ appropriate salts and
buffers to render delivery vehicles stable and allow for uptake by
target cells. Aqueous compositions of the present invention
comprise an effective amount of the delivery vehicle comprising the
inhibitor polynucleotides or miRNA polynucleotide sequences (e.g.
liposomes or other complexes or expression vectors) dissolved or
dispersed in a pharmaceutically acceptable carrier or aqueous
medium. The phrases "pharmaceutically acceptable" or
"pharmacologically acceptable" refers to molecular entities and
compositions that do not produce adverse, allergic, or other
untoward reactions when administered to an animal or a human. As
used herein, "pharmaceutically acceptable carrier" includes
solvents, buffers, solutions, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like acceptable for use in formulating
pharmaceuticals, such as pharmaceuticals suitable for
administration to humans. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredients of the present invention, its use in therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions, provided they do not
inactivate the vectors or polynucleotides of the compositions.
[0076] The active compositions of the present invention may include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention may be via any
common route so long as the target tissue is available via that
route. This includes oral, nasal, or buccal. Alternatively,
administration may be by intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection, or by direct injection
into cardiac tissue. Pharmaceutical compositions comprising miRNA
inhibitors, polynucleotides encoding miRNA sequence or expression
constructs comprising miRNA sequences may also be administered by
catheter systems or systems that isolate coronary circulation for
delivering therapeutic agents to the heart. Various catheter
systems for delivering therapeutic agents to the heart and coronary
vasculature are known in the art. Some non-limiting examples of
catheter-based delivery methods or coronary isolation methods
suitable for use in the present invention are disclosed in U.S.
Pat. No. 6,416,510; U.S. Pat. No. 6,716,196; U.S. Pat. No.
6,953,466, WO 2005/082440, WO 2006/089340, U.S. Patent Publication
No. 2007/0203445, U.S. Patent Publication No. 2006/0148742, and
U.S. Patent Publication No. 2007/0060907, which are all herein
incorporated by reference in their entireties. Such compositions
would normally be administered as pharmaceutically acceptable
compositions as described herein.
[0077] The active compounds may also be administered parenterally
or intraperitoneally. By way of illustration, solutions of the
active compounds as free base or pharmacologically acceptable salts
can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations generally contain a preservative to prevent the growth
of microorganisms.
[0078] The pharmaceutical forms suitable for injectable use or
catheter delivery include, for example, sterile aqueous solutions
or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions.
Generally, these preparations are sterile and fluid to the extent
that easy injectability exists. Preparations should be stable under
the conditions of manufacture and storage and should be preserved
against the contaminating action of microorganisms, such as
bacteria and fungi. Appropriate solvents or dispersion media may
contain, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial an antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0079] Sterile injectable solutions may be prepared by
incorporating the active compounds in an appropriate amount into a
solvent along with any other ingredients (for example as enumerated
above) as desired, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the desired other ingredients, e.g., as
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation include vacuum-drying and freeze-drying techniques
which yield a powder of the active ingredient(s) plus any
additional desired ingredient from a previously sterile-filtered
solution thereof.
[0080] The compositions of the present invention generally may be
formulated in a neutral or salt form. Pharmaceutically-acceptable
salts include, for example, acid addition salts (formed with the
free amino groups of the protein) derived from inorganic acids
(e.g., hydrochloric or phosphoric acids, or from organic acids
(e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups of the protein can also be
derived from inorganic bases (e.g., sodium, potassium, ammonium,
calcium, or ferric hydroxides) or from organic bases (e.g.,
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0081] Upon formulation, solutions are preferably administered in a
manner compatible with the dosage formulation and in such amount as
is therapeutically effective. The formulations may easily be
administered in a variety of dosage forms such as injectable
solutions, drug release capsules and the like. For parenteral
administration in an aqueous solution, for example, the solution
generally is suitably buffered and the liquid diluent first
rendered isotonic for example with sufficient saline or glucose.
Such aqueous solutions may be used, for example, for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
Preferably, sterile aqueous media are employed as is known to those
of skill in the art, particularly in light of the present
disclosure. By way of illustration, a single dose may be dissolved
in 1 ml of isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
[0082] Any of the compositions described herein may be comprised in
a kit. In one embodiment, the kit contains a first pharmaceutical
composition comprising a miR-208a or miR-208b inhibitor and a
second pharmaceutical composition comprising a miR-499 inhibitor.
In another embodiment, the kit contains a single pharmaceutical
composition comprising a miR-208a or miR-208b inhibitor and a
miR-499 inhibitor. In another embodiment, the kit contains a first
pharmaceutical composition comprising a miR-208a or miR-208b
agonist and a second pharmaceutical composition comprising a
miR-499 agonist. In still another embodiment, the kit contains a
single pharmaceutical composition comprising a miR-208a or miR-208b
agonist and a miR-499 agonist. In some embodiments, the kit may
also include one or more transfection reagent(s) to facilitate
delivery of the miRNA agonists or inhibitors to cells.
[0083] The components of the kits may be packaged either in aqueous
media or in lyophilized form. The container means of the kits will
generally include at least one vial, test tube, flask, bottle,
syringe or other container means, into which a component may be
placed, and preferably, suitably aliquoted. Where there is more
than one component in the kit, the kit also will generally contain
a second, third or other additional container into which the
additional components may be separately placed (e.g. sterile,
pharmaceutically acceptable buffer and/or other diluents). However,
various combinations of components may be comprised in a vial. The
kits of the present invention also will typically include a means
for containing the nucleic acids, and any other reagent containers
in close confinement for commercial sale. Such containers may
include injection or blow molded plastic containers into which the
desired vials are retained.
[0084] When the components of the kit are provided in one and/or
more liquid solutions, the liquid solution is an aqueous solution,
with a sterile aqueous solution being particularly preferred.
[0085] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0086] Such kits may also include components that preserve or
maintain the miRNA agonist or miRNA inhibitors or that protect
against their degradation. Such components may be DNAse-free,
RNAse-free or protect against nucleases (e.g. RNAses and DNAses).
Such kits generally will comprise, in suitable means, distinct
containers for each individual reagent or solution.
[0087] A kit will also include instructions for employing the kit
components as well the use of any other reagent not included in the
kit. Instructions may include variations that can be implemented. A
kit may also include utensils or devices for administering the
miRNA agonist or inhibitor by various administration routes, such
as parenteral or catheter administration.
[0088] It is contemplated that such reagents are embodiments of
kits of the invention. Such kits, however, are not limited to the
particular items identified above and may include any reagent used
for the manipulation or characterization of miRNA.
[0089] The following examples are included solely to illustrate
various aspects of the invention. The reference to miR208 in the
Examples and figure refers to miR208a in mice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that the invention is equally applicable to any human or
other animal, and encompasses modulating either miR208a and/or
miR208b.
EXAMPLES
Example 1
MiR-208 Knockout Mice Exhibit Reduced Cardiac Hypertrophy and
Fibrosis in Response to Pressure Overload
[0090] Encoded within an intron of the .alpha.-MHC gene is miR-208.
Like .alpha.-MHC, miR-208 is expressed specifically in the heart
with trace expression in the lung miR-208 is processed out of the
.alpha.-MHC pre-mRNA rather than being transcribed as a separate
transcript. Intriguingly, however, miR-208 displays a remarkably
long half-life of at least 14 days, and can thereby exert functions
even when .alpha.-MHC mRNA expression has been down-regulated.
[0091] MiR-208 knockout mice were created by generating a miR-208
targeting vector by digesting a 0.4 kb fragment (5' arm) extending
upstream of the miR-208 coding region with SacII and NotI and
ligating the fragment into the pGKneoF2L2dta targeting plasmid
upstream of the loxP sites and the Frt-flanked neomycin cassette. A
3.3 kb fragment (3' arm) was digested with SalI and HindIII and
ligated into the vector between the neomycin resistance and Dta
negative selection cassettes. Targeted ES-cells carrying the
disrupted allele were identified by Southern blot analysis with 5'
and 3' probes. Three miR-208 targeted ES clones were identified and
used for blastocyst injection. The resulting chimeric mice were
bred to C57BL/6 to obtain germline transmission of the mutant
allele.
[0092] Although genetic deletion of miR-208 in mice failed to
induce an overt phenotype, microarray analysis on hearts from
wild-type and miR-208-/- animals at 2 months of age revealed
removal of miR-208 to result in pronounced expression of numerous
fast skeletal muscle contractile protein genes, which are normally
not expressed in the heart. Thus, these results suggest that under
normal conditions miR-208 is co-expressed with the sole
cardiac-specific MHC gene to maintain cardiomyocyte identity by
repressing the expression of skeletal muscle genes in the
heart.
[0093] The most remarkable function of miR-208 was revealed by the
aberrant response of miR-208 null mice to cardiac stress (van Rooij
et al., (2007) Science, Vol. 316: 575-579). In response to pressure
overload by thoracic aortic banding (TAB) that drives pathological
remodeling of the heart, histological sections of hearts from
miR-208 knockout mice showed virtually no hypertrophy of
cardiomyocytes or fibrosis as compared to sections from wild-type
mice (FIG. 1A). In addition, miR-208 knockout animals were unable
to up-regulate (3-MHC expression in response to pressure overload
(FIGS. 1B and C). In contrast, other stress responsive genes, such
as those encoding ANF and BNP, were strongly induced in miR-208
mutant animals (FIG. 1B), demonstrating that miR-208 is dedicated
specifically to the control of .beta.-MHC expression, which can be
uncoupled from other facets of the cardiac stress response.
Example 2
Knockdown of miR-208 Phenocopies miR-208 Knockout Animals in
Response to Stress
[0094] To examine the specificity of the effect of the absence of
miR-208 on the cardiac stress response, animals were injected
intravenously daily with either an antagomir having a sequence
complementary to the mature miR-208 sequence (anti 208; SEQ ID NO:
16) or a mismatched sequence (mm; SEQ ID NO: 17). All nucleosides
were 2'-OMe modified, and the 5' terminal two and 3' terminal four
bases contained a phosphorothioate internucleoside. Cholesterol was
attached to the 3' end of the passenger strand through a
hydroxyprolinol linker (FIG. 2A). Realtime PCR analysis of hearts
of animals injected with the miR-208 antagomir two months after
treatment showed efficient knockdown of miR-208 (FIG. 2B).
[0095] To test the effect of in vivo miR-208 downregulation on the
cardiac stress response, animals receiving either the anti-miR-208
antagomir or the mismatched control were subject to a sham
procedure or a thoracic aortic banding procedure to induce pressure
overload. Animals that were treated with the mismatched control
exhibited a typical stress response with upregulation of .beta.-MHC
as well as other stress genes (ANF and BNP). In contrast, animals
that were treated with the anti-miR-208 antagomir failed to show an
upregulation of .beta.-MHC in response to the stress stimulus.
However, an increase in expression of the other stress genes (ANF
and BNP) was observed (FIG. 2C). The stress response of animals
treated with the anti-miR-208 antagomir was remarkably similar to
that of miR-208 knockout animals suggesting that miR-208 plays a
critical role in the regulation of .beta.-MHC expression in
response to stress.
Example 3
MiR-208 is Required for Expression of miR-499
[0096] To further explore the mechanism of action of miR-208 in the
heart, the inventors defined the miRNA expression patterns in
hearts from wild type and miR-208 knockout mice by microarray
analysis. Among several miRNAs that were up- and down-regulated in
miR-208 knockout hearts, the inventors discovered that miR-499 was
highly abundant in normal hearts, but was not expressed above
background levels in miR-208 knockout animals. These findings were
confirmed by Northern blot (FIG. 3). Analysis of the genomic
location of the miR-499 gene showed it to be contained within the
20.sup.th intron of the Myh7b gene, a homolog of the .alpha.-MHC
gene. MiR-208 appears to regulate Myh7b and thereby miR-499
expression at the level of transcription since RT-PCR for Myh7b
indicates that the mRNA of the host gene is dose-dependently
abrogated in the absence of miR-208 (FIG. 3).
[0097] The Myh7b gene is conserved in vertebrates and is expressed
solely in the heart and slow skeletal muscle (e.g. soleus) (FIG.
4A). Similarly, miR-499 has the same expression pattern as its host
gene as confirmed by real-time PCR analysis (FIGS. 4A & B). In
situ hybridization using a probe directed against the 3' end of the
Myh7b gene, indicated that this myosin (and miR-499) was expressed
in heart as early as E10.5 (FIG. 4C). Later during embryogenesis,
Myh7b/miR-499 is also expressed in the somites. These data indicate
that miR-208 is required to drive an additional myosin, Myh7b,
which gives rise to related miR-499. In addition, miR-499 is
down-regulated during cardiac hypertrophy.
[0098] To further explore the role of miR-499 in pathological
cardiac hypertrophy and regulation of muscle contractility, miR-499
knockout animals were generated. Genetic deletion of miR-499 had no
effect on expression of its host gene, Myh7b (FIG. 5A). Western
blot analysis of hearts from miR-499 mutant and wild-type animals
for both .alpha.- and .beta.-MHC showed that deletion of miR-499
does not affect the expression of either gene at the protein level
(FIG. 5B). To examine whether miR-499 had an effect on .beta.-MHC
regulation, wild-type and miR-499 knockout animals received
propylthiouracil (PTU), which induces hypothyroidism and
upregulates .beta.-MHC. Both wild-type and miR-499 knockout animals
exhibited a decrease in .alpha.-MHC and an increase in .beta.-MHC
in response to PTU (FIG. 5C). Surprisingly, unlike miR-208, miR-499
is not required for the regulation of expression of either .alpha.-
or .beta.-MHC.
Example 4
Dual Targeting of miR-208 and miR-499
[0099] MiR-208 regulates the expression of miR-499 as shown by the
dose-dependent decrease in miR-499 expression in miR-208
heterozygote and miR-208 knockout animals (FIG. 3 and Example 3).
To further elucidate the interaction between miR-208 and miR-499,
wild-type animals were injected intravenously with saline or one of
four doses (20 mg/kg, 40 mg/kg, 80 mg/kg, and 160 mg/kg) of a
synthetic oligonucleotide (e.g. an antagomir) having a sequence
complementary to the mature miR-208 sequence (anti-miR-208; SEQ ID
NO: 16). Northern analysis of heart tissue three days after tail
vein injection revealed a dose-dependent decrease in the expression
of mature miR-208, while leaving the expression of the pre-miR-208
intact (FIG. 6A). However, unlike in the genetic deletion model,
the expression of miR-499 remained unchanged. In addition,
expression levels of .beta.-MHC were also unaffected three days
after injection of an anti-miR-208 antagomir (data not shown).
[0100] In a second series of experiments, wild-type animals were
injected intravenously either with a single dose of anti-miR-208
(80 mg/kg), two sequential doses (80 mg/kg) of anti-miR-208 on two
consecutive days, or two sequential doses (80 mg/kg) of a
mismatched control oligonucleotide (SEQ ID NO: 17) on two
consecutive days. Northern analysis of cardiac tissue two months
after treatment showed that both miR-208 and miR-499 expression was
reduced in animals treated with anti-miR-208 (FIG. 6B). Realtime
PCR analysis confirmed these results (FIG. 6C). In addition, a
decrease in expression of miR-208b, which is encoded within an
intron of .beta.-MHC and co-expressed with .beta.-MHC, was also
observed. Realtime PCR analysis for the corresponding host myosin
genes revealed that knockdown of miR-208 does not affect the
expression of .alpha.-MHC, but induces a decrease in the expression
of .beta.-MHC and Myh7b (FIG. 6C). A decrease in .beta.-MHC protein
was also observed two months after treatment with anti-miR-208
(FIG. 6D). These results indicate miR-208 regulation of miR-499 and
.beta.-MHC expression occurs after a delay suggesting that miR-208
is upstream of miR-499, which in turn is upstream of .beta.-MHC.
Thus, both miR-208 and miR-499 need to be downregulated to obtain
an expedited reduction of .beta.-MHC expression. MiR-208
downregulation alone leads to an eventual decrease in miR-499
expression, which in turn induces a decrease in (3-MHC expression.
To obtain a more immediate effect on .beta.-MHC expression, both
miR-499 and miR-208 can be targeted for downregulation.
[0101] To examine the combined effect of downregulating both
miR-208 and miR-499, miR-499 knockout animals were administered
anti-miR-208 oligonucleotides prior to receiving propylthiouracil
(PTU), an inducer of .beta.-MHC expression. Similar to previous
results, PTU induced a decrease in .alpha.-MHC expression and an
increase in .beta.-MHC/miR-208b expression (miR-208b is
co-expressed with .beta.-MHC) in both wild-type and miR-499
knockout animals in the absence of treatment with anti-miR-208
oligonucleotides (FIG. 7A, B). Such effects are characteristic of
the cardiac stress response. In contrast, northern and realtime PCR
analysis of cardiac tissue from miR-499 knockout animals treated
with anti-miR-208 oligonucleotides two weeks after treatment showed
that an increase in .beta.-MHC/miR-208b expression in response to
PTU was not observed (FIG. 7A,B). The response of the miR-499
knockout animals treated with anti-miR-208 resembled the response
of miR-208 knockout animals (FIG. 7B). These results suggest that
efficient and rapid downregulation of .beta.-MHC can be achieved by
targeting both miR-208 and miR-499. The dosage of anti-miR-208
oligonucleotides that were administered to the animals produced a
60% reduction in miR-208 expression. This percentage reduction was
sufficient to suppress the induction of .beta.-MHC by PTU in the
absence of miR-499 (FIG. 7B). These findings indicate that
reduction of both miR-499 and miR-208 may be an efficient
therapeutic strategy for the treatment of cardiac disorders, such
as pathological cardiac hypertrophy and heart failure.
Example 5
Knockdown of miR-208 and miR-499 Inhibits the Cardiac Stress
Response
[0102] To further assess the therapeutic value of targeting miR-208
and miR-499 for treating cardiac disorders, mice are injected
intravenously with an antisense oligonucleotide having a sequence
complementary to the mature miR-208a sequence (anti-208), an
antisense oligonucleotide having a sequence complementary to the
mature miR-499 sequence (anti-499), or both anti-208 and anti-499
oligonucleotide sequences. Both anti-208 and anti-499 contain a
combination of locked nucleic acids (LNA) and deoxyribonucleic
acids (DNA) linked by phosphorothioate internucleoside linkages.
Realtime PCR analysis of hearts of animals injected with the
antisense oligonucleotides three weeks up to two months after
treatment is used to assess knockdown of miR-208 and miR-499.
[0103] To test the effect of in vivo miR-208 and miR-499
downregulation on the cardiac stress response, animals receiving
the anti-208, anti-499, or both the anti-208 and anti-499 oligos
are subject to a sham procedure or a thoracic aortic banding
procedure to induce pressure overload. Animals that are untreated
are expected to exhibit a typical stress response with upregulation
of (3-MHC as well as other stress genes (ANF and BNP). In contrast,
animals that are treated with both anti-208 and anti-499 are
expected to exhibit a reduced upregulation of (3-MHC in response to
the stress stimulus that is more pronounced than animals receiving
either antisense oligo alone.
[0104] All publications, patents and patent applications discussed
and cited herein are incorporated herein by reference in their
entireties. It is understood that the disclosed invention is not
limited to the particular methodology, protocols and materials
described as these can vary. It is also understood that the
terminology used herein is for the purposes of describing
particular embodiments only and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
[0105] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
19171DNAHomo sapiens 1acgggcgagc ttttggcccg ggttatacct gatgctcacg
tataagacga gcaaaaagct 60tgttggtcag a 71271DNAMus sp. 2acgggtgagc
ttttggcccg ggttatacct gactctcacg tataagacga gcaaaaagct 60tgttggtcag
a 71371DNARattus sp. 3acgggtgagc ttttggcccg ggttatacct gactctcacg
tataagacga gcaaaaagct 60tgttggtcag a 71471DNACanis sp. 4acgcatgagc
ttttggctcg ggttatacct gatgctcacg tataagacga gcaaaaagct 60tgttggtcag
a 71522RNAUnknownmature miR-208 5auaagacgag caaaaagcuu gu
22676DNAMus sp. 6tccctgtgtc ttgggtgggc agctgttaag acttgcagtg
atgtttagct cctctgcatg 60tgaacatcac agcaag 76774DNARattus sp.
7tccctgtctt gggtgggcag ctgttaagac ttgcagtgat gtttagctcc tctccatgtg
60aacatcacag caag 74876DNAHomo sapiens 8cccctgtgcc ttgggcgggc
ggctgttaag acttgcagtg atgtttaact cctctccacg 60tgaacatcac agcaag
76976DNACanis sp. 9cccttgcacc ctgggcgggc ggccgttaag acttgcagtg
atgtttaact cctctccacg 60tgaacatcac agcaag 761076DNADidelphis sp.
10cccctgcctc cccggcgggc agctgttaag acttgcagtg atgtttaatt cttctctatg
60tgaacatcac aacaag 761162DNAGallus sp. 11ggagcggcag ttaagacttg
tagtgatgtt tagataatgt attacatgga catcacttta 60ag 621268DNAXenopus
tropicalis 12gtcttagcga ggcagttaag acttgcagtg atgtttagtt aaaatctttt
catgaacatc 60actttaag 681379RNAMus sp. 13gggugggcag cuguuaagac
uugcagugau guuuagcucc ucugcaugug aacaucacag 60caagucugug cugcugccu
791421RNAUnknownmature miR-499 14uuaagacuug cagugauguu u
211522RNAArtificial SequencemiR-208 antagomir 15acaagcuuuu
ugcucgucuu au 221622RNAArtificial SequenceantimiR-208
oligonucleotide 16acaagcuuuu ugcucgucuu au 221722RNAArtificial
Sequencemismatch miR-208 oligonucleotide 17accagcuuug ugcucguaug au
221843DNAUnknownpre-miR-208b 18tttctgatcc gaatataaga cgaacaaaag
gtttgtctga ggg 431922RNAUnknownmature miR-208b 19auaagacgaa
caaaagguuu gu 22
* * * * *