U.S. patent application number 16/985779 was filed with the patent office on 2020-12-31 for compounds of chemically modified oligonucleotides and methods of use thereof.
The applicant listed for this patent is City of Hope. Invention is credited to Mitsuo Kato, Rama Natarajan.
Application Number | 20200407721 16/985779 |
Document ID | / |
Family ID | 1000005090234 |
Filed Date | 2020-12-31 |
United States Patent
Application |
20200407721 |
Kind Code |
A1 |
Natarajan; Rama ; et
al. |
December 31, 2020 |
COMPOUNDS OF CHEMICALLY MODIFIED OLIGONUCLEOTIDES AND METHODS OF
USE THEREOF
Abstract
The present disclosure relates to isolated compounds including a
nucleic acid sequence capable of hybridizing to an RNA sequence 10
to 270 nucleobases downstream of the transcription start site of a
mammalian microRNA-379 transcript; method of treating diabetic
nephropathy in a subject with the compounds; and method of
inhibiting expression of a mammalian microRNA-379 megacluster with
the compounds.
Inventors: |
Natarajan; Rama; (Duarte,
CA) ; Kato; Mitsuo; (Duarte, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
City of Hope |
Duarte |
CA |
US |
|
|
Family ID: |
1000005090234 |
Appl. No.: |
16/985779 |
Filed: |
August 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15163816 |
May 25, 2016 |
10787664 |
|
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16985779 |
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62166533 |
May 26, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/341 20130101;
C12N 2310/10 20130101; C12N 2310/346 20130101; C12N 2310/3231
20130101; C12N 2310/315 20130101; C12N 2310/20 20170501; C12N
2310/113 20130101; C12N 15/113 20130101; C12N 2310/14 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under Grant
No. NIH R01 DK081705 awarded by the National Institute of Diabetes
and Digestive and Kidney Diseases (NIDDK). The Government has
certain rights to this invention.
Claims
1.-10. (canceled)
11. A method of treating diabetic nephropathy in a subject in need
thereof, the method comprising administering to said subject an
effective amount of a compound comprising a nucleic acid sequence
capable of hybridizing to an RNA sequence 10 to 270 nucleobases
downstream of the transcription start site of a mammalian
microRNA-379 transcript, wherein said nucleic acid sequence
comprises a nucleobase analog or internucleotide linkage.
12. The method of claim 11, wherein said compound inhibits
expression of a long non-coding RNA (lncMGC) comprising
microRNA-376a, microRNA-299, microRNA-376c, microRNA-410,
microRNA-494, microRNA-380-5p, microRNA-369-3p, microRNA-300,
microRNA-541, microRNA-329, microRNA-381, microRNA-411,
microRNA-134, microRNA-379, microRNA-154, microRNA-382,
microRNA-376b, microRNA-496, microRNA-409-5p, microRNA-543,
microRNA-377, microRNA-380-3p, or microRNA-495, in said
subject.
13. The method of claim 11, wherein said compound inhibits
expression of a microRNA-379 gene cluster.
14. The method of claim 11, wherein said treating diabetic
nephropathy is at an early stage of the disease.
15. The method of claim 11, wherein said nucleobase analog is at
the 5'-end or the 3'-end of said nucleic acid sequence.
16. The method of claim 15, wherein said nucleic acid sequence
comprises three nucleobase analogs at the 5'-end or the 3'-end of
said nucleic acid sequence.
17. The method of claim 16, wherein said nucleobase analog is a
Locked Nucleic Acid (LNA), 2'-O-alkyl nucleobase, 2'-Fluoro
nucleobase, or 2'-OMe nucleobase.
18. The method of claim 11, wherein said RNA sequence is 11 to 27,
61 to 93, 115 to 139, or 246 to 265 nucleobases downstream of said
transcription start site.
19. The method of claim 11, wherein said nucleic acid sequence
comprises modified internucleotide linkage.
20. The method of claim 19, wherein said modified internucleotide
linkage is a phosphorothioate linkage.
21. The method of claim 11, wherein the nucleic acid sequence has
at least 90% sequence identity with a continuous 10 nucleobase
sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.
22. The method of claim 11, wherein the compound inhibits renal
glomerular podocyte death, glomerular mesangial expansion,
glomerular hypertrophy, glomerular extracellular matrix
accumulation.
23. The method of claim 11, wherein: (a) said nucleic acid sequence
(i) is perfectly complementary to at least 15 continuous
nucleobases from nucleobases 11 to 27, or 115 to 139 of SEQ ID NO:
25, 118, 26 or 119, or (ii) is selected from SEQ ID NOs: 10-21; (b)
said nucleic acid sequence comprises at least one nucleobase analog
or at least one modified internucleotide linkage; and (c) said
nucleic acid is 15 to 20 nucleobases in length.
24. The method of claim 11, wherein said nucleic acid sequence
comprises three nucleobase analogs at the 5'-end or the 3'-end of
said nucleic acid sequence.
25. The method of claim 11, wherein said nucleic acid sequence is
perfectly complementary to at least 16 continuous nucleobases from
nucleobases 11 to 27, or 115 to 139 of SEQ ID NO: 25, 118, 26 or
119.
26. The method of claim 11, wherein said nucleic acid sequence is
selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, and 21.
27. The method of claim 11, wherein said nucleic acid sequence is
perfectly complementary to at least 15 continuous nucleobases from
nucleobases 11 to 27, or 115 to 139 of SEQ ID NO: 25, 118, 26 or
119.
28. The method of claim 11, wherein said nucleic acid sequence is
perfectly complementary to a sequence that is at least 90%
identical to the entire length of at least 18 continuous
nucleobases from nucleobases 11 to 27, or 115 to 139 of SEQ ID NO:
25, 118, 26 or 119.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) to U.S. Provisional Application No.
62/166,533, filed May 26, 2015 which is incorporated herein by
reference in its entirety for all purposes.
SEQUENCE SUBMISSION
[0003] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is entitled
48440-565001US_ST25.txt, was created on May 23, 2016, and is 30,720
bytes in size. The information in the electronic format of the
Sequence Listing is part of the present application and is
incorporated herein by reference in its entirety.
BACKGROUND OF THE DISCLOSURE
[0004] Diabetic nephropathy (DN) is one of the most common
complications of diabetes and a major cause of renal failure, which
requires painful dialysis. Although key therapeutic interventions
have been implemented to treat DN, diabetic patients continue to
reach end-stage renal disease at alarming proportions. It is
therefore imperative to identify new targets. Key features of DN
include the expansion and hypertrophy of glomerular mesangial cells
(MCs), increased accumulation of extracellular matrix (ECM)
proteins such as collagen 1 alpha1 (Col1.alpha.1), Col1.alpha.2,
Col4.alpha.1 and fibronectin, and tubulointerstitial fibrosis,
podocyte dysfunction and proteinuria.
BRIEF SUMMARY OF THE DISCLOSURE
[0005] The current disclosure provides, inter alia, an isolated
compound including a nucleic acid sequence capable of hybridizing
to an RNA sequence 10 to 270 nucleobases downstream of the
transcription start site of a mammalian microRNA-379 transcript;
method of treating diabetic nephropathy in a subject with the
compound; method of inhibiting expression of a mammalian
microRNA-379 megacluster with the compound.
[0006] In embodiments, the compound includes a nucleic acid
sequence having a nucleobase analog. In embodiments, the nucleic
acid sequence includes Locked Nucleic Acid (LNA), 2'-O-alkyl, 2'
O-Methyl, 2'-deoxy-2'fluoro, 2'-deoxy, a universal base,
5-C-methyl, an inverted deoxy abasic residue incorporation, or any
combination thereof. In embodiments, the nucleic acid sequence may
include analogs with positive backbones; non-ionic backbones,
modified sugars, and non-ribose backbones (e.g. phosphorodiamidate
morpholino oligos).
[0007] The current disclosure provides an isolated compound
including a nucleic acid sequence having at least 90% sequence
identity with a continuous 10 nucleobase sequence of SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or 24. The current disclosure further provides a
pharmaceutical composition including a compound of this disclosure,
and a pharmaceutically acceptable diluent, carrier, salt or
adjuvant.
[0008] Other features and advantages of the disclosure will be
apparent from the following detailed description and claims.
[0009] Unless noted to the contrary, all publications, references,
patents and/or patent applications reference herein are hereby
incorporated by reference in their entirety for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 depicts an alignment of the upstream sequences of
mouse and human microRNA-379 region. Sequence legend: mouse (SEQ ID
NO:49); human (SEQ ID NO:50); consensus (SEQ ID NO:51). The
transcript of mouse miR-379 is SEQ ID NO: 118, and that of human
miR-379 is SEQ ID NO: 119.
[0011] FIGS. 2A-2B are a diagram (2A) and a histogram (2B). FIG. 2A
is a diagram showing the mega cluster of microRNAs (miRNAs) and
their upstream promoter region. FIG. 2B depicts bar graphs showing
up-regulation of cluster miRNAs and the host long noncoding RNA
(lnc-RNA) in glomerular mesangial cells (MC) obtained from
glomeruli of diabetic and control mice.
[0012] FIG. 3 is a bar graph showing key cluster miRNAs and the
host IncRNA-MGC induced in MC treated with high glucose (HG) or
transforming growth factor beta (TGF.beta.1).
[0013] FIG. 4 is a diagram showing 5'RACE (Rapid Amplification of
cDNA Ends) to clone the cluster and LncRNA-MGC (lncMGC) upstream
region.
[0014] FIG. 5 is a bar graph showing that the expression of key
indicated targets of miRNAs of the cluster is down-regulated in
glomeruli of diabetic mice.
[0015] FIG. 6 is a bar graph showing that Tnrc6b and ER
(endoplasmic reticulum) degredation-enhancing
alpha-mannosidase-like 3 (EDEM) expression levels are decreased by
miR-379 mimic (M) and increased by miR-379 inhibitor (I) oligos in
MMCs.
[0016] FIG. 7 is a bar graph showing that the expression levels of
lnc-MGC and key cluster miRNAs are reduced in MMC transfected with
an siRNA mixture targeting the lncMGC (silnc-MGC).
[0017] FIG. 8 is a bar graph depicting that diabetes induced
increase in expression of key miRNAs and the lnc-MGC in mice
glomeruli is ameliorated in CHOP knockout (KO) mice relative to
wild type (WT).
[0018] FIG. 9 is a bar graph depicting that expression levels of
key miRNA target genes are higher in the glomeruli of CHOP KO mice
versus WT mice.
[0019] FIG. 10 is a diagram showing the scheme by which microRNA
mega cluster and lnc-MGC can promote the progression of diabetic
nephropathy (DN).
[0020] FIG. 11A is a schematic of genome editing using the
RNA-guided Cas9 nucleases from the microbial CRISPR (clustered
regularly interspaced short palindromic repeat)-Cas systems for
generating miR-379 knock-out mouse. FIG. 11B depicts the miR-379
genomic region (SEQ ID NO: 45) complementary regions of five guide
RNAs. S1, S2, and S3 are sense guide RNAs; and AS1 and AS2 are
antisense guide RNAs.
[0021] FIGS. 12A-12F are bar graphs of normalized expression of
microRNAs in the Lnc-MGC, the host of microRNA-379 cluster, under
normal (CTR, db/+) and diabetic conditions (db/db or streptozotocin
(STZ)). FIGS. 12G-12J are bar graphs of normalized expression of
Linc-MGC, Mirg (another lncRNA located in the locus of miRNA-379
cluster) under normal (CTR, db/+) and diabetic conditions (db/db or
streptozotocin (STZ)). FIGS. 12K-12L are bar graphs of Mirg
expression under when treated with normal glucose (NG), serum
depletion (SD), TGF.beta., or high glucose (HG) conditions.
[0022] FIGS. 13A-13D are graphs of normalized mRNA expression of
the putative target genes under different treatment conditions.
[0023] FIGS. 14A-14B are bar graphs of normalized Luciferase (Luc)
activity of the EDEM3 expression vectors after cotransfection to
MMC with miR-379 mimic. The data suggests that miR200 family and
miR-379 cluster may collaborate to inhibit EDEM3 expression. The
data also suggests that miR-379 cluster and miR-200b upregulated in
diabetic conditions induces DN through hypertrophy and fibrosis
mediated by EDEM3 (ER stress).
[0024] FIGS. 15A-15W are bar graphs of normalized RNA expression.
FIGS. 15C, 15K, and 15O depict western blots. MMC was treated with
tunicamycin (TM), a known ER stress inducer, and the expression of
miR-379 cluster was tested.
[0025] FIGS. 16A-16B depict schematics of representative Gapmer
designs. The basic design of the Gapmers is three LNAs at both 5'
and 3' ends of oligonucleotides and backbone is phosphorothioated.
Sequence legend (FIG. 16B, in order of appearance): SEQ ID NOS:
52-54. FIGS. 16C-16L are bar graphs of normalized RNA expression.
MGC10 transfection inhibited expression of lnc-MGC significantly at
48 hours after MGC10 transfection. MCG10 also reduced the
expression of lnc-MGC even after TGF.beta. treatment. Some miRNAs
in miR-379 cluster were reduced by MCG10 in MMC. Several targets
(EDEME3, Tnrc6b, and Phf21a) of miR-379 cluster were also
upregulated by MGC10, suggesting that downregulation of miR-379
cluster restores the target expression.
[0026] FIGS. 17A-17F demonstrates that MGC10 inhibits miR-379
cluster miRNAs in the mouse kidney in vivo. FIG. 17A is a diagram
of mouse receiving subcutaneous injection of 5 mg/kg MGC10. FIGS.
17B-17F are bar graphs of normalized RNA expression after
subcutaneous injection of 5 mg/kg MGC10, which consistently
inhibited the expression of lnc-MGC in kidney cortex at 24-72 hours
after injection. Three mice in each group were injected. The
expression of lnc-MGC (FIG. 17B) and miRNAs in the miR-379 cluster,
miR-379 (FIG. 17C), miR-495 (FIG. 17D), miR-377 (FIG. 17E) were
inhibited by subcutaneous injection of 5 mg/kg MGC10, while miR-882
outside of the cluster was not (FIG. 17F). Three mice were injected
for each condition and each time point. Gene expression quantified
in cortical samples are shown. Results are mean+SE in triplicate
PCRs from each mouse, *, P<0.05. These results suggest that
MGC10 is effective to reduce the expression of lnc-MGC and miR-379
cluster miRNAs.
[0027] FIG. 18A shows PAS staining images of WT-NS, WT-STZ, STZ-C
and STZ-MGC10. PAS staining showed mesangial expression and
increased glomerular size in diabetic mice compared to that in
non-diabetic mice and those were reduced in diabetic mice injected
with MGC10. FIG. 18B is a bar graph of PAS positive area. FIG. 18C
is a bar graph of glomerular area. FIG. 18D is an image of NS, STZ,
STZ+C and STZ+MGC10. FIG. 18E is a bar graph of percent area. These
results showed that MGC10 inhibits miR-379 and restore the EDEM3
and suppress the ER-stress in diabetic kidney.
[0028] FIGS. 19A-19F are bar graphs of normalized RNA expression
showing HMGC10 inhibiting the effects of HG or TGF.beta.. FIGS. 19A
and 19B depict Human homologue of lnc-MGC (hlnc-MGC) and its
inhibition by HMGC10 in human MC. Significant increase of hlnc-MGC,
miR-379, miR-494, miR495, miR-377 in human MC (HMC) treated with
TGF-.beta.1 (FIG. 19A) or HG (FIG. 19B) relative to respective
controls (SD or NG), but not miR-882 (outside of miR-379 cluster).
These increases were significantly reduced in HMC transfected with
HMGC10 compared to control oligo. FIG. 19C depicts HMGC10 mediated
restoration of miR-370 cluster targets, EDEM3, ATF3, CUGBP2 and
CPEB4 which were inhibited by TGF-.beta.1 in HMC. FIG. 19D depicts
significant increase of pro-fibrotic genes, TGF-.beta.1, COL1A2,
COL4A1, F1 and CTGF in HMC treated with TGF-B1 and their
significant inhibition ion HMC transfected with HMGC10. FIG. 19E
depicts HMGC10 mediated restoration of miR-379 cluster targets,
EDEM3, ATF3, CGBP2 and CPEB4 which were inhibited by HG in HMC.
FIG. 19F depicts significant increase of pro-fibrotic genes,
TGF-.beta.1, COL1A2, COL4A1, FN1 and CTGF in HMC treated with HG
which was significantly inhibited by HMGC10. Result are mean+SE in
triplicate PCRs from three-four independent culture experiments. *,
P<0.05.
[0029] FIGS. 20A-20D are depictions of genomic region of mouse with
a 36 base pair deletion in the miR-379 locus, generated using the
CRISPR/CAS9 system as described in FIG. 11. FIG. 20A represents SEQ
ID NO: 46; FIG. 20B represents SEQ ID NO: 47; FIG. 20C represents
SEQ ID NO: 47 with the guide RNAs; and FIG. 20D represents SEQ ID
NO: 48.
[0030] FIGS. 21A-21B are bar graphs depicting expression levels of
miR-379 (FIG. 21A) and EDEM3 (FIG. 21B) from miR-379 knockout
(miR-379KO) mice. FIG. 21A depicts relative expression of miR-379
in kidney mesangial cells from three miR-379KO mice compared to
wild type mice. FIG. 21B depicts relative protein expression of
EDEM3 (western blot (left panel) and bar graph (right panel)), a
target of miR-379, from miR-379KO mice compared to wild type
mice.
[0031] FIG. 22 depicts a schematic of the strategy for replacing a
human miR-379 region with a poly(A) signal to terminate
transcription of the human miR-379. Sequence legend: SEQ ID NO:
117.
[0032] FIG. 23A depicts a bar graph of glomerular basement membrane
(GBM) thickness, showing that the GBM thickness was significantly
increased in diabetic mice (STZ and STZ-C) compared to non-diabetic
mice (NS), and this was attenuated in diabetic mice injected with
MGC10 (STZ-MGC10). *, P<0.05. FIG. 23B depicts a bar graph of
cell death measured by Terminal deoxynucleotidyl transferase (TdT)
dUTP Nick-End Labeling (TUNEL) assay, showing that cell death was
significantly increased (increase in TUNEL positive cells) in
diabetic mice (STZ and STZ-C) compared to non-diabetic mice (NS),
and this was attenuated in diabetic mice injected with MGC10
(STZ-MGC10). *, P<0.05.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0033] Provided herein is, inter alia, an isolated compound
including a nucleic acid sequence capable of hybridizing to an RNA
sequence 10 to 270 nucleobases downstream of the transcription
start site of a mammalian microRNA-379 transcript; method of
treating diabetic nephropathy in a subject with the compound;
method of inhibiting expression of a mammalian microRNA-379
megacluster.
[0034] In embodiments, the compound includes a nucleic acid
sequence having a nucleobase analog. In embodiments, the nucleic
acid sequence includes Locked Nucleic Acid (LNA), 2'-O-alkyl, 2'
O-Methyl, 2'-deoxy-2'fluoro, 2'-deoxy, a universal base,
5-C-methyl, an inverted deoxy abasic residue incorporation, or any
combination thereof. In embodiments, the nucleic acid sequence may
include analogs with positive backbones; non-ionic backbones,
modified sugars, and non-ribose backbones (e.g. phosphorodiamidate
morpholino oligos).
[0035] The current disclosure provides an isolated compound
including a nucleic acid sequence having at least 90% sequence
identity with a continuous 10 nucleobase sequence of SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or 24. The current disclosure further provides a
pharmaceutical composition including a compound of this disclosure,
and a pharmaceutically acceptable diluent, carrier, salt or
adjuvant.
[0036] The following definitions are included for the purpose of
understanding the present subject matter and for constructing the
appended patent claims. Abbreviations used herein have their
conventional meaning within the chemical and biological arts.
[0037] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by a
person of ordinary skill in the art. See, e.g., Singleton et al.,
DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley
& Sons (New York, N.Y. 1994); Sambrook et al., MOLECULAR
CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold
Springs Harbor, N.Y. 1989). Any methods, devices and materials
similar or equivalent to those described herein can be used in the
practice of this disclosure. The following definitions are provided
to facilitate understanding of certain terms used frequently herein
and are not meant to limit the scope of the present disclosure.
[0038] "Nucleic acid" or "oligonucleotide" or "polynucleotide" or
grammatical equivalents used herein means at least two nucleotides
covalently linked together. The term "nucleic acid" includes
single-, double-, multiple-stranded or branched DNA, RNA and
analogs (derivatives) thereof.
[0039] The term "modified internucleotide linkage" or
"internucleotide linkage analogue" and the like refers, in the
usual and customary sense, to a non-physiologic linkage between
nucleotides. For example, the term "phosphorothioate nucleic acid"
refers to a nucleic acid in which one or more internucleotide
linkages are through a phosphorothioate moiety (thiophosphate)
moiety. The phosphorothioate moiety may be a monothiophosphate
(--P(O).sub.3(S).sup.3---) or a dithiophosphate
(--P(O).sub.2(S).sub.2.sup.3---). In embodiments, one or more of
the nucleosides of a phosphorothioate nucleic acid are linked
through a phosphorothioate moiety (e.g. monothiophosphate) moiety,
and the remaining nucleosides are linked through a phosphodiester
moiety (--P(O).sub.4.sup.3---). In embodiments, one or more of the
nucleosides of a phosphorothioate nucleic acid are linked through a
phosphorothioate moiety (e.g. monothiophosphate) moiety, and the
remaining nucleosides are linked through a methylphosphonate
linkage. In embodiments, all the nucleosides of a phosphorothioate
nucleic acid are linked through a phosphorothioate moiety (e.g. a
monothiophosphate) moiety.
[0040] As used herein, phosphorothioate oligonucleotides
(phosphorothioate nucleic acids) are from about 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 nucleotides in length. In embodiments, the
phosphorothioate nucleic acids herein contain one or more
phosphodiester bonds. In other embodiments, the phosphorothioate
nucleic acids include alternate backbones (e.g., mimics or analogs
of phosphodiesters as known in the art, such as, boranophosphate,
methylphosphonate, phosphoramidate, or O-methylphosphoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press).
[0041] In embodiments, the phosphorothioate nucleic acids may
include one or more nucleic acid analog monomers known in the art,
such as, peptide nucleic acid monomer or polymer, locked nucleic
acid monomer or polymer, morpholino monomer or polymer, glycol
nucleic acid monomer or polymer, or threose nucleic acid monomer or
polymer. Other analog nucleic acids include those with positive
backbones; non-ionic backbones, and nonribose backbones, including
those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and
Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate
Modifications in Antisense Research, Sanghui & Cook, eds.
Nucleic acids containing one or more carbocyclic sugars are also
included within one definition of nucleic acids. Modifications of
the ribose-phosphate backbone may be done for a variety of reasons,
e.g., to increase the stability and half-life of such molecules in
physiological environments or as probes on a biochip. Mixtures of
naturally occurring nucleic acids and analogs can be made;
alternatively, mixtures of different nucleic acid analogs, and
mixtures of naturally occurring nucleic acids and analogs may be
made. Phosphorothioate nucleic acids and phosphorothioate polymer
backbones can be linear or branched. For example, the branched
nucleic acids are repetitively branched to form higher ordered
structures such as dendrimers and the like.
[0042] The terms "analog," "nucleobase analog" and the like, in the
context of nucleic acid bases refer, in the usual and customary
sense, to chemical moieties that can substitute for normal (i.e.,
physiological) nucleobases (i.e., A, T, G, C and U) in nucleic
acids. Nucleobase analogs can be categorized as purine analogs and
pyrimidine analogs. Purine analogs have a core purine ring
structure which is substituted to form a purine analog. Pyrimidine
analogs have a core pyrimidine ring structure which is substituted
to form a pyrimidine analog. Substitution may be endocyclic (i.e.,
within the purine or pyrimidine ring structure) or exocyclic (i.e.,
attached to the purine or pyrimidine ring structure). Exemplary
nucleobase analogs include, but are not limited to:
1,5-dimethyluracil, 1-methyluracil, 2-amino-6-hydroxyaminopurine,
2-aminopurine, 3-methyluracil, 5-(hydroxymethyl)cytosine,
5-bromouracil, 5-carboxycytosine, 5-fluoroorotic acid,
5-fluorouracil, 5-formylcytosine, 5-formyluracil, 6-azathymine,
6-azauracil, 8-azaadenine, 8-azaguanine, N6-carbamoylmethyladenine,
N6-hydroxyadenine, allopurinol, hypoxanthine, thiouracil, locked
nucleic acid (LNA), 2'-O-alkyl nucleobase, 2'-Fluoro nucleobase,
and 2'-OMe nucleobase.
[0043] As used herein, locked nucleic acid (LNA) is a modified RNA
nucleotide. LNAs are RNA molecules which possess an extra bridge
connecting the 2' oxygen and 4' carbon of the ribose moiety. The
ribose becomes locked in the 3'-endo (North) conformation. Base
stacking and backbone pre-organization are enhanced by the locked
ribose conformation. In embodiments, LNA modification has several
advantages, including reduced toxicity, lower dosing, higher
affinity and efficient targeting.
[0044] As used herein the term "nucleobases" refers to the
naturally occurring compounds, which form the differentiating
component of nucleotides; five bases occur in nature, three of
which are common to RNA and DNA (uracil replaces thymine in RNA).
Bases are divided into two groups, purines and pyrimidines, based
on their chemical structure. Purines are larger, double-ring
molecules comprising adenine and guanine, whereas pyrimidines have
only a single-ring structure and comprise cytosine and
thymine/uracil. Because of the different size of the two types of
nucleobases, purines can only base pair with pyrimidines in order
to preserve the DNA molecule's constant width. More specifically,
the only base pairs that will fit the structure of the particular
molecule are adenine-thymine and cytosine-guanine.
[0045] The term "cell" as used herein also refers to individual
cells, cell lines, or cultures derived from such cells. A "culture"
refers to a composition comprising isolated cells of the same or a
different type.
[0046] Diabetic nephropathy (DN) is typically defined by
macroalbuminuria--that is, a urinary albumin excretion of more than
300 mg in a 24-hour collection--or macroalbuminuria and abnormal
renal function as represented by an abnormality in serum
creatinine, calculated creatinine clearance, or glomerular
filtration rate (GFR). Clinically, diabetic nephropathy is
characterized by a progressive increase in proteinuria and decline
in GFR, hypertension, and a high risk of cardiovascular morbidity
and mortality.
[0047] As used herein, "early stage DN" or "incipient DN" is
characterized by microalbuminuria, which is defined as levels of
albumin ranging from 30 to 300 mg in a 24-h urine collection.
Microalbuminuria progresses to overt nephropathy. Renal disease is
suspected to be secondary to diabetes in the clinical setting of
long-standing diabetes. This is supported by the history of
diabetic retinopathy, particularly in type 1 diabetics, in whom
there is a strong correlation. The natural history of diabetic
nephropathy is a process that progresses gradually over years.
[0048] Renal biopsy findings consistent with diabetic nephropathy
in the early stages of DN are mesangial expansion and glomerular
basement membrane thickening. Eventual progression of diabetic
nephropathy can lead to nodular glomerulosclerosis, also referred
to as Kimmelstiel-Wilson disease.
[0049] Early diabetes is heralded by glomerular hyperfiltration and
an increase in GFR. This is believed to be related to increased
cell growth and expansion in the kidneys, possibly mediated by
hyperglycemia itself. Microalbuminuria typically occurs after 5
years in type 1 diabetes. Overt nephropathy, with urinary protein
excretion higher than 300 mg/day, often develops after 10 to 15
years. ESRD develops in 50% of type 1 diabetics, with overt
nephropathy within 10 years.
[0050] Type 2 diabetes has a more variable course. Patients often
present at diagnosis with microalbuminuria because of delays in
diagnosis and other factors affecting protein excretion. Fewer
patients with microalbuminuria progress to advanced renal disease.
Without intervention, approximately 30% progress to overt
nephropathy and, after 20 years of nephropathy, approximately 20%
develop ESRD. Because of the high prevalence of type 2 compared
with type 1 diabetes, however, most diabetics on dialysis are type
2 diabetics.
[0051] Long-standing hyperglycemia is known to be a significant
risk factor for the development of diabetic nephropathy.
Hyperglycemia may directly result in mesangial expansion and injury
by an increase in the mesangial cell glucose concentration. The
glomerular mesangium expands initially by cell proliferation and
then by cell hypertrophy. Increased mesangial stretch and pressure
can stimulate this expansion, as can high glucose levels.
Transforming growth factor .beta. (TGF-.beta.) is particularly
important in the mediation of expansion and later fibrosis via the
stimulation of collagen and fibronectin. Glucose can also bind
reversibly and eventually irreversibly to proteins in the kidneys
and circulation to form advanced glycosylation end products (AGEs).
AGEs can form complex cross-links over years of hyperglycemia and
can contribute to renal damage by stimulation of growth and
fibrotic factors via receptors for AGEs. In addition, mediators of
proliferation and expansion, including platelet-derived growth
factor, TGF-.beta., and vascular endothelial growth factor (VEGF)
that are elevated in diabetic nephropathy can contribute to further
renal and microvascular complications.
[0052] Proteinuria, a marker and potential contributor to renal
injury, accompanies diabetic nephropathy. Increased glomerular
permeability will allow plasma proteins to escape into the urine.
Some of these proteins will be taken up by the proximal tubular
cells, which can initiate an inflammatory response that contributes
to interstitial scarring eventually leading to fibrosis.
Tubulointerstitial fibrosis is seen in advanced stages of diabetic
nephropathy and is a better predictor of renal failure than
glomerular sclerosis. Hyperglycemia, angiotensin II, TGF-.beta.,
and likely proteinuria itself all play roles in stimulating this
fibrosis. There is an epithelial-mesenchymal transition that takes
place in the tubules, with proximal tubular cell conversion to
fibroblast-like cells. These cells can then migrate into the
interstitium and produce collagen and fibronectin.
[0053] In diabetic nephropathy, the activation of the local
renin-angiotensin system occurs in the proximal tubular epithelial
cells, mesangial cells, and podocytes. Angiotensin II (ATII) itself
contributes to the progression of diabetic nephropathy. ATII is
stimulated in diabetes despite the high-volume state typically seen
with the disease, and the intrarenal level of ATII is typically
high, even in the face of lower systemic concentrations. ATII
preferentially constricts the efferent arteriole in the glomerulus,
leading to higher glomerular capillary pressures. In addition to
its hemodynamic effects, ATII also stimulates renal growth and
fibrosis through ATII type 1 receptors, which secondarily
upregulate TGF-.beta. and other growth factors.
[0054] Control of hypertension has clearly shown to be an important
and powerful intervention in decreasing the progression of diabetic
nephropathy. In diabetics who have disordered autoregulation at the
level of the kidney, systemic hypertension can contribute to
endothelial injury. Human studies of type 2 diabetics have shown
that blood pressure lowering, regardless of the agent used, retards
the onset and progression of diabetic nephropathy. In animal
studies, the degree and severity of the diabetic nephropathy were
strongly linked to systemic blood pressure.
[0055] The fact that most types 1 and 2 diabetics do not develop
diabetic nephropathy (DN) suggests that other factors may be
involved. Genetic factors clearly play a role in the predisposition
to diabetic nephropathy in family members who have DN, and linkage
to specific areas on the human genome is evolving. The theory of a
reduction in nephron number at birth indicates that individuals
born with a reduced number of glomeruli may be predisposed to
subsequent renal injury and progressive nephropathy. This has been
shown in animal studies in which the mother was exposed to
hyperglycemia at the time of pregnancy. If this linkage is true in
humans, that would have important implications concerning the role
of maternal factors in the eventual development of kidney
disease.
[0056] Diabetic nephropathy (DN) include the expansion and
hypertrophy of glomerular mesangial cells (MCs), increased
accumulation of extracellular matrix (ECM) proteins such as
collagen 1alpha1 (Col1.alpha.1), Col1.alpha.2, Col4.alpha.1 and
fibronectin, and tubulointerstitial fibrosis, podocyte dysfunction
and proteinuria. Levels of transforming growth factor-beta1
(TGF-.beta.1) are increased in MCs and other renal cells in
diabetics and TGF-.beta.1 mediates many of the adverse effects.
Several biochemical mechanisms of action have been reported for
TGF-.beta.1. Factors relevant to the pathogenesis of DN such as
angiotensin II, and high glucose (HG), increase TGF-.beta.1
expression in MCs in vitro and in vivo. Signals from the activated
TGF-.beta.1 receptor complex are transduced to the nucleus by Smad
proteins, including Smad2/3/4, which regulate TGF-.beta.-induced
genes, including PAI-1, collagen and p21cip1/waf1. However, the
molecular mechanisms by which diabetic conditions and TGF-.beta.1
regulate the genes that increase the hypertrophy, protein synthesis
and fibrosis associated with DN are not fully clear. A few
microRNAs (miRNAs or miRs, in short) are involved in mediating the
pro-fibrotic effects of TGF-.beta.1 in MCs in vitro and diabetic
conditions in vivo.
[0057] microRNAs (miRNA) are endogenously produced, short
single-stranded non-coding RNAs (.about.20-23 nucleotides) that
play key roles in post-transcriptional regulation of gene
expression to silence genes by repressing the translation or
inducing the degradation of target mRNAs. There are more than 1000
mammalian miRNAs that can target nearly 60% of mRNAs in the genome,
and therefore, they regulate many key cellular functions. The terms
microRNA, miRNA, and miR are interchangeable.
[0058] Long ncRNAs (lncRNAs) are long transcripts that range from
>200 nucleotides up to .about.100 kb, and are similar to
messenger RNAs (mRNAs) but lack protein coding (translation)
potential. LncRNAs can regulate the expression of local and distal
genes by various mechanisms that include recruiting histone
modifying complexes and modulating the activities of transcription
factors (TFs). LncRNAs also serve as hosts for miRNAs and/or a
miRNA megacluster. LncRNAs have cell-specific expression, and
function in various biological processes including transcription,
differentiation, and the immune response.
[0059] As used herein, the microRNA megacluster is a region of the
genome where more than 10 microRNA genes are encoded. In
embodiments, 35-60 microRNAs are encoded in the region. In
embodiments, some of these clustered miRNA genes may be encoded by
a single-copy DNA sequence. Alternatively, the miRNA genes may be
arranged in tandem arrays of closely related sequences.
[0060] As used herein, the microRNA-379 transcript is a RNA
sequence transcribed from a microRNA-379 gene of a mammalian
genome, e.g., a human genome. In its ordinary meaning, a
"transcript" in molecular biology or similar context is a product
of transcription. miRNAs are transcribed as much larger primary
transcripts (pri-miRNAs). The vast majority of mature miRNAs are
produced from primary transcripts of microRNAs (pri-miRNAs) by a
multi-step pathway. In mammals, miRNAs are first transcribed as
longer primary transcripts called primary miRNA (pri-miRNA). The
transcript may contain multiple miRNA stem loops and is capped at
the 5' end through polyadenylation. Drosha, a nuclear RNase III, is
recruited to crop the pri-miRNA transcript into a hairpin-shaped
structure, about 70 nt long, known as precursor-miRNA (pre-miRNA).
This cleavage event is critical and site-specific, as it determines
the mature miRNA sequence. The pre-miRNA is then exported out of
the nucleus for further cleavage into a 22 nt duplex. The
complementary strand becomes degraded leaving one fully mature
miRNA strand. Mature miRNA then associate with several members of
the Argonaute protein family to form the RNA-induced silencing
complex which then binds to specific protein-coding mRNA
transcripts, directing mRNA inactivation by translational
repression, deadenylation, or degradation.
[0061] As used herein, plasminogen activator inhibitor-1 (PAI-1) is
an endothelial plasminogen activator inhibitor or serpin E1 is a
protein that in humans is encoded by the SERPINE1 gene. PAI-1 is a
serine protease inhibitor (serpin) that functions as the principal
inhibitor of tissue plasminogen activator (tPA) and urokinase
(uPA), the activators of plasminogen and hence fibrinolysis (the
physiological breakdown of blood clots). It is a serine protease
inhibitor (serpin) protein (SERPINE1). Other PAI, plasminogen
activator inhibitor-2 (PAI-2) is secreted by the placenta and only
present in significant amounts during pregnancy. In addition,
protease nexin acts as an inhibitor of tPA and urokinase. PAI-1,
however, is the main inhibitor of the plasminogen activators.
[0062] As used herein, connective-tissue growth factor (CTGF) is a
secreted protein implicated in multiple cellular events including
angiogenesis, skeletogenesis and wound healing.
[0063] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over
a specified region when compared and aligned for maximum
correspondence over a comparison window or designated region) as
measured using a BLAST or BLAST 2.0 sequence comparison algorithms
with default parameters described below, or by manual alignment and
visual inspection (see, e.g., NCBI web site or the like). Such
sequences are then said to be "substantially identical." This
definition also refers to, or may be applied to, the compliment of
a test sequence. The definition also includes sequences that have
deletions and/or additions, as well as those that have
substitutions. As described below, the preferred algorithms can
account for gaps and the like. Preferably, identity exists over a
region that is at least about 10 amino acids or 20 nucleotides in
length, or more preferably over a region that is 10-50 amino acids
or 20-50 nucleotides in length. As used herein, percent (%) amino
acid sequence identity is defined as the percentage of amino acids
in a candidate sequence that are identical to the amino acids in a
reference sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity. Alignment for purposes of determining percent sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign
(DNASTAR) software. Appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full-length of the sequences being compared can be determined
by known methods.
[0064] For sequence comparisons, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0065] "Patient," "subject," "patient in need thereof," and
"subject in need thereof" are herein used interchangeably and refer
to a living organism suffering from or prone to a disease or
condition that can be treated by administration using the methods
and compositions provided herein. Non-limiting examples include
humans, other mammals, bovines, rats, mice, dogs, monkeys, goat,
sheep, cows, deer, and other non-mammalian animals. In some
embodiments, a patient is human. Tissues, cells and their progeny
of a biological entity obtained in vitro or cultured in vitro are
also contemplated.
[0066] The terms "treat," "treating" or "treatment," and other
grammatical equivalents as used herein, include alleviating,
abating, ameliorating, or preventing a disease, condition or
symptoms, preventing additional symptoms, ameliorating or
preventing the underlying metabolic causes of symptoms, inhibiting
the disease or condition, e.g., arresting the development of the
disease or condition, relieving the disease or condition, causing
regression of the disease or condition, relieving a condition
caused by the disease or condition, or stopping the symptoms of the
disease or condition, and are intended to include prophylaxis. The
terms further include achieving a therapeutic benefit and/or a
prophylactic benefit. By therapeutic benefit is meant eradication
or amelioration of the underlying disorder being treated. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the patient, notwithstanding that the patient may still
be afflicted with the underlying disorder.
[0067] The terms "prevent," "preventing," or "prevention," and
other grammatical equivalents as used herein, include to keep from
developing, occur, hinder or avert a disease or condition symptoms
as well as to decrease the occurrence of symptoms. The prevention
may be complete (i.e., no detectable symptoms) or partial, so that
fewer symptoms are observed than would likely occur absent
treatment. The terms further include a prophylactic benefit. For a
disease or condition to be prevented, the compositions may be
administered to a patient at risk of developing a particular
disease, or to a patient reporting one or more of the physiological
symptoms of a disease, even though a diagnosis of this disease may
not have been made.
[0068] The term "inhibiting" also means reducing an effect (disease
state or expression level of a gene/protein/mRNA) relative to the
state in the absence of a compound or composition of the present
disclosure.
[0069] A "test compound" as used herein refers to an experimental
compound used in a screening process to identify activity,
non-activity, or other modulation of a particularized biological
target or pathway.
[0070] "Control" or "control experiment" is used in accordance with
its plain ordinary meaning and refers to an experiment in which the
subjects or reagents of the experiment are treated as in a parallel
experiment except for omission of a procedure, reagent, or variable
of the experiment. In some instances, the control is used as a
standard of comparison in evaluating experimental effects. In some
embodiments, a control is the measurement of the activity of a
protein in the absence of a compound as described herein (including
embodiments and examples).
[0071] "Disease" or "condition" refer to a state of being or health
status of a patient or subject capable of being treated with the
compounds or methods provided herein. In some instances, "disease"
or "condition" refers to diabetes nephropathy (DN).
[0072] "Contacting" is used in accordance with its plain ordinary
meaning and refers to the process of allowing at least two distinct
species (e.g. chemical compounds including biomolecules or cells)
to become sufficiently proximal to react, interact or physically
touch. It should be appreciated; however, the resulting reaction
product can be produced directly from a reaction between the added
reagents or from an intermediate from one or more of the added
reagents which can be produced in the reaction mixture. In some
embodiments contacting includes allowing a compound described
herein to interact with a protein or enzyme.
[0073] The terms "phenotype" and "phenotypic" as used herein refer
to an organism's observable characteristics such as onset or
progression of disease symptoms, biochemical properties, or
physiological properties.
[0074] The word "expression" or "expressed" as used herein in
reference to a DNA nucleic acid sequence (e.g. a gene) means the
transcriptional and/or translational product of that sequence. The
level of expression of a DNA molecule in a cell may be determined
on the basis of either the amount of corresponding mRNA that is
present within the cell or the amount of protein encoded by that
DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning:
A Laboratory Manual, 18.1-18.88). When used in reference to
polypeptides, expression includes any step involved in the
production of a polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion. Expression can be
detected using conventional techniques for detecting protein (e.g.,
ELISA, Western blotting, flow cytometry, immunofluorescence,
immunohistochemistry, etc.).
[0075] The term "gene" means the segment of DNA involved in
producing a protein; it includes regions preceding and following
the coding region (leader and trailer) as well as intervening
sequences (introns) between individual coding segments (exons). The
leader, the trailer as well as the introns include regulatory
elements that are necessary during the transcription and the
translation of a gene. Further, a "protein gene product" is a
protein expressed from a particular gene.
[0076] The term "promoter" and the like in the usual and customary
sense, is a region of DNA that initiates transcription of a
particular gene. Promoters are located near the transcription start
sites of genes, on the same strand and upstream on the DNA (towards
the 5' region of the sense strand). Upstream and downstream in the
usual and customary sense both refer to a relative position in DNA
or RNA. Each strand of DNA or RNA has a 5' end and a 3' end, so
named for the carbon position on the deoxyribose (or ribose) ring.
By convention, upstream and downstream relate to the 5' to 3'
direction in which RNA transcription takes place. Upstream is
toward the 5' end of the RNA molecule and downstream is toward the
3' end. When considering double-stranded DNA, upstream is toward
the 5' end of the coding strand for the gene in question and
downstream is toward the 3' end. Due to the anti-parallel nature of
DNA, this means the 3' end of the template strand is upstream of
the gene and the 5' end is downstream.
[0077] The term "an amount of" in reference to a polynucleotide or
polypeptide, refers to an amount at which a component or element is
detected. The amount may be measured against a control, for
example, wherein an increased level of a particular polynucleotide
or polypeptide in relation to the control, demonstrates enrichment
of the polynucleotide or polypeptide. The term refers to
quantitative measurement of the enrichment as well as qualitative
measurement of an increase or decrease relative to a control.
[0078] Throughout the description and claims of this specification
the word "comprise" and other forms of the word, such as
"comprising" and "comprises," means including but not limited to,
and is not intended to exclude, for example, other components.
[0079] "Analog," "analogue," or "derivative" is used in accordance
with its plain ordinary meaning within Chemistry and Biology and
refers to a chemical agent that is structurally similar to another
agent (i.e., a so-called "reference" agent) but differs in
composition, e.g., in the replacement of one atom by an atom of a
different element, or in the presence of a particular functional
group, or the replacement of one functional group by another
functional group, or the absolute stereochemistry of a chiral
center of the reference agent. In some embodiments, a derivative
may be a conjugate with a pharmaceutically acceptable agent, for
example, phosphate or phosphonate.
[0080] As used herein, the term "salt" refers to acid or base salts
of the agents used herein. Illustrative but non-limiting examples
of acceptable salts are mineral acid (hydrochloric acid,
hydrobromic acid, phosphoric acid, sulfuric acid, and the like)
salts, organic acid (acetic acid, propionic acid, glutamic acid,
citric acid, and the like) salts, and quaternary ammonium (methyl
iodide, ethyl iodide, and the like) salts.
[0081] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds that are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the present disclosure contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the present disclosure contain
relatively basic functionalities, acid addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, e.g.,
Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)).
Certain specific compounds of the present disclosure contain both
basic and acidic functionalities that allow the compounds to be
converted into either base or acid addition salts. Other
pharmaceutically acceptable carriers known to those of skill in the
art are suitable for the present disclosure. Salts tend to be more
soluble in aqueous or other protonic solvents that are the
corresponding free base forms. In other cases, the preparation may
be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,
2%-7% mannitol at a pH range of 4.5 to 5.5, which is combined with
buffer prior to use.
[0082] Thus, the compounds of the present disclosure may exist as
salts, such as with pharmaceutically acceptable acids. The present
disclosure includes such salts. Examples of such salts include
hydrochlorides, hydrobromides, sulfates, methanesulfonates,
nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.,
(+)-tartrates, (-)-tartrates, or mixtures thereof including racemic
mixtures), succinates, benzoates, and salts with amino acids such
as glutamic acid. These salts may be prepared by methods known to
those skilled in the art.
[0083] An "adjuvant" (from Latin, adiuvare: to aid) is a
pharmacological and/or immunological agent that modifies the effect
of other agents.
[0084] A "diluent" (also referred to as a filler, dilutant or
thinner) is a diluting agent. Certain fluids are too viscous to be
pumped easily or too dense to flow from one particular point to the
other. This can be problematic, because it might not be
economically feasible to transport such fluids in this state. To
ease this restricted movement, diluents are added. This decreases
the viscosity of the fluids, thereby also decreasing the
pumping/transportation costs.
[0085] The terms "administration" or "administering" refer to the
act of providing an agent of the current embodiments or
pharmaceutical composition including an agent of the current
embodiments to the individual in need of treatment.
[0086] By "co-administer" it is meant that a composition described
herein is administered at the same time, just prior to, or just
after the administration of additional therapies. The compound or
the composition of the disclosure can be administered alone or can
be co-administered to the patient. Co-administration is meant to
include simultaneous or sequential administration of the compound
individually or in combination (more than one compound or agent).
The preparations can also be combined, when desired, with other
active substances (e.g. to reduce metabolic degradation).
[0087] As used herein, "sequential administration" includes that
the administration of two agents (e.g., the compounds or
compositions described herein) occurs separately on the same day or
do not occur on a same day (e.g., occurs on consecutive days).
[0088] As used herein, "concurrent administration" includes
overlapping in duration at least in part. For example, when two
agents (e.g., any of the agents or class of agents described herein
that has bioactivity) are administered concurrently, their
administration occurs within a certain desired time. The agents'
administration may begin and end on the same day. The
administration of one agent can also precede the administration of
a second agent by day(s) as long as both agents are taken on the
same day at least once. Similarly, the administration of one agent
can extend beyond the administration of a second agent as long as
both agents are taken on the same day at least once. The bioactive
agents/agents do not have to be taken at the same time each day to
include concurrent administration.
[0089] As used herein, "intermittent administration includes the
administration of an agent for a period of time (which can be
considered a "first period of administration"), followed by a time
during which the agent is not taken or is taken at a lower
maintenance dose (which can be considered "off-period") followed by
a period during which the agent is administered again (which can be
considered a "second period of administration"). Generally, during
the second phase of administration, the dosage level of the agent
will match that administered during the first period of
administration but can be increased or decreased as medically
necessary.
[0090] As used herein, the term "administering" means oral
administration, administration as a suppository, topical contact,
intravenous, parenteral, intraperitoneal, intramuscular,
intralesional, intrathecal, intranasal or subcutaneous
administration, or the implantation of a slow-release device, e.g.,
a mini-osmotic pump, to a subject. Administration is by any route,
including parenteral and transmucosal (e.g., buccal, sublingual,
palatal, gingival, nasal, vaginal, rectal, or transdermal).
Parenteral administration includes, e.g., intravenous,
intramuscular, intra-arteriole, intradermal, subcutaneous,
intraperitoneal, intraventricular, and. Other modes of delivery
include, but are not limited to, the use of liposomal formulations,
intravenous infusion, transdermal patches, etc.
[0091] The compositions disclosed herein can be delivered
transdermally, by a topical route, formulated as applicator sticks,
solutions, suspensions, emulsions, gels, creams, ointments, pastes,
jellies, paints, powders, and aerosols. Oral preparations include
tablets, pills, powder, dragees, capsules, liquids, lozenges,
cachets, gels, syrups, slurries, suspensions, etc., suitable for
ingestion by the patient. Solid form preparations include powders,
tablets, pills, capsules, cachets, suppositories, and dispersible
granules. Liquid form preparations include solutions, suspensions,
and emulsions, for example, water or water/propylene glycol
solutions. The compositions of the present disclosure may
additionally include components to provide sustained release and/or
comfort. Such components include high molecular weight, anionic
mucomimetic polymers, gelling polysaccharides and finely-divided
drug carrier substrates. These components are discussed in greater
detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and
4,861,760. The entire contents of these patents are incorporated
herein by reference in their entirety for all purposes. The
compositions disclosed herein can also be delivered as microspheres
for slow release in the body. For example, microspheres can be
administered via intradermal injection of drug-containing
microspheres, which slowly release subcutaneously (see Rao, J.
Bioniater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and
injectable gel formulations (see, e.g., Gao Phann. Res. 12:857-863,
1995); or, as microspheres for oral administration (see, e.g.,
Eyles, J. Phann. Pharmacol. 49:669-674, 1997).
[0092] As used herein, an "effective amount" or "therapeutically
effective amount" is that amount sufficient to affect a desired
biological effect, such as beneficial results, including clinical
results. As such, an "effective amount" depends upon the context in
which it is being applied. An effective amount may vary according
to factors known in the art, such as the disease state, age, sex,
and weight of the individual being treated. Several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation. In
addition, the compositions/formulations of this disclosure can be
administered as frequently as necessary to achieve a therapeutic
amount.
[0093] Pharmaceutical compositions may include compositions wherein
the therapeutic drug (e.g., agents described herein, including
embodiments or examples) is contained in a therapeutically
effective amount, i.e., in an amount effective to achieve its
intended purpose. The actual amount effective for a particular
application will depend, inter alia, on the condition being
treated. When administered in methods to treat a disease, such
compositions will contain an amount of therapeutic drug effective
to achieve the desired result, e.g., modulating the activity of a
target molecule, and/or reducing, eliminating, or slowing the
progression of disease symptoms.
[0094] The dosage and frequency (single or multiple doses)
administered to a mammal can vary depending upon a variety of
factors, for example, whether the mammal suffers from another
disease, and its route of administration; size, age, sex, health,
body weight, body mass index, and diet of the recipient; nature and
extent of symptoms of the disease being treated, kind of concurrent
treatment, complications from the disease being treated or other
health-related problems. Other therapeutic regimens or agents can
be used in conjunction with the methods and agents of this
disclosure. Adjustment and manipulation of established dosages
(e.g., frequency and duration) are well within the ability of those
skilled in the art.
[0095] For any therapeutic agent described herein, the
therapeutically effective amount can be initially determined from
cell culture assays. Target concentrations will be those
concentrations of therapeutic drug(s) that are capable of achieving
the methods described herein, as measured using the methods
described herein or known in the art.
[0096] As is well known in the art, therapeutically effective
amounts for use in humans can also be determined from animal
models. For example, a dose for humans can be formulated to achieve
a concentration that has been found to be effective in animals. The
dosage in humans can be adjusted by monitoring agent's
effectiveness and adjusting the dosage upwards or downwards, as
described above. Adjusting the dose to achieve maximal efficacy in
humans based on the methods described above and other methods is
well within the capabilities of the ordinarily skilled artisan.
[0097] Dosages may be varied depending upon the requirements of the
patient and the therapeutic drug being employed. The dose
administered to a patient should be sufficient to effects a
beneficial therapeutic response in the patient over time. The size
of the dose also will be determined by the existence, nature, and
extent of any adverse side-effects. Determination of the proper
dosage for a particular situation is within the skill of the
practitioner. Generally, treatment is initiated with smaller
dosages which are less than the optimum dose of the agent.
Thereafter, the dosage is increased by small increments until the
optimum effect under circumstances is reached. Dosage amounts and
intervals can be adjusted individually to provide levels of the
administered agent effective for the particular clinical indication
being treated. This will provide a therapeutic regimen that is
commensurate with the severity of the individual's disease
state.
[0098] A weight percent of a component, unless specifically stated
to the contrary, is based on the total weight of the formulation or
composition in which the component is included.
[0099] "Excipient" is used herein to include any other agent that
may be contained in or combined with a disclosed agent, in which
the excipient is not a therapeutically or biologically active
agent/agent. As such, an excipient should be pharmaceutically or
biologically acceptable or relevant (for example, an excipient
should generally be non-toxic to the individual). "Excipient"
includes a single such agent and is also intended to include a
plurality of excipients. For the purposes of the present disclosure
the term "excipient" and "carrier" are used interchangeably in some
embodiments of the present disclosure and said terms are defined
herein as, "ingredients which are used in the practice of
formulating a safe and effective pharmaceutical composition."
[0100] The term "about" refers to any minimal alteration in the
concentration or amount of an agent that does not change the
efficacy of the agent in preparation of a formulation and in
treatment of a disease or disorder. The term "about" with respect
to concentration range of the agents (e.g., therapeutic/active
agents) of the current disclosure also refers to any variation of a
stated amount or range which would be an effective amount or
range.
[0101] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it is understood that the particular value
forms another aspect. It is further understood that the endpoints
of each of the ranges are significant both in relation to the other
endpoint, and independently of the other endpoint. It is also
understood that there are a number of values disclosed herein, and
that each value is also herein disclosed as "about" that particular
value in addition to the value itself. It is also understood that
throughout the application, data are provided in a number of
different formats and that this data represent endpoints and
starting points and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point "15" are disclosed, it is understood that greater than,
greater than or equal to, less than, less than or equal to, and
equal to 10 and 15 are considered disclosed as well as between 10
and 15. It is also understood that each unit between two particular
units are also disclosed. For example, if 10 and 15 are disclosed,
then 11, 12, 13, and 14 are also disclosed.
Compound
[0102] The present disclosure includes an isolated compound
including a nucleic acid sequence capable of hybridizing to an RNA
sequence 10 to 270 nucleobases downstream of the transcription
start site of a mammalian microRNA-379 transcript or a microRNA-379
megacluster transcript. In embodiments, the present disclosure
includes an isolated compound including a nucleic acid sequence
capable of hybridizing to at least one nucleic acid base of a
downstream region of the transcription start site of a mammalian
microRNA-379 transcript or a microRNA-379 megacluster transcript.
In embodiments, the transcript is as exists immediately after
transcription, e.g., primary transcript mRNA or pre-mRNA. In
embodiments, the compound includes a nucleic acid sequence having a
nucleobase analog or modified internucleotide linkage.
[0103] In embodiments, the compound includes a nucleic acid
sequence having a nucleobase analog. In embodiments, the nucleic
acid sequence includes Locked Nucleic Acid (LNA), 2'-O-alkyl, 2'
O-Methyl, 2'-deoxy-2'fluoro, 2'-deoxy, a universal base,
5-C-methyl, an inverted deoxy abasic residue incorporation, or any
combination thereof. In embodiments, the nucleic acid sequence may
include analogs with positive backbones; non-ionic backbones,
modified sugars, and non-ribose backbones (e.g. phosphorodiamidate
morpholino oligos), including those described in U.S. Pat. Nos.
5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series
580, Carbohydrate Modifications in Antisense Research, Sanghui
& Cook, eds. Nucleic acids containing one or more carbocyclic
sugars are also included within one definition of nucleic
acids.
[0104] In embodiments, the nucleic acid sequence includes at least
one nucleic acid analog. In embodiments, the nucleic acid sequence
includes at least one nucleic acid analog having an alternate
backbone (e.g. phosphodiester derivative (e.g. phosphoramidate,
phosphorodiamidate, phosphorothioate, phosphorodithioate,
phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic
acid, phosphonoformic acid, methyl phosphonate, boron phosphonate,
or O-methylphosphoroamidite), peptide nucleic acid backbone(s),
LNA, or linkages). In embodiments, a nucleic acid sequence includes
or is DNA. In embodiments, a nucleic acid sequence includes or is
RNA. In embodiments, a nucleic acid sequence includes or is a
nucleic acid having internucleotide linkages selected from
phosphodiesters and phosphodiester derivatives (e.g.,
phosphoramidate, phosphorodiamidate, phosphorothioate,
phosphorodithioate, phosphonocarboxylic acids,
phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,
methyl phosphonate, boron phosphonate, O-methylphosphoroamidite, or
combinations thereof). In embodiments, a nucleic acid sequence
consists of a nucleic acid having internucleotide linkages selected
from phosphodiesters and phosphorothioates. In embodiments, a
nucleic acid sequence includes or is a nucleic acid having backbone
linkages selected from phosphodiesters and phosphorodithioates. In
embodiments, a nucleic acid sequence includes or is a nucleic acid
having phosphodiester backbone linkages. In embodiments, a nucleic
acid sequence includes or is a nucleic acid having phosphorothioate
backbone linkages. In embodiments, a nucleic acid sequence includes
or is a nucleic acid having phosphorodithioate backbone
linkages.
[0105] In embodiments, a nucleic acid sequence in the compound
includes a nucleic acid analog (e.g. LNA, 2'-O-alkyl, 2'-Fluoro, or
2' O-Methyl (2'-OMe)) at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
nucleobases. In embodiments, the compound includes a nucleic acid
sequence capable of hybridizing to an RNA sequence 10 to 270
nucleobases downstream of the transcription start site of a
mammalian microRNA-379 transcript, where the nucleic acid sequence
has an analog (e.g., LNA, 2'-O-alkyl, 2'-Fluoro, or 2' O-Methyl
(2'-OMe)) at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases. In
embodiments, the compound includes a nucleic acid sequence with an
analog (e.g., LNA, 2'-O-alkyl, 2'-Fluoro, or 2' O-Methyl (2'-OMe))
at 3 nucleobases.
[0106] In embodiments, the nucleobase analog is at the 5'-end or
the 3'-end of the nucleic acid sequence. In embodiments, the
nucleobase analog (e.g., LNA, 2'-O-alkyl, 2'-Fluoro, or 2' O-Methyl
(2'-OMe)) is at the 5'-end or the 3'-end of the nucleic acid
sequence. In embodiments, the nucleobase analog (e.g., LNA,
2'-O-alkyl, 2'-Fluoro, or 2'-OMe) is at the 5'-end and the 3'-end
of the nucleic acid sequence.
[0107] In embodiments, the nucleic acid sequence includes three,
four or five nucleobase analogs (e.g., LNA, 2'-O-alkyl, 2'-Fluoro,
or 2' O-Methyl (2'-OMe)) at the 5'-end or the 3'-end of the nucleic
acid sequence. In embodiments, the nucleic acid sequence includes
three, four or five nucleobase analogs (e.g., LNA, 2'-O-alkyl,
2'-Fluoro, or 2' O-Methyl (2'-OMe)) at the 5'-end and the 3'-end of
the nucleic acid sequence. In embodiments, the nucleic acid
sequence includes three nucleobase analogs (e.g., LNA, 2'-O-alkyl,
2'-Fluoro, or 2' O-Methyl (2'-OMe)) at the 5'-end or the 3'-end of
the nucleic acid sequence. In embodiments, the nucleic acid
sequence includes three nucleobase analogs (e.g., LNA, 2'-O-alkyl,
2'-Fluoro, or 2' O-Methyl (2'-OMe)) at the 5'-end and the 3'-end of
the nucleic acid sequence
[0108] In embodiments, the compound includes a nucleic acid
sequence with a modified internucleotide linkage. In embodiments,
the modified internucleotide linkage is a phosphorothioate (also
known as phosphothioate) linkage. In other embodiments, nucleic
acid analogs are included that may have alternate backbones (e.g.
phosphodiester derivatives), including, e.g., phosphoramidate,
phosphorodiamidate, phosphorodithioate, phosphonocarboxylic acids,
phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,
methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press); and peptide nucleic acid
backbones and linkages. Modifications of the ribose-phosphate
backbone may be done for a variety of reasons, e.g., to increase
the stability and half-life of such molecules in physiological
environments or as probes on a biochip. Mixtures of naturally
occurring nucleic acids and analogs can be made; alternatively,
mixtures of different nucleic acid analogs, and mixtures of
naturally occurring nucleic acids and analogs may be made. In
embodiments, the internucleotide linkages in DNA are
phosphodiester, phosphodiester derivatives, or a combination of
both.
[0109] In embodiments, the compound includes a nucleic acid
sequence with internal modified internucleotide linkage between
nucleobases at one or more positions. In embodiments, the compound
includes a nucleic acid sequence with internal modified
internucleotide linkage between nucleobases at one or more
positions, and one, two, three, or four nucleobase analogs at the
5'- or the 3'-ends of the nucleic acid sequence. In embodiments,
the compound includes a nucleic acid sequence with internal
internucleotide phosphorothioate linkage between nucleobases at one
or more positions, and one, two, three, or four nucleobase LNA
analogs at the 5'- or the 3'-ends of the nucleic acid sequence. In
embodiments, the compound includes a nucleic acid sequence with
internal modified internucleotide linkage between nucleobases at
one or more positions, and one, two, three, or four nucleobase
analogs at the 5'- and the 3'-ends of the nucleic acid sequence. In
embodiments, the compound includes a nucleic acid sequence with
internal internucleotide phosphorothioate linkage between
nucleobases at one or more positions, and one, two, three, or four
nucleobase LNA analogs at the 5'- and the 3'-ends of the nucleic
acid sequence.
[0110] Structures of exemplary molecules for internucleotide
analogyes, such as an LNA monomer, and internucleotide linkages,
such as phosphodiester linkage and phosphorothioate linkage, are
depicted below.
##STR00001##
[0111] In embodiments, the RNA sequence to which a nucleic acid
sequence of the present disclosure hybridizes to includes 11 to 27,
61 to 93, 115 to 139, or 246 to 265 nucleobases downstream of the
transcription start site of the gene. In embodiments, the target
site on the RNA sequence to which a nucleic acid sequence of the
present disclosure hybridizes to is listed in Table 1. The target
site range listed in Table 1 (middle column) reflects the
nucleobase positions counting from the transcription start site at
+1 of a target RNA.
TABLE-US-00001 TABLE 1 Nu- cleic Acid Iden- Target tity site
Nucleic acid sequence MGC8 +11 to +26 TGAAGGCCACACTAAC (SEQ ID NO:
1) MGC12 +12 to +27 ATGAAGGCCACACTAA (SEQ ID NO: 2) MGC15 +11 to
+25 GAAGGCCACACTAAC (SEQ ID NO: 3) MGC5 +64 to +79 CACGGTGCTGAAAGAG
(SEQ ID NO: 4) MGC6 +63 to +78 ACGGTGCTGAAAGAGA (SEQ ID NO: 5)
MGC13 +63 to +77 CGGTGCTGAAAGAGA (SEQ ID NO: 6) MGC14 +78 to +93
TCCTTGAATGGTTGCA (SEQ ID NO: 7) MGC18 +75 to +90 TTGAATGGTTGCACGG
(SEQ ID NO: 8) MGC20 +62 to +77 CGGTGCTGAAAGAGAG (SEQ ID NO: 9)
MGC10 +117 to +132 ATTTGGCAGTGGGAAG (SEQ ID NO: 10) MGC17 +116 to
+131 TTTGGCAGTGGGAAGC (SEQ ID NO: 11) MGC19 +115 to +130
TTGGCAGTGGGAAGCA (SEQ ID NO: 12) MGC1 +246 to +261 TCAAAAACATAACGCC
(SEQ ID NO: 13) MGC2 +247 to +262 GTCAAAAACATAACGC (SEQ ID NO: 14)
MGC3 +248 to +262 GGTCAAAAACATAACGC (SEQ ID NO: 15) MGC4 +248 to
+263 GGTCAAAAACATAACG (SEQ ID NO: 16) MGC7 +249 to +264
AGGTCAAAAACATAAC (SEQ ID NO: 17) MGC9 +249 to +263
AGGTCAAAAACATAACG (SEQ ID NO: 18) MGC11 +251 to +265
TAGGTCAAAAACATA (SEQ ID NO: 19) MGC16 +246 to +260 CAAAAACATAACGCC
(SEQ ID NO: 20) HMGC10 +124 to +139 GATTTGGCATTGGAAG (SEQ ID NO:
21) HMGC8 +12 to +27 GGAAGGCCATGTCAAC (SEQ ID NO: 22) HMGC5 +61 to
+76 GGCATTGATGGGGGAA (SEQ ID NO: 23) HMGC1 +249 to +265
TCAGAAATCATAACGCC (SEQ ID NO: 24)
[0112] In embodiments, compound includes, e.g., GATTTGGCATTGGAAG
(SEQ ID NO: 21) with internal internucleotide phosphorothioate
linkage between one or more nucleobases, and one, two, three, or
four nucleobase LNA analogs at the 5'- and/or the 3'-ends of the
nucleic acid sequence. The LNA analogs at the 5' and/or the 3'-ends
of the sequence are underlined, e.g., GATTTGGCATTGGAAG (SEQ ID NO:
21). In embodiments, the remaining internal internucleotide
linkages between nucleobases (italicized in the above sequence) are
phosphorothioate linkages. In embodiments, a compound is, e.g.,
GATTTGGCATTGGAAG (SEQ ID NO: 21), with internal internucleotide
phosphorothioate linkage between one or more nucleobases.
[0113] In embodiments, the compound includes a nucleic acid that
binds to the mouse miR-379 transcript including the upstream region
of mouse miR-379 and the mouse miR-379 sequence. The sequence of
the mouse miR-379 transcript including the upstream region of mouse
miR-379 and the mouse miR-379 sequence is shown in SEQ ID NO: 118.
The nucleic acid sequences of SEQ ID NOs: 1-20 hybridize to a
region of mouse miR-379 transcript of SEQ ID NO: 25 (the
transcription start site indicated with "+1"), i.e., SEQ ID NO:
118; in SEQ ID NO: 118, a uracil ("U") replaces each thymine ("T")
of SEQ ID NO: 25.
TABLE-US-00002 +1 (SEQ ID NO: 25)
ATTTTTCTGAGTTAGTGTGGCCTTCATCTGGTAATGTACTACCTGAGG
GGGGAGGTGCCGCCTCTCTTTCAGCACCGTGCAACCATTCAAGGAGGG
TGTGTTGTTCACCACATCTGCTTCCCACTGCCAAATCAGGCCTCAGAA
AAGCTTTCTGGAAGTGACGCCAGCTTCAGGGACAAGGCCCAAGTTTCT
AGGGGTCAACACCGTTCCATGGTTCCTGAAGAGATGGTAGACTATGGA
ACGTAGGCGTTATGTTTTTGACCTATGTAACATGGTCCACTAACTCT +1 (SEQ ID NO: 118)
AUUUUUCUGAGUUAGUGUGGCCUUCAUCUGGUAAUGUACUACCUGAGG
GGGGAGGUGCCGCCUCUCUUUCAGCACCGUGCAACCAUUCAAGGAGGG
UGUGUUGUUCACCACAUCUGCUUCCCACUGCCAAAUCAGGCCUCAGAA
AAGCUUUCUGGAAGUGACGCCAGCUUCAGGGACAAGGCCCAAGUUUCU
AGGGGUCAACACCGUUCCAUGGUUCCUGAAGAGAUGGUAGACUAUGGA
ACGUAGGCGUUAUGUUUUUGACCUAUGUAACAUGGUCCACUAACUCU
[0114] In embodiments, the compound includes a nucleic acid that
binds to the human miR-379 transcript including the upstream region
of human miR-379 and the human miR-379 sequence. The sequence of
the human miR-379 transcript including the upstream region of human
miR-379 and the human miR-379 sequence is shown in SEQ ID NO: 119.
The nucleic acid sequences of SEQ ID NOs: 21-24 hybridize to a
region of human miR-379 transcript of SEQ ID NO: 26 (the
transcription start site indicated with "+1"), i.e., SEQ ID NO:
119; in SEQ ID NO: 119, a uracil ("U") replaces each thymine ("T")
of SEQ ID NO: 26.
[0115] In embodiments, a nucleic acid sequence having 90-91%,
91-92%, 92-93%, 93-94%, 94-95%, 95-96%, 96-97%, 97-98%, or 98-99%
sequence identity with a continuous 10 nucleobase sequence of SEQ
ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 hybridizes to a region of human miR-379 transcript of
SEQ ID NO: 26, i.e., SEQ ID NO: 119; in SEQ ID NO: 119, a uracil
("U") replaces each thymine ("T") of SEQ ID NO: 26.
TABLE-US-00003 +1 (SEQ ID NO: 26)
AGTCTTTCCAAGTTGACATGGCCTTCCTGGAGGAATTACCACTTAG
GGTAGAGGCACCCCTTCCCCCATCAATGCCACTGCCCCACATTGGA
GGAGGGGTTGTTTATGTTCACCATGTGCCTGCTTCCAATGCCAAAT
CCAGCCTCAGAAAGCTTTCTGGAAGTGACGCCAACTTCAGGGGCAA
GGCCCTGGTTCTGGGGTCAGCACCATTCCGTGGTTCCTGAAGAGAT
GGTAGACTATGGAACGTAGGCGTTATGATTTCTGACCTATGTAACA TGGTCCACTAACTCT. +1
(SEQ ID NO: 119) AGUCUUUCCAAGUUGACAUGGCCUUCCUGGAGGAAUUACCACUUAG
GGUAGAGGCACCCCUUCCCCCAUCAAUGCCACUGCCCCACAUUGGA
GGAGGGGUUGUUUAUGUUCACCAUGUGCCUGCUUCCAAUGCCAAAU
CCAGCCUCAGAAAGCUUUCUGGAAGUGACGCCAACUUCAGGGGCAA
GGCCCUGGUUCUGGGGUCAGCACCAUUCCGUGGUUCCUGAAGAGAU
GGUAGACUAUGGAACGUAGGCGUUAUGAUUUCUGACCUAUGUAA CAUGGUCCACUAACUCU
[0116] The consensus sequence of the mouse and human miR-379
transcript corresponds to a transcript of the consensus sequence
provided in SEQ ID NO: 51.
[0117] In embodiments, the compound includes a nucleic acid
sequence that is 10 to 30 nucleobases in length. In embodiments,
the compound includes a nucleic acid sequence capable of
hybridizing at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases
within a RNA sequence 10 to 270 nucleobases downstream of the
transcription start site of a mammalian microRNA-379 transcript. In
embodiments, the mammalian microRNA-379 transcript is a human
microRNA-379 transcript. In embodiments, the compound includes a
nucleic acid sequence capable of hybridizing at least 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 nucleobases within the sequence of SEQ ID NO:
118 or 119. In embodiments, the compound includes a nucleic acid
sequence capable of hybridizing at least 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30 nucleobases within 10-20, 10-30, 20-40, 20-50, 40-60, 40-70,
60-80, 60-90, 80-100, 80-110, 100-120, 100-130, 120-140, 120-150,
140-160, 140-170, 160-180, 160-190, 180-200, 180-210, 200-220,
200-230, 220-240, 220-230, 240-260, or 240-270 nucleobases
downstream of the transcription start site (indicated with "+1") of
the transcript sequence of SEQ ID NO: 25 or 26 (i.e., transcript
sequence SEQ ID NO: 118 or 119), a sequence including the
transcript of SEQ ID NO: 25 or 26 (i.e., transcript sequence SEQ ID
NO: 118 or 119), or a variation thereof. In embodiments, the
compound includes a nucleic acid sequence capable of hybridizing at
least 5 nucleobases within the sequence of 10-20, 10-30, 20-40,
20-50, 40-60, 40-70, 60-80, 60-90, 80-100, 80-110, 100-120,
100-130, 120-140, 120-150, 140-160, 140-170, 160-180, 160-190,
180-200, 180-210, 200-220, 200-230, 220-240, 220-230, 240-260, or
240-270 nucleobases downstream of the transcription start site
(indicated with "+1") of the transcript of SEQ ID NO: 25 or 26
(i.e., transcript sequence SEQ ID NO: 118 or 119).
[0118] In embodiments, the compound includes a nucleic acid
sequence capable of hybridizing at least 5 nucleobases within a RNA
sequence 10 to 270 nucleobases downstream of the transcription
start site of a mammalian microRNA-379 transcript. In embodiments,
the compound includes a nucleic acid sequence capable of
hybridizing at least 5 nucleobases within a RNA sequence 10-20,
10-30, 20-40, 20-50, 40-60, 40-70, 60-80, 60-90, 80-100, 80-110,
100-120, 100-130, 120-140, 120-150, 140-160, 140-170, 160-180,
160-190, 180-200, 180-210, 200-220, 200-230, 220-240, 220-230,
240-260, or 240-270 nucleobases downstream of the transcription
start site of a mammalian microRNA-379 transcript.
[0119] In embodiments, the present disclosure includes a compound
including a nucleic acid sequence having 90-91%, 91-92%, 92-93%,
93-94%, 94-95%, 95-96%, 96-97%, 97-98%, 98-99%, or 99-100% sequence
identity with a continuous 10 nucleobase sequence of SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or 24, or analogues or derivatives thereof.
[0120] In embodiments, the compound includes a nucleic acid
sequence having 90-91%, 91-92%, 92-93%, 93-94%, 94-95%, 95-96%,
96-97%, 97-98%, 98-99%, or 99-100% sequence identity with a
continuous 10 nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or
24, with internal modified internucleotide linkage between
nucleobases and/or terminal nucleobase analogs at the 5'- and/or
the 3'-ends of the nucleic acid sequence.
[0121] In embodiments, the compound includes a nucleic acid
sequence having 90-91%, 91-92%, 92-93%, 93-94%, 94-95%, 95-96%,
96-97%, 97-98%, 98-99%, or 99-100% sequence identity with a
continuous 10 nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or
24, with internal internucleotide phosphorothioate linkage between
nucleobases and/or terminal nucleobase LNA analogs at the 5'-
and/or the 3'-ends of the nucleic acid sequence. In embodiments,
the nucleobase analogs at the 5'- and/or the 3' ends may be
2'-O-alkyl nucleobase, 2'-Fluoro nucleobase, or 2'-OMe
nucleobase.
[0122] In embodiments, the present disclosure includes a compound
including a nucleic acid sequence having at least 90% sequence
identity with a continuous 11, 12, 13, 14, 15, 16, or 17 nucleobase
sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.
[0123] In embodiments, the compound includes a nucleic acid
sequence having at least 90% sequence identity with a continuous
11, 12, 13, 14, 15, 16, or 17 nucleobase sequence of SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or 24, with internal modified internucleotide linkage
between nucleobases and/or terminal nucleobase analogs at the 5'-
and/or the 3'-ends of the nucleic acid sequence.
[0124] In embodiments, the compound includes a nucleic acid
sequence having at least 90% sequence identity with a continuous
11, 12, 13, 14, 15, 16, or 17 nucleobase sequence of SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or 24, with internal internucleotide phosphorothioate
linkage between nucleobases and/or nucleobase LNA analogs at the
5'- and/or the 3'-ends of the nucleic acid sequence. In
embodiments, the nucleobase analogs at the 5'- and the 3' ends may
be 2'-O-alkyl nucleobase, 2'-Fluoro nucleobase, or 2'-OMe
nucleobase, and any combination thereof.
[0125] In embodiments, the present disclosure includes a compound
including a nucleic acid sequence having 90-91%, 91-92%, 92-93%,
93-94%, 94-95%, 95-96%, 96-97%, 97-98%, 98-99%, or 99-100% sequence
identity with a continuous 11, 12, 13, 14, 15, 16, or 17 nucleobase
sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.
[0126] In embodiments, the compound includes a nucleic acid
sequence having 90-91%, 91-92%, 92-93%, 93-94%, 94-95%, 95-96%,
96-97%, 97-98%, 98-99%, or 99-100% sequence identity with a
continuous 11, 12, 13, 14, 15, 16, or 17 nucleobase sequence of SEQ
ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23 or 24, with internal modified
internucleotide linkage between nucleobases and/or terminal
nucleobase analogs at the 5'- and/or the 3'-ends of the nucleic
acid sequence.
[0127] In embodiments, the compound includes a nucleic acid
sequence having 90-91%, 91-92%, 92-93%, 93-94%, 94-95%, 95-96%,
96-97%, 97-98%, 98-99%, or 99-100% sequence identity with a
continuous 11, 12, 13, 14, 15, 16, or 17 nucleobase sequence of SEQ
ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23 or 24, with internal internucleotide
phosphorothioate linkage between nucleobases and/or nucleobase LNA
analogs at the 5'- and/or the 3'-ends of the nucleic acid sequence.
In embodiments, the nucleobase analogs at the 5'- and/or the 3'
ends may be 2'-O-alkyl nucleobase, 2'-Fluoro nucleobase, or 2'-OMe
nucleobase, and any combination thereof.
Complex
[0128] In embodiments, the present disclosure provides a complex of
a compound including a nucleic acid sequence described in this
disclosure hybridized to an RNA sequence 10 to 270 nucleobases
downstream of the transcription start site of a mammalian
microRNA-379 transcript or a microRNA-379 megacluster
transcript.
[0129] In embodiments, the present disclosure includes a nucleic
acid sequence of SEQ ID NOs: 1-20 hybridized to a region of mouse
miR-379 transcript of SEQ ID NO: 25 (i.e., transcript sequence SEQ
ID NO: 118) to form a complex. In embodiments, the present
disclosure includes a nucleic acid sequence of SEQ ID NOs: 21-24
hybridized to a region of human miR-379 transcript of SEQ ID NO: 26
(i.e., transcript sequence SEQ ID NO: 119) to form a complex. In
embodiments, the present disclosure includes a nucleic acid
sequence having 90-91%, 91-92%, 92-93%, 93-94%, 94-95%, 95-96%,
96-97%, 97-98%, or 98-99% sequence identity with a continuous 10
nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or analogues thereof,
hybridized to a region of human miR-379 transcript of SEQ ID NO: 26
(i.e., transcript sequence SEQ ID NO: 119) to form a complex.
[0130] In embodiments, the present disclosure includes a complex of
a nucleic acid sequence having 90-91%, 91-92%, 92-93%, 93-94%,
94-95%, 95-96%, 96-97%, 97-98%, 98-99%, or 99-100% sequence
identity with a continuous 10, 11, 12, 13, 14, 15, 16, or 17
nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, with
internal internucleotide phosphorothioate linkage between
nucleobases and/or nucleobase LNA analogs at the 5'- and/or the
3'-ends of the nucleic acid sequence, hybridized to a RNA sequence
10 to 270 nucleobase downstream of the transcription start site of
microRNA-379 transcript.
[0131] In embodiments, the present disclosure includes a complex of
a nucleic acid sequence having 90-91%, 91-92%, 92-93%, 93-94%,
94-95%, 95-96%, 96-97%, 97-98%, 98-99%, or 99-100% sequence
identity with a continuous 10, 11, 12, 13, 14, 15, 16, or 17
nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, with
internal internucleotide phosphorothioate linkage between
nucleobases and/or nucleobase LNA analogs at the 5'- and/or the
3'-ends of the nucleic acid sequence, hybridized to a RNA sequence
transcript at 10-20, 10-30, 20-40, 20-50, 40-60, 40-70, 60-80,
60-90, 80-100, 80-110, 100-120, 100-130, 120-140, 120-150, 140-160,
140-170, 160-180, 160-190, 180-200, 180-210, 200-220, 200-230,
220-240, 220-230, 240-260, or 240-270 nucleobases downstream of the
transcription start site of microRNA-379.
Method of Treatment or Use
[0132] The present disclosure provides a method of treating
diabetic nephropathy in a subject in need thereof, the method
including administering to the subject an effective amount of a
compound of the present disclosure. The present disclosure includes
a method of treating diabetic nephropathy in a subject by
administering to the subject about 0.001 mg/kg to about 100 mg/kg
of a compound of the present disclosure. In embodiments, a compound
of the present disclosure inhibits renal glomerular podocyte death,
glomerular mesangial expansion, glomerular hypertrophy, glomerular
extracellular matrix accumulation, thereby treating DN.
[0133] In embodiments, the method of treating diabetic nephropathy
in a subject includes administering to the subject a compound or a
pharmaceutical composition including a nucleic acid sequence having
90-91%, 91-92%, 92-93%, 93-94%, 94-95%, 95-96%, 96-97%, 97-98%,
98-99%, or 99-100% sequence identity with a continuous 10, 11, 12,
13, 14, 15, 16, or 17 nucleobase sequence of SEQ ID NO: 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23 or 24, or analogues thereof.
[0134] In embodiments, the method of treating diabetic nephropathy
in a subject includes administering to the subject a compound or a
pharmaceutical composition including a nucleic acid sequence having
a nucleobase analog. In embodiments, the nucleic acid sequence
includes Locked Nucleic Acid (LNA), 2'-O-alkyl, 2' O-Methyl,
2'-deoxy-2'fluoro, 2'-deoxy, a universal base, 5-C-methyl, an
inverted deoxy abasic residue incorporation, or any combination
thereof. In embodiments, the nucleic acid sequence may include
analogs with positive backbones; non-ionic backbones, modified
sugars, and non-ribose backbones (e.g. phosphorodiamidate
morpholino oligos).
[0135] In embodiments, the present disclosure includes a method of
treating diabetic nephropathy by administering a compound to a
subject in need of such treatment, where the compound inhibits
expression of a long non-coding RNA (lncMGC) in the subject. The
method of treating diabetic nephropathy is by administering a
compound to a subject in need of such treatment, where the compound
inhibits expression of a long non-coding RNA (lncMGC) in the
subject, which includes microRNA-376a, microRNA-299, microRNA-376c,
microRNA-410, microRNA-494, microRNA-380-5p, microRNA-369-3p,
microRNA-300, microRNA-541, microRNA-329, microRNA-381,
microRNA-411, microRNA-134, microRNA-379, microRNA-154,
microRNA-382, microRNA-376b, microRNA-496, microRNA-409-5p,
microRNA-543, microRNA-377, microRNA-380-3p, and/or
microRNA-495.
[0136] In embodiments, the present disclosure includes a method of
treating diabetic nephropathy by administering a compound to a
subject, where the compound inhibits expression of a microRNA gene
cluster. In embodiments, expression of the microRNA gene cluster
that is inhibited for treating diabetic nephropathy is microRNA-379
gene cluster. In embodiments, the microRNA gene cluster expression
of which is inhibited expresses microRNAs such as microRNA-376a,
microRNA-299, microRNA-376c, microRNA-410, microRNA-494,
microRNA-380-5p, microRNA-369-3p, microRNA-300, microRNA-541,
microRNA-329, microRNA-381, microRNA-411, microRNA-134,
microRNA-379, microRNA-154, microRNA-382, microRNA-376b,
microRNA-496, microRNA-409-5p, microRNA-543, microRNA-377,
microRNA-380-3p, and/or microRNA-495.
[0137] The sequence of the nucleic acid that inhibits the microRNA
for treating diabetic nephropathy is complementary to the microRNA
sequence, or complementary to a transcript that includes the
targeted microRNA and binds downstream of the transcription start
site.
[0138] Human microRNAs targeted for treating diabetic nephropathy
are listed in Table 2.
TABLE-US-00004 TABLE 2 Human microRNAs Name Sequence SEQ ID NO:
microRNA-376a UGCACCUAAAAGGAGAUACUA 83 microRNA-299-3p
UAUGUGGGAUGGUAAACCGCUU 84 microRNA-376c UGCACCUUAAAGGAGAUACAA 85
microRNA-410 UGUCCGGUAGACACAAUAUAA 86 microRNA-494
CUCCAAAGGGCACAUACAAAGU 87 microRNA-380-5p AUGGUUGACCAUAGAACAUGCG 88
microRNA-369-3p AAUAAUACAUGGUUGAUCUUU 89 microRNA-300
UCUCUCUCAGACGGGAACAUAU 90 microRNA-541 AAAGGAUUCUGCUGUCGGUCCCACU 91
microRNA-329 UUUCUCCAAUUGGUCCACACAA 92 microRNA-381
UGUCUCUCGAACGGGAACAUAU 93 microRNA-411 GCAUGCGAUAUGCCAGAUGAU 94
microRNA-134 GGGGAGACCAGUUGGUCAGUGU 95 microRNA-379
GGAUGCAAGGUAUCAGAUGGU 96 microRNA-154 UAGGUUAUCCGUGUUGCCUUCG 97
microRNA-382 GAAGUUGUUCGUGGUGGAUUCG 98 microRNA-376b
UUGUACCUAAAAGGAGAUACUA 99 microRNA-496 CUCUAACCGGUACAUUAUGAGU 100
microRNA-409-5p AGGUUACCCGAGCAACUUUGCAU 101 microRNA-543
UUCUUCACGUGGCGCUUACAAA 102 microRNA-377 UGUUUUCAACGGAAACACACUA 103
microRNA-380-3p UAUGUAAUAUGGUCCACAUCUU 104 microRNA-495
UUCUUCACGUGGUACAAACAAA 105
[0139] In embodiments, mouse microRNA targeted for inhibition are
listed in Table 3.
TABLE-US-00005 TABLE 3 Mouse microRNAs: Name Sequence SEQ ID NO:
microRNA-299-3p UAUGUGGGAUGGUAAACCGCUU 106 microRNA-376c
UGCACUUUAAAGGAGAUACAA 107 microRNA-410 UGUCCGGUAGACACAAUAUAA 108
microRNA-494 CUCCAAAGGGCACAUACAAAGU 109 microRNA-380-5p
AUGGUUGACCAUAGAACAUGCG 110 microRNA-369-3p AAUAAUACAUGGUUGAUCUUU
111 microRNA-541 AAGGGAUUCUGAUGUUGGUCACACU 112 microRNA-329
UUUUUCCAAUCGACCCACACAA 113 microRNA-381 UGUCUCUCGAACGGGAACAUAU 114
microRNA-411 GCAUGCGAUAUGCCAGAUGAU 115 microRNA-134
UGUUUUCAACGGAAACACACUA 116 microRNA-379 GGAUGCAAGGUAUCAGAUGGU 65
microRNA-154 UAGGUUAUCCGUGUUGCCUUCG 66 microRNA-382
GAAGUUGUUCGUGGUGGAUUCG 67 microRNA-376b UUCACCUACAAGGAGAUACUA 68
microRNA-496 CUCUAACCGGUACAUUAUGAGU 69 microRNA-409-5p
AGGUUACCCGAGCAACUUUGCAU 70 microRNA-543 UUCUUCACGUGGCGCUUACAAA 71
microRNA-377 UGUUUUCAACGGAAACACACUA 74 microRNA-380-3p
UAUGUAGUAUGGUCCACAUCUU 75 microRNA-495 UUCUUCACGUGGUACAAACAAA 76
miR-3072-5p AGGGACCCCGAGGGAGGGCAGG 77 miR-3072-3p
UGCCCCCUCCAGGAAGCCUUCU 78
[0140] In embodiments, the present disclosure includes a method of
treating diabetic nephropathy by administering a compound of the
present disclosure, which upregulates microRNA target genes and
down-regulates expression of profibrotic genes. In embodiments, the
compound of the present disclosure up-regulates and down-regulates
in kidney mesangial cells.
[0141] In embodiments, the compound of the present disclosure
up-regulates target genes, for example, Tnrc6, CUGBP2, CPEB4,
Pumillio2, BHC80 EDEM3, and any combination(s) thereof. In
embodiments, the compound of the present disclosure down-regulates
profibrotic genes, for example, pro-fibrotic genes Col1.alpha.2,
TGF-.beta.1, Col1.alpha.4, Plasminogen activator inhibitor-1
(PAI-1), fibronectin, connective tissue growth factor (CTGF), and
any combination(s) thereof. In embodiments, the compound of the
present disclosure treats diabetic nephropathy at an early stage of
the disease. In embodiments, the diabetic nephropathy is
characterized as having glomerular lesions including glomerular
basement membrane thickening, mild mesangial expansion, severe
mesangial expansion, nodular sclerosis (Kimmelstiel-Wilson
lesions), or advanced diabetic glomerulosclerosis.
Method of Inhibiting Expression of a Mammalian MicroRNA-379
Cluster
[0142] The present disclosure provides a method of inhibiting
expression of a mammalian microRNA-379, the method includes
hybridizing a compound of the present disclosure to an RNA sequence
10 to 270 nucleobases downstream of the transcription start site of
a mammalian microRNA-379 transcript. In embodiments, the method of
inhibiting expression of a mammalian microRNA-370 cluster includes
contacting a cell or tissue with a nucleic acid sequence of SEQ ID
NOs: 1-24. In embodiments, the method of inhibiting expression of a
mammalian microRNA-370 cluster includes contacting a cell or tissue
with a nucleic acid sequence having 90-91%, 91-92%, 92-93%, 93-94%,
94-95%, 95-96%, 96-97%, 97-98%, 98-99%, or 99-100% sequence
identity with a continuous 10, 11, 12, 13, 14, 15, 16, or 17
nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, with
internal internucleotide phosphorothioate linkage between
nucleobases and/or nucleobase LNA analogs at the 5'- and/or the
3'-ends of the nucleic acid sequence.
[0143] In embodiments, the method of inhibiting expression of a
mammalian microRNA-370 cluster includes contacting a kidney
mesangial cell with a nucleic acid sequence of SEQ ID NOs: 1-24. In
embodiments, the method of inhibiting expression of a mammalian
microRNA-370 cluster includes contacting a kidney mesangial cell
with a nucleic acid sequence having 90-91%, 91-92%, 92-93%, 93-94%,
94-95%, 95-96%, 96-97%, 97-98%, 98-99%, or 99-100% sequence
identity with a continuous 10, 11, 12, 13, 14, 15, 16, or 17
nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, with
internal internucleotide phosphorothioate linkage between
nucleobases and/or nucleobase LNA analogs at the 5'- and/or the
3'-ends of the nucleic acid sequence.
Pharmaceutical Composition
[0144] The present disclosure provides a pharmaceutical composition
including a compound of the present disclosure and a
pharmaceutically acceptable diluent, carrier, salt, and/or
adjuvant.
[0145] In embodiments, the pharmaceutical composition of the
present disclosure includes a nucleic acid sequence of SEQ ID NOs:
1-24. In embodiments, the pharmaceutical composition of the present
disclosure includes a nucleic acid sequence having 90-91%, 91-92%,
92-93%, 93-94%, 94-95%, 95-96%, 96-97%, 97-98%, 98-99%, or 99-100%
sequence identity with a continuous 10, 11, 12, 13, 14, 15, 16, or
17 nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, with
internal internucleotide phosphorothioate linkage between
nucleobases and/or nucleobase LNA analogs at the 5'- and/or the
3'-ends of the nucleic acid sequence.
[0146] In embodiments, the present disclosure includes
administering to an individual, a composition of a therapeutically
effective amount of a compound including a nucleic acid sequence of
SEQ ID NOs: 1-24, alone or in combination with a diabetic and/or
diabetic nephropathic agent. The effective dose of the composition
may be between about 0.001 mg/kg to about 100 mg/kg of compound. In
embodiments, the compositions may have between about 0.1% to about
20% of the pharmaceutical composition. In embodiments, the
compositions may include pharmaceutically acceptable diluent(s),
excipient(s), and/or carrier(s).
[0147] The composition of a compound including a nucleic acid
sequence of SEQ ID NOs: 1-24 may be administered with a suitable
pharmaceutical carrier. The administration can be local or
systemic, including oral, parenteral, intraperitoneal, intrathecal
or topical application. The release profiles of such composition
may be rapid release, immediate release, controlled release or
sustained release. For example, the composition may comprise a
sustained release matrix and a therapeutically effective amount.
Alternatively, a composition of a compound including a nucleic acid
sequence of SEQ ID NOs: 1-24 can be secreted by genetically
modified cells that are implanted, either free or in a capsule, at
the gut of a subject. In embodiments, a composition of a compound
including a nucleic acid sequence of SEQ ID NOs: 1-24 may be
administered to a subject via subcutaneous route. In embodiments,
the composition may be administered as an oral nutritional
supplement.
[0148] Oral compositions may include an inert diluent or an edible
pharmaceutically acceptable carrier. They can be enclosed in
gelatin capsules or compressed into tablets. For the purpose of
oral administration, a composition of a compound including a
nucleic acid sequence of SEQ ID NOs: 1-24 can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the agent in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents or adjuvant materials
can be included as part of the composition. The tablets, pills,
capsules, troches and the like can contain any of the following
ingredients, or agents of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic
acid, primogel, or corn starch; a lubricant such as magnesium
stearate or sterotes; a glidant such as colloidal silicon dioxide;
a sweetening agent such as sucrose or saccharin; or a flavoring
agent such as peppermint, methyl salicylate, or orange
flavoring.
[0149] In embodiments, a composition of a compound including a
nucleic acid sequence of SEQ ID NOs: 1-24 in combination with
another pharmaceutically active agent (small molecule or a large
biological molecule) formulated for parenteral (including
subcutaneous, intramuscular, and intravenous), inhalation, buccal,
sublingual, nasal, rectal, topical, or oral administration for
treating a viral infection, for inducing immune response, for
treating neuroinflammation. The compositions may be conveniently
presented in unit dosage form, and prepared by any of the methods
well known to one skilled in the art.
[0150] In embodiments, the composition of the present disclosure
may be formulated in dosage unit form for ease of administration
and uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
individual to be treated; each unit containing a predetermined
quantity of agent calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms are dictated by and
directly dependent on the unique characteristics of the agent and
the particular therapeutic effect to be achieved.
[0151] The following examples are provided as illustrations of
various embodiments of the disclosure but are not meant to limit
the disclosure in any manner.
EXAMPLES
Example 1: Small RNA (miRNA)-Sequencing Revealed Mega Cluster of
miRNAs was Up-Regulated Under Diabetic Conditions
[0152] RNA obtained from the glomeruli of streptozotocin (STZ)
diabetic and control mice was profiled by miRNA-sequence and the
Illumina sequencing data analyzed. In the diabetic mice glomeruli,
a significant increase in the expression of several miRNAs was
found (e.g. miR-379, -495, -377) (FIG. 2B) that are among the mega
cluster of miRNAs within mouse Chr12 (FIG. 2A). Genome organization
showed that the miRNA mega cluster is hosted downstream of the
lncRNA termed lnc-MGC, with another reported lncRNA, Mirg, present
in this locus (FIG. 2A). Lnc-MGC expression (5' and 3') was also
increased under these diabetic conditions (FIG. 2B), p<0.05.
Furthermore, apart from the region upstream of lnc-MGC, no clear
putative promoter was evident throughout the cluster, indicating
that the miRNA cluster and lncRNA may be co-transcribed together as
one unit, i.e., lnc-MGC may be induced under diabetic conditions
and this up-regulates key miRNAs of the, cluster as one transcript,
which encompasses a long ncRNA that functionally serves as host for
the mega miRNA cluster.
[0153] Mouse MC (MMC) was treated with high glucose (HG, 25 mM),
mannitol or TGF-.beta.1 and several miRNAs within the cluster, (and
lnc-MGC), was examined by amplifying these regions with specific
PCR primers. TGF-.beta.1 and HG significantly up-regulated the
expression of miR-379 (the first miRNA in the cluster), miR-495 (in
the middle) and miR-377 (downstream), as well as lnc-MGC (FIG. 3),
further supporting the sequencing and in vivo data (*, p<0.05)
(NG=normal glucose, SD=serum depletion control for TGF-.beta.1).
miR-882 outside the cluster was unaffected. These results indicated
that an miRNA mega cluster and its host lncRNA are concomitantly
up-regulated in the glomeruli and MCs under diabetic
conditions.
[0154] Small RNA (sm-RNA) sequencing was performed as previously
described. Scatter plot of miRNAs in kidney glomeruli from control
(CTR) and diabetic mice (STZ) were generated. The expression of
each detectable miRNA in the form of log scaled reads was plotted
with x-axis for CTR and y-axis for STZ. Each dot represents one
miRNA. All the miRNAs in the miR-379 cluster were presented in red.
Among them, the upregulated miRNAs with fold change .gtoreq.2) were
highlighted with bigger size dots and labeled with the
corresponding miRNA names. miR-882 was plotted in blue as a
negative control (outside of miR-379 cluster). The expression of
each detectable miRNA within the miR-379 cluster in the two samples
(CTR and STZ) were ordered by log 2 fold change from low to high,
mean-centered and shown in the heatmap. Green represents lower than
average expression in the 2 samples and red presents higher than
average expression level. The expression of the detected miRNAs in
this cluster was higher in STZ than CTR. The mouse miR-379
megacluster is located within the largest miRNA cluster currently
identified in the genome. It maps within the DLK-DIO 3 genomic
region (mouse chr 12, human chr14), which is home to several miRNAs
and lncRNAs. TSS, transcription start site. miR-882 is located
far-upstream of miR-379 cluster and not covered by lnc-MGC. All the
miRNAs detected by smRNA-seq with at least 5 scaled reads in at
least one sample are ranked by log 2 fold change between STZ and
CTR samples to generate ranked list and all the detectable miRNAs
in cluster are considered as a gene set. Pre-ranked gene set
analysis (GSEA) applied on the gene set using the ranked list of
all the miRNAs revealed that miRNAs in the miR-379 cluster were
significantly enriched within the miRNAs upregulated in the STZ
diabetic mice, with normalized enrichment score of 1.56
(p=0.004).
Example 2: The Upstream Promoter of the Mega Cluster and
Lnc-MGC
[0155] To identify a putative promoter that could drive the genomic
region, 5' RACE experiments using primers from the miR-379 upstream
region were carried out (FIG. 4). Results revealed that this region
has promoter-like features with TATA box and an initiator (INR)
site. The binding sites for the C/EBP homologous protein (CHOP), a
transcription factor (TF) associated with the ER and stress
response, and an overlapping E-box was identified. Data for this
promoter location was further supported by results showing that
this region upstream of miR379 (lncMGC 5' RACE (rapid amplification
of cDNA ends)) is up-regulated in vivo in the glomeruli of diabetic
mice (STZ-injected and db/db (the db/db mouse is a model of
obesity, diabetes, and dyslipidemia wherein leptin receptor
activity is deficient because the mice are homozygous for a point
mutation in the gene for the leptin receptor)), and in vitro in
MMCs treated with HG or TGF-.beta.1 (*, p<0.05). These results
identify the promoter of the lnc-RNA-MGC-miRNA cluster but also
indicate that CHOP may be a key TF that regulates their coordinate
expression.
Regulatory Role of CHOP: CHOP Protein Levels are Up-Regulated in
the Glomeruli of Diabetic Db/Db and STZ-Injected Mice
[0156] Experiments were performed to test whether CHOP regulates
this genomic region. Western blots showed that CHOP protein levels
were up-regulated in the glomeruli of diabetic db/db and
STZ-injected mice, compared to the respective controls. CHOP was
also induced in MMCs treated with TGF-.beta.1, HG or osmotic
control, mannitol, indicating that diabetic conditions and cellular
stress can upregulate CHOP which in turn, increases transcription
of lncRNA-MGC and the miRNA cluster.
CHOP Enrichment at the miR379 Promoter.
[0157] Chromatin immunoprecipitation (ChIP) assays were performed
to evaluate CHOP enrichment at the miR379 promoter. A significant
increase in CHOP occupancy at the CHOP binding site of the miR-379
promoter (arrows) in MMCs treated with HG or TGF-.beta.1, versus
control normal glucose (NG) or serum-depleted (SD) was
observed.
CHOP-siRNA Effects
[0158] Efficacy of CHOP siRNA (relative to a negative control
siRNA, NC) for down-regulating CHOP in MMCs was evaluated. CHOP
siRNA significantly attenuated HG- and TGF-.beta.1-induced
expression of miR-379 and lnc-MGC. Mir-495 and -377 expression were
also inhibited. In addition to these miRNAs, similar trends were
observed for 29 other cluster miRNAs that were tested, i.e. these
29 were significantly up-regulated in diabetic mice glomeruli and
in response to TGF-.beta.1, and increases of the most of them were
inhibited by CHOP siRNA in MC. Cumulatively, these results indicate
that CHOP is a key transcriptional regulator of the miRNA cluster
and related lncRNA-MGC.
Example 3: Targets of miRNA Cluster
[0159] miRNA target prediction algorithms predicted that numerous
miRNAs in the mega cluster collectively targeted similar genes.
Therefore, the 3' UTRs of at least 25 genes were similarly targeted
by 8-13 cluster miRNAs, with some genes having more than two miRNA
target sites. Furthermore, it was found that these target genes had
functions already related to DN, namely protein synthesis, ER
stress, RNA binding proteins and protein translation (as determined
by in silico GSEA (Gene Set Enrichment Analysis), IPA (Ingenuity
Pathway Analysis), as well as Gene ontology analyses). This
indicated that miRNAs in the mega cluster work in unison to alter
the expression of groups of similar genes that can functionally
modulate DN progression.
Identification and Validation of Putative Targets of the miR-379
Cluster.
[0160] Several potential targets of key cluster miRNAs that have
functions relevant to DN were identified. These included RNA
binding proteins and translational regulators (CUGBP2), Pumilio2,
Tnrc6b, CPEB4, HuR, TFs and co-factors (Arid2, BHC80), Nf1a/b), an
ER stress modulator (EDEM3), and the cell growth gene phosphatase
and tensin homolog (Pten). To validate, down-regulation under
diabetic condition was evaluated in the target genes. It was found
that TGF-.beta.1 or HG can reduce the mRNA expression levels of
Tnrc6, CUGBP2, CPEB4, Pumilio2, BHC80 and EDEM3 in MCs in vitro,
and in the glomeruli of diabetic mice in vivo (db/db and STZ),
relative to controls (FIG. 5; *p<0.05). These genes are known to
regulate protein translation, protein synthesis, mRNA stability
(e.g., CUGBP2) and miRNA processing (Tnrc6b). BHC80 interacts with
histone deacetylases (HDACs) and the Ets TFs to promote chromatin
condensation (86). Therefore, it is indicated that these targets
mediate the downstream effects of the corresponding miRNAs in a
cooperative and synergistic manner, to augment renal hypertrophy
and fibrosis, and thereby enhance renal dysfunction in DN (FIG.
10).
Example 4: Effect of CHOP siRNA on Key miRNA Targets, MC
Hypertrophy, Protein Synthesis and Fibrotic Genes
[0161] CHOP siRNA treatment significantly prevented a key miRNA
target (TNRC6) from being down-regulated by HG and TGF-.beta..
Furthermore, in parallel, transfection of CHOP siRNA into MMCs also
prevented increases in the TGF-.beta.1 induced expression of key
pro-fibrotic genes Col1.alpha.2, TGF-.beta.1, Col1.alpha.4 and
PAI-1 which are associated with DN. Consistently, CHOP siRNA also
prevented TGF-.beta.1 induced MC hypertrophy and protein content.
These data indicate that inducing key mega cluster miRNAs can
regulate MC genes and functions associated with DN pathogenesis
through CHOP.
miRNAs and siRNAs
[0162] Oligonucleotide mimics and inhibitors of miRNAs, and siRNAs
and corresponding control oligos were obtained from Integrated DNA
technologies (IDT) or Thermo Fisher Scientific Inc. (Waltham,
Mass.), as described. Wild-type (WT) MMCs (from WT C57BL/6 mice),
MMC transfected with CHOP siRNA, and MMCs from CHOP-KO mice were
treated with HG, mannitol and TGF-.beta.1 as described. Briefly,
cells (.about.10.sup.6/transfection) were transfected with siRNA or
miRNA oligonucleotides using an Amaxa Nucleofector (Lonza, Basel,
Switzerland) according to the manufacturer's protocols as described
previously. siRNAs (double-stranded oligos of three pairs of sense
(S) and antisense (AS) synthesized oligos,
TABLE-US-00006 S1 [SEQ ID NO: 59]
rCrArUrCrUrGrCrUrUrCrCrCrArCrUrGrCrCrArArArUrCAG and AS1 [SEQ ID
NO: 60] rCrUrGrArUrUrUrGrGrCrArGrUrGrGrGrArArGrCrArGrArUr GrUrG; S2
[SEQ ID NO:61] rUrCrArGrCrArCrCrGrUrGrCrArArCrCrArUrUrCrArArGGA and
AS2 [SEQ ID NO: 62]
rUrCrCrUrUrGrArArUrGrGrUrUrGrCrArCrGrGrUrGrCrUrGr ArArA; S3 [SEQ ID
NO: 63] rCrUrUrCrArUrCrUrGrGrUrArArUrGrUrArCrUrArCrCrUGA and AS3
[SEQ ID NO: 64] rUrCrArGrGrUrArGrUrArCrArUrUrArCrCrArGrArUrGrArAr
GrGrC; (r, ribose) against mouse upstream region of miR-379 were
obtained from IDT.
[0163] Non-targeting siRNA controls were obtained from Thermo
Fisher Scientific Inc. MMC were trypsinized and resuspended in
Basic Nucleofection Solution at 1.times.10.sup.7/ml. Subsequently,
100 .mu.l of cell suspension (1.times.10.sup.6 cells) was mixed
with miRNA mimic, hairpin inhibitor oligonucleotides, or ON-TARGET
plus siRNA or negative controls (Thermo Fischer Scientific Inc.,
Waltham, Mass.). Transfected cells were harvested for RNA and
protein extraction. RNA was extracted from the cells and the
expression of lncRNA-MGC, and all 40 miRNAs within the cluster were
systematically examined using primers designed for each of the
mature miRNAs. MMCs were transfected with oligonucleotide mimics,
siRNAs of candidate miRNAs or negative control (NC) oligos to
determine if manipulating their levels can influence TGF-.beta.1
and HG responses. At 48-72 hr post-transfection, the expression of
miRNA target genes, fibrosis and hypertrophy related genes, and
proteins induced by TGF-.beta.1/HG were determined by RT-qPCR and
Western blotting using our published methods.
EDEM3 as a Direct Target of miR-379
[0164] Oligo mimics of miR-379 directly decreased the mRNA levels
of its predicted targets Tnrc6b and EDEM3 in MMCs, whereas miR-379
inhibitor oligos (miR-379I) enhanced their mRNA levels in MMCs
(FIG. 6). Protein levels of EDEM3 were also decreased in MMCs
treated with TGF-.beta.1 or transfected with miR-379 mimic,
indicating that direct effects of a key cluster miRNA (miR-379) on
MC gene expression.
[0165] Whether EDEM3 is a direct target of miR-379 was tested.
3'UTR of mouse EDEM3 gene was cloned into psiCheck2 vector and
co-transfected to MMC with miR-379 mimic. miR-379 inhibited
luciferase activity of this reporter, which suggested EDEM3 is a
direct target of miR-379. A potential target site of miR-200b/c was
found in the 3'UTR of EDEM3. miR-200b inhibited the reporter
activity. Because miR-200b/c are also known to be upregulated in
glomeruli from diabetic mouse and MMC treated with TGF-.beta.1,
miR200 family and miR-379 cluster may collaborate to inhibit EDEM3
expression. Because miR-379 and miR-200b had no effect on the
reporter with partial deletion of region which includes miR-379 and
miR-200b/c sites from 3'UTR of EDEM3, those sites are real targets
of miR-379 and miR-200b. Those results also suggest that miR-379
cluster and miR-200b upregulated in diabetic conditions induces DN
through hypertrophy and fibrosis mediated by EDEM3 (ER stress).
[0166] To confirm if ER stress is involved in the upregulation of
miR-379 cluster, MMC was treated with tunicamycin (TM), a known ER
stress inducer, and the expression of miR-379 cluster was tested.
Initially, to test what dose of TM is best, the expression of HSPA5
(heat shock 70 kDa protein 5), also known as GRP78
(glucose-regulated protein, 78 kDa), in MMC after treatment of TM
(FIG. 15A). 50 ng/ml was the minimum and significant dose to induce
the expression of HSPA5 and similar induction of CHOP was observed
(FIGS. 15A-15C).
[0167] The expression of lncMGC and miRNAs in the cluster was
increased by the TM (.about.50 ng/ml) (FIGS. 15D-15I), suggesting
that TM (ER stress) induces this cluster expression in MMC. Targets
of miR-379 cluster including EDEM3, a target of miR-379, were
decreased by TM as expected (FIGS. 15J-15R). Faster migrating
isoform of EDEM3 protein was detected in TM-treated cells by
western blot while no such isoform was detected in MMC treated with
TGF-.beta. (FIG. 15K). TM is an inhibitor of N-glycosylation and
faster-migrating form is an un-glycosylated form of EDEM3 which
loses the activity to protect the cells from ER stress (FIG. 15K).
TGF-.beta. treatment decreased the expression of EDEM3 through
induction of miR-379 and miR-200b (FIG. 15K). Thus, there are two
independent regulations of EDEM3, loss of N-glycosylation and
decrease of expression through miR-379 (miR-200b) induction by TM
treatment (FIGS. 15K-15M). Other potential targets were
down-regulated by TM (FIGS. 15N-15S). Pro-fibrotic genes, such as
Col1.alpha.2, Col4.alpha.1, FN and TGF-.beta.1 were also
upregulated by TM treatment in MMC (FIGS. 15T-15W). These results
suggest that TM (ER stress) induces miR-379 cluster expression and
enhances ER stress and DN phenotypes (hypertrophy and fibrosis) by
inhibiting the miR-379 cluster targets.
Approach to Interrupt Lnc-MGC Expression
[0168] To inhibit the expression of lnc-MGC, a mixture of siRNAs to
target lnc-MGC, which are located upstream of miR-379 was designed.
Notably, transfecting this silnc-MGC into MMCs decreased both basal
and TGF-.beta.-induced expression not only of lncRNA-MGC, but also
of the key cluster miRNAs 379, -495 and -377, without affecting
miR-882, located outside the cluster (FIG. 7 p<0.05). This
indicated that silnc-MGC RNAs down-regulate lnc-MGC, and thereby
down-regulate the component cluster miRNAs and their functions.
Genome-Wide miRNA Target Identification
[0169] MCs were serum starved and tested with or without
TGF-.beta.1 for 24 hours. Cells were then UV crosslinked, sonicated
and immunoprecipitated using an Ago2 antibody. Ago-associated RNA
was purified then sequenced for RNA and miRNA on the Illumina Hiseq
platform. The levels of 3' UTR RNAs of the Col1.alpha.1 gene, which
were enriched in control Ago2-IP samples (CTR), were decreased by
TGF-.beta.1. The results indicate that, under CTR conditions, Ago2
miRNA complexes bind to the 3' UTR of Col1.alpha.1 RNAs. This is
attenuated by TGF-.beta., which decreases specific miRNAs such as
miR-29 to up-regulate Col1.alpha.1. These data suggest the
feasibility of CLIP-seq experiments.
Example 5: Inhibition of lncMGC by Gapmers In Vivo
[0170] To knockdown the expression of lnc-MGC in vivo (mouse
kidney), four LNA-modified Gapmers (MGC1, MGC5, MGC8 and MGC10)
were designed (FIG. 16A). GapmeRs were synthesized and obtained
from Exiqon (Vedbaek, Denmark) according to the following design
strategy: MGC1, TCAaaaacataacGCC [SEQ ID NO:55]; MGC5,
CACggtgctgaaaGAG [SEQ ID NO:56]; MGC8, TGAaggccacactAAC [SEQ ID
NO:57]; MGC10, ATTtggcagtgggAAG [SEQ ID NO: 58], (uppercase: LNA;
lowercase: DNA, full phosphorothioate). LNA modification has
several advantages, including less toxicity, lower dosing and
efficient targeting. Basic design of these Gapmers is three LNAs at
both 5' and 3' ends of oligonucleotides and backbone is
phosphorothioated (FIG. 16B). MMC were transfected with those
Gapmers and the expression of lnc-MGC was examined. MGC10
consistently inhibited expression of lnc-MGC significantly at 48
hours after MGC10 transfection in two independent experiments
although others did not with that consistency (FIGS. 16C-16D).
MGC10 also reduced the expression of lnc-MGC even after TGF-.beta.
treatment (FIG. 16E). Some miRNAs in miR-379 cluster were confirmed
to be reduced by MGC10 in MMC (FIGS. 16F-16H). Several targets
(EDEME3, Tnrc6b and Phf21a) of miR-379 cluster were also
upregulated by MGC10 (FIGS. 16I-16L) suggesting that
down-regulation of miR-379 cluster restores the target
expression.
[0171] Because MGC10 was effective to reduce the expression of
miR-379 cluster in MMC in vitro, it was also tested in mouse kidney
in vivo (FIGS. 17A-17F). Subcutaneous injection of 5 mg/kg MGC10
significantly reduced expression of lnc-MGC in the mouse kidney
(24-72 hours) (FIG. 17B). miRNAs in the cluster (miR-379, miR-495,
and miR-377) were also reduced in the same samples (FIGS. 17C-F).
Those results suggest that MGC10 is effective in vivo in mouse
kidney to reduce the expression of lnc-MGC and miR-379 cluster. To
confirm the delivery of MGC10, the antisense in situ LNA modified
probe was designed. Clear accumulation of MGC10 was observed in the
kidney injected with MGC10 although very week background in the
kidney injected with vehicle (PBS). Phosphorothioated
oligonucleotides can be transported into nucleus by a protein
complex (TCP1 complex). Because MGC10 is fully phosphorothioated,
it may be efficiently transported into nucleus and cleaved lncMGC
RNA and suppressed the expression of miR-379 cluster.
[0172] Next, MGC10 was tested in STZ injected diabetic mice. Five
non-diabetic mice, five diabetic without injection, six diabetic
mice with injection of negative control oligonucleotides and six
diabetic mice with MGC10 injection were examined. lnc-MGC
expression was higher in the kidney from diabetic mice than that
from non-diabetic mice and interestingly, its expression was
reduced in kidney from diabetic mice injected with MGC10,
suggesting that MGC10 is effective even in diabetic mice. miRNAs in
the miR-379 cluster behaved the similar patterns to lnc-MGC while
no significant change of the expression of miR-882 (outside of
miR-379 cluster) was observed. Targets (EDEM3, TRNC6B, CPEB4,
Pumilio2) of miR-379 cluster were reduced in kidney from diabetic
mice and it was restored in the kidney from diabetic mice injected
with MGC10. Profibrotic genes, TGF-.beta.1, Col1a2, Col4a1, CTGF,
which were upregulated in the kidney from diabetic mice, were
reduced in the kidney from diabetic mice injected with MGC10. These
results show that MGC10 is effective to reduce the expression of
lnc-MGC and miR-379 cluster miRNAs and restore targets and inhibits
profibrotic genes even in diabetic mice.
[0173] PAS staining showed mesangial expansion and increased
glomerular size in diabetic mice compared to that in non-diabetic
mice and those were reduced in diabetic mice injected with MGC10
(FIGS. 18A-18C). Those results suggest that MGC10 can prevent
glomerular fibrosis and hypertrophy in diabetic mice. Regarding to
ER stress, IHC of EDEM3, a target of miR-379, showed the
significant decrease in kidney glomeruli from diabetic mice and
that was restored in that from diabetic mice injected with MGC10
(FIGS. 18D-18E). Those results demonstrate that MGC10 inhibits
miR-379 and restore the EDEM3 and suppress the ER-stress in
diabetic kidney. Serum profiling of those mice showed no
significant difference in liver or kidney toxicity by MGC10
injection.
Example 6: Human Version of lncMGC and miRNA-379 Cluster and the
Inhibition by Humanized Gapmer HMGC10
[0174] To test if miR-379 cluster is regulated by the same way even
in human cells, human mesangial cells (HMC) was purchased from
Lonza and treated with TGF-.beta. or HG. Although genomic sequence
of the miR-379 cluster region is conserved from human to mouse,
because there are some minor mismatches in the genomic sequences,
the expression of human version of lnc-MGC (hlnc-MGC) was examined
in HMC by PCR using human specific primers. Similar to MMC, the
expression of hlnc-MGC and miRNA-379 cluster miRNAs was increased
by TGF-.beta. or HG even in HMC although miR-882 (outside of
miR-379 cluster) showed no significant difference. Decrease of some
targets and increase of pro-fibrotic genes were confirmed in
HMC.
[0175] Because target sequence in human of MGC10 has two base
mismatches, human version of MGC10 (HMGC10) was designed based on
human sequence. Basic chemistry was the same as mouse version of
MGC10. The condition of transfection was optimized and D33 was the
best (regarding viability and transfection efficiency). hlnc-MGC
and miR-379 cluster miRNAs were reduced by transfection of HMGC10
in HMC, suggesting that the same strategy can be useful to
suppressed cluster miRNAs in human cells and may be useful to treat
DN patients.
[0176] Next, whether HMGC10 can inhibit the effects by HG or
TGF-.beta. in HMC was tested. Human lncMGC expression was
significantly inhibited by HMGC10 even after TGF-.beta. treatment
(FIG. 19A). Similar trends were observed in the expression of
miR-379, miR-495 and miR-377 but not miR-822 which is out of
miR-379 cluster, suggesting that inhibition of hlncMGC (host RNA)
by HMGC10 resulted in reduction of the expression of miRNAs in the
cluster in HMC treated with TGF-.beta. (FIGS. 19A-19B). Targets
(EDEM3, CPEB4, Pumilio2 and CUGBP2) of the cluster miRNAs were also
examined and reduction of target expression was attenuated by
HMGC10 treatment in HMC treated with TGF-.beta. (FIGS. 19C and
19E). Induction of profibrotic genes (TGF-b1, COL1A2, COL4A1, FN1
and CTGF) by TGF-.beta. was also attenuated by HMGC10 in HMC (FIGS.
19D and 19F). Those results suggest that reduction of hlncMGC by
HMGC10 suppressed the expression of miR-379 cluster miRNAs and
restored the expression of targets and also inhibited the
expression of profibrotic genes in HMC even after treatment of
TGF-.beta.. Similar to TGF-.beta. results, hlncMGC expression was
significantly inhibited by HMGC10 in HMC treated with HG. The
expression of miR-379, miR-495 and miR-377 but not miR-822 was
inhibited by HMGC10 in HMC treated with HG. The reduction of target
expression (EDEM3, CPEB4 and CUGBP2) was attenuated by HMGC10
treatment in HMC treated with HG. Induction of profibrotic genes
(TGF-b1, COL1A2, COL4A1, FN1 and CTGF) by HG was also attenuated by
HMGC10 in HMC. Again, those results suggest that reduction of
hlncMGC by HMGC10 suppressed the expression of miR-379 cluster
miRNAs and restored the expression of targets and also inhibited
the expression of profibrotic genes in HMC even after treatment of
HG. These results demonstrated that inhibition of lncMGC by Gapmer
is useful also in human cells, which could be applied for human
patient therapy.
Expression of miR-379 Cluster miRNAs in Human Kidney Tissue
[0177] Glomeruli of patients with diabetic kidney disease were
studied for cluster miRNA expression. Several cluster miRNAs were
examined by qRT-PCR and small RNA-sequencing in RNA isolated from
micro-dissected glomeruli of kidney biopsies from 46 Southwestern
American (Pima) Indians with documented type-2 diabetes. Total RNA
was isolated using spin-columns; miRNA expression was quantified
using qRT-PCR performed using TaqMan Array Human MicroRNA Card
(Applied Biosystems) and small RNA-sequencing. Samples were
normalized to geometric mean of reference RNAs. Expression of miRNA
precursors was determined in micro-dissected glomeruli of
nephrectomy samples using Affymetrix Human Gene 2.1 ST 24-Array.
The cluster miRNAs were expressed robustly in these diabetic
patient samples with read frequency comparable to miR-192, which
are highly enriched in the kidney and mediate important mechanisms
in diabetic nephropathy (DN).
[0178] In humans, DN is associated with glomerular hypertrophy,
mesangial expansion and loss of podocytes leading to
glomerulosclerosis. As described herein, increased expression of
the precursors of some of the cluster miRNAs is associated with
morphometric parameters of increased glomerular damage in
micro-dissected glomeruli of human nephrectomy tissue samples that
showed various stages of glomerular pathology similar to early
stages diabetic glomerulopathy. These include decreased podocyte
density and increased podocyte and glomerular volume as well as
mesangial index, suggesting that cluster miRNA expression increases
with glomerular damage. These associations suggest that inhibition
of cluster miRNAs may also ameliorate human glomerular diseases
including DN.
[0179] The experiments in this example show that diabetic
conditions (HG) induces TGF-.beta.1 which upregulates miR-379
cluster targeting ER stress regulators and protein synthesis that
resulted in hypertrophy and ER stress in mouse kidney related to DN
(FIG. 10). A host noncoding RNA (lnc-MGC) is regulated by CHOP
which is activated by ER stress. The expression of miRNA-379
cluster depends on the expression of lnc-MGC from its promoter.
CHOP siRNA inhibited the induction of lnc-MGC and miR-379 cluster
miRNAs and the early features (ECM expression and cellular
hypertrophy) of DN. miRNAs in this cluster target several groups of
genes, transcription factors, RNA binding proteins regulating gene
expression and protein synthesis and ER stress, which results in
hypertrophy by increased protein synthesis and fibrosis by
accumulation of ECM (profibrotic genes). Induction of those miRNAs
was inhibited in the kidney from diabetic CHOPKO mice compared to
those from WT mice. Similarly induction of those miRNAs and
profibrotic genes by TGF-.beta. was prevented in MMC from CHOPKO
mice. A known ER stress inducer TM also induces lnc-MGC and miR-379
cluster miRNAs in MMC through reduction of N-glycosylation of
EDEM3. Therefore, induction of lnc-MGC in diabetes may also be
mediated by ER stress. Inhibition of those miRNAs by gapmer (MGC10)
knocking down lnc-MGC ameliorated DN features (ECM accumulation and
glomerular hypertrophy) in the early stage of mouse model of DN.
This is the one of the critical mechanisms in DN and potential
target to prevent DN. Gapmer inhibiting lnc-MGC (MGC10) can be
developed as new drugs to prevent or treat the early stage of
DN.
[0180] TGF-.beta. or diabetic conditions (as well as ER stress)
induce glomerular podocyte dysfunction and death. In order to
determine whether MGC10 confers any protection on podocytes in
diabetes, podocyte effacement and glomerular basement membrane
(GBM) thickness using electron microscopy was assessed (summarized
in the bar graph depicted in FIG. 23A). Clear protection from
diabetes induced podocyte effacement and GBM thickening was
observed in diabetic mice treated with the MGC10 compared to
control oligo. Cell death measured by Terminal deoxynucleotidyl
transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was
increased in glomeruli of diabetic mice compared to non-diabetic
mice, which was attenuated by MGC10 (summarized in the bar graph
depicted in FIG. 23B). These results indicate that MGC10 is
effective in reducing the expression of not only lnc-MGC and
miR-379 cluster miRNAs in vivo in diabetic mice, but also restores
the expression of the cluster miRNA target genes, inhibits
profibrotic genes, and prevents glomerular fibrosis, podocyte
death, and hypertrophy in diabetic mice.
Example 7: Expression of Key miRNAs and lncRNA-MGC in CHOP KO
Mice
[0181] Diabetes was induced in CHOP KO and control WT mice by
administering STZ injections following standard protocols. CHOP KO
mice developed diabetes at the same rates as WT mice. Four weeks
after diabetes induction, mice were sacrificed. Diabetes-induced
mice increased in the key cluster miRNAs, as well as in the
lnc-MGC, which occurred in the glomeruli of WT mice, were abrogated
in the glomeruli of diabetic CHOP KO mice (FIG. 8). The expression
of profibrotic genes Col1.alpha.2, Col4.alpha.1 and TGF-.beta.1
were also attenuated in glomeruli from diabetic CHOP KO mice
compared to diabetic WT mice. Histological analysis showed that PAS
staining and glomerular hypertrophy were clearly reduced in
diabetic CHOP KO mice compared to diabetic WT. Conversely,
glomeruli of CHOP KO mice showed increased expression of key target
genes of the cluster miRNAs compared to WT (FIG. 9).
[0182] MMCs were cultured from non-diabetic WT and CHOP KO mice,
with or without TGF-.beta.1. TGF-.beta.1 induced increases in
cellular hypertrophy were also ameliorated in MMCs from CHOP KO
compared to WT mice. In addition, both the basal and TGF-.beta.1
induced expression of fibrotic genes, Col1.alpha.1, Col4.alpha.1
and TGF-.beta.1 (relevant to DN) were significantly decreased in
MMC from the CHOP KO mice compared to WT. Under these conditions,
both the basal and TGF-.beta.1-induced increases in tree key
cluster miRNAs and the lnc-RNA-MGC were also ameliorated in MMC
derived from CHOP KO mice, compared to MMCs from WT mice.
Use of TALENs (Transcription Activator-Like Effector Nuclease) to
Target the Cluster miRNAs
[0183] TALENs designed and generated to target and delete two
genomic regions in the miRNA cluster are used. The first is
knockout of miR-379 and the upstream promoter, aimed to interrupt
both miR-379 and lnc-MGC expression. The second is to delete the
entire mega cluster region. These TALENs are tested in vitro for
their readiness to inject directly into mouse embryos, for the
rapid and efficient generation of KO mice.
Example: 8: Identify the Molecular Mechanisms by which Diabetic
Conditions (HG and TGF-.beta.1) Upregulate the miRNA Mega Cluster
and its Host Transcript, lncRNA-MGC
[0184] Diabetic conditions lead to increased transcription of the
promoter for the miRNA cluster and host gene lncRNA-MGC
[0185] The CHOP binding sites are cloned and nearby regions of the
promoter into luciferase reporters. These constructs are
transfected into MMC and treated with or without HG (25 mM),
mannitol (19.5 mM) and incubated for 24-72 hours; TGF-.beta. (10
ng/mL is added, and is incubated for 6-24 hours). MMCs pre-treated
with or without CHOP specific siRNAs or negative control siRNAs, or
MMC from WT versus CHOP KO mice is treated with HG, mannitol or
TGF-.beta.1 to determine if losing CHOP reduces promoter
transactivation. Chromatin immunoprecipitation (ChIP) assays with a
CHOP antibody are used to evaluate CHOP occupancy at the promoter
regions in response to TGF-.beta.1 and HG in MMCs. ChIP-qPCRs
amplify the desired promoter genomic region and an unrelated
control region. Western blots are used to determine CHOP protein
levels.
Diabetic Conditions Increase the Expression of Multiple Mega
Cluster Component Key miRNAs, and are Regulated by CHOP
[0186] Wild type (WT) MMCs, MMC transfected with CHOP siRNA and
MMCs from CHOP KO mice are treated with HG, mannitol and
TGF-.beta.1. RNA is extracted from the cells and the expression of
lncRNA-MGC, and all 40 miRNAs within the cluster are systematically
examined using primers designed for each of the mature miRNAs. This
is supported by data indicating nearly 30 of the cluster miRNAs
were induced in MMCs by TGF-.beta.1 but inhibited by CHOP
siRNAs.
Chromatin Features of this Genome Region
[0187] Known and novel transcripts, including miRNAs and many
lncRNAs, can be identified by combining RNA-seq with ChIP-seq to
identify domains marked with H3K4me3 (promoter mark), H3K36me3
(gene body), and enhancers (H3K4me1). Therefore, antibodies for
these three chromatin marks are used to perform ChIP, and then use
the ChIP-enriched DNA to sequence (ChIP-seq) this specific genomic
region. The results verify whether the lncRNA and miRNAs within the
cluster have their own promoters, or if they are transcribed as one
unit. Initial observations indicate the presence of an H3K4me1 mark
at the promoter start site, but nowhere else along the cluster, and
lack of H3K4me3 marks, supporting the hypothesis, there is a single
transcription unit. If the ChIP-seq data indicates potential
intermediate promoters/transcription, it will be verified by
cloning longer transcripts that include the majority of the
miRNAs.
Influence of the Host lncRNA-MGC
[0188] MMCs are pre-treated with a mixture of siRNAs designed to
target the lnc-MGC, and then treat them .+-.TGF-.beta.1 or HG.
Determining expression levels for all 40 miRNAs reveals if some or
all are down regulated by the siRNAs.
Example 9: Determine the Functional Significance of MCs
Up-Regulating Key Component miRNAs of the Mega Cluster and
Down-Regulating their Key Targets in Response to HG and
TGF-.beta.1
[0189] Validate Targets of miRNAs within the Mega Cluster that are
Upregulated by TGF-.beta.1 Predicted Computationally
[0190] Putative target genes that are targeted by multiple miRNAs
within the cluster and have functions related to DN include TNRC6B,
CUGBP2, CPEB4, Pumilio2 (RNA binding proteins that regulate
translation) and BHC80 (a transcription factor) and are common
targets of multiple miRNAs including miR-379. Others, including HuR
(RNA binding proteins), FoxP2, NF1A, Arid2 (TFs and co-factors)
have binding sites in their 3' UTRs for 9 to 18 of the cluster
miRNAs. EDEM3, a protein related to ER-associated degradation, is a
miR-379 target with interesting functions. Decreasing EDEM3 through
miR-379 will cause unfolded proteins to accumulate resulting in
glomerular hypertrophy. Hence ER stress may induce further ER
stress through the miR-379-EDEM3 pathway (FIG. 10).
[0191] 3'UTRs of the genes listed above are cloned downstream of a
luciferase reporter gene. MMCs are transfected with the constructs
(sense and antisense), and negative control oligos, and treated
with or without TGF-.beta.1, or with mimics or inhibitors of key
selected miRNAs that target the genes discussed above (including
miR-379, -377 and -495). It is expected that miRNA mimics will
decrease, and inhibitors will increase, the luciferase activity of
reporters that contain 3'-UTRs from bona fide target genes. The
miRNA binding sites in the 3' UTRs of targets identified
experimentally are mutated to verify that the inhibitory effects of
the mimics are lost. The protein and mRNA levels of these genes are
also determined by Western blotting and RT-qPCR, respectively, as
described.
In Silico Analysis of the Biological Functions of the Predicted
Targets of the Mega Cluster miRNAs
[0192] Select candidate megacluster miRNAs is selected, whose
expression, according to miRNA-seq, is increased at least
1.5-2-fold under diabetic conditions, e.g., the 28-30 miRNAs
identified in the studies. mRNA expression profiles induced by
TGF-.beta.1 in MMCs is compared (transcriptome profiling by
RNA-seq) with the micro RNA profiles, to identify genes whose mRNA
expression patterns inversely correlate with miRNA expression. The
3'-UTRs of these mRNAs (in the USCS and ENSEMBLE genome browsers)
is aligned with the differentially expressed miRNAs to identify
potential targets. The results are compared with publically
available miRNA target prediction sites in TargetScan, miRBase,
PicTar and DIANA-microT 3.0. Targets predicted by two or more
databases and conserved among rat, human and mouse species are
pooled. Particular attention is paid to those miRNA, whose
predicted targets have functions related to MC dysfunction and DN.
These targets are imported into GO, Pathway analyses software (IPA)
and GSEA to determine their potential biological functions, and
changes they cause in functional disease networks, which are
supported by initial data showing that multiple miRNAs target the
same genes related to MC dysfunction.
Ago-2-CLIP-Seq Based miRNA Target Profiling
[0193] The ClIP-seq (HITS-CLIP) method can generate sequencing data
for both miRNAs and their complementary (target) mRNAs bound to
Ago2 in RISCs (RNA-inducing silencing complex), genome-wide.
Ago2-CLIP-Seq coupled with bioinformatics is used to identify
specific megacluster miRNAs and their targets bound to Ago2 in
control and TGF-.beta.1-treated MMC. The results reveal mechanisms
of action and the in vivo functional roles of the miRNA cluster.
MMCs are treated with and without TGF-.beta.1 for 24 hours, then UV
crosslinked and immunoprecipitated with Ago2 antibody. Ago2
associated mRNAs and miRNAs are isolated, transcribed into cDNAs,
and libraries prepared for small and regular RNAs for sequencing
(Iluminia). From the smRNA-seq CLIP data, the abundance of each
mouse miRNA (miRBase v20) in each sample is determined by
processing the raw reads. For target mRNA-seq CLIP data, reads in
each sample are first aligned to the mouse genome assembly (NCBI
GRCm38) using TopHat2.
[0194] Ago2-binding clusters (containing reads that are
significantly greater than background signal) are identified using
the pooled, uniquely-aligned reads from all samples. Reads are
summarized and normalized to obtain Ago2-binding cluster levels in
each sample. Ago2 binding clusters across all the samples and
replicates for each condition, and regions that differentially bind
Ago2 between the two conditions are identified. The CLIP data and
potential miRNA target sites predicted using base pair searches and
existing tools like TargetScan are analyzed together to identify
bona fide miRNA-mRNA interactions in control and
TGF-.beta.1-treated MCs.
[0195] In addition, the abundance of each Refseq mRNA is determined
by regular RNA-seq of the MC samples without IP, summarized,
normalized and compared between the two conditions using
Bioconductor package "edgeR." The miRNA-mRNA interactions are
integrated and identified by CLIP assays with the related changes
in gene expression to further verify the interactions and reveal
the potential effects of up-regulating cluster miRNAs on target
gene expression levels. Furthermore, the potential functions of
target mRNAs is determined with respect to DN using in silico
analyses, including GO, WA, network and motif analysis of the
identified targets. Mega cluster miRNAs whose expression was
co-modulated, or mega cluster target genes that contain multiple,
closely related miRNA binding sites, are also identified by
comparing data obtained under the two conditions. The expression
levels of target mRNAs and corresponding proteins is assessed by
qPCR and Western blots respectively.
Functional Roles of Candidate Megacluster miRNAs tat Respond to
TGF-.beta.1/HG and their Targets in MC Dysfunction
[0196] "Gain of function" and "loss of function" approaches are
used. MMCs are transfected with oligonucleotide mimics, inhibitors
of candidate miRNAs (miR-379, -495, -377), or NC oligos to
determine whether manipulating the levels of their putative target
genes can influence TGF-.beta.1 and HG responses. At 48 to 72 hours
post-transfection, the expression of miRNA target genes, fibrosis
and hypertrophy related genes and proteins induced by
TGF-.beta.1/HG is determined by RT-qPCR and Western blotting.
Cellular hypertrophy, oxidant and ER stress markers are assayed.
Similarly, the gain and loss of function of key target genes
(including EDEM3, CUGB2, Tnrc6, BHC80) are tested.
Role of CHOP and lncRNA-MGC on the Expression of miRNA Targets
[0197] The down-regulating of target genes, which augments DN
pathogenesis is tested by determining that CHOP and lncRNA-MGC
drive the expression of the miRNA cluster. The effect of CHOP siRNA
and lncRNA-MGC siRNAs on the expression of the same miRNA targets,
and potential new targets identified by CLIP-seq is determined in
control and TGF-.beta.1-treated MMCs. Targets are also examined in
glomeruli and MMC derived from CHOP KO and WT mice.
Example 10: Functional Roles of the miRNA Cluster and Lnc-MGC in DN
Progression In Vivo
[0198] Dysregulation of key megacluster miRNAs and their targets in
mouse models of DN
[0199] miRNAs and their targets identified in MCs in vitro are
regulated in mouse models of T1D- and T2D-associated DN in vivo
using: 1) STZ-injected T1D mouse models of DN in which increases in
expression f 30 of the cluster miRNAs was observed, 2) Male T2D
leptin receptor deficient db/db mice (Strain BKS. CG-m+/+lepr db/J)
at 10-14 weeks of age (101-103). db/db mice are obese, insulin
resistant and diabetic by 6-8 weeks, and develop renal dysfunction
by 10-12. After confirming hyperglycemia (glucose >300 mg/dL),
db/db mice are compared with age-matched controls (db/+, glucose
<150 mg/dL). 3) Akita T1D mice in the DBA2J background, which
develop features of DN that are more overt than the STZ model. 4)
Mice diet-induced obese (DIO) mice (C57BL/6J) on 14 week high fat
diets (60 kcal percent fat), and controls on standard diets. DIO
mice are insulin resistant, have mild hyperglycemia, elevated
triglycerides and fibrosis/inflammation in glomeruli. Blood
pressure, changes in physiology (blood glucose, urinary protein,
albumin, creatinine), and the histology and morphology of the renal
cortex are monitored regularly and at sacrifice. At the end of the
indicated time periods, kidneys are dissected from all the mice,
and cortical tissues and glomeruli isolated, then either flash
frozen for RNA/protein extraction, or used to prepare MMCs. RT-QPCR
is used to compare the basal levels of the miRNAs and their
candidate target genes in glomeruli.
Reduced Levels of Mega Cluster miRNAs and Lnc-MGC in CHOP-KO Mice;
Protection (CHOP KO Mice) from DN Development
[0200] CHOP KO mice and their controls are made diabetic by STZ
injections. At 4, 12 and 16-20 weeks post-diabetes development, key
features of DN are assessed in these mice. The expression levels of
fibrotic genes, TGF-.beta.1, 40 of the mega cluster miRNAs, and
lnc-MGC, in glomeruli are determined at each time point. At least
10 mice per group are studied per time point and experiments are
performed in triplicate.
Treating T1D Mice with LNA Anti-miR-379 Alters Course of DN, and
LNA-Gapmers that Target the Lnc-MGC
[0201] LNA modification has several advantages, including less
toxicity, lower dosing and efficient targeting. LNA-modified
anti-miR-379 is designed, and the control LNAs target miR-239b,
which is expressed in Caenorhabditis elegans. To target lnc-MGC in
vivo Gapmer technology is adopted/used. LNA.TM. longRNA GapmeRs are
newly available antisense oligos that are used to functionally
analyze mRNAs and lncRNAs. They contain a central stretch (gap) of
DNA monomers flanked by blocking of LNA-modified nucleotides
(http://www.exiqon.com/gamper). The LNA blocks increase the oligos'
target affinity and nuclease resistance and the DNA gap induces
RNase H cleavage of the target RNA after binding. The gapmers are
14-16 nucleotides in length and are fully phosphorothioated.
Exiqon's advanced design algorithms design the most potent LNA.TM.
longRNA GapmeRs that have minimal off-target effects and high
success rates. LNA-anti-miR-379 or LNA-anti-lnc-MGC are injected
subcutaneously twice weekly into control and STZ-injected DBA2J
mice, which develop more sever DN than C57BL6 mice. The progression
of key structural, histological and molecular features of DN is
followed for up to 20 weeks as described. The anti-miR-379 and
anti-lnc-MGC are evaluated in these in vivo translational
studies.
Example 11: Genetic Knockout of the Megacluster miRNA379
[0202] To study the function of miR-379 in vivo, miR-379 knockout
(KO) mouse was generated. The genome editing using the RNA-guided
Cas9 nucleases from the microbial CRISPR (clustered regularly
interspaced short palindromic repeat)-Cas systems was used to
generate gene knockout mouse. A paired nickase strategy was used
for engineering a system to ameliorate off-target activity. Paired
nicking method can reduce off-target activity by 50- to 1,500-fold
in cell lines and to facilitate gene knockout in mouse zygotes
without sacrificing on-target cleavage efficiency.
[0203] A double nickase strategy was used to delete mouse miR-379
genomic region with less off-target. Five guide RNAs, three sense
(S1, S2, and S3) and two antisense (AS1 and AS2) were designed
(shown in FIG. 11B). Positions and actual sequences of
oligonucleotides to construct expression vectors (e.g., PX461,
EGFP; PX462, or Puromycine (PX461 and PX462 are plasmid names; the
double strand DNA made from designed and synthesized sense and
antisense oligos were cloned into the plasmid vectors. The guide
RNAs are expressed from the plasmids. PX461 has EGFP gene to
monitor the transfection by GFP. PX462 has Puromycine resistance
gene to select the cells by drug (Puromycine) resistance) are
listed below.
[0204] Potential Targets
TABLE-US-00007 S1 (SEQ ID NO: 27) CCTGAAGAGATGGTAGACTATGG S1S (SEQ
ID NO: 28) CACCgCCTGAAGAGATGGTAGACTA S1AS (SEQ ID NO: 29)
AAACTAGTCTACCATCTCTTCAGGc S2 (SEQ ID NO: 30)
GATGGTAGACTATGGAACGTAGG S2S (SEQ ID NO: 31)
CACCgGATGGTAGACTATGGAACGT S2AS (SEQ ID NO: 32)
AAACACGTTCCATAGTCTACCATCc S3 (SEQ ID NO: 33)
TGTTTTTGACCTATGTAACATGG S3S (SEQ ID NO: 34)
CACCgTGTTTTTGACCTATGTAACA S3AS (SEQ ID NO: 35)
AAACTGTTACATAGGTCAAAAACAc AS1 (SEQ ID NO: 36)
CCTATGTAACATGGTCCACTAAC AS1S (SEQ ID NO: 37)
CACCgGTTAGTGGACCATGTTACAT AS1AS (SEQ ID NO: 38)
AAACATGTAACATGGTCCACTAACc AS2 (SEQ ID NO: 39)
CCACTAACTCTCAGTATCCAATC AS2S (SEQ ID NO: 40)
CACCgGATTGGATACTGAGAGTTAG AS2AS (SEQ ID NO: 41)
AAACCTAACTCTCAGTATCCAATCc
[0205] In the above sequences, S1 is one of the target sequences.
S15 is synthesized oligonucleotides for sense strand with extra 5'
overhang (CACC) for cloning. S1AS is synthesized oligonucleotides
for antisense strand with extra 5' overhang (AAAC) for cloning. S1S
and S1AS are complimentary and annealed double strand DNA
(S1S/S1AS) has 5' overhang for cloning. Small "g" is transcription
start site in the expression vector. Likewise, in the remaining
target sequences listed above.
[0206] Using TCMK cells (mouse kidney cell line) in vitro, activity
of genome editing at miR-370 locus was tested with several
combinations of those guide RNAs (sense and antisense). Since the
combination S2 and AS1 showed the best activity, this combination
was injected into fertilized eggs to make mutant mice.
[0207] Two strategies are used to inject guide RNAs. First, guide
RNAs are injected into pronuclei of fertilized eggs with plasmids
expressing guide RNAs and nickase. Second, the guide RNA is
transcribed in vitro and nickase RNA is injected into cytoplasm of
fertilized eggs. The first strategy required injecting the same
plasmids used for cell line transfection in vitro. For the second
strategy, T7-transcribed guide RNAs were made. For this purpose,
guide RNA part in the plasmids was amplified with primers with
T7-promoter and reverse primer for guide RNA (below).
TABLE-US-00008 T7-S2 (SEQ ID NO: 42)
TTAATACGACTCACTATAGGGATGGTAGACTATGGAACGT T7-AS1 (SEQ ID NO: 43)
TTAATACGACTCACTATAGGGTTAGTGGACCATGTTACAT gRNA-R (SEQ ID NO: 44)
AAAAGCACCGACTCGGTGCC
[0208] The amplified PCR products were used as template for in
vitro transcription of guide RNAs using T7 in vitro transcription
kit. Seven mice were obtained from plasmid injection and fourteen
mice were obtained from RNA injection experiments. The short
deletion of miR-379 genomic region was tested by fragment analysis
in the PCR fragment amplified from tail DNA.
[0209] Deletion found in 8F was 36 bp at miR-379 locus. FIG. 20A-B
shows the 36 base-pair deletion in the miR-379 locus of a mouse
generated using the CRISPR/CAS9 system described in this
disclosure.
[0210] A colony of mice possessing the 8F deletion was generated by
crossing heterozygotes to produce homozygotes. The colony was
expanded and mice were used for characterization of miR-379
knockout as well as for experiments of type 1 diabetes
(STZ-induced) and high fat diet (HFD). Kidney mesangial cells (MC)
from three miR-379 KO mice were cultured in vitro and expression of
miR-379 was significantly reduced compared to MC from wild type
mice (FIG. 21A). One of the targets of miR-379 was confirmed
significantly increased in miR-370K0 mice compared to MC from wild
type mice (FIG. 21B). These mice were also used for in vitro
experiments in diabetic conditions (high glucose, TGF-.beta., and
other treatments.
Example 12: Replacement of miR-379 Region with Poly(A) Signal (to
Terminate Transcription)
[0211] Another method to stop the transcription of lnc-MGC (miR-379
cluster) was also established, as depicted in FIG. 22. For
replacement of miR-379 region with poly(A) signal sequence,
.about.200 bases oligonucleotide was designed (below) which
includes 5' and 3' homologous sequences to miR-379 region
(underline) and minimum BGH poly(A) sequence in the middle
(italics). DNA ligase inhibitor (SCR7) was also used to enhance
recombination.
Minimum bGH polyA (52 Bases)
TABLE-US-00009 [SEQ ID NO: 72]
GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTA GGTGTC
Any available poly(A) sites from any of the mammalian transcripts
can be used as well.
Synthesized Oligonucleotide (Single Stranded DNA) for Targeting
TABLE-US-00010 [0212] [SEQ ID NO: 73]
AAGTGACGCCAGCTTCAGGGACAAGGCCCAAGTTTCTAGGGGTCAACACC
GTTCCATGGTTCCTGAAGAGAGTCCTTTCCTAATAAAATGAGGAAATTGC
ATCGCATTGTCTGAGTAGGT GACATGTAACATGGTCCACTAACTCTCA
GTATCCAATCCATCCTCGGAGGGCACCCCGGAGGTGTTACCAACAGC
[0213] For the replacement, .about.100 eggs were injected with Cas9
nickase mRNA, two guide RNAs (S2 & AS1) and SCR7 (ligase
inhibitor inhibiting end joining and enhancing replacement). Twenty
two (22) mice were born and twenty-one (21) mice survived. By PCR
screening, five founders were identified as four full insertion
(7F, 9M, 10M, 18M) and one partial insertion (8F). These mice were
crossed with wild-type B6 mice and confirmed replacement. The line
with partial insertion was designated as 51536 and full insertion
was S1543. These lines were expanded for further experiment. The
method to insert poly(A) signal are applicable to stop any kinds of
transcripts (coding or noncoding) if guide RNAs are designed for
specific targets.
Example 13: Strategy for Treating Human Patients
[0214] Because the disclosed CRSPR-CAS system works to delete
(inhibit) miR-397 cluster in mouse tissue, miR-379 cluster in human
patients is inhibited by the same strategy. Therefore, human
versions of guide RNAs for miR-379 region were designed (below).
Six guide RNAs (4 sense (S) and 2 antisense (AS)) were designed.
Positions and actual sequences of oligonucleotides for guide RNAs
on human miR379 [SEQ ID NO: 79] are shown below:
##STR00002##
[0215] Design of Human miR379 guide RNAs
[0216] Sequences of potential targets (e.g., 51 below) and
sequences of oligos for cloning (e.g., S1S and S1AS) are listed
below (the oligos for cloning are listed below the target
sequences):
TABLE-US-00011 [SEQ ID NO: 80] S1: TTCCGTGGTTCCTGAAGAGATGG [SEQ ID
NO: 120] S1S: CACCgTTCCGTGGTTCCTGAAGAGA [SEQ ID NO: 121] S1AS:
AAACTCTCTTCAGGAACCACGGAAc [SEQ ID NO: 27] S2:
CCTGAAGAGATGGTAGACTATGG (same as mouse S1) [SEQ ID NO: 28] S2S:
CACCgCCTGAAGAGATGGTAGACTA [SEQ ID NO: 29] S2AS:
AAACTAGTCTACCATCTCTTCAGGc [SEQ ID NO: 30] S3:
GATGGTAGACTATGGAACGTAGG (same as mouse S2) [SEQ ID NO: 31] S3S:
CACCgGATGGTAGACTATGGAACGT [SEQ ID NO: 32] S3AS:
AAACACGTTCCATAGTCTACCATCc [SEQ ID NO: 82] S4:
GATTTCTGACCTATGTAACATGG [SEQ ID NO: 122] S4S:
CACCgGATTTCTGACCTATGTAACA [SEQ ID NO: 123] S4AS:
AAACTGTTACATAGGTCAGAAATCc [SEQ ID NO: 81] AS1:
CCGTGGTTCCTGAAGAGATGGTA [SEQ ID NO: 124] AS1S:
CACCgTACCATCTCTTCAGGAACCA [SEQ ID NO: 125] AS1AS:
AAACTGGTTCCTGAAGAGATGGTAc [SEQ ID NO: 27] AS2:
CCTGAAGAGATGGTAGACTATGG (same target as S2) [SEQ ID NO: 126] AS2S:
CACCgCCATAGTCTACCATCTCTTC [SEQ ID NO: 127] AS2AS:
AAACGAAGAGATGGTAGACTATGGc
[0217] S1, S2, S3, S4, AS1, and AS2 are sequences of potential
targets. Sequences of oligos for cloning are under the each target
sequences. For example, S1S is sequence of sense oligo for S1 and
S1AS is sequence of antisense oligo for S1. They are designed for
constructing expression vectors for miR-379 target genes (e.g.,
PX461, EGFP; PX462, or Puromycine (PX461 and PX462 are plasmid
names; the double strand DNA made from designed and synthesized
sense and antisense oligos were cloned into the plasmid vectors.
The guide RNAs are expressed from the plasmids. PX461 has EGFP gene
to monitor the transfection by GFP. PX462 has Puromycine resistance
gene to select the cells by drug (Puromycine) resistance). Possible
combinations include: S1-AS2, S2-AS1, S3-AS1, S3-AS2, and S4-AS2.
Additional sets of possible combinations include S1S-S1AS,
S4S-S4AS, AS1S-AS1AS, AS2S-AS2AS. Delivery of plasmids expressing
these guide RNAs and CAS9 nickase (or other CAS9 derivatives, for
example, fusion with repressor) into human kidney reduces human
lnc-MGC and miRNA-379 cluster and prevents DN.
Other Embodiments
[0218] While the disclosure has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the disclosure
following, in general, the principles of the disclosure and
including such departures from the present disclosure within known
or customary practice within the art to which the disclosure
pertains and may be applied to the essential features hereinbefore
set forth, and follows in the scope of the appended claims.
Sequence CWU 1
1
127116DNAArtificial SequenceSynthetic polynucleotide 1tgaaggccac
actaac 16216DNAArtificial SequenceSynthetic polynucleotide
2atgaaggcca cactaa 16315DNAArtificial SequenceSynthetic
polynucleotide 3gaaggccaca ctaac 15416DNAArtificial
SequenceSynthetic polynucleotide 4cacggtgctg aaagag
16516DNAArtificial SequenceSynthetic polynucleotide 5acggtgctga
aagaga 16615DNAArtificial SequenceSynthetic polynucleotide
6cggtgctgaa agaga 15716DNAArtificial SequenceSynthetic
polynucleotide 7tccttgaatg gttgca 16816DNAArtificial
SequenceSynthetic polynucleotide 8ttgaatggtt gcacgg
16916DNAArtificial SequenceSynthetic polynucleotide 9cggtgctgaa
agagag 161016DNAArtificial SequenceSynthetic polynucleotide
10atttggcagt gggaag 161116DNAArtificial SequenceSynthetic
polynucleotide 11tttggcagtg ggaagc 161216DNAArtificial
SequenceSynthetic polynucleotide 12ttggcagtgg gaagca
161316DNAArtificial SequenceSynthetic polynucleotide 13tcaaaaacat
aacgcc 161416DNAArtificial SequenceSynthetic polynucleotide
14gtcaaaaaca taacgc 161517DNAArtificial SequenceSynthetic
polynucleotide 15ggtcaaaaac ataacgc 171616DNAArtificial
SequenceSynthetic polynucleotide 16ggtcaaaaac ataacg
161716DNAArtificial SequenceSynthetic polynucleotide 17aggtcaaaaa
cataac 161817DNAArtificial SequenceSynthetic polynucleotide
18aggtcaaaaa cataacg 171915DNAArtificial SequenceSynthetic
polynucleotide 19taggtcaaaa acata 152015DNAArtificial
SequenceSynthetic polynucleotide 20caaaaacata acgcc
152116DNAArtificial SequenceSynthetic polynucleotide 21gatttggcat
tggaag 162216DNAArtificial SequenceSynthetic polynucleotide
22ggaaggccat gtcaac 162316DNAArtificial SequenceSynthetic
polynucleotide 23ggcattgatg ggggaa 162417DNAArtificial
SequenceSynthetic polynucleotide 24tcagaaatca taacgcc 1725287DNAMus
musculus 25atttttctga gttagtgtgg ccttcatctg gtaatgtact acctgagggg
ggaggtgccg 60cctctctttc agcaccgtgc aaccattcaa ggagggtgtg ttgttcacca
catctgcttc 120ccactgccaa atcaggcctc agaaaagctt tctggaagtg
acgccagctt cagggacaag 180gcccaagttt ctaggggtca acaccgttcc
atggttcctg aagagatggt agactatgga 240acgtaggcgt tatgtttttg
acctatgtaa catggtccac taactct 28726291DNAHomo sapiens 26agtctttcca
agttgacatg gccttcctgg aggaattacc acttagggta gaggcacccc 60ttcccccatc
aatgccactg ccccacattg gaggaggggt tgtttatgtt caccatgtgc
120ctgcttccaa tgccaaatcc agcctcagaa agctttctgg aagtgacgcc
aacttcaggg 180gcaaggccct ggttctgggg tcagcaccat tccgtggttc
ctgaagagat ggtagactat 240ggaacgtagg cgttatgatt tctgacctat
gtaacatggt ccactaactc t 2912723DNAArtificial SequenceSynthetic
polynucleotide 27cctgaagaga tggtagacta tgg 232825DNAArtificial
SequenceSynthetic polynucleotide 28caccgcctga agagatggta gacta
252925DNAArtificial SequenceSynthetic polynucleotide 29aaactagtct
accatctctt caggc 253023DNAArtificial SequenceSynthetic
polynucleotide 30gatggtagac tatggaacgt agg 233125DNAArtificial
SequenceSynthetic polynucleotide 31caccggatgg tagactatgg aacgt
253225DNAArtificial SequenceSynthetic polynucleotide 32aaacacgttc
catagtctac catcc 253323DNAArtificial SequenceSynthetic
polynucleotide 33tgtttttgac ctatgtaaca tgg 233425DNAArtificial
SequenceSynthetic polynucleotide 34caccgtgttt ttgacctatg taaca
253525DNAArtificial SequenceSynthetic polynucleotide 35aaactgttac
ataggtcaaa aacac 253623DNAArtificial SequenceSynthetic
polynucleotide 36cctatgtaac atggtccact aac 233725DNAArtificial
SequenceSynthetic polynucleotide 37caccggttag tggaccatgt tacat
253825DNAArtificial SequenceSynthetic polynucleotide 38aaacatgtaa
catggtccac taacc 253923DNAArtificial SequenceSynthetic
polynucleotide 39ccactaactc tcagtatcca atc 234025DNAArtificial
SequenceSynthetic polynucleotide 40caccggattg gatactgaga gttag
254125DNAArtificial SequenceSynthetic polynucleotide 41aaacctaact
ctcagtatcc aatcc 254240DNAArtificial SequenceSynthetic
polynucleotide 42ttaatacgac tcactatagg gatggtagac tatggaacgt
404340DNAArtificial SequenceSynthetic polynucleotide 43ttaatacgac
tcactatagg gttagtggac catgttacat 404420DNAArtificial
SequenceSynthetic polynucleotide 44aaaagcaccg actcggtgcc
204595DNAMus musculus 45ggttcctgaa gagatggtag actatggaac gtaggcgtta
tgtttttgac ctatgtaaca 60tggtccacta actctcagta tccaatccat cctcg
954659DNAArtificial SequenceSynthetic polynucleotide 46ggttcctgaa
gagatggtag aacatggtcc actaactctc agtatccaat ccatcctcg 594795DNAMus
musculus 47ggttcctgaa gagatggtag actatggaac gtaggcgtta tgtttttgac
ctatgtaaca 60tggtccacta actctcagta tccaatccat cctcg
954859DNAArtificial SequenceSynthetic polynucleotide 48ggttcctgaa
gagtggtaga aacatggtcc actaactctc agtatccaat ccatcctcg 5949287DNAMus
musculus 49atttttctga gttagtgtgg ccttcatctg gtaatgtact acctgagggg
ggaggtgccg 60cctctctttc agcaccgtgc aaccattcaa ggagggtgtg ttgttcacca
catctgcttc 120ccactgccaa atcaggcctc agaaaagctt tctggaagtg
acgccagctt cagggacaag 180gcccaagttt ctaggggtca acaccgttcc
atggttcctg aagagatggt agactatgga 240acgtaggcgt tatgtttttg
acctatgtaa catggtccac taactct 28750291DNAHomo sapiens 50agtctttcca
agttgacatg gccttcctgg aggaattacc acttagggta gaggcacccc 60ttcccccatc
aatgccactg ccccacattg gaggaggggt tgtttatgtt caccatgtgc
120ctgcttccaa tgccaaatcc agcctcagaa agctttctgg aagtgacgcc
aacttcaggg 180gcaaggccct ggttctgggg tcagcaccat tccgtggttc
ctgaagagat ggtagactat 240ggaacgtagg cgttatgatt tctgacctat
gtaacatggt ccactaactc t 29151297DNAArtificial SequenceSynthetic
polynucleotidemisc_feature(1)..(1)Residue is a or
absentmisc_feature(27)..(27)Residue is a or
absentmisc_feature(28)..(28)Residue is t or
absentmisc_feature(69)..(69)Residue is c or
absentmisc_feature(80)..(80)Residue is a or
absentmisc_feature(87)..(87)Residue is a or
absentmisc_feature(107)..(107)Residue is t or
absentmisc_feature(108)..(108)Residue is a or
absentmisc_feature(109)..(109)Residue is t or
absentmisc_feature(121)..(121)Residue is g or
absentmisc_feature(122)..(122)Residue is c or
absentmisc_feature(133)..(133)Residue is c or
absentmisc_feature(155)..(155)Residue is a or
absentmisc_feature(199)..(199)Residue is t or
absentmisc_feature(202)..(202)Residue is a or
absentmisc_feature(268)..(268)Residue is c or absent 51nrtytttcyr
agttrryrtg gccttcnnct ggwrrwrtwm ymmctkaggg krgaggyrcc 60scytcycynw
tcaryrccrn tgcmmcncat tsraggaggg kktgttnnng ttcaccayrt
120nnctgcttcc mantgccaaa tcmrgcctca gaaangcttt ctggaagtga
cgccarcttc 180agggrcaagg cccwrgttnc tnggggtcar caccrttccr
tggttcctga agagatggta 240gactatggaa cgtaggcgtt atgwtttntg
acctatgtaa catggtccac taactct 29752225DNAMus musculus 52tatagtcagc
acagtggttc atttttctga gttagtgtgg ccttcatctg gtaatgtact 60acctgagggg
ggaggtgccg cctctctttc agcaccgtgc aaccattcaa ggagggtgtg
120ttgttcacca catctgcttc ccactgccaa atcaggcctc agaaaagctt
tctggaagtg 180acgccagctt cagggacaag gcccaagttt ctaggggtca acacc
2255316DNAArtificial SequenceSynthetic polynucleotide 53cttcccactg
ccaaat 165416DNAArtificial SequenceSynthetic polynucleotide
54atttggcagt gggaag 165516DNAArtificial SequenceSynthetic
polynucleotidemodified_base(1)..(16)Residues modified with
phosphorothioatemodified_base(1)..(3)Residues are
LNAmodified_base(14)..(16)Residues are LNA 55tcaaaaacat aacgcc
165616DNAArtificial SequenceSynthetic
polynucleotidemodified_base(1)..(16)Residues modified with
phosphorothioatemodified_base(1)..(3)Residues are
LNAmodified_base(14)..(16)Residues are LNA 56cacggtgctg aaagag
165716DNAArtificial SequenceSynthetic
polynucleotidemodified_base(1)..(16)Residues modified with
phosphorothioatemodified_base(1)..(3)Residues are
LNAmodified_base(14)..(16)Residues are LNA 57tgaaggccac actaac
165816DNAArtificial SequenceSynthetic
polynucleotidemodified_base(1)..(16)Residues modified with
phosphorothioatemodified_base(1)..(3)Residues are
LNAmodified_base(14)..(16)Residues are LNA 58atttggcagt gggaag
165925RNAArtificial SequenceSynthetic
polynucleotidemisc_feature(24)..(24)Residue is
DNAmisc_feature(25)..(25)Residue is DNA 59caucugcuuc ccacugccaa
aucag 256027RNAArtificial SequenceSynthetic polynucleotide
60cugauuuggc agugggaagc agaugug 276125RNAArtificial
SequenceSynthetic polynucleotidemisc_feature(24)..(24)Residue is
DNAmisc_feature(25)..(25)Residue is DNA 61ucagcaccgu gcaaccauuc
aagga 256227RNAArtificial SequenceSynthetic polynucleotide
62uccuugaaug guugcacggu gcugaaa 276325RNAArtificial
SequenceSynthetic polynucleotidemisc_feature(24)..(24)Residue is
DNAmisc_feature(25)..(25)Residue is DNA 63cuucaucugg uaauguacua
ccuga 256427RNAArtificial SequenceSynthetic polynucleotide
64ucagguagua cauuaccaga ugaaggc 276521RNAArtificial
SequenceSynthetic polynucleotide 65ggaugcaagg uaucagaugg u
216622RNAArtificial SequenceSynthetic polynucleotide 66uagguuaucc
guguugccuu cg 226722RNAArtificial SequenceSynthetic polynucleotide
67gaaguuguuc gugguggauu cg 226821RNAArtificial SequenceSynthetic
polynucleotide 68uucaccuaca aggagauacu a 216922RNAArtificial
SequenceSynthetic polynucleotide 69cucuaaccgg uacauuauga gu
227023RNAArtificial SequenceSynthetic polynucleotide 70agguuacccg
agcaacuuug cau 237122RNAArtificial SequenceSynthetic polynucleotide
71uucuucacgu ggcgcuuaca aa 227252DNAArtificial SequenceSynthetic
polynucleotide 72gtcctttcct aataaaatga ggaaattgca tcgcattgtc
tgagtaggtg tc 5273198DNAArtificial SequenceSynthetic polynucleotide
73aagtgacgcc agcttcaggg acaaggccca agtttctagg ggtcaacacc gttccatggt
60tcctgaagag agtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
120gtcgacatgt aacatggtcc actaactctc agtatccaat ccatcctcgg
agggcacccc 180ggaggtgtta ccaacagc 1987422RNAArtificial
SequenceSynthetic polynucleotide 74uguuuucaac ggaaacacac ua
227522RNAArtificial SequenceSynthetic polynucleotide 75uauguaguau
gguccacauc uu 227622RNAArtificial SequenceSynthetic polynucleotide
76uucuucacgu gguacaaaca aa 227722RNAArtificial SequenceSynthetic
polynucleotide 77agggaccccg agggagggca gg 227822RNAArtificial
SequenceSynthetic polynucleotide 78ugcccccucc aggaagccuu cu
227977DNAArtificial SequenceSynthetic polynucleotide 79ttccgtggtt
cctgaagaga tggtagacta tggaacgtag gcgttatgat ttctgaccta 60tgtaacatgg
tccacta 778023DNAArtificial SequenceSynthetic polynucleotide
80ttccgtggtt cctgaagaga tgg 238123DNAArtificial SequenceSynthetic
polynucleotide 81ccgtggttcc tgaagagatg gta 238223DNAArtificial
SequenceSynthetic polynucleotide 82gatttctgac ctatgtaaca tgg
238321RNAArtificial SequenceSynthetic polynucleotide 83ugcaccuaaa
aggagauacu a 218422RNAArtificial SequenceSynthetic polynucleotide
84uaugugggau gguaaaccgc uu 228521RNAArtificial SequenceSynthetic
polynucleotide 85ugcaccuuaa aggagauaca a 218621RNAArtificial
SequenceSynthetic polynucleotide 86uguccgguag acacaauaua a
218722RNAArtificial SequenceSynthetic polynucleotide 87cuccaaaggg
cacauacaaa gu 228822RNAArtificial SequenceSynthetic polynucleotide
88augguugacc auagaacaug cg 228921RNAArtificial SequenceSynthetic
polynucleotide 89aauaauacau gguugaucuu u 219022RNAArtificial
SequenceSynthetic polynucleotide 90ucucucucag acgggaacau au
229125RNAArtificial SequenceSynthetic polynucleotide 91aaaggauucu
gcugucgguc ccacu 259222RNAArtificial SequenceSynthetic
polynucleotide 92uuucuccaau ugguccacac aa 229322RNAArtificial
SequenceSynthetic polynucleotide 93ugucucucga acgggaacau au
229421RNAArtificial SequenceSynthetic polynucleotide 94gcaugcgaua
ugccagauga u 219522RNAArtificial SequenceSynthetic polynucleotide
95ggggagacca guuggucagu gu 229621RNAArtificial SequenceSynthetic
polynucleotide 96ggaugcaagg uaucagaugg u 219722RNAArtificial
SequenceSynthetic polynucleotide 97uagguuaucc guguugccuu cg
229822RNAArtificial SequenceSynthetic polynucleotide 98gaaguuguuc
gugguggauu cg 229922RNAArtificial SequenceSynthetic polynucleotide
99uuguaccuaa aaggagauac ua 2210022RNAArtificial SequenceSynthetic
polynucleotide 100cucuaaccgg uacauuauga gu 2210123RNAArtificial
SequenceSynthetic polynucleotide 101agguuacccg agcaacuuug cau
2310222RNAArtificial SequenceSynthetic polynucleotide 102uucuucacgu
ggcgcuuaca aa 2210322RNAArtificial SequenceSynthetic polynucleotide
103uguuuucaac ggaaacacac ua 2210422RNAArtificial SequenceSynthetic
polynucleotide 104uauguaauau gguccacauc uu
2210522RNAArtificial SequenceSynthetic polynucleotide 105uucuucacgu
gguacaaaca aa 2210622RNAArtificial SequenceSynthetic polynucleotide
106uaugugggau gguaaaccgc uu 2210721RNAArtificial SequenceSynthetic
polynucleotide 107ugcacuuuaa aggagauaca a 2110821RNAArtificial
SequenceSynthetic polynucleotide 108uguccgguag acacaauaua a
2110922RNAArtificial SequenceSynthetic polynucleotide 109cuccaaaggg
cacauacaaa gu 2211022RNAArtificial SequenceSynthetic polynucleotide
110augguugacc auagaacaug cg 2211121RNAArtificial SequenceSynthetic
polynucleotide 111aauaauacau gguugaucuu u 2111225RNAArtificial
SequenceSynthetic polynucleotide 112aagggauucu gauguugguc acacu
2511322RNAArtificial SequenceSynthetic polynucleotide 113uuuuuccaau
cgacccacac aa 2211422RNAArtificial SequenceSynthetic polynucleotide
114ugucucucga acgggaacau au 2211521RNAArtificial SequenceSynthetic
polynucleotide 115gcaugcgaua ugccagauga u 2111622RNAArtificial
SequenceSynthetic polynucleotide 116uguuuucaac ggaaacacac ua
22117169DNAArtificial SequenceSynthetic polynucleotide
117ggtcaacacc gttccatggt tcctgaagag aggttcctga agagatggta
gactatggaa 60cgtaggcgtt atgtttttga cctatgtaac atggtccact aactctcagt
atccaatcca 120tcctcgatgt aacatggtcc actaactctc agtatccaat ccatcctcg
169118287RNAMus musculus 118auuuuucuga guuagugugg ccuucaucug
guaauguacu accugagggg ggaggugccg 60ccucucuuuc agcaccgugc aaccauucaa
ggagggugug uuguucacca caucugcuuc 120ccacugccaa aucaggccuc
agaaaagcuu ucuggaagug acgccagcuu cagggacaag 180gcccaaguuu
cuagggguca acaccguucc augguuccug aagagauggu agacuaugga
240acguaggcgu uauguuuuug accuauguaa caugguccac uaacucu
287119291RNAHomo sapiens 119agucuuucca aguugacaug gccuuccugg
aggaauuacc acuuagggua gaggcacccc 60uucccccauc aaugccacug ccccacauug
gaggaggggu uguuuauguu caccaugugc 120cugcuuccaa ugccaaaucc
agccucagaa agcuuucugg aagugacgcc aacuucaggg 180gcaaggcccu
gguucugggg ucagcaccau uccgugguuc cugaagagau gguagacuau
240ggaacguagg cguuaugauu ucugaccuau guaacauggu ccacuaacuc u
29112025DNAArtificial SequenceSynthetic polynucleotide
120caccgttccg tggttcctga agaga 2512125DNAArtificial
SequenceSynthetic polynucleotide 121aaactctctt caggaaccac ggaac
2512225DNAArtificial SequenceSynthetic polynucleotide 122caccggattt
ctgacctatg taaca 2512325DNAArtificial SequenceSynthetic
polynucleotide 123aaactgttac ataggtcaga aatcc 2512425DNAArtificial
SequenceSynthetic polynucleotide 124caccgtacca tctcttcagg aacca
2512525DNAArtificial SequenceSynthetic polynucleotide 125aaactggttc
ctgaagagat ggtac 2512625DNAArtificial SequenceSynthetic
polynucleotide 126caccgccata gtctaccatc tcttc 2512725DNAArtificial
SequenceSynthetic polynucleotide 127aaacgaagag atggtagact atggc
25
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References