U.S. patent application number 16/002845 was filed with the patent office on 2019-05-02 for mir-29 mimics and uses thereof.
The applicant listed for this patent is MIRAGEN THERAPEUTICS, INC.. Invention is credited to Christina M. Dalby, Corrie Gallant-Behm, Rusty L. Montgomery, Eva Van Rooij.
Application Number | 20190127734 16/002845 |
Document ID | / |
Family ID | 55436959 |
Filed Date | 2019-05-02 |
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United States Patent
Application |
20190127734 |
Kind Code |
A1 |
Montgomery; Rusty L. ; et
al. |
May 2, 2019 |
MIR-29 MIMICS AND USES THEREOF
Abstract
The present invention relates to synthetic oligonucleotide
mimetics of miRNAs. In particular, the present invention provides
double-stranded, chemically-modified oligonucleotide mimetics of
miR-29. Pharmaceutical compositions comprising the mimetics and
their use in treating or preventing conditions associated with
dysregulation of extracellular matrix genes, such as tissue
fibrotic conditions, are also described.
Inventors: |
Montgomery; Rusty L.;
(Boulder, CO) ; Dalby; Christina M.; (Boulder,
CO) ; Van Rooij; Eva; (Utrecht, NL) ;
Gallant-Behm; Corrie; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MIRAGEN THERAPEUTICS, INC. |
Boulder |
CO |
US |
|
|
Family ID: |
55436959 |
Appl. No.: |
16/002845 |
Filed: |
June 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15175636 |
Jun 7, 2016 |
9994847 |
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16002845 |
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14848085 |
Sep 8, 2015 |
9376681 |
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15175636 |
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62047562 |
Sep 8, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 27/02 20180101;
A61P 19/02 20180101; C12N 2310/141 20130101; C12N 15/113 20130101;
C12N 2310/3231 20130101; C12N 2310/346 20130101; A61K 31/7105
20130101; C12N 2310/113 20130101; C12N 2310/315 20130101; A61P
11/00 20180101; A61K 31/713 20130101; C12N 2320/51 20130101; A61P
9/00 20180101; A61K 9/0078 20130101; A61K 9/0073 20130101; A61P
43/00 20180101; C12N 2310/343 20130101; A61P 1/16 20180101; A61P
13/12 20180101; A61P 17/02 20180101; A61K 9/0075 20130101; C12N
2310/321 20130101; C12N 2310/3521 20130101; C12N 2310/322 20130101;
C12N 2310/3533 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713; A61K 9/00 20060101
A61K009/00; A61K 31/7105 20060101 A61K031/7105 |
Claims
1. A miR-29 mimetic compound comprising: a first strand of about 23
to about 26 ribonucleotides comprising a mature miR-29a, miR-29b,
or miR-29c sequence; and a second strand of about 22 to about 23
ribonucleotides comprising a sequence that is substantially
complementary to the first strand and having at least one modified
nucleotide, wherein the first strand has a 3' nucleotide overhang
relative to the second strand.
2.-29. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present Application is a continuation of U.S.
application Ser. No. 15/175,636, filed Jun. 7, 2016 (which issued
as U.S. Pat. No. 9,994,847), which is a continuation of U.S.
application Ser. No. 14/848,085, filed Sep. 8, 2015 (which issued
as U.S. Pat. No. 9,376,681), which claims the benefit of priority
to U.S. Provisional Application No. 62/047,562, filed on Sep. 8,
2014, the contents of each of which are hereby incorporated by
reference in their entireties for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to synthetic miRNA mimics or
promiRs that increase miRNA activity in vivo. In particular, the
present invention relates to mimics of miR-29 and their use in
reducing collagen deposition and associated conditions, such as
fibrosis.
BACKGROUND OF THE INVENTION
[0003] Based on gain- or loss-of-function data collected in animal
disease models using genetics or pharmacological modulation of
microRNAs (miRNAs), it is now well accepted that miRNAs are
important players during disease. These studies, combined with
recent positive clinical efficacy data (Janssen et al, 2013),
underscore the relevance of miRNAs and the viability for miRNAs to
become the next class of therapeutics. Indeed, miRNAs have several
advantages as therapeutic intervention points in that they are
small and comprise a known sequence. Additionally, since a single
miRNA can regulate numerous target mRNAs within biological
pathways, modulation of a miRNA in principle allows for influencing
an entire gene network and modifying complex disease phenotypes
(van Rooij & Olson, 2012).
[0004] While many studies have shown therapeutic efficacy using
single-stranded miRNA inhibitors called antimiRs, efforts to
restore or increase the function of a miRNA have been lagging
behind (van Rooij et al, 2012). Currently, miRNA function can be
increased either by viral overexpression or by using synthetic
double-stranded miRNAs. So far the use of adeno-associated viruses
(AAV) to drive expression of a given miRNA for restoring its
activity in vivo has shown to be effective in a mouse model of
hepatocellular and lung carcinoma (Kasinski & Slack, 2012; Kota
et al, 2009) and spinal and bulbar muscular atrophy (Miyazaki et
al, 2012), while the use of unformulated synthetic
oligonucleotide-based approaches to increase miRNA levels has not
been well explored.
[0005] The microRNA-29 (miR-29) family is well characterized for
their ability to regulate extracellular matrix proteins (He et al,
2013). The family consists of miR-29a, -29b and -29c, which are
expressed as two bicistronic clusters (miR-29a/-29b-1 and
miR-29b-2/-29c), and are largely homologous in sequence with only a
few mismatches between the different members in the 3' regions of
the mature miRNA (van Rooij et al, 2008). All three members are
reduced in different types of tissue fibrosis and therapeutic
benefit of increasing miR-29 levels has been shown for heart (van
Rooij et al, 2008), kidney (Qin et al, 2011; Wang et al, 2012; Xiao
et al, 2012), liver (Roderburg et al, 2011; Sekiya et al, 2011;
Zhang et al, 2012), lung (Cushing et al, 2011; Xiao et al, 2012)
and systemic sclerosis (Maurer et al, 2010).
[0006] There is a need in the art for synthetic oligonucleotide
mimics of miR-29 that can effectively increase miR-29 activity in
vivo. Such miR-29 mimics or miR-29 promiRs are useful for treating
various tissue fibrotic conditions.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, on the discovery
that miRNA mimics with modifications for stability and cellular
uptake can be used to replicate endogenous functions of miR-29. For
instance, therapeutic treatment with a miR-29b mimic in the setting
of pulmonary fibrosis restores the bleomycin-induced reduction of
miR-29 and blocks and reverses pulmonary fibrosis, which coincides
with a repression of miR-29 target genes that are induced during
the disease process. Similarly, treatment of skin incisions with a
miR-29b mimic down-regulates the expression of extra-cellular
matrix genes and other genes involved in the fibrotic process.
Accordingly, the present invention provides double-stranded RNA
miR-29 mimetic compounds.
[0008] In some embodiments, the miR-29 mimetic compound comprises
(a) a first strand of about 23 to about 26 ribonucleotides
comprising a mature miR-29a, miR-29b, or miR-29c sequence; and (b)
a second strand of about 22 to about 23 ribonucleotides comprising
a sequence that is substantially complementary to the first strand,
wherein the first strand has a 3' nucleotide overhang relative to
the second strand. In certain embodiments, the first strand and
second strand contain one or more modified nucleotides. The
modified nucleotides may be 2' sugar modifications, such as
2'-alkyl (2'-O-methyl) or 2'-fluoro modifications. In one
embodiment, the first strand has one or more 2'-fluoro
modifications. In another embodiment, the second strand has one or
more 2'-O-methyl modifications. In some embodiments, the second
strand has one, two, three, or more mismatches relative to the
first strand. In one embodiment, the second strand of a miR-29
mimetic compound contains mismatches at positions 4, 13, and/or 16
from the 3' end (of the second strand) relative to the first
strand.
[0009] In one embodiment, the second strand of the miR-29 mimetic
compound is linked to a cholesterol molecule at its 3' terminus. In
certain embodiments, the cholesterol molecule is linked to the
second strand through at least a six carbon linker. The linker may
be a cleavable linker.
[0010] In one embodiment, the nucleotides comprising the 3'
overhang in the first strand are linked by phosphorothioate
linkages.
[0011] In one embodiment, the miR-29 mimetic compound comprises a
first strand comprising the sequence of SEQ ID NO: 27 and a second
strand comprising the sequence of SEQ ID NO: 5.
[0012] In another embodiment, the miR-29 mimetic compound comprises
a first strand comprising the sequence of SEQ ID NO: 19 and a
second strand comprising the sequence of SEQ ID NO: 1.
[0013] In yet another embodiment, the miR-29 mimetic compound
comprises a first strand comprising the sequence of SEQ ID NO: 19
and a second strand comprising the sequence of SEQ ID NO: 15.
[0014] In yet another embodiment, the miR-29 mimetic compound
comprises a first strand comprising the sequence of SEQ ID NO: 33
and a second strand comprising the sequence of SEQ ID NO: 1.
[0015] In yet another embodiment, the miR-29 mimetic compound
comprises a first strand comprising the sequence of SEQ ID NO: 34
and a second strand comprising the sequence of SEQ ID NO: 1.
[0016] In yet another embodiment, the miR-29 mimetic compound
comprises a first strand comprising the sequence of SEQ ID NO: 35
and a second strand comprising the sequence of SEQ ID NO: 24.
[0017] The present invention provides a pharmaceutical composition
comprising an effective amount of the miR-29 mimetic compounds
described herein or a pharmaceutically-acceptable salt thereof, and
a pharmaceutically-acceptable carrier or diluent. In certain
embodiments, the pharmaceutical composition is formulated for
pulmonary, nasal, intranasal or ocular delivery and can be in the
form of powders, aqueous solutions, aqueous aerosols, nasal drops,
aerosols, and/or ocular drops. In some embodiments, the
pharmaceutical composition is administered with an inhalation
system such as a nebulizer, a metered dose inhaler, a dry powder
inhaler, or a soft mist inhaler.
[0018] The present invention also includes a method of regulating
an extracellular matrix gene in a cell comprising contacting the
cell with the miR-29 mimetic compounds described herein. In one
embodiment, the expression or activity of the extracellular matrix
gene is reduced following contact with the miR-29 mimetic compound
or composition. In some embodiments, the extracellular matrix gene
is a collagen gene, such as Col1a1 and Col3a1. The cell may be in
vitro, in vivo, or ex vivo.
[0019] The present invention provides a method of treating or
preventing tissue fibrosis in a subject in need thereof. In one
embodiment, the method comprises administering to the subject a
miR-29 mimetic compound described herein. In certain embodiments,
the tissue fibrosis is cardiac fibrosis, pulmonary fibrosis, renal
fibrosis, hepatic fibrosis, ocular fibrosis, cutaneous fibrosis
including hypertrophic scarring and keloids, hand, joint or tendon
fibrosis, Peyronie's disease or scleroderma. In one embodiment, the
tissue fibrosis is idiopathic pulmonary fibrosis (IPF).
[0020] In certain embodiments, the method for treating or
preventing tissue fibrosis comprises administering a miR-29 mimetic
compound or a composition described herein via the pulmonary,
nasal, or intranasal route. In one embodiment, the miR-29 mimetic
compound or the composition is delivered via inhalation.
[0021] The present invention also includes a method of regulating
non-extracellular matrix genes in a cell comprising contacting the
cell with the miR-29 mimetic compounds described herein. In one
embodiment, the expression or activity of the non-extracellular
matrix gene is increased following contact with the miR-29 mimetic
compound or composition. In some embodiments, the non-extracellular
matrix gene is a gene such as Itga3 or Numb. The cell may be in
vitro, in vivo, or ex vivo.
[0022] The present invention also provides a method for assessing
the efficacy of a treatment with a miR-29 agonist or miR-29
antagonist, the method comprising determining a level of expression
of one or more genes in cells or a fibrotic tissue of a subject
prior to the treatment with the miR-29 agonist or miR-29
antagonist, wherein the one or more genes are selected from a set
of genes modulated by miR-29; determining the level of expression
of the same one or more genes in cells/fibrotic tissue of the
subject after treatment with the miR-29 agonist or miR-29
antagonist; and determining the treatment to be effective, less
effective, or not effective based on the expression levels prior to
and after the treatment. In one embodiment, the one or more genes
modulated by miR-29 are selected from Table 5.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1A. The double-stranded miR-29 mimics design contains a
`guide strand` or `antisense strand` that is identical to the
miR-29b, with a UU overhang on the 3' end, modified to increase
stability, and chemically phosphorylated on the 5' end and a
`passenger strand` or `sense strand` that contains 2'-O-Me
modifications to prevent loading into RNA-induced silencing complex
(RISC) as well as increase stability and is linked to cholesterol
for enhanced cellular uptake. Several mismatches are introduced in
the sense strand to prevent this strand from functioning as an
antimiR.
[0024] FIG. 1B. Transfection experiments in NIH 3T3 show a
dose-dependent decrease in Col1a1 with increasing amount of miR-29b
mimic compared to either untreated or mock treated cells. An siRNA
directly targeting Col1a1 was taken along as a positive control. *
p<0.05 versus mock, # p<0.05 versus untreated.
[0025] FIG. 1C. Northern blot analysis for miR-29b in different
tissues 4 days after intravenous injection with 10, 50, 100, or 125
mpk miR-29b mimic indicates delivery to all tissues at the highest
dose, with the most effective delivery taking place to the lungs
and spleen compared to saline injected mice. U6 is used as a
loading control.
[0026] FIG. 1D. Real-time quantification of miR-29b mimicry
indicates an increased level of miR-29b at the higher dose levels
with the most efficient delivery to the lungs and spleen (n=4 per
group). *p<0.05 versus Saline injected animals.
[0027] FIG. 1E. Northern blot analysis for miR-29b in different
tissues 1, 2, 4 and 7 days after intravenous injection with 125 mpk
of mimic indicates the presence of miR-29b mimic in all tissues
examined, with a longer detection in lung and spleen. U6 is used as
a loading control.
[0028] FIG. 1F. Real-time quantification of miR-29b mimicry
indicates an increased level of miR-29b in all tissues measured
which is maintained the longest in lungs and spleen (n=4 per
group). *p<0.05 versus Saline injected animals.
[0029] FIG. 2A. Real-time PCR analysis indicates a reduction in all
miR-29 family members in response to bleomycin, while miR-29 mimic
treatment resulted in the increased detection of miR-29b levels
compared to either control or saline injected animals. *p<0.05
vs Saline/Saline
[0030] FIG. 2B. Real-time PCR analysis indicated a comparable
decline in miR-29 levels in pulmonary biopsies of patients with
idiopathic pulmonary fibrosis (IPF) compared to normal controls.
*p<0.05 vs Normal
[0031] FIG. 2C. Histological examination by trichrome staining
showing pronounced fibrotic and inflammatory response in response
to bleomycin, which was blunted by miR-29b mimic treatment. Scale
bar indicates 100 .mu.m.
[0032] FIG. 2D. Hydroxyproline measurements to assay for total
collagen content showed a significant increase following bleomycin
treatment in both saline and control treated groups, while there
was no statistical difference in the miR-29 mimic treated group
between Saline and bleomycin treated mice.
[0033] FIGS. 2E-2G. Cytokine measurements on bronchoalveolar lavage
(BAL) fluids indicated a significantly higher concentrations of
IL-12 (FIG. 2E), IL-4 (FIG. 2F) and G-CSF (FIG. 2G) were detectable
in BAL fluids from lungs from bleomycin treated mice, which was
reduced with miR-29b mimic. (n=4), *p<0.05.
[0034] FIG. 2H. Bleomycin treatment increases the detection of
immune cells in BAL fluids which was significantly reduced in the
presence of miR-29b mimic while the Control mimic had no effect.
(n=4), * p<0.05 vs Saline/Bleo, Ap<0.05 vs Control/Bleo
[0035] FIGS. 3A-3B. Bleomycin treatment increases the expression of
Col1a1 (FIG. 3A) and Col3a1 (FIG. 3B) and the presence of miR-29b
mimic inhibits Col1a1 (FIG. 3A) and Col3a1 (FIG. 3B) as measured by
real-time PCR. MiR-29b mimicry has no effect on target repression
under baseline conditions. (n=6-8), * p<0.05
[0036] FIG. 3C. IGF1 levels in BAL fluids increase following
bleomycin treatment which were significantly blunted in the
presence of miR-29 mimic compared to both Saline and Control mimic
treated mice. (n=4), * p<0.05.
[0037] FIG. 3D. Immunohistochemistry demonstrated robust detection
of IGF1 after bleomycin treatment, which was reduced in the miR-29b
mimic treated group compared to Saline or Control mimic treated
mice. Scale bar indicates 50 .mu.m.
[0038] FIG. 4A. Hydroxyproline assessment showed a significant
increase following bleomycin treatment in both saline and control
treated groups, however, there was no statistical difference in the
miR-29 mimic treated group between Saline and bleomycin treated
mice. *P<0.05 (n=8)
[0039] FIGS. 4B-4C. Real-time PCR analysis for Col1a1 (FIG. 4B) and
Col3a1 (FIG. 4C) showed a significant increase with bleomycin
treatment. miR-29b mimic treatment normalized both Col1a1 and
Col3a1 to vehicle treated expression levels. *P<0.05 (n=8)
[0040] FIG. 4D. Histological examination by trichrome staining
showing robust fibrosis in response to bleomycin, which was blunted
by miR-29b mimic treatment.
[0041] FIGS. 4E-4F. Primary pulmonary fibroblasts from patients
with IPF were treated with vehicle or TGF-.beta. and transfected
with control mimic or miR-29b mimic. Real-time PCR was performed
for Col1a1 (FIG. 4E) and Col3a1 (FIG. 4F). miR-29b mimic treatment
showed a dose-dependent reduction in both collagens.
[0042] FIGS. 4G-4H. A549 cells were treated with vehicle or
TGF-.beta. and transfected with control mimic or miR-29b mimic.
Real-time PCR was performed for Col1a1 (FIG. 4G) and Col3a1 (FIG.
4H). miR-29b mimic treatment showed a dose-dependent reduction in
expression of both Col1a1 and Col3a1.
[0043] FIG. 5. miR-29b mimic does not induce general signs of
toxicity. MiR-29b mimic treatment does not induce any overt signs
of liver or kidney toxicity as indicated by the lack of change in
aspartate or alanine transaminases (AST and ALT). n=4 per
group.
[0044] FIGS. 6A-6B. Increasing doses of miR-29b mimic fail to
induce overt changes in gene expression under baseline conditions.
Real-time PCR analysis indicates there to be no significant changes
in expression in the different tissue 4 days after treatment with
increasing doses of miR-29b mimic for Col1a1 (FIG. 6A) and Col3a1
(FIG. 6B) compared to Saline injected mice. n=4 per group, *
p<0.05 compared to Saline injected.
[0045] FIGS. 7A-7B. miR-29b mimic specifically increases miR-29b.
MiR-29b mimicry specifically increases the level of miR-29b without
affecting the level of miR-29a (FIG. 7A) or miR-29c (FIG. 7B)
compared to Saline injected mice. The increase in miR-29c at day 1
might be due to some cross-reactivity of the real-time probe. n=4
per group.
[0046] FIGS. 8A-8B. miR-29b mimic does not induce any target
changes in baseline conditions. Real-time PCR analysis showed the
absence of significant target changes at the indicated timepoints
after injecting 125 mpk of miR-29b mimic for Col1a1 (FIG. 8A) and
Col3a1 (FIG. 8B) compared to Saline injected mice. n=4 per
group.
[0047] FIG. 9. miR-29b mimic effects on gene expression in RAW
cells. Real-time PCR analysis showed significant increases in Csf3,
Igf1, and Kc expression after miR-29b mimic treatment compared to
vehicle or control mimic. * p<0.05 compared to Vehicle
injected.
[0048] FIG. 10A. miR29b mimic and antimiR effects on gene
expression in mouse skin. A heatmap of the microarray data is
presented, where upregulated genes are represented in red and
downregulated genes are represented in blue, and the intensity of
the color is representative of the fold change relative to PBS
control. The top portion of the heatmap (finely dashed box)
contains genes which are repressed by miR-29b mimic treatment and
upregulated by antimiR-29 treatment, and the bottom portion of the
heatmap (wide dashed box) contains genes which are upregulated by
miR-29b mimic treatment and repressed by antimiR-29 treatment. All
fold change and significance values are presented in Table 5.
[0049] FIG. 10B. miR29b mimic and antimiR effects on gene
expression in mouse skin. DAVID analysis (NCBI) of functional terms
that are enriched in the two groups shown in FIG. 10A are
presented. Gene Ontology (GO) terms of Extracellular Matrix, (Skin)
Function, Adhesion/Cell Signaling and Cell
Differentiation/Apoptosis are the top negatively regulated pathways
following miR-29b mimic treatment and Cellular (Nuclear) Structure
and RNA Processing are the top positively regulated pathways
following miR-29b mimic treatment. The microarray analysis of skin
and acute skin wounds in C57BL/6 mice shows reciprocal regulation
of 228 genes by intradermal treatment with a miR-29b mimic
comprising SEQ ID NO: 2 and SEQ ID NO: 1, and antimiR-29 (SEQ ID
NO: 36).
[0050] FIG. 11A. Quantitative real-time (RT)-PCR analysis of mouse
incisional wounds treated with intradermal miR-29b mimic (SEQ ID
NO: 2/SEQ ID NO: 1) or PBS control. Data are presented as a grouped
bar graph where the first bar in each treatment group represents
the expression level of the first gene in the list on the right,
and so on. RT-PCR confirmed that collagens, other extracellular
matrix genes and other direct and indirect target genes previously
shown to be repressed by miR-29b mimic treatment (FIG. 10a, upper
half of the heatmap) show a dose-dependent reduction in expression
with miR-29b mimic treatment in acute skin wounds. **** p<0.0001
versus PBS treated incisions, using a 2-way ANOVA with treatment
and gene as the two factors assessed.
[0051] FIG. 11B. Genes previously identified as being de-repressed
with miR-29b mimic treatment (FIG. 10A, lower half of the heatmap)
show a dose-dependent increase in expression with miR-29b mimic
treatment by quantitative RT-PCR. *** p<0.001 and ****
p<0.0001 versus PBS treated incisions, using a 2-way ANOVA with
treatment and gene as the two factors assessed. The miR-29b mimic
significantly affects the expression of miR-29 target genes in
acute skin wounds.
[0052] FIG. 12. Activity of miR-29 mimics and effects of nucleotide
modifications. Transfection experiments in IMR-90 human lung
fibroblasts show that different miR-29 mimics have different levels
of activity as measured by repression of collagen gene expression.
* p<0.05, *** p<0.001 and **** p<0.0001 versus mock
transfection, using a 2-way ANOVA with treatment and gene as the
two factors assessed.
[0053] FIG. 13. In vivo activity of miR-29b mimics with linker
modifications. Mice with incisional wounds were treated with 20
nmol of various miR-29b mimics that differ only in the linkage
between the cholesterol moiety and the second/sense strand.
Activity of the miR-29b mimics was determined by measuring the
expression of five collagen synthesis genes.
[0054] FIG. 14. Effect of 5' phosphorylation on the activity of
miR-29b mimics. RAB-9 skin fibroblasts were transfected with
varying concentrations of miR-29b mimics with (SEQ ID NO: 2/SEQ ID
NO: 1) and without (SEQ ID NO: 19/SEQ ID NO: 1) 5' phosphorylation
on the antisense strand. Activity of the miR-29b mimics was
determined by measuring the expression of three collagen synthesis
genes.
DETAILED DESCRIPTION OF THE INVENTION
[0055] Over the last decade great enthusiasm has evolved for
microRNA (miRNA) therapeutics. Part of the excitement stems from
the fact that a miRNA often regulates numerous related mRNAs. As
such, modulation of a single miRNA allows for parallel regulation
of multiple genes involved in a particular disease. While many
studies have shown therapeutic efficacy using miRNA inhibitors,
efforts to restore or increase the function of a miRNA have been
lagging behind.
[0056] The miR-29 family has gained a lot of attention for its
clear function in tissue fibrosis. This fibroblast-enriched miRNA
family is downregulated in fibrotic diseases which induces a
coordinate increase of many extracellular matrix genes. The present
inventors have found that administration of synthetic RNA duplexes
can increase miR-29 levels in vivo for several days. Moreover,
therapeutic delivery of these miR-29 mimics during
bleomycin-induced pulmonary fibrosis restores endogenous miR-29
function thereby decreasing collagen expression and blocking and
reversing pulmonary fibrosis. Furthermore, administration of miR-29
mimics of the present invention to skin incisions downregulates
extracellular matrix genes and other genes involved in the fibrotic
process. These data support the feasibility of designing effective
miRNA mimics to therapeutically increase miRNAs and indicate miR-29
to be a potent therapeutic miRNA for treating various fibrotic
conditions and disorders involving increased collagen production.
Accordingly, the present invention provides miR-29 mimics,
compositions and uses thereof.
[0057] A microRNA mimetic compound according to the invention
comprises a first strand and a second strand, wherein the first
strand comprises a mature miR-29a, miR-29b, or miR-29c sequence and
the second strand comprises a sequence that is substantially
complementary to the first strand and has at least one modified
nucleotide. Throughout the disclosure, the term "microRNA mimetic
compound" may be used interchangeably with the terms "promiR-29,"
"miR-29 agonist," "microRNA agonist," "microRNA mimic," "miRNA
mimic," or "miR-29 mimic;" the term "first strand" may be used
interchangeably with the terms "antisense strand" or "guide
strand"; the term "second strand" may be used interchangeably with
the term "sense strand" or "passenger strand;" and the term "miR-29
antagonist" may be used interchangeably with the terms
"oligonucleotide inhibitor," "antimiR-29," "antisense
oligonucleotide," "miR-29 antagomir" or "anti-microRNA
oligonucleotide."
[0058] In one embodiment, the first strand of the microRNA mimetic
compound comprises from about 23 to about 26 nucleotides comprising
a sequence of mature miR-29a, miR-29b, or miR-29c and the second
strand comprises from about 22 to about 23 nucleotides comprising a
sequence that is partially, substantially, or fully complementary
to the first strand. In various embodiments, the first strand may
comprise about 23, 24, 25, or 26 nucleotides and the second strand
may comprise about 22 or 23 nucleotides.
[0059] The nucleotides that form the first and the second strand of
the microRNA mimetic compounds may comprise ribonucleotides,
deoxyribonucleotides, modified nucleotides, and combinations
thereof. In certain embodiments, the first strand and the second
strand of the microRNA mimetic compound comprise ribonucleotides
and/or modified ribonucleotides. The term "modified nucleotide"
means a nucleotide where the nucleobase and/or the sugar moiety is
modified relative to unmodified nucleotides.
[0060] In certain embodiments, the microRNA mimetic compounds have
a first strand or an antisense strand, whose sequence is identical
to all or part of a mature miR-29a, miR-29b, or miR-29c sequence,
and a second strand or a sense strand whose sequence is about 70%
to about 100% complementary to the sequence of the first strand. In
one embodiment, the first strand of the miRNA mimetic compound is
at least about 75, 80, 85, 90, 95, or 100% identical, including all
integers there between, to the entire sequence of a mature,
naturally occurring miR-29a, miR-29b, or miR-29c sequence. In
certain embodiments, the first strand is about or is at least about
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the
sequence of a mature, naturally-occurring miRNA, such as the mouse,
human, or rat miR-29a, miR-29b, or miR-29c sequence. Alternatively,
the first strand may comprise 20, 21, 22, or 23 nucleotide
positions in common with a mature, naturally-occurring miRNA as
compared by sequence alignment algorithms and methods well known in
the art.
[0061] It is understood that the sequence of the first strand is
considered to be identical to the sequence of a mature miR-29a,
miR-29b, or miR-29c even if the first strand includes a modified
nucleotide instead of a naturally-occurring nucleotide. For
example, if a mature, naturally-occurring miRNA sequence comprises
a cytidine nucleotide at a specific position, the first strand of
the mimetic compound may comprise a modified cytidine nucleotide,
such as 2'-fluoro-cytidine, at the corresponding position or if a
mature, naturally-occurring miRNA sequence comprises a uridine
nucleotide at a specific position, the miRNA region of the first
strand of the mimetic compound may comprise a modified uridine
nucleotide, such as 2'-fluoro-uridine, 2'-O-methyl-uridine,
5-fluorouracil, or 4-thiouracil at the corresponding position.
Thus, as long as the modified nucleotide has the same base-pairing
capability as the nucleotide present in the mature,
naturally-occurring miRNA sequence, the sequence of the first
strand is considered to be identical to the mature,
naturally-occurring miRNA sequence. In some embodiments, the first
strand may include a modification of the 5'-terminal residue. For
example, the first strand may have a 5'-terminal monophosphate. In
some other embodiments, the first strand does not contain a
5'-terminal monophosphate.
[0062] In some embodiments, the second strand of the microRNA mimic
is partially complementary to the sequence of the first strand. For
example, the sequence of the second strand is at least about 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, inclusive of all
values therebetween, complementary to the sequence of the first
strand. In some other embodiments, the second strand is
substantially complementary to the sequence of the first strand.
For example, the second strand is at least about 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,
inclusive of all values therebetween, complementary to the sequence
of the first strand. In yet some other embodiments, the sequence of
the second strand may be fully complementary to the first strand.
In certain embodiments, about 19, 20, 21, 22, or 23 nucleotides of
the complementary region of the second strand may be complementary
to the first strand.
[0063] It is understood that the sequence of the second strand is
considered to be complementary to the first strand even if the
second strand includes a modified nucleotide instead of a
naturally-occurring nucleotide. For example, if the first strand
sequence comprises a guanosine nucleotide at a specific position,
the second strand may comprise a modified cytidine nucleotide, such
as 2'-0-methyl-cytidine, at the corresponding position.
[0064] In some embodiments, the second strand comprises about 1, 2,
3, 4, 5, or 6 mismatches relative to the first strand. That is, up
to 1, 2, 3, 4, 5, or 6 nucleotides between the first strand and the
second strand may not be complementary. In one embodiment, the
mismatches are not consecutive and are distributed throughout the
second strand. In another embodiment, the mismatches are
consecutive and may create a bulge. In one embodiment, the second
strand contains 3 mismatches relative to the first strand. In
certain embodiments, the second strand of a miR-29a mimic or a
miR-29c mimic contains mismatches at positions 4, 13, and/or 16
from the 3' end (of the second strand) relative to the first
strand. In one embodiment, the second strand of a miR-29b mimic
contains mismatches at positions 4, 13, and/or 16 from the 3' end
(of the second strand) relative to the first strand. In another
embodiment, the second strand of a miR-29b mimic contains
mismatches at positions 4, 9, 10, 11, 13 and/or 16 from the 3' end
(of the second strand) relative to the first strand.
[0065] In some embodiments, the first and/or the second strand of
the mimetic compound may comprise an overhang on the 5' or 3' end
of the strands. In certain embodiments, the first strand comprises
a 3' overhang, i.e., a single-stranded region that extends beyond
the duplex region, relative to the second strand. The 3' overhang
of the first strand may range from about one nucleotide to about
four nucleotides. In certain embodiments, the 3' overhang of the
first strand may comprise 1 or 2 nucleotides. In some embodiments,
the nucleotides comprising the 3' overhang in the first strand are
linked by phosphorothioate linkages. The nucleotides comprising the
3' overhang in the first strand may include ribonucleotides,
deoxyribonucleotides, modified nucleotides, or combinations
thereof. In certain embodiments, the 3' overhang in the first
strand comprises two ribonucleotides. In one embodiment, the 3'
overhang of the first strand comprises two uridine nucleotides
linked through a phosphorothioate linkage. In some embodiments, the
first strand may not contain an overhang.
[0066] In one embodiment, the nucleotides in the second/sense
strand of miR-29 mimics of the invention are linked by
phosphodiester linkages and the nucleotides in the first/antisense
strand are linked by phosphodiester linkages except for the last
three nucleotides at the 3' end which are linked to each other via
phosphorothioate linkages.
[0067] In various embodiments, miR-29 mimics of the present
invention comprise modified nucleotides. For instance, in one
embodiment, the first strand of the mimic comprises one or more
2'-fluoro nucleotides. In another embodiment, the first strand may
not include any modified nucleotide. In one embodiment, the second
strand comprises one or more 2'-O-methyl modified nucleotides.
[0068] In various embodiments, miR-29 mimics according to the
present invention comprise first and second strands listed in the
Tables below. Definitions of the modifications are presented in
Table 4. These miR-29 mimetic compounds are useful for regulating
the expression of extracellular matrix genes in a cell and treating
associated conditions, such as tissue fibrosis, dermal fibrosis,
including the uses and conditions described in WO 2009/018493,
which is hereby incorporated by reference in its entirety.
TABLE-US-00001 TABLE 1 miR-29a mimics SEQ ID Modified Sequence NO.
Second/sense/passenger strands
5'-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rG.rA.rU.rG.rG.rU.rG.rC.rU.rA.rU.rU-
-3' 3
5'-mU.mA.rA.mC.mC.rG.mU.mU.mU.rA.mC.rA.rG.rA.mU.rG.rG.mU.mC.mC.mU.rA-3'
4
5'-mU.mA.rA.mC.mC.rG.mU.mU.mU.rA.mC.rA.rG.rA.mU.rG.rG.mU.mC.mC.mU.rA.chol6-
-3' 5
5'-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rG.rA.rU.rG.rG.rU.rG.rC.rU.rAs.rUs.-
rUs. 11 chol6-3'
5'-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rG.rA.rU.rG.rG.rU.rG.rC.rU.rA-3'
37 First/antisense/guide strands
5'-p.rU.rA.rG.rC.rA.rC.rC.rA.rU.rC.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.-
rU-3' 6
5'-p.fU.rA.rG.fC.rA.fC.fC.rA.fU.fC.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rU-
s.rU-3' 7
5'-fU.rA.rG.fC.rA.fC.fC.rA.fU.fC.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rUs.-
rU-3' 27
5'-rU.rA.rG.rC.rA.rC.rC.rA.rU.rC.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.rU-
-3' 38
TABLE-US-00002 TABLE 2 miR-29b mimics SEQ ID Modified Sequence NO.
Second/sense/passenger strands
5'-mA.mA.rC.rA.rC.rU.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA.rU-
.rU-3' 8
5'-mA.mA.mC.rA.mC.mU.rG.rA.mU.mU.mU.mC.rA.rA.rA.mU.rG.rG.mU.rG.mC.mU.rA.ch-
ol6-3' 9
5'-mA.mA.rC.rA.rC.rU.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rAs.r-
Us.rUs. 10 chol6-3'
5'-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rG.rG.rG.mU.rG.rG.mU.mC.mC.mU.rA-3'
13
5'-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rG.rG.rG.mU.rG.rG.mU.mC.mC.mU.rA.ch-
ol6-3' 14
5'-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.ch-
ol6-3' 1 5'- 15
mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.dT.dT-
.cho16-3' 5'- 16
C6Chol.dT.dT.mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.m-
C.mU.rA-3'
5'-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.ch-
ol9-3' 17
5'-A.mA.rC.mA.rC.mU.rG.mA.rU.mU.rU.mC.rA.mA.rA.mU.rG.mG.rU.mG.rC.mU.rA.cho-
l6-3' 28
5'-rA.mA.rC.mA.rC.mU.rG.mA.rU.mU.rU.mC.rA.mA.rA.mU.rG.mG.rU.mG.rC.mU.rAs.r-
Us.rU.chol6- 29 3'
5'-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.ch-
olTEG-3' 30
5'-mA.mA.rC.rA.rC.rU.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA-3'
39 First/antisense/guide strands
5'-p.rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rA.rG.rU.rG.rU.rU.-
rU.rU-3' 18
5'-p.fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs-
.rUs.rU-3' 2
5'-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.r-
Us.rU-3' 19
5'-p.fU.rA.rG.fC.rA.fC.fC.rA.fC.fC.fC.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs-
.rUs.rU-3' 20
5'-rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rA.rG.rU.rG.rU.rU.rU-
.rU-3' 21
5'-mU.rA.mG.rC.mA.rC.mC.rA.mU.rU.mU.rG.mA.rA.mA.rU.mC.rA.mG.rU.mG.rU.mU-3'
31
5'mU.rA.mG.rC.mA.rC.mC.rA.mU.rU.mU.rG.mA.rA.mA.rU.mC.rA.mG.rU.mG.rU.mUs.rU-
s.rU-3' 32
5'-mU.rA.rG.mC.rA.mC.mC.rA.mU.mU.mU.rG.rA.rA.rA.mU.mC.rA.rG.mU.rG.mU.mUs.r-
Us.rU-3' 33
5'-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fU-3'
34
5'-rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rA.rG.rU.rG.rU.rU.rU-
.rU-3' 40
TABLE-US-00003 TABLE 3 miR-29c mimics SEQ ID Modified Sequence NO.
Second/sense/passenger strands
5'-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA.rU.rU-
-3' 22
5'-mU.mA.rA.mC.mC.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA-3'
23
5'-mU.mA.rA.mC.mC.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.chol6-
-3' 24
5'-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rAs.rUs.-
rUs.chol6-3' 12
5'-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA-3'
41 First/antisense/guide strands
5'-p.rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.-
rU-3' 25
5'-p.fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rU-
s.rU-3' 26
5'-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rUs.-
rU-3' 35
5'-rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.rU-
-3' 42
TABLE-US-00004 TABLE 4 Definitions of Abbreviations Nucleotide unit
or Nucleotide unit or modification Abbreviation modification
Abbreviation ribo A rA ribo A P.dbd.S rAs ribo G rG ribo G P.dbd.S
rGs ribo C rC ribo C P.dbd.S rCs ribo U rU ribo U P.dbd.S rUs
O-methyl A mA O-methyl A P.dbd.S mAs O-methyl G mG O-methyl G
P.dbd.S mGs O-methyl C mC O-methyl C P.dbd.S mCs O-methyl U mU
O-methyl U P.dbd.S mUs fluoro C fC fluoro C P.dbd.S fCs fluoro U fU
fluoro U P.dbd.S fUs deoxy A dA deoxy A P.dbd.S dAs deoxy G dG
deoxy G P.dbd.S dGs deoxy C dC deoxy C P.dbd.S dCs deoxy T dT deoxy
T P.dbd.S dTs monophosphate p Cholesterol conjugate Chol6/C6 chol
with a 6 carbon linker Cholesterol conjugate Chol9 with a 9 carbon
linker
[0069] In certain embodiments, a miR-29a mimic comprises a first
strand comprising SEQ ID NO: 27 and a second strand comprising SEQ
ID NO: 5. In other embodiments, a miR-29a mimic comprises a first
strand comprising SEQ ID NO: 7 and a second strand comprising SEQ
ID NO: 5.
[0070] In some embodiments, a miR-29b mimic comprises a first
strand comprising SEQ ID NO: 19 and a second strand comprising SEQ
ID NO: 1. In some other embodiments, a miR-29b mimic comprises a
first strand comprising SEQ ID NO: 2 and a second strand comprising
SEQ ID NO: 1. In yet some other embodiments, a miR-29b mimic
comprises a first strand comprising SEQ ID NO: 19 and a second
strand comprising SEQ ID NO: 15. In yet some other embodiments, a
miR-29b mimic comprises a first strand comprising SEQ ID NO: 33 and
a second strand comprising SEQ ID NO: 1. In yet some other
embodiments, a miR-29b mimic comprises a first strand comprising
SEQ ID NO: 34 and a second strand comprising SEQ ID NO: 1. In yet
some other embodiments, a miR-29b mimic comprises a first strand
comprising SEQ ID NO: 19 and a second strand comprising SEQ ID NO:
30.
[0071] In certain embodiments, a miR-29c mimic comprises a first
strand comprising SEQ ID NO: 35 and a second strand comprising SEQ
ID NO: 24. In other embodiments, a miR-29c mimic comprises a first
strand comprising SEQ ID NO: 26 and a second strand comprising SEQ
ID NO: 24.
[0072] The modified nucleotides that may be used in the microRNA
mimetic compounds of the invention can include nucleotides with a
base modification or substitution. The natural or unmodified bases
in RNA are the purine bases adenine (A) and guanine (G), and the
pyrimidine bases cytosine (C) and uracil (U) (DNA has thymine (T)).
In contrast, modified bases, also referred to as heterocyclic base
moieties, include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and
cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo (including
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines), 7-methylguanine and 7-methyladenine, 2-F-adenine,
2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
[0073] In some embodiments, the microRNA mimetic compounds can have
nucleotides with modified sugar moieties. Representative modified
sugars include carbocyclic or acyclic sugars, sugars having
substituent groups at one or more of their 2',3' or 4' positions
and sugars having substituents in place of one or more hydrogen
atoms of the sugar. In certain embodiments, the sugar is modified
by having a substituent group at the 2' position. In additional
embodiments, the sugar is modified by having a substituent group at
the 3' position. In other embodiments, the sugar is modified by
having a substituent group at the 4' position. It is also
contemplated that a sugar may have a modification at more than one
of those positions, or that an RNA molecule may have one or more
nucleotides with a sugar modification at one position and also one
or more nucleotides with a sugar modification at a different
position.
[0074] Sugar modifications contemplated in the miRNA mimetic
compounds include, but are not limited to, a substituent group
selected from: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl;
O--, S-- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,
alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to
C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl.
[0075] In some embodiments, miRNA mimetic compounds have a sugar
substituent group selected from the following: C.sub.1 to C.sub.10
lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl,
aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, Cl, Br, CN, OCN,
CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2,
NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, or similar substituents. In one embodiment, the
modification includes 2'-methoxyethoxy
(2'-O-CH.sub.2CH.sub.2OCH.sub.3, which is also known as
2'-O-(2-methoxyethyl) or 2'-MOE), that is, an alkoxyalkoxy group.
Another modification includes 2'-dimethylaminooxyethoxy, that is, a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE
and 2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), that is,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2.
[0076] Sugar substituent groups on the 2' position (2'-) may be in
the arabino (up) position or ribo (down) position. One 2'-arabino
modification is 2'-F. Other similar modifications may also be made
at other positions on the sugar moiety, particularly the 3'
position of the sugar on the 3' terminal nucleoside or in 2'-5'
linked oligonucleotides and the 5' position of 5' terminal
nucleotide.
[0077] In certain embodiments, the sugar modification is a
2'-O-alkyl (e.g. 2'-O-methyl, 2'-O-methoxyethyl), 2'-halo (e.g.,
2'-fluoro, 2'-chloro, 2'-bromo), and 4' thio modifications. For
instance, in some embodiments, the first strand of the miR-29a,
miR-29b, or miR-29c mimetic compound comprises one or more 2'
fluoro nucleotides. In another embodiment, the first strand of the
mimetic compounds has no modified nucleotides. In yet another
embodiment, the second strand of miR-29a, miR-29b, or miR-29c
mimetic compound comprises one or more 2'-O-methyl modified
nucleotides.
[0078] The first and the second strand of microRNA mimetic
compounds of the invention can also include 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 example, in some embodiments, the
nucleotides comprising the 3' overhang in the first strand are
linked by phosphorothioate linkages.
[0079] In some embodiments, the microRNA mimetic compounds are
conjugated to a carrier molecule such as a steroid (cholesterol), a
vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or
other small molecule ligand to facilitate in vivo delivery and
stability. Preferably, the carrier molecule is attached to the
second strand of the microRNA mimetic compound at its 3' or 5' end
through a linker or a spacer group. In various embodiments, the
carrier molecule is cholesterol, a cholesterol derivative, cholic
acid or a cholic acid derivative. The use of carrier molecules
disclosed in U.S. Pat. No. 7,202,227, which is incorporated by
reference herein in its entirety, is also envisioned. In certain
embodiments, the carrier molecule is cholesterol and it is attached
to the 3' or 5' end of the second strand through at least a six
carbon linker. In one embodiment, the carrier molecule is attached
to the 3' end of the second strand through a six or nine carbon
linker. In some embodiments, the linker is a cleavable linker. In
various embodiments, the linker comprises a substantially linear
hydrocarbon moiety. The hydrocarbon moiety may comprise from about
3 to about 15 carbon atoms and may be conjugated to cholesterol
through a relatively non-polar group such as an ether or a
thioether linkage. In certain embodiments, the hydrocarbon
linker/spacer comprises an optionally substituted C2 to C15
saturated or unsaturated hydrocarbon chain (e.g. alkylene or
alkenylene). A variety of linker/spacer groups described in U.S.
Pre-grant Publication No. 2012/0128761, which is incorporated by
reference herein in its entirety, can be used in the present
invention.
[0080] In various embodiments, the present invention provides
methods of treating, ameliorating, or preventing fibrotic
conditions in a subject in need thereof comprising administering to
the subject a therapeutically effective amount of at least one of a
miR-29a, miR-29b, and/or miR-29c mimic described herein. Fibrotic
conditions that may be treated using miR-29 mimics of the invention
include, but are not limited to, tissue fibrosis such as pulmonary
fibrosis, cardiac fibrosis, hepatic fibrosis, kidney fibrosis,
diabetic fibrosis, skeletal muscle fibrosis, and ocular fibrosis;
and dermal/cutaneous fibrosis such as keloids, cutaneous sclerosis,
systemic sclerosis (scleroderma), hypertrophic scars,
hand/joint/tendon fibrosis, and Peyronie's disease. In one
embodiment, the fibrotic condition treated using the miR-29 mimics
of the invention is idiopathic pulmonary fibrosis. Use of miR-29
agonists in treating certain fibrotic conditions is described in
U.S. Pat. No. 8,440,636, which is hereby incorporated by reference
herein.
[0081] In one embodiment, administration of miR-29 mimics of the
present invention reduces the expression or activity one or more
extracellular matrix genes in cells of the subject. In another
embodiment, administration of miR-29 mimics of the present
invention reduces the expression or activity one or more collagen
synthesis genes in cells of the subject. In yet another embodiment,
administration of miR-29 mimics up-regulates the expression or
activity one or more genes involved in the skin development,
epidermis development, ectoderm development and cellular
homeostasis. Cells of the subject where the expression or activity
of various genes is regulated by miR-29 mimics of the invention
include fibroblasts and epidermal cells. In some embodiments,
administration of miR-29 mimics down-regulates inflammatory
responses associated with fibrosis. For example, administration of
miR-29 mimics reduces the levels of pro-inflammatory cytokines such
as IL-12, IL-4, GCSF, and TNF-a in fibrosis patients.
Administration of miR-29 mimics may also reduce infiltration of
immune effector cells such as neutrophils, lymphocytes, monocytes,
and macrophages in fibrotic tissues or organs.
[0082] In certain embodiments, the present invention provides
methods of regulating an extracellular matrix gene in a cell
comprising contacting the cell with a miR-29 mimic of the present
invention. In some embodiments, the invention provides methods of
regulating a collagen synthesis gene in a cell comprising
contacting the cell with a miR-29 mimic of the present invention.
Upon treatment or contact, the miR-29 mimic reduces the expression
or activity of the extracellular matrix gene or the collagen
synthesis gene.
[0083] As used herein, the term "subject" or "patient" refers to
any vertebrate including, without limitation, humans and other
primates (e.g., chimpanzees and other apes and monkey species),
farm animals (e.g., cattle, sheep, pigs, goats and horses),
domestic mammals (e.g., dogs and cats), laboratory animals (e.g.,
rabbits, rodents such as mice, rats, and guinea pigs), and birds
(e.g., domestic, wild and game birds such as chickens, turkeys and
other gallinaceous birds, ducks, geese, and the like). In some
embodiments, the subject is a mammal. In other embodiments, the
subject is a human.
[0084] The invention also provides methods for assessing the
efficacy of a treatment with miR-29 agonists (e.g. drugs or miR-29
mimics) or miR-29 antagonists (e.g. drugs or antimiR-29). For
instance, in one embodiment, the method for assessing the treatment
efficacy comprises determining a level of expression of one or more
genes in cells or a fibrotic tissue of a subject prior to the
treatment with miR-29 mimics or miR-29 antagonists, wherein the one
or more genes are selected from a set of genes modulated by miR-29,
e.g. genes listed in Table 5; determining the level of expression
of the same one or more genes in cells/fibrotic tissue of the
subject after treatment with miR-29 mimics or miR-29 antagonist;
and determining the treatment to be effective, less effective, or
not effective based on the expression levels prior to and after the
treatment. That is, in one embodiment, the genes listed in Table 5
serve as a biomarker for clinical efficacy of the miR-29 mimic or
miR-29 antagonist treatment. In one embodiment, a statistically
significant difference in the expression of the genes prior to and
after treatment indicates the treatment to be effective. In another
embodiment, at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold,
3.5-fold, or 4-fold difference in the expression of the genes prior
to and after treatment indicates the treatment to be effective.
[0085] The present invention also provides pharmaceutical
compositions comprising a therapeutically effective amount of one
or more microRNA mimetic compounds of miR-29a, miR-29b, and/or
miR-29c according to the invention or a pharmaceutically acceptable
salt thereof, and a pharmaceutically acceptable carrier or
excipient.
[0086] In one embodiment, the pharmaceutical composition comprises
a therapeutically effective amount of a miR-29a mimetic compound
and a pharmaceutically acceptable carrier or excipient, wherein the
first strand of the mimetic compound comprises a mature miR-29a
sequence and the second strand is substantially complementary to
the first strand. In another embodiment, the pharmaceutical
composition comprises a therapeutically effective amount of a
miR-29b mimetic compound and a pharmaceutically acceptable carrier
or excipient, wherein the first strand of the mimetic compound
comprises a mature miR-29b sequence and the second strand is
substantially complementary to the first strand. In yet another
embodiment, the pharmaceutical composition comprises a
therapeutically effective amount of a miR-29c mimetic compound and
a pharmaceutically acceptable carrier or excipient, wherein the
first strand of the mimetic compound comprises a mature miR-29c
sequence and the second strand is substantially complementary to
the first strand.
[0087] In some embodiments, the pharmaceutical composition
comprises a therapeutically effective amount of at least two
microRNA mimetic compounds of the invention and a pharmaceutically
acceptable carrier or excipient. For instance, a pharmaceutical
composition may comprise a combination of a miR-29a and a miR-29b
mimics; a miR-29a and a miR-29c mimics; or a miR-29b and a miR-29c
mimics. Alternatively, the composition may comprise two mimics of
the same microRNA. In yet some other embodiments, the invention
provides pharmaceutical compositions comprising a therapeutically
effective amount of three microRNA mimetic compounds of the
invention and a pharmaceutically acceptable carrier or excipient.
For instance, a pharmaceutical composition may comprise a
combination of a miR-29a, a miR-29b and a miR-29c mimics.
[0088] Preferably, in the pharmaceutical compositions comprising at
least two microRNA mimetic compounds according to the invention,
the first and the second mimetic compounds or the first, second and
the third mimetic compounds are present in equimolar
concentrations. Other mixing ratios such as about 1:2, 1:3, 1:4,
1:5, 1:2:1, 1:3:1, 1:4:1, 1:2:3, 1:2:4 are also envisioned for
preparing pharmaceutical compositions comprising at least two of
the miR-29a, miR-29b, and miR-29c mimetic compounds.
[0089] In some embodiments, one or more microRNA mimetic compounds
of the invention may be administered concurrently but in separate
compositions, with concurrently referring to mimetic compounds
given within a short period, for instance, within about 5, 10, 20,
or 30 minutes of each other. In some other embodiments, miR-29a,
miR-29b, and/or miR-29c mimetic compounds may be administered in
separate compositions at different times.
[0090] The invention also encompasses embodiments where additional
therapeutic agents may be administered along with miR-29a, miR-29b,
and/or miR-29c mimetic compounds. In one embodiment, the additional
therapeutic agent is a second anti-fibrotic agent. The additional
therapeutic agents may be administered concurrently but in separate
formulations or sequentially. In other embodiments, additional
therapeutic agents may be administered at different times prior to
after administration of miR-29a, miR-29b, and/or miR-29c mimetic
compounds. 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.
[0091] Colloidal dispersion systems, such as macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
liposomes and exosomes, may be used as delivery vehicles for
miR-29a, miR-29b, and/or miR-29c mimetic compounds. In some
embodiments, miR-29 mimics of the present invention may be
formulated into liposome particles, which can then be aerosolized
for inhaled delivery.
[0092] Commercially available fat emulsions that are suitable for
delivering the nucleic acids of the invention to target 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.
[0093] In certain embodiments, liposomes used for delivery are
amphoteric liposomes such SMARTICLES.RTM. (Marina Biotech, Inc.)
which are described in detail in U.S. Pre-grant Publication No.
20110076322. The surface charge on the SMARTICLES.RTM. is fully
reversible which make them particularly suitable for the delivery
of nucleic acids. SMARTICLES.RTM. can be delivered via injection,
remain stable, and aggregate free and cross cell membranes to
deliver the nucleic acids.
[0094] 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
miR-29 mimic (e.g. liposomes or other complexes) 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 polynucleotides of the compositions.
[0095] In one embodiment, pharmaceutical compositions of the
invention are formulated for pulmonary, nasal, intranasal or ocular
delivery and can be in the form of powders, aqueous solutions,
aqueous aerosols, nasal drops, aerosols, and/or ocular drops. Solid
formulations for nasal/intranasal administration may contain
excipients such as lactose or dextran. Liquid formulations for
nasal/intranasal administration may be aqueous or oily solutions
for use in the form of aerosols, nasal drops or metered spray.
Formulations for pulmonary/nasal/intranasal administration may also
include surfactants such as, for example, glycocholic acid, cholic
acid, taurocholic acid, ethocholic acid, deoxycholic acid,
chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid,
salts of these acids, and cyclodextrins.
[0096] In some embodiments, formulations for
pulmonary/nasal/intranasal administration via inhalation include,
but are not limited to a dry powder formulation, a liposomal
formulation, a nano-suspension formulation, or a microsuspension
formulation.
[0097] In some embodiments, pharmaceutical compositions for
pulmonary/nasal/intranasal delivery are administered using an
inhalation device. The term "inhalation device" refers to any
device that is capable of administering a miR-29 mimic composition
to the respiratory airways of the subject. Inhalation devices
include devices such as metered dose inhalers (MDIs), dry powder
inhalers (DPIs), jet nebulizers, ultrasonic wave nebulizers, heat
vaporizers, soft mist inhalers, thermal aerosol inhalers,
electrohydrodynamic-based solution misting inhaler. Inhalation
devices also include high efficiency nebulizers. In some
embodiments, a nebulizer is a jet nebulizer, an ultrasonic
nebulizer, a pulsating membrane nebulizer, a nebulizer comprising a
vibrating mesh or plate with multiple apertures, a nebulizer
comprising a vibration generator and an aqueous chamber, or a
nebulizer that uses controlled device features to assist
inspiratory flow of the aerosolized aqueous solution to the lungs
of the subject. Nebulizers, metered dose inhalers, and soft mist
inhalers deliver pharmaceuticals by forming an aerosol which
includes droplet sizes that can easily be inhaled.
[0098] In some embodiments, a composition administered with a high
efficiency nebulizer comprises one or more miR-29 mimics and
pharmaceutically acceptable excipients or carriers such as purified
water, mannitol, surfactants, and salts such as sodium chloride and
sodium EDTA, etc.
[0099] 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 (e.g. inhalational), ocular, or
buccal. Alternatively, administration may be by intravenous,
intradermal, subcutaneous, intraocular or intramuscular injection,
or by direct injection into pulmonary or cardiac tissue.
Pharmaceutical compositions comprising miRNA mimics 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.
[0100] In another embodiment of the invention, compositions
comprising miR-29 mimics as described herein may be formulated as a
coating for a medical device, such as a stent, balloon, or
catheter. Particularly useful in methods of treating cardiac
fibrosis in a subject, the miR-29 mimics can be used to coat a
metal stent to produce a drug-eluting stent. A drug-eluting stent
is a scaffold that holds open narrowed or diseased arteries and
releases a compound to prevent cellular proliferation and/or
inflammation. The mimetic compounds may be applied to a metal stent
imbedded in a thin polymer for release of the agonists or
inhibitors over time. Methods for device-based delivery and methods
of coating devices are well known in the art, as are drug-eluting
stents and other implantable devices. See, e.g., U.S. Pat. Nos.
7,294,329, 7,273,493, 7,247,313, 7,236,821, 7,232,573, 7,156,869,
7,144,422, 7,105,018, 7,087,263, 7,083,642, 7,055,237, 7,041,127,
6,716,242, and 6,589,286, and WO 2004/004602, which are herein
incorporated by reference in their entireties. Thus, the present
invention includes a medical device, such as a balloon, catheter,
or stent, coated with a miR-29 mimic.
[0101] 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.
[0102] 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).
[0103] 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, drug-eluting stents or other
coated vascular devices, 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, intradermal, intraocular, and
intraperitoneal administration.
[0104] This invention is further illustrated by the following
additional examples that should not be construed as limiting. Those
of skill in the art should, in light of the present disclosure,
appreciate that many changes can be made to the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
[0105] All patent and non-patent documents referenced throughout
this disclosure are incorporated by reference herein in their
entirety for all purposes.
EXAMPLES
Example 1
In Vitro Activity of miR-29 mimic
[0106] To test for functional efficacy, a miR-29b mimic containing
SEQ ID NO: 2 as the first strand and SEQ ID NO: 1 as the second
strand was transfected into a mouse fibroblast cell line (NIH 3T3)
and the effect on Collagen1a1 (Col1a1) expression, a known direct
target gene of miR-29 (van Rooij et al, 2008), was measured using
qPCR. Increasing amount of miR-29b mimic showed a dose-dependent
decrease in Col1a1 , compared to untreated, mock transfected
(without any oligo) or non-targeting control (NTC) oligo treated
cells, indicating the miR-29b mimic to be functional. A siRNA
directly targeting Col1a1 was used as a positive control (FIG.
1B).
Example 2
In Vivo Distribution, Stability and Clearance of miR-29 mimic
[0107] To explore the in vivo applicability and distribution of the
miR-29 mimic, mice were injected intravenously with 10, 50, 100, or
125 mg per kg of the miR-29b mimic containing SEQ ID NO: 2 as the
first strand and SEQ ID NO: 1 as the second strand and sacrificed
four days later.
[0108] Total RNA was isolated from cardiac tissue samples by using
TRIzol.RTM. reagent (solution of phenol and guanidine
isothiocyanate) (Gibco/BRL). Northern blots to detect microRNAs
were performed as previously described (van Rooij et al, 2008). A
U6 probe served as a loading control (IDT). 10 .mu.g of total RNA
from the indicated tissues was loaded on 20% acrylamide denaturing
gels and transferred to Zeta-probe GT genomic blotting membranes
(Bio-Rad) by electrophoresis. After transfer, the blots were
cross-linked and baked at 80.degree. C. for 1 hr. To maximize the
sensitivity of miRNA detection, oligonucleotide probes were labeled
with the Starfire Oligos Kit (IDT, Coralville, Iowa) and
.alpha.-.sup.32P dATP (Amersham or Perkin Elmer). Probes were
hybridized to the membranes overnight at 39.degree. C. in Rapid-hyb
buffer (Amersham), after which they were washed twice for 10
minutes at 39.degree. C. with 0.5.times.SSC containing 0.1% SDS.
The blots were exposed and quantified by Phosphorlmager analysis
(GE HealthCare Life Sciences) and a U6 probe served as a loading
control (ABI). The intensity of the radioactive signal was used to
quantify the fold change in expression using a phosphorimager and
ImageQuant (Bio-Rad).
[0109] For real-time PCR analysis, RNA was extracted from cardiac
tissue using Trizol (Invitrogen) after which one to two .mu.g RNA
from each tissue sample was used to generate cDNA using Super
Script II reverse transcriptase per manufacturer's specifications
(Invitrogen). Taqman MicroRNA assay (Applied Biosystems, ABI) was
used to detect changes in miRNAs or genes according the
manufacturer's recommendations, using 10-100 ng of total RNA. U6
was used a control for miRNA analysis and GAPDH was used as a
control for gene analysis.
[0110] Northern blot analysis on multiple tissues indicated little
to no increase in miR-29b in kidney or liver samples compared to
saline control. Cardiac distribution was detected; however this
appeared to be quite variable and spleen delivery could be observed
at the highest dose only. However, delivery to the lungs could be
observed at all 3 of the highest doses four days after injection
(FIG. 1C). No effects on liver function (transaminase, ALT) were
observed in the plasma, indicating that these miRNA mimics are well
tolerated at these doses (FIG. 5). Real-time PCR demonstrated
similar results with robust dose-dependent distribution of the
miR-29b mimic to the lung compared to saline injected animals (FIG.
1D). Additionally, real-time PCR analysis of miR-29 targets showed
no regulation at the mRNA level in the treated animals except for
Col3a1 at the highest dose in the spleen (FIGS. 6A-B). This
suggests that the target genes are either at steady-state in
non-stressed animals and that mimics lower target genes when they
are elevated, or that functional delivery was inadequate or
insufficient.
[0111] To gain more insights into the in vivo stability of miRNA
mimics, 125 mpk of the miR-29b mimic was injected into mice and
they were sacrificed 1, 2, 4, or 7 days later. Robust presence of
miR-29b mimic could be detected by both Northern blot and real-time
PCR analysis one day after injection in all tissues examined,
however tissue clearance greatly differed thereafter (FIGS. 1E and
F). Liver and kidney rapidly cleared miR-29b mimic with minimal
detection after day 1. Lung and spleen demonstrated the most
pronounced detection of miR-29b mimic over time, which was
sustained at least 4 days post-treatment (FIGS. 1E and 1F). The
increase was specific for miR-29b without any effect on miR-29a and
miR-29c levels as measured by real-time PCR (FIGS. 7A-B). Also here
real-time PCR analysis of miR-29 targets showed no downregulation
at the mRNA level in non-stressed animals (FIGS. 8A-B).
[0112] Together these data indicate that unformulated miR-29b mimic
can increase the miRNA level with tissue-dependent clearance and
delivery efficiency, without any clear effect on gene expression
under baseline conditions.
Example 3
miR-29b Mimic Blunts Bleomycin-Induced Pulmonary Fibrosis
[0113] Current treatments of tissue fibrosis mostly rely on
targeting the inflammatory response; however these treatments are
ultimately ineffective in preventing progression of the disease,
underscoring the need for new mechanistic insights and therapeutic
approaches (Friedman et al, 2013). Recent studies indicate the
involvement of miRNAs in pulmonary fibrosis (Pandit et al,
2011).
[0114] Due to the preferential lung distribution of the miR-29b
mimic, the question of whether stress and subsequent induction of
target gene expression would allow for detectable changes in mRNA
target genes and downstream therapeutic effects in response to
treatment with miR-29b mimic was explored. To test this, the
bleomycin-induced model of pulmonary fibrosis was used as
previously described (Pandit et al, 2010). Specifically, mice were
anesthetized by placing them in a chamber having paper towels
soaked with 40% isoflurane solution. 0.0375 U of bleomycin
(Hospira, Ill.) was administered intratracheally in 50 .mu.l of
0.9% saline. To determine the effect of miR-29b mimicry on early
fibrosis, control (saline-treated) and bleomycin-treated mice were
injected with 100 mg per kg of the miR-29b mimiccontaining SEQ ID
NO: 2 as the first strand and SEQ ID NO: 1 as the second strand,
control mimic or a comparable volume of saline at two time-points:
3 and 10 days after bleomycin treatment; the animals were
sacrificed and lungs harvested at day 14. To determine the effect
of miR-29b mimicry on established fibrosis, the miR-29b mimic was
administered at days 10, 14 and 17 after bleomycin or saline and
the mice were sacrificed at day 21. In both protocols, the lungs
were harvested for histological analysis, hydroxyproline assay and
RNA extraction.
[0115] As expected, 14 days after bleomycin treatment, miR-29
levels were reduced, while miR-29b mimic treatment resulted in the
increased detection of miR-29b levels compared to either control or
saline injected animals as measured by real-time PCR, albeit with a
high level of variation (FIG. 2A). To determine whether a similar
decline in miR-29 levels is observed in humans, sixteen lung tissue
samples were obtained from surgical remnants of biopsies or lungs
explanted from patients with idiopathic pulmonary fibrosis (IPF)
who underwent pulmonary transplantation. Samples were obtained from
University of Pittsburgh Health Sciences Tissue Bank. A comparable
decline in miR-29 levels was observed in pulmonary biopsies of
patients with idiopathic pulmonary fibrosis (IPF) compared to
normal controls (FIG. 2B).
[0116] For histological examination, lung tissue sections (4 .mu.m)
were stained with Masson Trichrome (collagen/connective tissue),
two slices per animal, two animals per group. Immune staining was
performed after paraffin removal, hydration, and blocking,
following the recommendation of the manufacturer (ABC detection
system form Vector's lab, USA). Sections were incubated overnight
at 4.degree. C. with the primary antibody (Igf1, diluted 1:100 in
PBS) and during 1 hour at room temperature with the secondary
antibodies (Invitrogen, USA). The sections were counterstained with
hematoxylin. The primary antibody was replaced by nonimmune serum
for negative controls. Finally, sections were mounted with mounting
medium (DAKO, USA) and analyzed using a Nikon microscope.
Histological analysis showed a clear and robust fibrotic and
inflammatory response to bleomycin treatment, which was blunted by
miR-29b mimic treatment (FIG. 2C).
[0117] Additionally, lung hydroxyproline was analyzed for total
collagen content with hydroxyproline colorimetric assay kit from
Biovision (Milpitas, CA) following manufacturer's instruction.
Briefly, the lungs from control and experimental mice were dried
until constant weight and hydrolyzed in 12N HCl for 3 hours at
120.degree. C. The digestions reacted with Chloramine T reagent and
visualized in DMAB reagent. The absorbance was measured at 560 nm
in a microplate reader. Data are expressed as .mu.g of
hydroxyproline/right lung.
[0118] The hydroxyproline analysis indicated a significant increase
following bleomycin treatment in both saline and control treated
groups, while there was no statistical difference in the miR-29
mimic treated group between saline and bleomycin-treated mice,
indicating that miR-29b mimic treatment blunts bleomycin-induced
pulmonary fibrosis (FIG. 2D).
[0119] Innate immune effector signaling pathways act as important
drivers of myofibroblast transdifferentiation by provoking
fibrosis. To further characterize the therapeutic effects of
miR-29b mimic in the setting of bleomycin-induced pulmonary
fibrosis, bronchoalveolar lavage (BAL) was performed on these mice
and cytokine levels were assessed using a human Cytokine/Chemokine
Panel from Bio-Rad. The entire procedure was performed following
manufacturer's instruction. Briefly, BALs were diluted five-fold
and assay was performed in 96-well filter plates. For the detection
step, samples were incubated for 30 min with streptavidin
conjugated to R-phycoerythrin and analyzed in the Bio-Plex
suspension array system (Bio-Rad). Raw data was analyzed using
Bioplex Manager software 6.0 (Bio-Rad). The cytokine standards
supplied by the manufacturer were used to calculate the
concentrations of the samples. The analytes that were below the
detection range were not included in date interpretation. Also,
samples that had a particular analyte below the detection range
were excluded while calculating the median value.
[0120] Significantly higher concentrations of IL-12, IL-4 and G-CSF
were detectable in BAL fluids from lungs from bleomycin-treated
mice, which were reduced with miR-29b mimic (FIGS. 2E to 2G).
Additionally, the bleomycin-induced elevation of detectable immune
cells in BAL fluids was significantly reduced in the presence of
miR-29b mimic (FIG. 2H), indicating an inhibitory effect on the
immune response by miR-29b, which is likely secondary to the
antifibrotic-effect. To determine if miR-29 mimicry has a direct
effect on macrophages, miR-29b mimic or control was transfected
into macrophage cells, RAW 264.7, and the cell supernatant was
harvested at 24 and 48 hours after transfection. IFN-r, IL-1B,
IL-2, IL-4, IL-5, IL-6, KC, IL-10, IL-12P70, and TNF-a were
assessed, with no significant differences observed between miR-29b
mimic and control (data not shown). By real-time PCR analysis,
there were no significant differences in Tgfb1, Ctgf, FGF1, or PDGF
expression; however, a significant difference in Csf3, Igf1, and Kc
expression was observed (FIG. 9 and data not shown).
[0121] Since it has been well validated that miR-29 functions
through the regulation of many different extracellular matrix
related genes (van Rooij & Olson, 2012), the regulation of a
subset of these target genes was confirmed. While a significant
increase in Col1a1 and a trend increase in Col3a1 expression were
observed with bleomycin treatment in both saline and
control-treated groups, the detection of Col1a1 and Col3a1 was
significantly blunted in the presence of miR-29b mimic in the
bleomycin-treated mice (FIGS. 3A and 3B). Interestingly, the
increase in Igf1 levels in BAL fluids following bleomycin treatment
were significantly blunted in the presence of miR-29 mimic compared
to both saline and control-treated mice (FIG. 3C). Furthermore,
immunohistochemistry for Igf1 demonstrated robust reductions in
Igf1 after bleomycin in miR-29b mimic-treated groups compared to
saline or controls (FIG. 3D).
[0122] After establishing that early (day 3 and day 10) miR-29
mimicry was sufficient to prevent bleomycin induced fibrosis, the
ability of miR-29 mimicry to affect established fibrosis was
investigated. For that purpose, the miR-29b mimic administration
was started at day 10 post bleomycin, and the doses were repeated
at days 14 and 17, after which the lungs were harvested at day 21.
Hydroxyproline assessment of the right lung showed a significant
increase with bleomycin in both saline and control-treated lungs;
however miR-29b mimic treatment blunted this effect (FIG. 4A).
Furthermore, bleomycin treatment resulted in significant increases
in Col1a1 and Col3a1 expression, which was also normalized with
miR-29b mimic treatment (FIGS. 4B and 4C). Histological assessment
by trichrome staining corroborated this effect, whereby bleomycin
induced significant fibrosis with saline or control treatment which
was blunted with miR-29b mimicry (FIG. 4D).
[0123] While it was believed that these observed effects were
mediated through regulation of collagen production from lung
fibroblasts, the effects from other collagen producing cells could
not be ruled out. To address this issue, miR-29b mimic effects were
assessed in vitro from different lung cells, including primary
fibroblasts from IPF patients and A549 cells, a lung epithelial
cell line. As expected, primary pulmonary fibroblasts from IPF
patients show an increase in Col1a1 and Col3a1 in response to
TGF-.alpha. (FIGS. 4E and 4F). This effect was dose-dependently
blunted with miR-29b mimic treatment at both 24 and 48 hours (FIGS.
4E, 4F and data not shown). Similarly, A549 cells respond to TGF-a
with robust increases in Col1a1 and Col3a1 expression (FIGS. 4G and
4H). Again, miR-29b mimic treatment is able to block collagen
induction, in both TGF-a treated as well as baseline conditions
(FIGS. 4G and 4H). The effects on collagen induction are much more
robust in the A549 cells compared to primary IPF cells. However,
this is likely due to the already high expression in primary
fibroblasts from IPF patients. Additionally, miR-29 effects in the
macrophage line, Thp-1, were also examined, but no collagen
expression could be observed in the cells, regardless of
stimulation (data not shown). These data suggest miR-29b mimicry is
able to blunt collagen-induced expression in fibroblasts and
epithelial cells. These data are in agreement with a paper by Xiao
et al., in which they showed that gene transfer of miR-29 using a
Sleeping Beauty-transposon system was capable of preventing and
treating bleomycin-induced pulmonary fibrosis (Xiao et al, 2012),
further underscoring the therapeutic potential for increasing
miR-29.
[0124] The data described herein shows the feasibility of using
microRNA mimics to restore the function of lost or down-regulated
miRNAs. However, it is important to note that because RISC
incorporation is required for appropriate miRNA function, careful
design of the structural features of the synthetic oligonucleotide
mimics is required. In addition, the data in the present
application shows that delivery to the appropriate cell type or
tissue provides effective miRNA mimicry. For instance, in the case
of pulmonary fibrosis, direct delivery through the inhaled route
provides better treatment efficacy compared to traditional routes
of administration.
Example 4
miR-29b Mimic Blunts Extracellular Matrix Production in the
Skin
[0125] In addition to organ fibrosis, a number of studies have
shown a role for miR-29 in dermal fibrosis such as in hypertrophic
scars and keloids, and in cutaneous and other forms of systemic
sclerosis (scleroderma). For example, fibroblasts and skin sections
obtained from patients with systemic sclerosis showed a dramatic
reduction in miR-29a levels compared to healthy controls (Maurer et
al 2010). When overexpressed in systemic sclerosis fibroblasts,
miR-29a was able to robustly reduce the expression of type I and
III collagens, at both the RNA and protein level. Conversely,
inhibition of miR-29 in normal fibroblasts resulted in an increase
in these collagens. Further, bleomycin-treated skin showed a
significant reduction in miR-29 as well, suggesting miR-29
down-regulation is broadly applicable in multiple indications of
fibrosis (Maurer et al 2010). These results were further validated
in another study where miR-29a transfection in healthy control
dermal fibroblasts significantly down-regulated collagen
expression. Additionally, miR-29a overexpression in dermal
fibroblasts decreased secreted TIMP-1 and increased collagen gel
degradation. These results were validated in fibroblasts from
patients with systemic sclerosis as well (Ciechomska et al 2014).
Collectively, these studies point to miR-29 mimicry as a
therapeutic potential in local forms of fibrosis, including dermal
fibrosis and systemic sclerosis.
[0126] To determine the effect of miR-29 agonism or antagonism in
skin, a mouse incisional wound model was used. Specifically, male
C57BL/6 mice were anesthetized with 2-5% inhaled isofluorane using
an inhalation chamber and were maintained under 1% isofluorane on a
nose cone for all procedures. Buprenorphine (0.1 mg/kg) was used as
an analgesic for all surgical procedures. Animals were
anesthetized, depilated by shaving and administration of Nair hair
removal cream, and the skin site was prepared for incision by
betadine and alcohol surgical scrub. One to two 1 cm long skin
incisions were made on the backs of the mice. Incisions were closed
with 2-5 interrupted sutures and were covered with Tegaderm
transparent semi-occlusive dressing (3M). Mice with or without
incisional skin wounds were treated with intradermal injection with
vehicle (PBS), 100 nmol miR-29b mimic (comprising SEQ ID NO: 2 as
the first strand and SEQ ID NO: 1 as the second strand) or 100 nmol
antimiR-29, 3 days after incisional wound creation, by intradermal
injection of oligonucleotide compounds or vehicle (PBS) in two 50
.mu.L volumes located on either side of the incision midline, or by
injection of a single 100 volume into adjacent, unwounded skin. The
antimiR-29 has the sequence of
5'-lGs.dAs.dTs.dTs.lTs.lCs.dAslAs.dAs.lTs.lGs.dGs.lTs.dGs.lCs.lTs-3'
(SEQ ID NO: 36) where "l" represents a locked nucleotide, "d"
represents a deoxyribonucleotides and "s" represents a
phosphorothioate linkage. Mice were sacrificed 24 hours after
administration of oligonucleotide compounds and skin at the
treatment site(s) was harvested and snap frozen in liquid nitrogen
for analysis of mRNA expression.
[0127] Total RNA was extracted from skin using Trizol (Invitrogen)
and real-time PCR analysis was performed as described above except
that GAPDH, B2M, HPRT1 and PPIB were used as controls for gene
analysis of skin samples.
[0128] For microarray analysis, one .mu.g total RNA per sample was
sent to MOgene for microarray analysis as compared to a mouse
universal reference RNA (Agilent) on an Agilent array (Mouse GE
(V2), 4.times.44k-026655). Analysis was performed using ArrayStudio
and heatmaps were generated using the R software program.
[0129] Using microarray analysis, positively and negatively
regulated genes were identified relative to PBS controls. 228 genes
were reciprocally regulated (p<0.05 or fold change >1.5)
between the miR-29b mimic and antimiR-29 (FIG. 10 and Table 5).
These miR-29 regulated genes and their orthologs in other species
may be utilized as translational biomarkers to indicate response to
treatment with a miR-29 agonist or miR-29 antagonist.
TABLE-US-00005 TABLE 5 miR-29b mimic antimiR-29 Gene Fold change p
value Fold change p value Cytl1 -3.21 0.000 1.53 0.034 Col3a1 -2.68
0.074 1.61 0.352 Col1a1 -2.43 0.051 2.16 0.081 Col1a2 -2.38 0.028
2.26 0.036 Fstl1 -2.25 0.015 2.15 0.019 Col5a2 -2.19 0.005 1.95
0.012 4930543N07Rik -2.13 0.005 3.10 0.000 Faim2 -2.13 0.083 2.42
0.049 Tmem213 -2.06 0.047 2.39 0.022 Tgfb3 -1.99 0.002 -1.29 0.145
Lzts1 -1.97 0.001 1.43 0.017 Eln -1.95 0.004 1.14 0.472 Slc5a2
-1.94 0.018 2.09 0.010 Tnp2 -1.93 0.073 4.05 0.002 Olfr1336 -1.87
0.001 1.75 0.002 Tdrd9 -1.82 0.113 2.85 0.014 Gm6602 -1.81 0.194
2.83 0.038 F830016B08Rik -1.78 0.098 2.16 0.037 Myo3b -1.78 0.034
1.82 0.030 Colec11 -1.77 0.010 1.80 0.009 Gm10428 -1.75 0.010 1.94
0.005 Vmn1r65 -1.70 0.042 3.21 0.001 Olfr67 -1.69 0.003 1.37 0.034
Bsnd -1.69 0.107 2.05 0.038 Slc10a5 -1.69 0.038 1.65 0.045 Defa26
-1.68 0.076 2.06 0.022 Serpinh1 -1.68 0.124 2.01 0.049 Gm5606 -1.67
0.018 1.77 0.011 Wfdc11 -1.67 0.252 2.64 0.048 Gimap7 -1.65 0.091
1.92 0.036 Nedd4l -1.64 0.001 1.34 0.012 Cacna1g -1.63 0.094 1.81
0.049 Prickle1 -1.63 0.001 1.25 0.036 4931415C17Rik -1.62 0.182
2.94 0.012 D730005E14Rik -1.61 0.156 2.30 0.025 Ccr10 -1.60 0.014
1.50 0.028 Gm22 -1.60 0.003 2.56 0.000 Ngp -1.60 0.075 1.83 0.030
Ascl1 -1.60 0.026 1.78 0.010 Tgfb2 -1.60 0.016 1.08 0.618 Cyp2c29
-1.59 0.009 1.42 0.030 Gm5797 -1.59 0.011 1.49 0.021 Col5a3 -1.59
0.035 1.05 0.779 A730093L10Rik -1.59 0.060 1.69 0.039 Fkbp10 -1.57
0.027 1.57 0.028 Mfap2 -1.56 0.032 1.83 0.008 Gm5485 -1.55 0.064
5.68 0.000 Slamf9 -1.54 0.204 2.08 0.048 Mab21l3 -1.54 0.005 1.53
0.006 Fam57b -1.53 0.008 1.50 0.010 Pcolce -1.53 0.091 1.62 0.060
Gm6760 -1.52 0.215 2.26 0.031 Gng13 -1.51 0.073 2.07 0.006
4933404M02Rik -1.50 0.361 3.49 0.018 C1qtnf6 -1.50 0.032 1.50 0.031
Tmem119 -1.49 0.019 1.46 0.023 Ubtd2 -1.49 0.001 1.48 0.001 Rasl11b
-1.48 0.045 1.77 0.009 Nr5a2 -1.47 0.011 1.36 0.031 Gm3727 -1.46
0.018 1.43 0.024 Gprasp2 -1.46 0.044 1.93 0.003 Syt10 -1.45 0.015
2.08 0.000 Otog -1.44 0.036 1.65 0.009 Bdh2 -1.43 0.011 1.81 0.001
Sema3b -1.42 0.045 1.56 0.017 Al118078 -1.42 0.032 1.69 0.005
Npc1l1 -1.41 0.032 1.40 0.036 Dnmt3a -1.41 0.015 1.30 0.046 Cxcr6
-1.40 0.024 1.99 0.000 Sh3pxd2a -1.37 0.044 1.42 0.029 Scarf2 -1.35
0.004 1.67 0.000 LOC100862627 -1.34 0.006 1.45 0.002 Selm -1.34
0.015 1.42 0.006 Col11a1 -1.34 0.509 -1.02 0.958 Slc12a5 -1.34
0.007 1.29 0.013 Pex11c -1.33 0.000 1.24 0.002 Gpr176 -1.32 0.041
1.68 0.002 Qprt -1.32 0.037 1.29 0.050 Phldb2 -1.31 0.042 1.52
0.006 Prl2c1 -1.31 0.007 1.37 0.003 Rab39 -1.30 0.018 1.61 0.001
Dact3 -1.30 0.003 1.60 0.000 Dlx3 -1.26 0.010 1.19 0.037 Sepw1
-1.25 0.021 1.26 0.017 Socs7 -1.25 0.019 1.22 0.030 Maged1 -1.24
0.008 1.26 0.006 Ckb -1.22 0.035 1.24 0.026 Mmp2 -1.21 0.288 1.55
0.031 Nfatc4 -1.20 0.043 1.21 0.039 Gm13623 -1.20 0.032 1.51 0.000
Trp53i13 -1.19 0.025 1.32 0.002 Lysmd4 -1.17 0.015 1.23 0.004
Polr2m -1.17 0.000 1.12 0.002 Pkd1 -1.17 0.041 1.17 0.035 Zdhhc1
-1.16 0.034 1.25 0.005 Nlgn2 -1.15 0.043 1.22 0.009 Gm9223 -1.15
0.005 1.30 0.000 Tox4 -1.14 0.016 1.16 0.007 Josd1 -1.10 0.039 1.14
0.009 Trip12 -1.10 0.017 1.28 0.000 Bet1l -1.10 0.020 1.24 0.000
Scaf1 -1.09 0.039 1.16 0.003 Dynlrb1 -1.07 0.015 1.07 0.024 Fam195b
-1.07 0.001 1.24 0.000 Smarca5 1.05 0.013 -1.05 0.023 Rae1 1.07
0.016 -1.17 0.000 Nhp2l1 1.07 0.013 -1.23 0.000 Pin1-ps1 1.07 0.038
-1.13 0.003 Ppp1r7 1.08 0.011 -1.16 0.000 Lars 1.08 0.012 -1.11
0.002 Wdsub1 1.09 0.031 -1.13 0.006 Fam120a 1.09 0.043 -1.10 0.032
3010027C24Rik 1.10 0.032 -1.15 0.005 Eif4a3 1.10 0.021 -1.14 0.004
Vprbp 1.10 0.042 -1.38 0.000 Naa20 1.10 0.008 -1.07 0.037 Smu1 1.11
0.006 -1.17 0.001 Tmed10 1.11 0.004 -1.06 0.039 Dus1l 1.11 0.033
-1.16 0.006 Ecd 1.11 0.026 -1.24 0.001 Naa10 1.11 0.015 -1.10 0.026
Ddx18 1.11 0.011 -1.11 0.014 Btbd9 1.11 0.002 -1.06 0.037 Ubap2l
1.11 0.008 -1.11 0.008 Pnkp 1.12 0.022 -1.11 0.023 Parl 1.12 0.022
-1.25 0.001 Tle4 1.12 0.011 -1.20 0.001 Wbp11 1.12 0.027 -1.12
0.026 Nek4 1.12 0.027 -1.26 0.001 Poc5 1.13 0.039 -1.12 0.046 Uchl5
1.13 0.002 -1.28 0.000 Recql 1.13 0.006 -1.09 0.030 Psmd3 1.13
0.011 -1.10 0.028 Asna1 1.13 0.033 -1.31 0.000 Polr1e 1.13 0.048
-1.20 0.009 Csrp2bp 1.13 0.023 -1.18 0.006 Parp1 1.13 0.049 -1.14
0.043 Abi1 1.13 0.003 -1.48 0.000 Tubgcp2 1.14 0.006 -1.47 0.000
Reps1 1.14 0.007 -1.16 0.003 Mon2 1.14 0.023 -1.13 0.032 Seh1l 1.14
0.034 -1.16 0.021 Mri1 1.14 0.000 -1.13 0.000 Ddx20 1.14 0.002
-2.18 0.000 Nup133 1.14 0.001 -1.09 0.006 Ubr7 1.15 0.006 -1.10
0.033 Fam32a 1.15 0.009 -1.16 0.007 Cct2 1.15 0.003 -1.26 0.000
Actl6a 1.15 0.027 -1.15 0.024 Snrpb 1.15 0.012 -1.21 0.002 Ino80
1.15 0.032 -1.14 0.038 1500002O20Rik 1.15 0.025 -1.28 0.001 Anxa7
1.15 0.008 -1.18 0.003 Wdr74 1.15 0.035 -1.32 0.001 Mrps27 1.16
0.015 -1.22 0.003 Trnau1ap 1.16 0.007 -1.18 0.004 Usp39 1.16 0.031
-1.19 0.015 Mbd2 1.17 0.010 -1.12 0.043 Akap10 1.17 0.016 -1.35
0.000 Rps19bp1 1.17 0.031 -1.23 0.008 Faf1 1.17 0.026 -1.25 0.004
Wdr55 1.17 0.002 -1.15 0.005 Gorasp2 1.17 0.049 -1.20 0.027 Nfe2l2
1.17 0.011 -1.23 0.002 Nup54 1.17 0.048 -1.70 0.000 Med6 1.17 0.001
-1.17 0.002 Mapkap1 1.17 0.034 -1.19 0.026 Nsmce2 1.18 0.002 -1.09
0.043 Nsun2 1.18 0.011 -1.17 0.016 Map3k3 1.19 0.030 -1.28 0.005
Stat6 1.19 0.021 -1.40 0.001 Yrdc 1.19 0.008 -1.25 0.002 Ap1m1 1.19
0.002 -1.15 0.005 Ccdc51 1.19 0.013 -1.17 0.019 Gins4 1.19 0.012
-1.24 0.004 Tmem165 1.19 0.027 -1.21 0.018 Txnl1 1.19 0.031 -1.71
0.000 Zfp608 1.19 0.000 -1.30 0.000 Mphosph10 1.19 0.019 -1.29
0.003 Spp1 1.20 0.014 -1.16 0.029 Wdr43 1.20 0.014 -1.16 0.028
Atpbd4 1.20 0.004 -1.11 0.044 Pafah1b2 1.20 0.050 -1.20 0.049
Exosc8 1.20 0.027 -1.21 0.021 Nop14 1.20 0.003 -1.48 0.000 Nop16
1.20 0.038 -1.22 0.030 Pdcd6ip 1.20 0.011 -1.90 0.000 Cbl 1.21
0.035 -1.83 0.000 Pcif1 1.21 0.020 -1.24 0.011 Rbm14 1.21 0.045
-1.22 0.036 Epb4.1l5 1.21 0.049 -1.38 0.004 Mtmr10 1.21 0.041 -1.48
0.001 Ttf2 1.21 0.030 -1.42 0.001 Cenpo 1.22 0.009 -1.31 0.002
Rreb1 1.22 0.049 -1.84 0.000 Depdc5 1.22 0.002 -1.39 0.000 Umps
1.23 0.012 -1.16 0.046 Zfp52 1.23 0.039 -1.55 0.001 BB070754 1.24
0.017 -1.28 0.010 Gnl3 1.24 0.036 -1.34 0.010 Rbbp5 1.25 0.003
-1.34 0.001 Fam178a 1.26 0.048 -1.35 0.015 Etv5 1.27 0.035 -1.55
0.002 Gins1 1.27 0.034 -1.32 0.019 Lbr 1.28 0.002 -1.45 0.000
Gm5039 1.29 0.030 -1.57 0.002 Pgam1 1.29 0.027 -1.26 0.036 Atg7
1.29 0.005 -1.24 0.011 9030425P06Rik 1.30 0.005 -1.34 0.003 Lyst
1.30 0.028 -2.43 0.000 Rgs19 1.31 0.003 -1.24 0.012 Numb 1.31 0.001
-1.63 0.000 Snx27 1.32 0.014 -2.48 0.000 Rnf130 1.32 0.021 -1.31
0.026 Pias3 1.33 0.014 -1.90 0.000 Pqlc3 1.33 0.009 -1.30 0.013
Chka 1.33 0.005 -1.44 0.001 A430105D02Rik 1.37 0.003 -3.13 0.000
Sdc4 1.38 0.004 -2.05 0.000 Rbm3 1.39 0.002 -1.56 0.000
5830468K08Rik 1.41 0.044 -1.89 0.002 Clcn5 1.41 0.026 -1.49 0.014
Fam65b 1.41 0.049 -1.50 0.026 Tgfa 1.46 0.004 -1.34 0.015 Fgd4 1.48
0.000 -1.18 0.036 3930401B19Rik 1.57 0.020 -1.46 0.043 Itga3 1.57
0.031 -1.63 0.024 2410137M14Rik 1.63 0.040 -1.64 0.039 Egr4 2.02
0.024 -1.86 0.040 Olfr663 2.06 0.022 -2.08 0.020
[0130] DAVID analysis (NCBI) of functional terms that are enriched
in the two groups are presented in FIG. 10B. Not surprisingly, the
Gene Ontology (GO) terms of Extracellular Matrix, (Skin) Function,
Adhesion/Cell Signaling and Cell Differentiation/Apoptosis are the
top negatively regulated pathways following miR-29b mimic
treatment. Cellular (Nuclear) Structure and RNA Processing are the
top positively regulated pathways following miR-29b mimic
treatment.
[0131] To further confirm the effect of miR-29b mimic treatment in
the skin, mice with acute incisional wounds were treated with
intradermal injection of PBS, 20, 50, or 100 nmol of miR-29b mimic
(comprising SEQ ID NO: 2 as the first strand and SEQ ID NO: 1 as
the second strand). Quantitative reverse-transcriptase PCR analysis
was performed on 24 genes identified as being direct or indirect
targets of miR-29 modulation in the skin (19 repressed by miR-29b
mimic and upregulated by antimiR-29, 5 upregulated by miR-29b mimic
and repressed by antimiR-29). Extracellular matrix genes
(collagens, ELN, etc.) and others involved in the fibrotic process
(e.g. MMP2, TGFB2) were shown to be repressed by miR-29b mimic
treatment in the skin (FIG. 11A), whereas selected cell surface
receptors (ITGA3, LBR, NUMB, SDC4) and factors associated with
receptor endocytosis (SNX27) were shown to be increased with
miR-29b mimic treatment in the skin (FIG. 11B). These studies
indicate that the miR-29b mimic is active when treated locally in
the skin, and suggests that in addition to its effect on organ
fibrosis, miR-29 mimicry could be an effective therapy for
cutaneous fibrosis of various etiologies. These studies also
identify the above-mentioned genes as translational biomarkers
whose expression correlates with the activity of a miR-29b mimic in
the skin. These translational biomarkers can be utilized in a
clinical trial, testing the safety and efficacy of miR-29b mimics
in normal healthy volunteers and patients with cutaneous
scleroderma.
Example 5
Activity of miR-29 Mimics and Effects of Nucleotide
Modifications
[0132] To determine the efficacy of miR-29a, b, and c mimics in
regulating the expression of target genes, different miR-29a, b and
c mimics were transfected into IMR-90 human lung fibroblasts at a
concentration of 10 nM, and collagen expression was measured by
quantitative RT-PCR. These studies demonstrate that a miR-29a mimic
comprising SEQ ID NO: 27 as the first strand and SEQ ID NO: 5 as
the second strand and a miR-29b mimic comprising SEQ ID NO: 19 as
the first strand and SEQ ID NO: 1 as the second strand are the most
effective at repressing expression of multiple collagen genes,
whereas a miR-29c mimic comprising SEQ ID NO: 35 as the first
strand and SEQ ID NO: 24 as the second strand is less effective
(FIG. 12). These effects may be cell-type or target gene specific,
but indicate that there are indeed differences in the ability of
the three mimics to repress extracellular matrix gene
expression.
[0133] In the same experiment, the effect of nucleotide
modifications on the efficacy of miR-29b mimics was also tested.
These studies indicate that the miR-29b mimic containing SEQ ID NO:
19 as the first strand and SEQ ID NO: 1 as the second strand
performs similarly to a miR-29b mimic comprising SEQ ID NO: 19 as
the first strand and SEQ ID NO: 30 as the second strand which has
the same first and second sequence and pattern, but a different
chemical linker between the sense (passenger) strand and
cholesterol. A checkerboard pattern of 2' O-Methyl modifications
makes miR-29 mimics (a mimic comprising SEQ ID NO: 31 as the first
strand and SEQ ID NO: 28 as the second strand and a mimic
comprising SEQ ID NO: 32 as the first strand and SEQ ID NO: 29 as
the second strand) completely ineffective. Modifications of all C
and U residues on the antisense (first/guide) strand (a mimic
comprising SEQ ID NO: 33 as the first strand and SEQ ID NO: 1 as
the second strand) partially reduces the mimic activity. Similarly,
removal of the 3' overhang on the antisense strand (a mimic
comprising SEQ ID NO: 24 as the first strand and SEQ ID NO: 1 as
the second strand) partially reduces the mimic activity. See FIG.
12.
[0134] These studies have allowed the stratification of miR-29
mimic compounds on the basis of in vitro activity in a specific
cell line (IMR-90) and using particular target genes (COL1A1,
COL3A1, COL4A5) as the readout for efficacy, independent on
compound uptake, and can be used as the basis for selecting
compounds to test via passive delivery in vitro or to test in
vivo.
Example 6
In Vivo Activity of miR-29b Mimics with Linker Modifications
[0135] Mice with incisional wounds were treated with 20 nmol of
various miR-29b mimics that differ only in the linkage between the
cholesterol moiety and the second/sense strand. The mimetic
compound that contained a six carbon linker between cholesterol and
the sense strand (the mimic comprising SEQ ID NO: 19 as the first
strand and SEQ ID NO: 1 as the second strand) and the compound that
contained the same six carbon linker between cholesterol and the
sense strand but connected through a cleavable moiety (dT.dT) (SEQ
ID NO: 19/SEQ ID NO: 15) showed similar activity in repressing
target genes (FIG. 13). N/S in FIG. 13 represents no significant
difference was observed in the activities of the miR-29b mimic
comprising SEQ ID NO: 19 as the first strand and SEQ ID NO: 1 as
the second strand and the mimic comprising SEQ ID NO: 19 as the
first strand and SEQ ID NO: 15 as the second strand. miR-29b mimics
containing a nine carbon linker (SEQ ID NO: 19/SEQ ID NO: 17) and a
linker at the 5' end (SEQ ID NO: 19/SEQ ID NO: 16) were not
effective in repressing target genes (FIG. 13).
Example 7
Effect of 5' Phosphorylation on the Activity of miR-29b Mimics
[0136] RAB-9 skin fibroblast cells (ATCC CRL-1414) were transfected
with varying concentrations of miR-29b mimics with (the mimic
containing SEQ ID NO: 2 as the first strand and SEQ ID NO: 1 as the
second strand) and without (the mimic containing SEQ ID NO: 19 as
the first strand and SEQ ID NO: 1 as the second strand) 5'
phosphorylation on the antisense strand. No significant differences
in target gene repression as measured by Col1a1 , Col1a2 or Col3a1
expression was observed in the activity of the two mimics
(represented as N/S in FIG. 14). Both miR-29b mimics significantly
(p<0.0001) repressed the expression of target genes compared to
vehicle, mock transfection or control mimic treatment. See FIG.
14.
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Sequence CWU 1
1
42123RNAArtificial SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl adenosineMOD_RES(2)..(2)May be
O-methyl adenosineMOD_RES(3)..(3)May be O-methyl
cytidineMOD_RES(5)..(5)May be O-methyl cytidineMOD_RES(6)..(6)May
be O-methyl uridineMOD_RES(8)..(8)May be O-methyl
uridineMOD_RES(9)..(9)May be O-methyl uridineMOD_RES(10)..(10)May
be O-methyl uridineMOD_RES(12)..(12)May be O-methyl
cytidineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(19)..(19)May be O-methyl uridineMOD_RES(20)..(20)May
be O-methyl cytidineMOD_RES(21)..(21)May be O-methyl
cytidineMOD_RES(22)..(22)May be O-methyl
uridineMISC_FEATURE(23)..(23)May be modified with a cholesterol
conjugate with a 6 carbon linker 1aacacuguuu acaaaugguc cua
23225RNAArtificial SequencemiR-29b mimic first-antisense-guide
strandMISC_FEATURE(1)..(1)May be modified with a monophosphate
moietyMOD_RES(1)..(1)May be fluoro uridineMOD_RES(4)..(4)May be
fluoro cytidineMOD_RES(6)..(6)May be fluoro
cytidineMOD_RES(7)..(7)May be fluoro cytidineMOD_RES(9)..(9)May be
fluoro uridineMOD_RES(10)..(10)May be fluoro
uridineMOD_RES(11)..(11)May be fluoro uridineMOD_RES(16)..(16)May
be fluoro uridineMOD_RES(17)..(17)May be fluoro
cytidineMOD_RES(20)..(20)May be fluoro uridineMOD_RES(22)..(22)May
be fluoro uridineMOD_RES(23)..(23)May be fluoro
uridineMISC_FEATURE(23)..(25)May be joined through phosphorothioate
bonds 2uagcaccauu ugaaaucagu guuuu 25324RNAArtificial
SequencemiR-29a mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl uridineMOD_RES(2)..(2)May be
O-methyl adenosine 3uaaccgauuu cagauggugc uauu 24422RNAArtificial
SequencemiR-29a mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl uridineMOD_RES(2)..(2)May be
O-methyl adenosineMOD_RES(4)..(4)May be O-methyl
cytidineMOD_RES(5)..(5)May be O-methyl cytidineMOD_RES(7)..(7)May
be O-methyl uridineMOD_RES(8)..(8)May be O-methyl
uridineMOD_RES(9)..(9)May be O-methyl uridineMOD_RES(11)..(11)May
be O-methyl cytidineMOD_RES(15)..(15)May be O-methyl
uridineMOD_RES(18)..(18)May be O-methyl uridineMOD_RES(19)..(19)May
be O-methyl cytidineMOD_RES(20)..(20)May be O-methyl
cytidineMOD_RES(21)..(21)May be O-methyl uridine 4uaaccguuua
cagauggucc ua 22522RNAArtificial SequencemiR-29a mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(4)..(4)May
be O-methyl cytidineMOD_RES(5)..(5)May be O-methyl
cytidineMOD_RES(7)..(7)May be O-methyl uridineMOD_RES(8)..(8)May be
O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(11)..(11)May be O-methyl
cytidineMOD_RES(15)..(15)May be O-methyl
uridineMOD_RES(18)..(18)May be O-methyl uridineMOD_RES(19)..(19)May
be O-methyl cytidineMOD_RES(20)..(20)May be O-methyl
cytidineMOD_RES(21)..(21)May be O-methyl
uridineMISC_FEATURE(22)..(22)May be modified with a cholesterol
conjugate with a 6 carbon linker 5uaaccguuua cagauggucc ua
22624RNAArtificial SequencemiR-29a mimic first-antisense-guide
strandMISC_FEATURE(1)..(1)May be modified with a monophosphate
moiety 6uagcaccauc ugaaaucggu uauu 24724RNAArtificial
SequencemiR-29a mimic first-antisense-guide
strandMISC_FEATURE(1)..(1)May be modified with a monophosphate
moietyMOD_RES(1)..(1)May be fluoro uridineMOD_RES(4)..(4)May be
fluoro cytidineMOD_RES(6)..(6)May be fluoro
cytidineMOD_RES(7)..(7)May be fluoro cytidineMOD_RES(9)..(9)May be
fluoro uridineMOD_RES(10)..(10)May be fluoro
cytidineMOD_RES(11)..(11)May be fluoro uridineMOD_RES(16)..(16)May
be fluoro uridineMOD_RES(17)..(17)May be fluoro
cytidineMOD_RES(20)..(20)May be fluoro uridineMOD_RES(21)..(21)May
be fluoro uridineMISC_FEATURE(22)..(24)May be joined through
phosphorothioate bonds 7uagcaccauc ugaaaucggu uauu
24825RNAArtificial SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl adenosineMOD_RES(2)..(2)May be
O-methyl adenosine 8aacacugauu ucaaauggug cuauu 25923RNAArtificial
SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl adenosineMOD_RES(2)..(2)May be
O-methyl adenosineMOD_RES(3)..(3)May be O-methyl
cytidineMOD_RES(5)..(5)May be O-methyl cytidineMOD_RES(6)..(6)May
be O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(10)..(10)May be O-methyl uridineMOD_RES(11)..(11)May
be O-methyl uridineMOD_RES(12)..(12)May be O-methyl
cytidineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(19)..(19)May be O-methyl uridineMOD_RES(21)..(21)May
be O-methyl cytidineMOD_RES(22)..(22)May be O-methyl
uridineMISC_FEATURE(23)..(23)May be modified with a cholesterol
conjugate with a 6 carbon linker 9aacacugauu ucaaauggug cua
231025RNAArtificial SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl adenosineMOD_RES(2)..(2)May be
O-methyl adenosineMISC_FEATURE(23)..(25)May be joined through
phosphorothioate bondsMISC_FEATURE(25)..(25)May be modified with a
cholesterol conjugate with a 6 carbon linker 10aacacugauu
ucaaauggug cuauu 251124RNAArtificial SequencemiR-29a mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(2)..(2)May be O-methyl
adenosineMISC_FEATURE(22)..(24)May be joined through
phosphorothioate bondsMISC_FEATURE(24)..(24)May be modified with a
cholesterol conjugate with a 6 carbon linker 11uaaccgauuu
cagauggugc uauu 241224RNAArtificial SequencemiR-29c mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(2)..(2)May be O-methyl
adenosineMISC_FEATURE(22)..(24)May be joined through
phosphorothioate bondsMISC_FEATURE(24)..(24)May be modified with a
cholesterol conjugate with a 6 carbon linker 12uaaccgauuu
caaauggugc uauu 241323RNAArtificial SequencemiR-29b mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
adenosineMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(3)..(3)May
be O-methyl cytidineMOD_RES(5)..(5)May be O-methyl
cytidineMOD_RES(6)..(6)May be O-methyl uridineMOD_RES(8)..(8)May be
O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(10)..(10)May be O-methyl uridineMOD_RES(12)..(12)May
be O-methyl cytidineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(19)..(19)May be O-methyl uridineMOD_RES(20)..(20)May
be O-methyl cytidineMOD_RES(21)..(21)May be O-methyl
cytidineMOD_RES(22)..(22)May be O-methyl uridine 13aacacuguuu
acgggugguc cua 231423RNAArtificial SequencemiR-29b mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
adenosineMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(3)..(3)May
be O-methyl cytidineMOD_RES(5)..(5)May be O-methyl
cytidineMOD_RES(6)..(6)May be O-methyl uridineMOD_RES(8)..(8)May be
O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(10)..(10)May be O-methyl uridineMOD_RES(12)..(12)May
be O-methyl cytidineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(19)..(19)May be O-methyl uridineMOD_RES(20)..(20)May
be O-methyl cytidineMOD_RES(21)..(21)May be O-methyl
cytidineMOD_RES(22)..(22)May be O-methyl
uridineMISC_FEATURE(23)..(23)May be modified with a cholesterol
conjugate with a 6 carbon linker 14aacacuguuu acgggugguc cua
231525DNAArtificial SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl adenosineMOD_RES(2)..(2)May be
O-methyl adenosineMOD_RES(3)..(3)May be O-methyl
cytidineMOD_RES(4)..(4)May be adenosineMOD_RES(5)..(5)May be
O-methyl cytidineMOD_RES(6)..(6)May be O-methyl
uridineMOD_RES(7)..(7)May be guanosineMOD_RES(8)..(8)May be
O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(10)..(10)May be O-methyl uridineMOD_RES(11)..(11)May
be adenosineMOD_RES(12)..(12)May be O-methyl
cytidineMOD_RES(13)..(13)May be adenosineMOD_RES(14)..(14)May be
adenosineMOD_RES(15)..(15)May be adenosineMOD_RES(16)..(16)May be
O-methyl uridineMOD_RES(17)..(17)May be
guanosineMOD_RES(18)..(18)May be guanosineMOD_RES(19)..(19)May be
O-methyl uridineMOD_RES(20)..(20)May be O-methyl
cytidineMOD_RES(21)..(21)May be O-methyl
cytidineMOD_RES(22)..(22)May be O-methyl
uridineMOD_RES(23)..(23)May be adenosineMISC_FEATURE(25)..(25)May
be modified with a cholesterol conjugate with a 6 carbon linker
15aacacuguuu acaaaugguc cuatt 251625DNAArtificial SequencemiR-29b
mimic second-sense-passenger strandMISC_FEATURE(1)..(1)May be
modified with a cholesterol conjugate with a 6 carbon
linkerMOD_RES(3)..(3)May be O-methyl adenosineMOD_RES(4)..(4)May be
O-methyl adenosineMOD_RES(5)..(5)May be O-methyl
cytidineMOD_RES(6)..(6)May be adenosineMOD_RES(7)..(7)May be
O-methyl cytidineMOD_RES(8)..(8)May be O-methyl
uridineMOD_RES(9)..(9)May be guanosineMOD_RES(10)..(10)May be
O-methyl uridineMOD_RES(11)..(11)May be O-methyl
uridineMOD_RES(12)..(12)May be O-methyl uridineMOD_RES(13)..(13)May
be adenosineMOD_RES(14)..(14)May be O-methyl
cytidineMOD_RES(15)..(15)May be adenosineMOD_RES(16)..(16)May be
adenosineMOD_RES(17)..(17)May be adenosineMOD_RES(18)..(18)May be
O-methyl uridineMOD_RES(19)..(19)May be
guanosineMOD_RES(20)..(20)May be guanosineMOD_RES(21)..(21)May be
O-methyl uridineMOD_RES(22)..(22)May be O-methyl
cytidineMOD_RES(23)..(23)May be O-methyl
cytidineMOD_RES(24)..(24)May be O-methyl
uridineMOD_RES(25)..(25)May be adenosine 16ttaacacugu uuacaaaugg
uccua 251723RNAArtificial SequencemiR-29b mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
adenosineMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(3)..(3)May
be O-methyl cytidineMOD_RES(5)..(5)May be O-methyl
cytidineMOD_RES(6)..(6)May be O-methyl uridineMOD_RES(8)..(8)May be
O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(10)..(10)May be O-methyl uridineMOD_RES(12)..(12)May
be O-methyl cytidineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(19)..(19)May be O-methyl uridineMOD_RES(20)..(20)May
be O-methyl cytidineMOD_RES(21)..(21)May be O-methyl
cytidineMOD_RES(22)..(22)May be O-methyl
uridineMISC_FEATURE(23)..(23)May be modified with a cholesterol
conjugate with a 9 carbon linker 17aacacuguuu acaaaugguc cua
231825RNAArtificial SequencemiR-29b mimic first-antisense-guide
strandMISC_FEATURE(1)..(1)May be modified with a monophosphate
moiety 18uagcaccauu ugaaaucagu guuuu 251925RNAArtificial
SequencemiR-29b mimic first-antisense-guide
strandMOD_RES(1)..(1)May be fluoro uridineMOD_RES(4)..(4)May be
fluoro cytidineMOD_RES(6)..(6)May be fluoro
cytidineMOD_RES(7)..(7)May be fluoro cytidineMOD_RES(9)..(9)May be
fluoro uridineMOD_RES(10)..(10)May be fluoro
uridineMOD_RES(11)..(11)May be fluoro uridineMOD_RES(16)..(16)May
be fluoro uridineMOD_RES(17)..(17)May be fluoro
cytidineMOD_RES(20)..(20)May be fluoro uridineMOD_RES(22)..(22)May
be fluoro uridineMOD_RES(23)..(23)May be fluoro
uridineMISC_FEATURE(23)..(25)May be joined through phosphorothioate
bonds 19uagcaccauu ugaaaucagu guuuu 252025RNAArtificial
SequencemiR-29b mimic first-antisense-guide
strandMISC_FEATURE(1)..(1)May be modified with a monophosphate
moietyMOD_RES(1)..(1)May be fluoro uridineMOD_RES(4)..(4)May be
fluoro cytidineMOD_RES(6)..(6)May be fluoro
cytidineMOD_RES(7)..(7)May be fluoro cytidineMOD_RES(9)..(9)May be
fluoro cytidineMOD_RES(10)..(10)May be fluoro
cytidineMOD_RES(11)..(11)May be fluoro cytidineMOD_RES(16)..(16)May
be fluoro uridineMOD_RES(17)..(17)May be fluoro
cytidineMOD_RES(20)..(20)May be fluoro uridineMOD_RES(22)..(22)May
be fluoro uridineMOD_RES(23)..(23)May be fluoro
uridineMISC_FEATURE(23)..(25)May be joined through phosphorothioate
bonds 20uagcaccacc cgaaaucagu guuuu 252125RNAArtificial
SequencemiR-29b mimic first-antisense-guide strand 21uagcaccauu
ugaaaucagu guuuu 252224RNAArtificial SequencemiR-29c mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(2)..(2)May be O-methyl adenosine 22uaaccgauuu
caaauggugc uauu 242322RNAArtificial SequencemiR-29c mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(4)..(4)May
be O-methyl cytidineMOD_RES(5)..(5)May be O-methyl
cytidineMOD_RES(7)..(7)May be O-methyl uridineMOD_RES(8)..(8)May be
O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(11)..(11)May be O-methyl
cytidineMOD_RES(15)..(15)May be O-methyl
uridineMOD_RES(18)..(18)May be O-methyl uridineMOD_RES(19)..(19)May
be O-methyl cytidineMOD_RES(20)..(20)May be O-methyl
cytidineMOD_RES(21)..(21)May be O-methyl uridine 23uaaccguuua
caaauggucc ua 222422RNAArtificial SequencemiR-29c mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(4)..(4)May
be O-methyl cytidineMOD_RES(5)..(5)May be O-methyl
cytidineMOD_RES(7)..(7)May be O-methyl uridineMOD_RES(8)..(8)May be
O-methyl uridineMOD_RES(9)..(9)May be O-methyl
uridineMOD_RES(11)..(11)May be O-methyl
cytidineMOD_RES(15)..(15)May be O-methyl
uridineMOD_RES(18)..(18)May be O-methyl uridineMOD_RES(19)..(19)May
be O-methyl cytidineMOD_RES(20)..(20)May be O-methyl
cytidineMOD_RES(21)..(21)May be O-methyl
uridineMISC_FEATURE(22)..(22)May be modified with a cholesterol
conjugate with a 6 carbon linker 24uaaccguuua caaauggucc ua
222524RNAArtificial SequencemiR-29c mimic first-antisense-guide
strandMISC_FEATURE(1)..(1)May be modified with a monophosphate
moiety 25uagcaccauu ugaaaucggu uauu 242624RNAArtificial
SequencemiR-29c mimic first-antisense-guide
strandMISC_FEATURE(1)..(1)May be modified with a monophosphate
moietyMOD_RES(1)..(1)May be fluoro uridineMOD_RES(4)..(4)May be
fluoro cytidineMOD_RES(6)..(6)May be fluoro
cytidineMOD_RES(7)..(7)May be fluoro cytidineMOD_RES(9)..(9)May be
fluoro uridineMOD_RES(10)..(10)May be fluoro
uridineMOD_RES(11)..(11)May be fluoro uridineMOD_RES(16)..(16)May
be fluoro uridineMOD_RES(17)..(17)May be fluoro
cytidineMOD_RES(20)..(20)May be fluoro uridineMOD_RES(21)..(21)May
be fluoro uridineMISC_FEATURE(22)..(24)May be joined through
phosphorothioate bonds 26uagcaccauu ugaaaucggu uauu
242724RNAArtificial SequencemiR-29a mimic first-antisense-guide
strandMOD_RES(1)..(1)May be fluoro uridineMOD_RES(4)..(4)May be
fluoro cytidineMOD_RES(6)..(6)May be fluoro
cytidineMOD_RES(7)..(7)May be fluoro cytidineMOD_RES(9)..(9)May be
fluoro uridineMOD_RES(10)..(10)May be fluoro
cytidineMOD_RES(11)..(11)May be fluoro uridineMOD_RES(16)..(16)May
be fluoro uridineMOD_RES(17)..(17)May be fluoro
cytidineMOD_RES(20)..(20)May be fluoro uridineMOD_RES(21)..(21)May
be fluoro uridineMISC_FEATURE(22)..(24)May be joined through
phosphorothioate bonds 27uagcaccauc ugaaaucggu uauu
242823RNAArtificial SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(4)..(4)May be
O-methyl adenosineMOD_RES(6)..(6)May be O-methyl
uridineMOD_RES(8)..(8)May be O-methyl adenosineMOD_RES(10)..(10)May
be O-methyl uridineMOD_RES(12)..(12)May be O-methyl
cytidineMOD_RES(14)..(14)May be O-methyl
adenosineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(18)..(18)May be O-methyl
guanosineMOD_RES(20)..(20)May be O-methyl
guanosineMOD_RES(22)..(22)May be O-methyl
uridineMISC_FEATURE(23)..(23)May be modified with a cholesterol
conjugate with a 6 carbon linker 28aacacugauu ucaaauggug cua
232925RNAArtificial SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(2)..(2)May be O-methyl adenosineMOD_RES(4)..(4)May be
O-methyl adenosineMOD_RES(6)..(6)May be O-methyl
uridineMOD_RES(8)..(8)May be O-methyl adenosineMOD_RES(10)..(10)May
be O-methyl uridineMOD_RES(12)..(12)May be
O-methyl cytidineMOD_RES(14)..(14)May be O-methyl
adenosineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(18)..(18)May be O-methyl
guanosineMOD_RES(20)..(20)May be O-methyl
guanosineMOD_RES(22)..(22)May be O-methyl
uridineMISC_FEATURE(23)..(25)May be joined through phosphorothioate
bondsMISC_FEATURE(25)..(25)May be modified with a cholesterol
conjugate with a 6 carbon linker 29aacacugauu ucaaauggug cuauu
253023RNAArtificial SequencemiR-29b mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl adenosineMOD_RES(2)..(2)May be
O-methyl adenosineMOD_RES(3)..(3)May be O-methyl
cytidineMOD_RES(5)..(5)May be O-methyl cytidineMOD_RES(6)..(6)May
be O-methyl uridineMOD_RES(8)..(8)May be O-methyl
uridineMOD_RES(9)..(9)May be O-methyl uridineMOD_RES(10)..(10)May
be O-methyl uridineMOD_RES(12)..(12)May be O-methyl
cytidineMOD_RES(16)..(16)May be O-methyl
uridineMOD_RES(19)..(19)May be O-methyl uridineMOD_RES(20)..(20)May
be O-methyl cytidineMOD_RES(21)..(21)May be O-methyl
cytidineMOD_RES(22)..(22)May be O-methyl
uridineMOD_RES(23)..(23)May be modified with a cholesterol
conjugate with a tetraethylene glycol moiety 30aacacuguuu
acaaaugguc cua 233123RNAArtificial SequencemiR-29b mimic
first-antisense-guide strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(3)..(3)May be O-methyl guanosineMOD_RES(5)..(5)May
be O-methyl adenosineMOD_RES(7)..(7)May be O-methyl
cytidineMOD_RES(9)..(9)May be O-methyl uridineMOD_RES(11)..(11)May
be O-methyl uridineMOD_RES(13)..(13)May be O-methyl
adenosineMOD_RES(15)..(15)May be O-methyl
adenosineMOD_RES(17)..(17)May be O-methyl
cytidineMOD_RES(19)..(19)May be O-methyl
guanosineMOD_RES(21)..(21)May be O-methyl
guanosineMOD_RES(23)..(23)May be O-methyl uridine 31uagcaccauu
ugaaaucagu guu 233225RNAArtificial SequencemiR-29b mimic
first-antisense-guide strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(3)..(3)May be O-methyl guanosineMOD_RES(5)..(5)May
be O-methyl adenosineMOD_RES(7)..(7)May be O-methyl
cytidineMOD_RES(9)..(9)May be O-methyl uridineMOD_RES(11)..(11)May
be O-methyl uridineMOD_RES(13)..(13)May be O-methyl
adenosineMOD_RES(15)..(15)May be O-methyl
adenosineMOD_RES(17)..(17)May be O-methyl
cytidineMOD_RES(19)..(19)May be O-methyl
guanosineMOD_RES(21)..(21)May be O-methyl
guanosineMISC_FEATURE(23)..(25)May be joined through
phosphorothioate bondsMOD_RES(23)..(23)May be O-methyl uridine
32uagcaccauu ugaaaucagu guuuu 253325RNAArtificial SequencemiR-29b
mimic first-antisense-guide strandMOD_RES(1)..(1)May be O-methyl
uridineMOD_RES(4)..(4)May be O-methyl cytidineMOD_RES(6)..(6)May be
O-methyl cytidineMOD_RES(7)..(7)May be O-methyl
cytidineMOD_RES(9)..(9)May be O-methyl uridineMOD_RES(10)..(10)May
be O-methyl uridineMOD_RES(11)..(11)May be O-methyl
uridineMOD_RES(16)..(16)May be O-methyl uridineMOD_RES(17)..(17)May
be O-methyl cytidineMOD_RES(20)..(20)May be O-methyl
uridineMOD_RES(22)..(22)May be O-methyl
uridineMISC_FEATURE(23)..(25)May be joined through phosphorothioate
bondsMOD_RES(23)..(23)May be O-methyl uridine 33uagcaccauu
ugaaaucagu guuuu 253423RNAArtificial SequencemiR-29b mimic
first-antisense-guide strandMOD_RES(1)..(1)May be fluoro
uridineMOD_RES(4)..(4)May be fluoro cytidineMOD_RES(6)..(6)May be
fluoro cytidineMOD_RES(7)..(7)May be fluoro
cytidineMOD_RES(9)..(9)May be fluoro uridineMOD_RES(10)..(10)May be
fluoro uridineMOD_RES(11)..(11)May be fluoro
uridineMOD_RES(16)..(16)May be fluoro uridineMOD_RES(17)..(17)May
be fluoro cytidineMOD_RES(20)..(20)May be fluoro
uridineMOD_RES(22)..(22)May be fluoro uridineMOD_RES(23)..(23)May
be fluoro uridine 34uagcaccauu ugaaaucagu guu 233524RNAArtificial
SequencemiR-29c mimic first-antisense-guide
strandMOD_RES(1)..(1)May be fluoro uridineMOD_RES(4)..(4)May be
fluoro cytidineMOD_RES(6)..(6)May be fluoro
cytidineMOD_RES(7)..(7)May be fluoro cytidineMOD_RES(9)..(9)May be
fluoro uridineMOD_RES(10)..(10)May be fluoro
uridineMOD_RES(11)..(11)May be fluoro uridineMOD_RES(16)..(16)May
be fluoro uridineMOD_RES(17)..(17)May be fluoro
cytidineMOD_RES(20)..(20)May be fluoro uridineMOD_RES(21)..(21)May
be fluoro uridineMISC_FEATURE(22)..(24)May be joined through
phosphorothioate bonds 35uagcaccauu ugaaaucggu uauu
243616DNAArtificial SequenceantimiR-29MISC_FEATURE(1)..(16)May be
joined through phosphorothioate bondsMOD_RES(1)..(1)May be locked
nucleic acid deoxyguanosineMOD_RES(5)..(5)May be locked nucleic
acid deoxythymidineMOD_RES(6)..(6)May be locked nucleic acid
deoxycytidineMOD_RES(8)..(8)May be locked nucleic acid
deoxyadenosineMOD_RES(10)..(10)May be locked nucleic acid
deoxythymidineMOD_RES(11)..(11)May be locked nucleic acid
deoxyguanosineMOD_RES(13)..(13)May be locked nucleic acid
deoxythymidineMOD_RES(15)..(15)May be locked nucleic acid
deoxycytidineMOD_RES(16)..(16)May be locked nucleic acid
deoxythymidine 36gatttcaaat ggtgct 163722RNAArtificial
SequencemiR-29a mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl uridineMOD_RES(2)..(2)May be
O-methyl adenosine 37uaaccgauuu cagauggugc ua 223824RNAArtificial
SequencemiR-29a mimic first-antisense-guide strand 38uagcaccauc
ugaaaucggu uauu 243923RNAArtificial SequencemiR-29b mimic
second-sense-passenger strandMOD_RES(1)..(1)May be O-methyl
adenosineMOD_RES(2)..(2)May be O-methyl adenosine 39aacacugauu
ucaaauggug cua 234025RNAArtificial SequencemiR-29b mimic
first-antisense-guide strand 40uagcaccauu ugaaaucagu guuuu
254122RNAArtificial SequencemiR-29c mimic second-sense-passenger
strandMOD_RES(1)..(1)May be O-methyl uridineMOD_RES(2)..(2)May be
O-methyl adenosine 41uaaccgauuu caaauggugc ua 224224RNAArtificial
SequencemiR-29c mimic first-antisense-guide strand 42uagcaccauu
ugaaaucggu uauu 24
* * * * *