U.S. patent application number 15/023300 was filed with the patent office on 2016-08-25 for treating diseases associated with pgc1-alpha by modulating micrornas mir-130a and mir-130b.
The applicant listed for this patent is ACADEMIA SINICA, Jyun-Yuan HUANG, Chiaho SHIH. Invention is credited to Jyun-Yuan Huang, Chiaho Shih.
Application Number | 20160244755 15/023300 |
Document ID | / |
Family ID | 52689454 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160244755 |
Kind Code |
A1 |
Shih; Chiaho ; et
al. |
August 25, 2016 |
TREATING DISEASES ASSOCIATED WITH PGC1-ALPHA BY MODULATING
MICRORNAS MIR-130A AND MIR-130B
Abstract
Methods of regulating PGC1.alpha. and/or PGC1.beta., or treating
diseases associated with PGC1.alpha. and/or PGC1.beta. using a
miR-130a RNA, miR-130b RNA, or a combination thereof, or using an
antagomiR of miR-130a, an antagomiR of miR-130b, or both, which can
be in the form of microRNA sponge or a locked nucleic acid
(LNA).
Inventors: |
Shih; Chiaho; (Houston,
TX) ; Huang; Jyun-Yuan; (Kaohsiung City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIH; Chiaho
HUANG; Jyun-Yuan
ACADEMIA SINICA; |
Houston
Kaohsiung City
Nankang, Taipei |
TX |
US
TW
TW |
|
|
Family ID: |
52689454 |
Appl. No.: |
15/023300 |
Filed: |
September 19, 2014 |
PCT Filed: |
September 19, 2014 |
PCT NO: |
PCT/US2014/056611 |
371 Date: |
March 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61880508 |
Sep 20, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/12 20180101;
C12N 2310/141 20130101; C12N 2320/30 20130101; A61K 45/06 20130101;
A61P 3/00 20180101; A61P 3/10 20180101; C12N 15/113 20130101; A61K
31/7088 20130101; C12N 2310/113 20130101; C12N 15/1131 20130101;
C12N 2310/531 20130101; C12N 7/00 20130101; C12N 2730/10111
20130101; A61P 31/00 20180101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1-28. (canceled)
29. A method for regulating PGC1.alpha. or PGC1.beta., comprising
contacting cells with an effective amount of: a miR-130a RNA, a
miR-130b RNA, or a combination thereof; or an antagomiR of
miR-130a, an antagomiR of miR-130b, or a combination thereof.
30. The method of claim 29, wherein the cells are contacted with
the miR-130a, the miR-130b, or a combination thereof in an amount
effective to down-regulate PGC1.alpha. or PGC1.beta..
31. The method of claim 30, wherein the miR-130a RNA, the miR-130b
RNA, or both are duplex RNA molecules, single-strand RNA molecules,
or encoded by expression vectors.
32. The method of claim 30, wherein the miR-130a RNA or the
miR-130b RNA has the nucleotide sequence of AGUGCAA.
33. The method of claim 32, wherein the miR-130a RNA has the
nucleotide sequence of CAGUGCAAUGUUAAAAGGGCAU (SEQ ID NO:1).
34. The method of claim 32, wherein the miR-130b RNA has the
nucleotide sequence of CAGUGCAAUGAUGAAAGGGCAU (SEQ ID NO:2).
35. The method of claim 29, wherein the cells are contacted with
the antagomiR of miR-130a, the antagomiR of miR-130b, or a
combination thereof in an amount effective to up-regulate
PGC1.alpha. or PGC1.beta..
36. The method of claim 35, wherein the antagomiR of miR-130a, the
antagomiR of miR-130b, or both are duplex RNA molecules,
single-strand RNA molecules, or encoded by expression vectors.
37. The method of claim 30, wherein the contacting step is
performed by administering to a subject in need thereof an
effective amount of the miR-130a, the miR-130b, or a combination
thereof.
38. The method of claim 37, wherein the subject has or is suspected
of having diabetes or steatosis.
39. The method of claim 37, wherein the miR-130a RNA has the
nucleotide sequence of CAGUGCAAUGUUAAAAGGGCAU (SEQ ID NO:1).
40. The method of claim 37, wherein the miR-130b RNA has the
nucleotide sequence of CAGUGCAAUGAUGAAAGGGCAU (SEQ ID NO:2).
41. The method of claim 37, wherein the miR-130a RNA, the miR-130b
RNA, or both are duplex RNA molecules, single-strand RNA molecules,
or encoded by expression vectors.
42. The method of claim 35, wherein the contacting step is
performed by administering to a subject in need thereof the
antagomiR of miR-130a, the antagomiR of miR-130b, or a combination
thereof.
43. The method of claim 42, wherein the subject has or is suspected
of having a disease associated with reactive oxygen species
(ROS).
44. The method of claim 43, wherein the disease associated with ROS
is selected from the group consisting of muscle dysfunction, heart
failure, and a neurodegenerative disease.
45. The method of claim 44, wherein the neurodegenerative disease
is Parkinson disease, Hungtinton disease, or Alzheimer disease.
46. The method of claim 43, wherein the subject has or is suspected
of having diabetes or inflammation.
47. The method of claim 42, wherein the antagomiR of miR-130a, the
antagomiR of miR-130b, or both are microRNA sponges or locked
nucleic acids.
Description
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 61/880,508,
filed Sep. 20, 2013, which is herein incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0002] Human peroxisome proliferator-activated receptor gamma
coactivator 1-alpha (PGC-1.alpha.) is a protein encoded by the
PPARGC1A gene. PGC-1.alpha. is a transcriptional coactivator that
regulates genes involved in energy metabolism. It also regulates
mitochondrial biogenesis and function. In addition to hepatic
gluconeogenesis (Yoon, et al. Nature 413:131-138; 2001),
PGC1.alpha. is known to be involved in brown adipose adaptive
thermogenesis (Puigserver, et al. Cell 92:829-839; 1998),
mitochondria biogenesis and respiration (Houten, et al. Cell 119:
5-7; 2004), and neurodegenerative diseases (St-Pierre, et al. Cell
127:397-408; 2006).
[0003] MicroRNAs (miRNAs) play an important regulatory role in
differentiation and development (Ambros, Curr Opin Genet Dev
21:511-517, 2011). Recently, microRNAs have emerged as an important
posttranscriptional regulator of metabolism (Rottiers, et al. Nat
Rev Mol Cell Biol 13:239-250, 2012).
SUMMARY OF THE INVENTION
[0004] The present disclosure is based on the unexpected
discoveries that miR-130a down regulates PGC1.alpha., PGC1.beta.,
and PPAR.gamma. by directly targeting the 3'UTR of their mRNAs.
[0005] Accordingly, one aspect of the present disclosure features a
method for regulating PGC1.alpha. or PGC1.beta., the method
comprising contacting cells with an effective amount of: (a) a
miR-130a RNA, a miR-130b RNA, or a combination thereof; or (b) an
antagomiR of the miR-130a RNA, an antagomiR of the miR-130b RNA, or
a combination thereof. In some examples, the cells are contacted
with the miR-130a, the miR-130b, or a combination thereof in an
amount effective to down-regulate PGC1.alpha. or PGC1.beta.. In
other examples, the cells are contacted with an antagomiR of
miR-130a, an antatomiR of miR-130b, or a combination thereof in an
amount effective to up-regulate PGC1.alpha. or PGC1.beta.. An
antagomiR of miR-130a, an antagomiR of miR-130b, or both may be
microRNA sponges or locked nucleic acid (LNA).
[0006] In any of the methods described herein, one or more of the
miR-130a RNA, the miR-130b RNA are duplex RNA molecules or
single-strand RNA molecules. The antagomiR of miR-130a, and
antagomiR of miR-130b can be duplex RNA molecules or in the form of
a microRNA sponge or LNA. In some embodiments, one or more of the
antagomiRs can be a microRNA sponge or an LNA. The miR-130a RNA,
the miR-130b RNA, or both may comprise the nucleotide sequence of
AGUGCAA. For example, the miR-130a RNA may comprise the nucleotide
sequence of CAGUGCAAUGUUAAAAGGGCAU (SEQ ID NO:1).
[0007] In another example, the miR-130b RNA may comprise the
nucleotide sequence of CAGUGCAAUGAUGAAAGGGCAU (SEQ ID NO:2).
[0008] In any of the methods described herein, an effective amount
of the miR-130a RNA, the miR-130b RNA, or their antagomiRs thereof
may be administered to a subject in need thereof for treating a
disease associated with PGC1.alpha. or PGC1.beta.. Optionally, the
disease is not associated with PPAR.gamma..
[0009] In some examples, the subject is a human patient having or
suspected of having diabetes or steatosis. The subject may be
administered with an effective amount of miR-130a, miR-130b, or
both for treating the disease.
[0010] In other examples, the subject is a human patient having or
suspected of having a disease associated with reactive oxygen
species (ROS), such as muscle dysfunction, heart failure, or a
neurodegenerative disease (e.g., Parkinson disease, Hungtinton
disease, or Alzheimer disease). Alternatively, the subject may be a
human patient having or suspected of having diabetes or
inflammation. Such a patient may be administered with an effective
amount of an antagomiR of miR-130a, an antagomiR of miR-130b, or a
combination thereof for treating the disease.
[0011] Also within the scope of the present disclosure are (a)
pharmaceutical compositions for use in treating a disease that is
associated with PGC1.alpha. and/or PGC1.beta., and may not be
associated with PPAR.gamma., the composition comprising a
pharmaceutically acceptable carrier and one or more of a miR-130a
RNA, a miR-130b RNA, or one or more of the antagomiRs of those
miRNAs as described herein; and (b) uses of any of the
pharmaceutical compositions or microRNA molecules for manufacturing
a medicament for treating any of the target diseases, including,
but not limited to, diabetes, steatosis, a disease associated with
reactive oxygen species (ROS), such as muscle dysfunction, heart
failure, or a neurodegenerative disease (e.g., Parkinson disease,
Hungtinton disease, or Alzheimer disease), and inflammation.
[0012] The details of one or more embodiments of the invention are
set forth in the description below. Other features or advantages of
the present invention will be apparent from the following drawings
and detailed description of several embodiments, and also from the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Expression of miR-130a was detected by Northern blot
analysis in HepG2 cell lines stably transfected with miR-130a
expression vector.
[0014] FIG. 2. Concurrent reduction of PGC1.alpha. and PPAR.gamma.
mRNAs in stable miR-130a expressing cell lines was measured by
Northern blot analysis.
[0015] FIG. 3. Western blot analysis detected the reduction of
PGC1.alpha., PPAR.gamma. and adiponectin proteins in stable
miR-130a expressing cells.
[0016] FIG. 4. is a diagram showing the reduction of PGC1.alpha.
and PPAR.gamma. mRNA in stable miR-130a expressing HepG2 cell lines
as measured by RT-qPCR analysis (upper panel). No appreciable
difference in the protein levels of SP1, PPAR.alpha., CEBPb, and
HNF4 by Western blot analysis (lower panel).
[0017] FIG. 5. The reduction of secreted adiponectin was also
detected by ELISA.
[0018] FIG. 6. HepG2 cells were transfected with LNA-miR-130a or a
LNA scramble control. Treatment by LNA-miR-130a increased
PGC1.alpha. mRNA and protein by real time RT-qPCR (upper panel) and
Western blot analysis (lower panel), respectively.
[0019] FIG. 7. Transgenic mice exhibited increased expression of
PGC1.alpha., PEPCK, G6Pase, PPAR.gamma. and adiponectin protein in
the liver (left panel) and adiponectin in sera (right panel).
[0020] FIG. 8. Cotransfection of both PGC1.alpha. and PPAR.gamma.
expression vectors resulted in a highly potent synergistic effect
on adiponectin secretion.
[0021] FIG. 9. is a diagram showing that MiR-130a significantly
reduced the luciferase activity in a reporter cotransfection assay
of the 3' UTR of PGC1.alpha. (NM_013261) and PPAR.gamma.
(NM_005037), but not the 3' UTR of SP1 (NM_003109).
[0022] FIG. 10. MiR-130a was shown to directly target at the 3' UTR
of PGC1.alpha. by compensatory mutagenesis. (*p<0.05). Mutation
sites were underlined in sequence alignment. hsa-miR-130a WT: SEQ
ID NO:1; hsa-PGC1.alpha. 3'UTR WT: SEQ ID NO: 68; hsa-miR-130a seed
sequence mutant: SEQ ID NO: 69; and hsa-PGC1.alpha. 3'UTR target
site mutant: SEQ ID NO:70.
[0023] FIG. 11. is a diagram showing the co-transfection of the
PGC1.alpha. 3'UTR luciferase reporter with increasing amounts of
miR-130a resulted in decreasing reporter activity in a dose
response manner (upper panel). Conversely, treatment with
increasing amounts of LNA-miR-130a plasmid resulted in increasing
reporter activity in a dose response manner (lower panel).
[0024] FIG. 12. This diagram highlights the key enzymes in glucose
metabolism. The mRNA expression of G6Pase, and PEPCK was reduced in
stable miR-130a expressing HepG2 cells by Northern blot
analysis.
[0025] FIG. 13. Reduced protein expression of PEPCK and G6Pase was
also detected by Western blot analysis. We noted an increased level
of glucokinase (GCK) in stable miR-130a expressing HepG2 cells, and
an increased level of pyruvate kinase (PKLR) in stable miR-130a
expressing Huh? cells.
[0026] FIG. 14. The levels of PKLR and GCK specific mRNAs were
increased in stable miR-130a expressing cells by RT-qPCR
analysis.
[0027] FIG. 15. The protein expression of PGC1.alpha. and
gluconeogenic enzymes PEPCK and G6Pase in HBV-producing HepG2 cells
(UP7-4 and UP7-7) was increased by Western blot analysis. However,
no significant change in PPAR.gamma. was noted.
[0028] FIG. 16. Stable expression of PPAR.gamma. in HepG2 cells
reduced glucose output, and conversely, expression of PGC1.alpha.
stimulated glucose production.
[0029] FIG. 17. Both stable HBV replication and a PPAR.gamma.
antagonist, GW9662 (20 uM), can increase glucose production in
HepG2 cells.
[0030] FIG. 18. The expression of miR-130a was not affected in two
stable PGC1.alpha.-expressing cell lines.
[0031] FIG. 19. This diagram summarizes the relationships among
PGC1.alpha., miR-130a and HBV. +/-: neither positive nor negative
effect.
[0032] FIG. 20. upper panel: Reduction of miR-130a was observed in
PPAR.gamma.-expressing HepG2 cell lines using stem-loop qPCR. U6
snRNA was used as an internal control. Rosiglitazone, but not
GW9662, further reduced the expression of miR-130a. lower panel:
Increased amounts of PPAR.gamma. protein in stable
PPAR.gamma.-expressing HepG2 cell lines were detected by Western
blot.
[0033] FIG. 21. This triad cartoon summarizes the relationships
among PPAR.gamma., PGC1.alpha., miR-130a and HBV. A feed-forward
amplification loop among HBV and PGC1.alpha. and PPAR.gamma. can be
mediated through a miR-130a intermediate.
[0034] FIG. 22 is a schematic illustration showing therapeutic
strategies.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present disclosure is based on the unexpected
discoveries that miR-130a regulates PGC1.alpha., PGC1.beta., and
PPAR.gamma. by direct targeting the 3' UTR of their messenger RNAs.
As such, miR-130 microRNAs (e.g., miR-130a and miR-130b), as well
as their antagomiRs can be used to modulate these proteins and thus
be effective in treating diseases associated with one or more of
these proteins (e.g., diseases associated with ROS or diseases
associated with dysfunction of mitochondria). Also, the
simultaneous effect of miR-130a on PGC1.alpha. (gluconeogenesis)
and PPAR.gamma. (inhibition of gluconeogenesis) may contribute to
glucose homeostasis.
[0036] A miR-130a RNA, a miR-130b RNA, or an antagomiR thereof may
be used of modulate the activity of the corresponding microRNA and
thus its target gene expression.
[0037] Modulating a microRNA means any approach that affects the
ultimate biological function of the microRNA in regulating its
target gene expression. In some examples, modulating a microRNA is
to regulate the cellular level of the microRNA. In other examples,
modulating a microRNA is to regulate (e.g., enhance or block) its
interaction with a target of the microRNA (e.g., a mRNA or a
gene).
[0038] Accordingly, described herein are methods for relating
PGC1.alpha., PGC1.beta., or both, or treating a disease associated
with one or more of the proteins using a miR-130a RNA, a miR-130b
RNA, or both, or an antagomiR of miR-130a or miR-130b, as well as
pharmaceutical compositions for use in the treatments described
herein and for use in manufacturing medicaments for those purposes.
miR-130b contains the same AGUGCAA sequence as miR-130a for base
pairing with a target gene.
MicroRNA Molecules
[0039] MicroRNAs are small non-coding RNA molecules (e.g., 22
nucleotides) found in many species, which regulates gene
expression. miR-130a and miR-130b are found in many species, e.g.,
human. The nucleotide sequences of exemplary miR-130a and miR-130b
(precursor and mature) can be found in MiRBase under accession
numbers MI0000448 (human miR-130a) and MI0000748 (human miR-130b).
Exemplary nucleotide sequences of these miRNA molecules are
provided below:
TABLE-US-00001 Human miR-130a: (SEQ ID NO: 3) ugcugcuggc cagagcucuu
uucacauugu gcuacugucu gcaccuguca cuagcagugc aauguuaaaa gggcauuggc
cguguagug Human miR-130b: (SEQ ID NO: 4) ggccugcccg acacucuuuc
ccuguugcac uacuauaggc cgcugggaag cagugcaaug augaaagggc aucggucagg
uc
[0040] A miR-130a RNA as described herein is an oligonucleotide
(e.g., an RNA molecule) that possesses the same bioactivity as a
wild-type miR-130a, such as the human miR-130a, e.g., regulating
the expression of PGC1.alpha., PGC1.beta., and/or PPAR.gamma.. Such
an oligonucleotide can comprise the nucleotide sequence of miR-130a
or a portion thereof (e.g., AGUGCAA or CAGUGCAAUGUUAAAAGGGCAU (SEQ
ID NO:1)). A miR-130a RNA can include up to 150 (e.g., 100, 80, 60,
50, 40, 30, or less) nucleotide residues. In some examples, the
miR-130a can be a duplex RNA molecule. In other examples, it can be
a hairpin molecule, which may include a 21-23 sense sequence (e.g.,
CAGUGCAAUGUUAAAAGGGCAU (SEQ ID NO:1)), a short linker, an antisense
sequence complementary to the sense sequence, and a polyT tail.
[0041] A miR-130b RNA as described herein is an oligonucleotide
such as an RNA molecule that possesses the same bioactivity as a
wild-type miR-130b, such as the human miR-130b. Since miR-130b
share the same sequence as miR-130a for base pairing with target
genes, miR-130b would possess the same biological functions as
miR-130a, e.g., regulating the expression of PGC1.alpha.,
PGC1.beta., and/or PPAR.gamma.. Such an oligonucleotide can
comprise the nucleotide sequence of miR-130b or a portion thereof
(e.g., AGUGCAA or CAGUGCAAUGAUGAAAGGGCAU (SEQ ID NO:2)). A miR-130b
RNA can include up to 150 (e.g., 100, 80, 60, 50, 40, 30, or less)
nucleotide residues. In some examples, the miR-130b can be a duplex
RNA molecule. In other examples, it can be a hairpin molecule,
which may include a 21-23 sense sequence (e.g.,
CAGUGCAAUGAUGAAAGGGCAU (SEQ ID NO:2)), a short linker, an antisense
sequence complementary to the sense sequence, and a polyT tail.
[0042] An antagomiR of miR-130a or miR-130b can be an engineered
oligonucletide capable of suppressing the activity of the target
(e.g., endogenous) miR-130a or miR-130b in a cell via, e.g.,
blocking the binding of the target miRNA to a target mRNA molecule.
An antagomiR can be a small synthetic oligonucleotide that is
completely or partially complementary to the target miRNA or a
portion thereof with either mis-pairing at, e.g., the cleavage site
of Ago2. It may also include some modifications to inhibit Ago
cleavage. In some examples, an antagomiR can include up to 150
(e.g., 100, 80, 60, 50, 40, 30, or less) nucleotide residues. In
some examples, an antagomiR can be a microRNA sponge, which can be
transcripts expressed from strong promoters and containing
multiple, tandem binding sites to a microRNA of interest. When
vectors encoding these sponges are transiently transfected into
cultured cells, sponges would suppress microRNA targets at least as
strongly as chemically modified antisense oligonucleotides. They
specifically inhibit microRNAs with a complementary heptameric
seed, such that a single sponge can be used to block an entire
microRNA seed family. RNA polymerase II promoter (Pol II)-driven
sponges contain a fluorescence reporter gene for identification and
sorting of sponge-treated cells. Stably expression of such microRNA
sponges may be used in disease treatment. Ebert et al., Nat
Methods. 2007 September; 4(9):721-6. Epub 2007 Aug. 12).
[0043] When necessary, the microRNA molecules or their antagomiRs
can include non-naturally-occurring nucleobases, sugars, or
covalent internucleoside linkages (backbones). Such a modified
oligonucleotide confers desirable properties such as enhanced
cellular uptake, improved affinity to the target nucleic acid, and
increased in vivo stability.
[0044] In one example, the oligonucleotide/RNA molecules described
herein has a modified backbone, including those that retain a
phosphorus atom (see, e.g., U.S. Pat. Nos. 3,687,808; 4,469,863;
5,321,131; 5,399,676; and 5,625,050) and those that do not have a
phosphorus atom (see, e.g., U.S. Pat. Nos. 5,034,506; 5,166,315;
and 5,792,608). Examples of phosphorus-containing modified
backbones include, but are not limited to, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates, 5'-alkylene phosphonates and
chiral phosphonates, phosphinates, phosphoramidates including
3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, selenophosphates and boranophosphates
having 3'-5' linkages, or 2'-5' linkages. Such backbones also
include those having inverted polarity, i.e., 3' to 3', 5' to 5' or
2' to 2' linkage. Modified backbones that do not include a
phosphorus atom are formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic
or heterocyclic internucleoside linkages. Such backbones include
those having morpholino linkages (formed in part from the sugar
portion of a nucleoside); siloxane backbones; sulfide, sulfoxide
and sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; riboacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0045] In another example, the microRNA molecules or antagomiR
molecules described herein includes one or more substituted sugar
moieties. Such substituted sugar moieties can include one of the
following groups at their 2' position: OH; F; O-alkyl, S-alkyl,
N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl,
N-alkynyl, and O-alkyl-O-alkyl. In these groups, the alkyl, alkenyl
and alkynyl can be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. They may also
include at their 2' position heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide.
Preferred substituted sugar moieties include those having
2'-methoxyethoxy, 2'-dimethylaminooxyethoxy, and
2'-dimethylaminoethoxyethoxy. See Martin et al., Helv. Chim. Acta,
1995, 78, 486-504.
[0046] In yet another example, the microRNA molecules or antagomiRs
described herein includes one or more modified native nucleobases
(i.e., adenine, guanine, thymine, cytosine and uracil). Modified
nucleobases include those described in U.S. Pat. No. 3,687,808, The
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990,
Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, CRC Press, 1993. Certain of these
nucleobases are particularly useful for increasing the binding
affinity of the microRNA molecules to their targeting sites. These
include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6
and O-6 substituted purines (e.g., 2-aminopropyl-adenine,
5-propynyluracil and 5-propynylcytosine). See Sanghvi, et al.,
eds., Antisense Research and Applications, CRC Press, Boca Raton,
1993, pp. 276-278).
[0047] In some examples, any of the antagomiR molecules are locked
nucleic acids (LNA). LNA is a modified RNA molecule containing at
least one modified RNA nucleotide, in which the ribose moiety is
modified with an extra bridge connecting the 2' oxygen and 4'
carbon so as to lock the ribose in the 3' endo confirmation. See,
e.g., Janssen et al., N Engl J Med. 368(18):1685-94 (2013), Lanford
et al., Science 327(5962):198-201 (2010); Hildebrandt-Eriksen et
al., Nucleic Acid Ther. 22(3):152-61 (2013); and Orom et al., Gene.
372:137-41 (2006). In some embodiments, an LNA as described herein
is a modified DNA phosphorothioate antisense oligonucleotide to
either miR-130a or miR-130b.
[0048] Any of the microRNAs and antagomiRs described herein can be
prepared by conventional methods, e.g., chemical synthesis or in
vitro transcription. Their intended bioactivity as described herein
can be verified by routine methods, e.g., those described in the
Examples below.
Use of miR-130 or AntagomiR Thereof in Modulating PGC1.alpha. or
PGC1.beta.
[0049] The miR-130a RNA, the miR-130b RNA, or an antagomiR of
miR-130a or miR-130b as described herein, either alone or in
combination, can be used in modulating PGC1.alpha. and/or
PGC1.beta. and in treating diseases associated with one or both of
the proteins. For example, miR-130a and/or miR-130b can be used to
down-regulate PGC1.alpha. and/or PGC1.beta., thereby be effective
in treating diseases where down-regulation of PGC1.alpha. and/or
PGC1.beta. is needed. Alternatively, an antagomiR of miR-130a, an
antagomiR of miR-130b, or both can be used to up-regulate
PGC1.alpha. and/or PGC1.beta., thereby be effective in treating
diseases where up-regulation of PGC1.alpha. and/or PGC1.beta. is
needed. PGC1.alpha. can function as a detoxifying enzyme for
reactive oxygen species (ROS), (St Pierre et al., (2006) Cell
127:page 397-408). ROS is responsible for a large number of human
diseases, such as cancer, neurodegenerative disease (Parkinson or
Hungtinton disease), heart failure, muscle dysfunction. Thus, an
antagomiR of miR-130a or miR-130b can be used for treating such
diseases. Examples of such diseases and their association with
PGC1.alpha. are illustrated in FIG. 23.
[0050] The term "treating" as used herein refers to the application
or administration of a composition including one or more active
agents to a subject, who has a disease associated with PGC1.alpha.,
PGC1.beta., or both, a symptom of the disease, or a predisposition
toward the disease, with the purpose to cure, heal, alleviate,
relieve, alter, remedy, ameliorate, improve, or affect the disease,
the symptoms of the disease, or the predisposition toward the
disease.
[0051] To perform the method described herein, an effective amount
of the miR-130a RNA, the miR-130b RNA, or both, or an effective
amount of an antagomiR of miR-130a, an antagomiR of miR-130b, or
both, can be in contact with cells, in which regulation of
PGC1.alpha. and/or PGC1.beta. is needed. In some embodiments, the
method can be performed in vitro, e.g., in cultured cells.
[0052] In other embodiments, an effective amount of one or more
microRNA molecules or antagomiRs as described herein can be
administered to a subject in need of the treatment via a suitable
route. Such a subject can be a human patient having, suspected of
having, or at risk for a disease associated with dysregulation of
PGC1.alpha., PGC1.beta., or both. In some embodiments, the disease
is not associated with PPAR.gamma.. The one or more microRNA
molecules or antagomiRs can be administered to a subject in need of
the treatment directly or indirectly (e.g., using naked
oligonucleotides or using expression vectors adapted for expressing
the microRNA molecules or antagomiRs). Such an expression vector
can be constructed by inserting one or more nucleotide sequences of
the microRNA(s) or one or more antagomiRs into a suitable
expression vector, in which the microRNA sequences are in operable
linkage with a suitable promoter.
[0053] One or more of the miR-130a RNA and miR-130b RNA, or one or
more of the antagomiRs of miR-130a and miR-130b, or one or more
expression vectors suitable for expressing such can be mixed with a
pharmaceutically acceptable carrier to form a pharmaceutical
composition. An "acceptable carrier" is a carrier compatible with
the active ingredient of the composition (and preferably,
stabilizes the active ingredient) and not deleterious to the
subject to be treated. Suitable carriers include, but are not
limited to, (a) salts formed with cations (e.g., sodium, potassium,
ammonium, magnesium, calcium) and polyamines (e.g., spermine and
spermidine); (b) acid addition salts formed with inorganic acids
(e.g., hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, nitric acid); (c) salts formed with organic acids
(e.g., acetic acid, oxalic acid, tartaric acid, succinic acid,
maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,
ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic
acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic
acid, p-toluenesulfonic acid, naphthalenedisulfonic acid,
polygalacturonic acid); and (d) salts formed from elemental anions
(e.g., chlorine, bromine, and iodine). Other suitable carriers
include microcrystalline cellulose, mannitol, glucose, defatted
milk powder, polyvinylpyrrolidone, starch, and a combination
thereof. See, e.g., Remington's Pharmaceutical Sciences, Edition
18, Mack Publishing Co., Easton, Pa. (1995); and Goodman and
Gilman's "The Pharmacological Basis of Therapeutics," Tenth
Edition, Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill
Press, 155-173, 2001.
[0054] To facilitate delivery, the microRNA molecules, the
antagomiRs, or the expression vectors thereof can be conjugated
with a chaperon agent. As used herein, "conjugated" means two
entities are associated, preferably with sufficient affinity that
the therapeutic benefit of the association between the two entities
is realized. Conjugated includes covalent or noncovalent bonding as
well as other forms of association, such as entrapment of one
entity on or within the other, or of either or both entities on or
within a third entity (e.g., a micelle).
[0055] The chaperon agent can be a naturally occurring substance,
such as a protein (e.g., human serum albumin, low-density
lipoprotein, or globulin), carbohydrate (e.g., a dextran, pullulan,
chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or
lipid. It can also be a recombinant or synthetic molecule, such as
a synthetic polymer, e.g., a synthetic polyamino acid. Examples of
polyamino acids include polylysine (PLL), poly L-aspartic acid,
poly L-glutamic acid, styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer
(HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, and polyphosphazine.
[0056] In one example, the chaperon agent is a micelle, liposome,
nanoparticle, or microsphere, in which the microRNA molecules or
expression vectors are encapsulated. Methods for preparing such a
micelle, liposome, nanoparticle, or microsphere are well known in
the art. See, e.g., U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
and 5,527,5285.
[0057] In another example, the chaperon agent serves as a substrate
for attachment of one or more of a fusogenic or condensing
agent.
[0058] A fusogenic agent is responsive to the local pH. For
instance, upon encountering the pH within an endosome, it can cause
a physical change in its immediate environment, e.g., a change in
osmotic properties which disrupts or increases the permeability of
the endosome membrane, thereby facilitating release of the microRNA
described herein into host cell's cytoplasm. A preferred fusogenic
agent changes charge, e.g., becomes protonated at a pH lower than a
physiological range (e.g., at pH 4.5-6.5). Fusogenic agents can be
molecules containing an amino group capable of undergoing a change
of charge (e.g., protonation) when exposed to a specific pH range.
Such fusogenic agents include polymers having polyamino chains
(e.g., polyethyleneimine) and membrane disruptive agents (e.g.,
mellittin). Other examples include polyhistidine, polyimidazole,
polypyridine, polypropyleneimine, and a polyacetal substance (e.g.,
a cationic polyacetal).
[0059] A condensing agent interacts with any of the microRNAs, any
of the antagomiRs, or any of the expression vectors for expressing
such, causing it to condense (e.g., reduce the size of the
oligonucleotide), thus protecting it against degradation.
Preferably, the condensing agent includes a moiety (e.g., a charged
moiety) that interacts with the oligonucleotide via, e.g., ionic
interactions. Examples of condensing agents include polylysine,
spermine, spermidine, polyamine or quarternary salt thereof,
pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer
polyamine, arginine, amidine, protamine, cationic lipid, cationic
porphyrin, and alpha helical peptide.
[0060] In some embodiments, an effective amount of a miR-130a RNA,
a miR-130b RNA, or a combination thereof is administered to a
subject (e.g., a human patient) suffering from, suspected of
having, or at risk for a disease associated with abnormally
up-regulated PGC1.alpha., PGC1.beta., or both. Such diseases
include, but are not limited to, diabetes and steatosis. See FIG.
22. In some examples, the amount of the microRNA or an expression
vector for producing such is effective to down-regulate
PGC1.alpha., PGC1.beta., or both.
[0061] In other embodiments, an effective amount of an antagomiR of
miR-130a or miR-130b, or an expression vector for producing such is
administered to a subject (e.g., a human patient) suffering from,
suspected of having, or at risk for a disease associated with
abnormally down-regulated PGC1.alpha., PGC1.beta., or both. Such
diseases include, but are not limited to a disease associated with
ROS (e.g., a neurodegenerative disease such as Parkinson disease,
Huntington disease, or Alzheimer disease, inflammation, muscle
dysfunction, a cardiovascular disease such as heart failure. See
FIG. 22 In some examples, the amount of the microRNA or an
expression vector for producing such is effective to up-regulate
PGC1.alpha., PGC1.beta., or both.
[0062] "An effective amount" as used herein refers to the amount of
a microRNA molecule, an antagomiR thereof, or an expression vector
for expressing such, that alone, or together with further doses or
one or more other active agents, produces the desired response,
e.g., enhancing or inhibiting PGC1.alpha., PGC1.beta., or both,
and/or alleviating one or more symptoms of a target disease (e.g.,
those described herein). This may involve only slowing the
progression of the disease temporarily, although more preferably,
it involves halting the progression of the disease permanently.
This can be monitored by routine methods, such as physical
examination and suitable lab tests. The desired response to
treatment of any of the target diseases disclosed herein also can
be delaying the onset or even preventing the onset of the
disease.
[0063] Effective amounts vary, as recognized by those skilled in
the art, depending on the particular condition being treated, the
severity of the condition, the individual patient parameters
including age, physical condition, size, gender and weight, the
duration of the treatment, the nature of concurrent therapy (if
any), the specific route of administration and like factors within
the knowledge and expertise of the health practitioner. These
factors are well known to those of ordinary skill in the art and
can be addressed with no more than routine experimentation. It is
generally preferred that a maximum dose of the individual
components or combinations thereof be used, that is, the highest
safe dose according to sound medical judgment. It will be
understood by those of ordinary skill in the art, however, that a
patient may insist upon a lower dose or tolerable dose for medical
reasons, psychological reasons or for virtually any other
reasons.
[0064] The interrelationship of dosages between animals and humans
(e.g., based on milligrams per meter squared of body surface or
milligrams per body weight) is well known in the art. See, e.g.,
Freireich et al., (1966) Cancer Chemother Rep 50: 219. Body surface
area may be approximately determined from height and weight of the
patient.
[0065] A subject in need of any of the above-described treatments
can be a subject (e.g., a human) suffering from, suspected of
having, or at risk for developing any of the target diseases
described herein. Examples include, but are not limited to,
diabetes (such as type I diabetes, type II diabetes, as well as
syndromes associated with diabetes such as retinopathy), a disease
associated with ROS such as muscle dysfunction, heart failure, and
neurodegenerative diseases (e.g., Parkinson's disease, Huntington's
disease). Such a subject can be identified via a routine medical
procedure, including, but are not limited to, physical examination
and pathological analysis. Common symptoms of diabetes include, but
are not limited to, frequent urination, feeling thirty, feeling
hungry, unusual weight loss or weight gain, fatigue, and blurred
vision. Such subjects can be identified via routine medical
procedures. A subject at risk for developing a disease as described
herein, such as a disease associated with ROS possesses one or more
risk factors associated with the disease or disorder.
[0066] Conventional methods, known to those of ordinary skill in
the art of medicine, can be used to administer to a subject in need
of the treatment the pharmaceutical composition described above.
For example, the pharmaceutical composition described above can be
delivered orally or parenterally. Parenteral administration
includes intravenous, intraarterial, subcutaneous, intraperitoneal
or intramuscular injection or infusion; or intracranial
administration (e.g., intrathecal or intraventricular).
[0067] An injectable composition containing an microRNA molecule
described herein, an antagomiR described herein, or an expression
vector thereof may contain various carriers such as vegetable oils,
dimethylactamide, dimethylormamide, ethyl lactate, ethyl carbonate,
isopropyl myristate, ethanol, and polyols (glycerol, propylene
glycol, liquid polyethylene glycol, and the like). For intravenous
injection, the oligonucleotide can be administered by the drip
method, whereby a pharmaceutical formulation containing the
oligonucleotide and a physiologically acceptable excipients is
infused. Physiologically acceptable excipients may include, for
example, 5% dextrose, 0.9% saline, Ringer's solution or other
suitable excipients. Intramuscular preparations, e.g., a sterile
formulation of a suitable soluble salt form of a peptide, can be
dissolved and administered in a pharmaceutical excipient such as
sterile water, 0.9% saline, or 5% glucose solution.
[0068] When oral administration is applied, it is preferred that
the oligonucleotide includes at least one 2'-O-methoxyethyl
modification. A composition for oral administration can be any
orally acceptable dosage form including, but not limited to,
capsules, tablets, emulsions and aqueous suspensions, dispersions
and solutions. In the case of tablets for oral use, carriers which
are commonly used include lactose and corn starch. Lubricating
agents, such as magnesium stearate, are also typically added. For
oral administration in a capsule form, useful diluents include
lactose and dried corn starch. When aqueous suspensions or
emulsions are administered orally, the active ingredient can be
suspended or dissolved in an oily phase combined with emulsifying
or suspending agents. If desired, certain sweetening, flavoring, or
coloring agents can be added. A nasal aerosol or inhalation
composition can be prepared according to techniques well known in
the art of pharmaceutical formulation. The pharmaceutical
composition described herein can also be administered in the form
of suppositories for rectal administration.
Kits
[0069] The present disclosure also provides kits for use in
regulating (e.g., inhibiting or enhancing) PGC1.alpha., PGC1.beta.,
and/or PPAR.gamma., or treating a disease associated with
PGC1.alpha., PGC1.beta., or both as those described herein. Such
kits can include one or more containers comprising one or more of
the microRNA molecules, the antagomiRs, or expression vectors
thereof.
[0070] In some embodiments, the kit can comprise instructions for
use in accordance with any of the methods described herein. The
included instructions can comprise a description of administration
of the microRNA(s), the antagomiRs, or the expression vector(s)
thereof to treat a desired target disease. The kit may further
comprise a description of selecting an individual suitable for
treatment based on identifying whether that individual has the
target disease.
[0071] The instructions relating to the use of an microRNA or an
antagomiR as described herein generally include information as to
dosage, dosing schedule, and route of administration for the
intended treatment. The containers may be unit doses, bulk packages
(e.g., multi-dose packages) or sub-unit doses. Instructions
supplied in the kits of the invention are typically written
instructions on a label or package insert (e.g., a paper sheet
included in the kit), but machine-readable instructions (e.g.,
instructions carried on a magnetic or optical storage disk) are
also acceptable.
[0072] The label or package insert indicates that the composition
is used for inhibiting or enhancing PGC1.alpha., PGC1.beta., or
both, and for treating any of the target diseases described herein
may be provided for practicing any of the methods described
herein.
[0073] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a microRNA molecule or its expression
vector as described herein.
[0074] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container. In some embodiments, the invention provides articles
of manufacture comprising contents of the kits described above.
[0075] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference for the purposes or subject matter referenced herein.
EXAMPLES
Regulation of PGC1-Alpha with MiR-130
Materials and Methods
[0076] Construction of miRNA Plasmids
[0077] The sequences of human miRNAs were retrieved from Ensembl
database and miRbase (Version 16) as noted above. The primer
sequences used in cloning the full length precursor miRNAs are
listed in Table 1 below:
TABLE-US-00002 TABLE 1 DNA sequences of synthetic oligonucleotides
and PCR primers used in this study SEQ Primer name sequences ID NO:
hsa-miR-31-F 5'-CATCTTCAAAAGCGGACACTC-3' 5 hsa-miR-31-R
5'-TCATGGAAATCCACATCCAA-3' 6 hsa-miR-130a-F
5'-GGCAAAAGGAAGAGTGGTGA-3' 7 hsa-miR-130a-R
5'-ACCAGGGTAGCTGACTGGTG-3' 8 HBV ayw nt 1521-2122 F
5'-AGCAGGTCTGGAGCAAACAT-3' 9 HBV ayw nt 1521-2122 R
5'-CACCCACCCAGGTAGCTAGA-3' 10 HBV ayw nt 1521-2122 mt F
5'-GGAGGAGTTGGGAGAGGAAATTAGGTTAAAGG-3' 11 HBV ayw nt 1521-2122 mt R
5'-CCTTTAACCTAATTTCCTCTCCCAACTCCTCC-3' 12 hsa-PGC1a 3' UTR-F
5'-ATATTCTAGAGCTTGTTCAGCGGTTCTTTC-3' 13 hsa-PGcla 3' UTR-R
5'-ATATTCTAGAAGCCATCAAGAAAGGACACA-3' 14 hsa-PGC1a 3'UTR mt-F
5'-GCAGTGTTTCTACTTGCTCAAGCATGGCCTCT-3' 15 hsa-PGc1a 3'UTR mt-R 5
'-AGAGGCCATGCTTGAGCAAGTAGAAACACTGC-3' 16 hsa-miR-130a mt-F
5'-GCACCTGTCACTAGCTGAGCAATGTTAAAAGG-3' 17 hsa-miR-130a mt-R
5'-CCTTTTAACATTGCTCAGCTAGTGACAGGTGC-3' 18 miR-130a sponge-F
5'-GCACTGCTCGAGATGCCCTTTTAACATTGCACTGGAATGCCCTTTT 19
AACATTGCACTGCTCGAGATGCC-3' miR-130a sponge-R
5'-GCATCTCGAGCAGTGCAATGTTAAAAGGGCATGAATTCCAGTGCAA 20
TGTTAAAAGGGCATCTCGAGCAGTGC-3' hsa-SP1 3'UTR-F
5'-ATATCTCGAGAGATGCATTCACAGGGGTTg-3' 21 hsa-SP1 3'UTR-R
5'-ATATCTCGAGGCTCAGAGCAGCTAATGAAG-3' 22 hsa-PPARg 3'UTR-F
5'-ATATCTCGAGCAGAGAGTCCTGAGCCACT-3' 23 hsa-PPARg 3'UTR-R
5'-ATATCTCGAGGGGTGGGAAACACACAAGA-3' 24 Q-PCR hsa-SOD2-F
5'-CCACTGCTGGGGATTGATGT-3' 25 Q-PCR hsa-SOD2-R
5'-GAGCTTAACATACTCAGCATAACG-3' 26 Q-PCR hsa-GPx1-F
5'-GCGGGGCAAGGTACTACTTAT-3' 27 Q-PCR hsa-GPx1-R
5'-CGTTCTTGGCGTTCTCCTGA-3' 28 Q-PCR hsa-CYCS-F
5'-GGAGCGAGTTTGGTTGCACT-3' 29 Q-PCR hsa-CYCS-R
5'-GTGGCACTGGGAACACTTCA-3' 30 Q-PCR hsa-Acly-F
5'-CCTGCCATGCCACAAGATTC-3' 31 Q-PCR hsa-Acly-R
5'-TCTGCATGCCCCACACAAT-3' 32 Q-PCR hsa-ApoE-F
5'-CCTTCCCCAGGAGCCGAC-3' 33 Q-PCR hsa-ApoE-R
5'-GCTCTGTCTCCACCGCTT-3' 34 Q-PCR hsa-GCK-F
5'-TACATGGAGGAGATGCAGAATg-3' 35 Q-PCR hsa-GCK-R
5'-ACTTGCCACCTATGAGCTTCTC-3' 36 Q-PCR hsa-PKLR-F
5'-GAGATCCCAGCAGAGAAGGTTT-3' 37 Q-PCR hsa-PKLR-R
5'-AGTCTCCCCTGACAGCATGA-3' 38 Q-PCR hsa-PPARg-F
5'-CATAAAGTCCTTCCCGCTGA-3' 39 Q-PCR hsa-PPARg-R
5'-TCTGTGATCTCCTGCACAGC-3' 40 Q-PCR hsa-HNF1-F
5'-ACCTCATCATGGCCTCACTT-3' 41 Q-PCR hsa-HNF1-R
5'-GTTGATGACCGGCACACTC-3' 42 Q-PCR hsa-HNF4-F
5'-GAGCTGCAGATCGATGACAA-3' 43 Q-PCR hsa-HNF4-R
5'-TACTGGCGGTCGTTGATGTA-3' 44 Q-PCR hsa-PPARa-F
5'-CCTCTCAGGAAAGGCCAGTA-3' 45 Q-PCR hsa-PPARa-R
5'-CACTTGATCGTTCAGGTCCA-3' 46 Q-PCR hsa-ESRRg-F
5'-GGAGAACAGCCCATACCTGA-3' 47 Q-PCR hsa-ESRRg-R
5'-GCCCATCCAATGATAACCAC-3' 48 Q-PCR hsa-CEBPb-F
5'-GACAAGCACAGCGACGAGTA-3' 49 Q-PCR hsa-CEBPb-R
5'-AGCTGCTCCACCTTCTTCTG-3' 50 Q-PCR hsa-CEBPa-F
5'-CAGACCACCATGCACCTG-3' 51 Q-PCR hsa-CEBPa-R
5'-CTCGTTGCTGTTCTTGTCCA-3' 52 Q-PCR hsa-SP1-F
5'-GCACCTGCCCCTACTGTAAA-3' 53 Q-PCR hsa-SP1-R
5'-GCGTTTCCCACAGTATGACC-3' 54 Q-PCR hsa-ESRRa-F
5'-TGGCTACCCTCTGTGACCTC-3' 55 Q-PCR hsa-ESRRa-R
5'-CCCCTCTTCATCCAGGACTA-3' 56 Q-PCR hsa-LRH-F
5'-ATCCTCGACCACATTTACCG-3' 57 Q-PCR hsa-LRH-R
5'-TGCCACTAACTCCTGTGCAT-3' 58 Q-PCR hsa-PGC1a-F
5'-TATCAGCACGAGAGGCTGAA-3' 59 Q-PCR hsa-PGC1a-R
5'-TCAAAACGGTCCCTCAGTTC-3' 60 Q-PCR hsa-Scd1-F
5'-GCAAACACCCAGCTGTCAAA-3' 61 Q-PCR hsa-Scd1-R
5'-GCACATCATCAGCAAGCCAG-3' 62 Q-PCR hsa-Acsl1-F
5'-GAGTGGGCTGCAGTGACA-3' 63 Q-PCR hsa-Acsl1-R
5'-GCACGTACTGTCGGAAGTCA-3' 64
[0078] The methods to construct the miRNA expression vectors are as
detailed elsewhere. Chen, H. L. et al. PloS one 7, e34116 (2012).
PCR products were sub-cloned from TA cloning vector (RBC) to pSuper
(OligoEngine, Inc) by Hind III digestion. All plasmids were
confirmed by sequencing. Approximately 8-400 fold higher level of
microRNA expression was detected by transfection and stem loop
RT-PCR analysis. The PPAR.gamma. and PGC1.alpha. expression vectors
were from GeneCopoeia.
Source of Antibodies
[0079] Anti-HBc (Dako), anti-PPAR.gamma. (Santa Cruz), anti-GAPDH,
anti-PKLR, anti-G6Pase, anti-tubulin, anti-adiponectin (GeneTex,
Taiwan), anti-PGC1.alpha. (Origene), anti-PCK1 (Abnova), anti-GCK
(Biovision), Secondary antibodies include mouse anti-rabbit-HRP,
goat anti-mouse-HRP (GeneTex, Taiwan) and donkey anti-goat-HRP
(Santa Cruz).
Synthetic RNA
[0080] The synthetic miRNAs (Genepharma) used are RNA duplexes
without modifications. The sense strand is the same as the mature
form of miRNAs. Mimic miR-130a (CAGUGCAAUGUUAAAAGGGCAU (SEQ ID
NO:1)), mimic miR-204 (UUCCCUUUGUCAUCCUAUGCCU (SEQ ID NO:65)),
mimic miR-1236 (CCUCUUCCCCUUGUCUCUCCAG (SEQ ID NO:66)), mimic
negative control (UUCUCCGAACGUGUCACGUTT (SEQ ID NO:67)). The siRNA
oligo against PGC1.alpha. was from Dharmacon.
MiR-130a Sponge
[0081] Each sense and antisense oligos (see Table 1 above) were
designed to contain four copies of synthetic target sites of
miR-130a. Annealed oligo product was gel purified before PCR
amplification. Gel-purified PCR product was subcloned into DsRedC1
vector at Hind III site. Colonies were screened by PCR and the
orientation of the insert and the copy numbers of target sites were
confirmed by sequencing.
Cell Culture
[0082] Human hepatoma Huh7 and HepG2 cells were maintained as
described previously Chua, P. K., et al. Journal of virology 84,
2340-2351 (2010), Le Pogam, S., et al., Journal of virology 79,
1871-1887 (2005). In general, the phenotype of viral replication
and the effect of microRNA are stronger in HepG2 than Huh7 cells.
However, Huh7 cells are easier to passage and transfect. Therefore,
we used these two cell lines interchangeably.
PPAR.gamma. Agonist and Antagonist
[0083] Rosiglitazone and GW9662 were from Sigma. HepG2 and Huh7
cells were seeded in 6-well tissue culture plates at 5.times.10,
Quasdorff, M. et al. Journal of viral hepatitis 17, 527-536 (2010)
cells/well. At 24 h post-transfection, Rosiglitazone or GW9662 in
0.1% DMSO was added to medium. Culture medium was changed ever two
days before harvest.
ELISA
[0084] The concentration of secreted adiponectin was measured by
ELISA (Biovision). ELISA of HBsAg and HBeAg was from General
Biologicals Co., Taiwan.
Measurement of Glucose Production
[0085] The glucose level of HBV transgenic mice was measured using
a kit of DRI-CHEM SLIDE GLU-PIII (FUJIFILM, JAPAN). Ten microliters
of mouse serum was deposited on a FUJI DRI-CHEM SLIDE GLU-PIII.
Glucose oxidase (GOD) catalyzes the oxidization of sample glucose
to generate hydrogen peroxide which then reacts with dye precursors
and forms red dye. The optical reflection density was measured at
505 nm by the FUJI DRI-CHEM analyzer and converted into the glucose
concentration (mg/L). For the measurement of glucose concentration
in cell culture, HBV producing cells (HepG2 and Q7 cells) were
treated with PPAR.gamma. antagonist, GW9662 at indicated
concentrations. Twenty-four hours before glucose measurement, the
medium was replaced with 1 ml of glucose-free DMEM, supplemented
with 2 mM sodium pyruvate. After 16 hrs incubation, 50 .mu.l of
medium was collected and the glucose concentration (mM) was
measured using the glucose colorimetric assay (Biovision).
Quantitative Real-Time PCR
[0086] Briefly, 2 .mu.g of total RNA was reverse transcribed into
cDNA using random primers and High Capacity cDNA Reverse
Transcription kit (Applied Biosystem) at 37.degree. C. for 120
minutes. The cDNA product was then diluted 100 times for real-time
PCR analysis using Power SYBR Green PCR master mix (Applied
Biosystem), and the default condition in a 20 .mu.l reaction volume
by Applied Biosystems 7500 Real-Time PCR System. Data were analyzed
by relative quantification methods (AACt methods) using 7500
software V2.0.1.
Stem-Loop qCR for miRNA
[0087] Taqman RT and stem-loop real-time assay were from Applied
Biosystems: miR-31 (assayID: 002279), miR-130a (assayID: 000454),
miR-204 (assayID: 000508) and miR-1236 (assayID: 002752). Briefly,
100 ng RNAs were reverse transcribed by specific stem-loop primer
and further analyzed by Taqman real-time PCR assay using default
setting. U6 snRNA (assayID: 001973) was used as an internal loading
control. Data were analyzed by Applied Biosystems 7500 software
V2.0.1.
Southern and Northern Blot
[0088] HBV core particle-associated DNA, total cellular cytoplasmic
RNA, and microRNA were analyzed by Northern blot as described
previously. Chua, P. K., et al. Journal of virology 84, 2340-2351
(2010), Le Pogam, S., et al., Journal of virology 79, 1871-1887
(2005).
Luciferase Reporter Assay
[0089] Assay for 3' UTR or enhancer/promoter was as described
previously. Chen, H. L., et al., PloS one 7, e34116 (2012).
Native Agarose Gel Electrophoresis and Western Blot
[0090] Native agarose gel electrophoresis and Western blot for
detecting HBV core particles were as described. Chua, P. K., et al.
Journal of virology 84, 2340-2351 (2010),
Stable miR-130a Expressing Cell Lines
[0091] Approximately one million Huh7 and HepG2 cells were
transiently transfected by 3 .mu.g plasmid DNA (pSuper and
pSuper-miR-130a) with Polyjet (SignaGen), followed by G418
selection for three weeks. The G418-resistant colonies were pooled
together.
LNA-miR-130a Knockdown
[0092] HepG2 and Huh7 cells were cotransfected with puromycin
resistamt plasmid (pTRE2pur) and LNA-scramble control or LNA
anti-miR-130a (Locked Nucleic Acid, Exiqon), using Lipofectamine
2000 (Invitrogen). Twelve hours post-transfection, transfected
culture was treated with puromycin (2 .mu.g/ml) for 2 days,
followed by reduced concentration of puromycin (0.5 .mu.g/ml) for
another 2 days before harvesting for Western blot analysis. Chen,
H. L., et al., PloS one 7, e34116 (2012).
Bioinformatic Analysis.
[0093] Computer-based programs including Targetscan
(http://www.targetscan.org/), Pictar
(http://pictar.mdc-berlin.de/), Microinspector
(http://bioinfo.uni-plovdiv.bg/microinspector/), RNAhybrid
(http://www.bibiserv.techfak.uni-bielefeld.de/) and DIANA
(http://diana.cslab.ece.ntua.gr) were used to predict potential
targets for miR-1236, miR-130a and miR-204. The minimal free energy
of binding less than -20 kcal/mol was used as the cut-off
value.
MicroRNA Taqman Low Density Array Analysis
[0094] The total RNA of HBV-producing cells were extracted by
Trizol (Invitrogen). The quality and quantity of RNA samples were
determined by Agilent 2100 Bioanalyzer using RNA 6000 Nano Kit
(Agilent Technologies, Inc.). The reverse transcription reactions
were performed using TaqMan MicroRNA Reverse Transcription kit
(Applied Biosystem). The expression of miRNA was detected by
TaqMan.RTM. Rodent MicroRNA Array A (Applied Biosystems), and
analyzed by Applied Biosystems 7900 HT Fast Real-Time PCR System
containing 381 rodent miRNA targets.
Statistics
[0095] Statistical significance was determined using the Student's
t test. In all figures, values were expressed as mean.+-.standard
deviation (SD) and statistical significance (p<0.05) was
indicated by an asterisk. The data represent results from at least
three independent experiments.
Results
MiR-130a Directly Targets at Both PGC1.alpha. and PPAR.gamma.
[0096] Four different target prediction algorithms were used to
identify potential target transcription factors of miR-130a in
hepatocytes. PPARGC1-.alpha. (PGC1.alpha.) was identified as such a
factor whose 3'UTR was consistently predicted by all four programs
(Table 2).
TABLE-US-00003 TABLE 2 Prediction of miR-130a target sites at the
3'UTR of human transcription factors known to influence HBV
transcription Transcription Target factors .English Pound. scan
PicTar DIANA RNAhybrid HNF1 + - - - HNF4.alpha. + - - -
C/EBP.alpha. - - - - C/EBP.beta. - - - - SP1 + - + - RXR.alpha. + -
+ - PPAR.alpha. + - - - PPAR.gamma. + - - + FoxA3 - - - -
(HNF3.gamma.) FoxO1 - - - - FXR .alpha. - - - - PGC1.alpha. + + + +
ERR.alpha. - - - - COUP-TF - - - - LRH - - - - .English Pound.
These transcription factors had been reported to be involved in HBV
RNA synthesis in literatures 5, 6, 38. MiR-130a was shown to target
at the 3' UTR of PPAR .gamma. in adipocytes. Lee, E. K. et al. Mol
Cell Biol 31, 626-638 (2011).
[0097] To address the potential relationship between miR-130a and
PGC1.alpha., a stable cell line expressing miR-130a was established
(FIG. 1). While the expression levels of most hepatic transcription
factors being examined here remained unchanged in miR-130a
expressing cell lines, simultaneous reductions of PPAR.gamma. and
PGC1.alpha. mRNAs and proteins were observed (FIGS. 2, 3 and 4),
suggesting that miR-130a can target both PPAR.gamma. and
PGC1.alpha.. PPAR.gamma. is known to stimulate adiponectin
production. Yu et al. The Journal of biological chemistry 278,
498-505 (2003). As expected, secreted adiponectin was significantly
reduced in miR-130a expressing cell lines (FIG. 5). In a reciprocal
experiment, HepG2 cells were treated with LNA-miR-130a antagomir,
and significant increase of PGC1.alpha. mRNA and protein was
observed (FIG. 6). In accordance with this finding miR-130a was
significantly reduced while PGC1.alpha., G6Pase, PEPCK, PPAR.gamma.
and serum adiponectin were all significantly increased the
transgenic mice described in (Chen, C. C. et al. Gene therapy 14,
11-19 (2007)) (FIG. 7). Therefore, miR-130a may reduce PPAR.gamma.
and PGC1.alpha. protein levels (FIG. 3) by reducing their
respective mRNA levels (FIGS. 2, 6). Interestingly, the combination
of both PPAR.gamma. and PGC1.alpha., in the absence of any
exogenous ligands exhibited a dramatic synergistic effect on
secretions of adiponectin (FIG. 8).
[0098] To distinguish between a direct and an indirect mechanism of
miR-130a on reducing PGC1.alpha. mRNA, a reporter assay was
performed using 3'UTR from either PPAR.gamma. or PGC1.alpha.. The
results support a functional interaction between miR-130a and the
3' UTR of PPAR.gamma. or PGC1.alpha. (FIG. 9). Next, compensatory
mutations were introduced into the seed sequences of miR-130a and
its evolutionarily conserved target site of PGC1.alpha. (FIG. 10).
By cotransfection assay, only the combination of a mutant miR-130a
and a mutant PGC1.alpha. could successfully restore the inhibitory
effect of miR-130a on the luciferase activity. In a dose-dependent
manner, miR-130a could reduce, while LNA-miR-130a could increase,
the luciferase activity of a reporter containing the 3'UTR of
PGC1.alpha. (FIG. 11). Taken together, miR-130a can directly target
at both PGC1.alpha. and PPAR.gamma..
MiR-130a in Hepatic Gluconeogenesis and Lipogenesis
[0099] The dual targets of PGC1.alpha. and PPAR.gamma. by miR-130a
strongly suggest its important role in energy metabolism. Several
key metabolic enzymes in glycolysis, gluconeogenesis, and
lipogenesis were examined using stable miR-130a expressing cells
(FIG. 12, upper panel). Both phosphoenolpyruvate carboxykinase
(PEPCK) and glucose-6-phosphatase (G6Pase) are rate-limiting
gluconeogenic enzymes known to be under the positive control of
PGC1.alpha., Yoon, J. C. et al. Nature 413, 131-138 (2001). Since
PGC1.alpha. mRNA and proteins were reduced in miR-130a expressing
cells (FIGS. 2 and 3), it was anticipated that PEPCK and G6Pase
should be reduced as well. Indeed, concurrent reductions of PEPCK
and G6Pase mRNAs (FIG. 12) and proteins (FIG. 13) were observed in
miR-130a expressing hepatocytes. While this result suggested a
glycolysis rather than a gluconeogenesis pathway, it would be more
certain if their glycolytic counterparts, pyruvate kinase (PKLR)
and glucokinase (GCK), was not reduced simultaneously (FIG. 14).
Indeed, the protein expression of GCK and PLKR was not reduced but
increased in HepG2 or Huh? cells (FIG. 13). Furthermore, increased
mRNA expression of both GCK and PLKR was observed in miR-130a
expressing hepatocytes (FIG. 14). These results suggest that
miR-130a can not only inhibit HBV replication, but also contribute
to downregulation of gluconeogenesis and upregulation of glycolysis
in hepatocytes via PGC1.alpha.. Thus, miR-130a would be effective
in diseases and disorders associated with dysregulation of
glycolysis, such as metabolic diseases.
A Metabolic Triad
[0100] HBV may exert an effect on PGC1.alpha. and PPAR.gamma..
Consistent with the reduction of miR-130a, PGC1.alpha., PEPCK,
G6Pase, PPAR.gamma. and secreted adiponectin proteins were all
significantly increased in HBV-producing UP7-4 and UP7-7 cells
(FIG. 15). Similarly, glucose production was significantly
increased in stable PGC1.alpha.-producing cells, and decreased in
PPAR.gamma.producing cells (FIG. 16). No significant difference in
glucose level was detected between control and miR-130a producing
cells. As expected, the glucose level was increased in
HBV-producing HepG2 cells, and further increased when HepG2 cells
were treated with a PPAR.gamma. antagonist, GW9662, irrespective of
its HBV-producing status (FIG. 17). Finally, there was no apparent
change in miR-130a expression in stable PGC1.alpha.-expressing cell
lines (FIG. 18).
[0101] A triad relationship among HBV, miR-130a, and PGC1.alpha. is
outlined in FIG. 19. In contrast to PGC1.alpha., expression of
miR-130a was reduced in stable PPAR.gamma.-expressing cell line,
and further reduced by Rosiglitazone treatment, but not by GW9662
(FIG. 20). These results suggest another triad diagram with a
positive feed-forward loop (FIG. 21). A primary weak signal
received by this loop may be amplified into a stronger phenotypic
outcome.
DISCUSSION
[0102] The most salient feature of miR-130a is its dual targets at
PPAR.gamma. and PGC1.alpha. (FIGS. 2, 3 and 5, 9, 10), leading to
reduced HBV replication. Conversely, HBV can reduce the expression
of miR-130a (Table 2,), leading to increased expression of
PPAR.gamma. (adiponectin) and PGC1.alpha. (PEPCK and G6Pase) (FIGS.
12, 13, and 15). Overexpression of PPAR.gamma. reduced the level of
miR-130a, and further reduction of miR-130a was observed by
Rosiglitazone (FIG. 20) or the combination of PPAR.gamma. and
PGC1.alpha.. Taken together, a positive feed-forward loop among
HBV, miR-130a, PGC1.alpha. and PPAR.gamma., was established (FIG.
21). This triad loop could in theory magnify a weak primary signal
by going through this loop repetitive rounds.
OTHER EMBODIMENTS
[0103] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0104] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
Sequence CWU 1
1
70122RNAArtificial SequenceSynthetic Polynucleotide 1cagugcaaug
uuaaaagggc au 22222RNAArtificial SequenceSynthetic Polynucleotide
2cagugcaaug augaaagggc au 22389RNAHomo sapiens 3ugcugcuggc
cagagcucuu uucacauugu gcuacugucu gcaccuguca cuagcagugc 60aauguuaaaa
gggcauuggc cguguagug 89482RNAHomo sapiens 4ggccugcccg acacucuuuc
ccuguugcac uacuauaggc cgcugggaag cagugcaaug 60augaaagggc aucggucagg
uc 82521DNAArtificial SequenceSynthetic Polynucleotide 5catcttcaaa
agcggacact c 21620DNAArtificial SequenceSynthetic Polynucleotide
6tcatggaaat ccacatccaa 20720DNAArtificial SequenceSynthetic
Polynucleotide 7ggcaaaagga agagtggtga 20820DNAArtificial
SequenceSynthetic Polynucleotide 8accagggtag ctgactggtg
20920DNAArtificial SequenceSynthetic Polynucleotide 9agcaggtctg
gagcaaacat 201020DNAArtificial SequenceSynthetic Polynucleotide
10cacccaccca ggtagctaga 201132DNAArtificial SequenceSynthetic
Polynucleotide 11ggaggagttg ggagaggaaa ttaggttaaa gg
321232DNAArtificial SequenceSynthetic Polynucleotide 12cctttaacct
aatttcctct cccaactcct cc 321330DNAArtificial SequenceSynthetic
Polynucleotide 13atattctaga gcttgttcag cggttctttc
301430DNAArtificial SequenceSynthetic Polynucleotide 14atattctaga
agccatcaag aaaggacaca 301532DNAArtificial SequenceSynthetic
Polynucleotide 15gcagtgtttc tacttgctca agcatggcct ct
321632DNAArtificial SequenceSynthetic Polynucleotide 16agaggccatg
cttgagcaag tagaaacact gc 321732DNAArtificial SequenceSynthetic
Polynucleotide 17gcacctgtca ctagctgagc aatgttaaaa gg
321832DNAArtificial SequenceSynthetic Polynucleotide 18ccttttaaca
ttgctcagct agtgacaggt gc 321973DNAArtificial SequenceSynthetic
Polynucleotide 19gcactgctcg agatgccctt ttaacattgc actggaattc
atgccctttt aacattgcac 60tgctcgagat gcc 732072DNAArtificial
SequenceSynthetic Polynucleotide 20gcatctcgag cagtgcaatg ttaaaagggc
atgaattcca gtgcaatgtt aaaagggcat 60ctcgagcagt gc
722130DNAArtificial SequenceSynthetic Polynucleotide 21atatctcgag
agatgcattc acaggggttg 302230DNAArtificial SequenceSynthetic
Polynucleotide 22atatctcgag gctcagagca gctaatgaag
302329DNAArtificial SequenceSynthetic Polynucleotide 23atatctcgag
cagagagtcc tgagccact 292429DNAArtificial SequenceSynthetic
Polynucleotide 24atatctcgag gggtgggaaa cacacaaga
292520DNAArtificial SequenceSynthetic Polynucleotide 25ccactgctgg
ggattgatgt 202624DNAArtificial SequenceSynthetic Polynucleotide
26gagcttaaca tactcagcat aacg 242721DNAArtificial SequenceSynthetic
Polynucleotide 27gcggggcaag gtactactta t 212820DNAArtificial
SequenceSynthetic Polynucleotide 28cgttcttggc gttctcctga
202920DNAArtificial SequenceSynthetic Polynucleotide 29ggagcgagtt
tggttgcact 203020DNAArtificial SequenceSynthetic Polynucleotide
30gtggcactgg gaacacttca 203120DNAArtificial SequenceSynthetic
Polynucleotide 31cctgccatgc cacaagattc 203219DNAArtificial
SequenceSynthetic Polynucleotide 32tctgcatgcc ccacacaat
193318DNAArtificial SequenceSynthetic Polynucleotide 33ccttccccag
gagccgac 183418DNAArtificial SequenceSynthetic Polynucleotide
34gctctgtctc caccgctt 183522DNAArtificial SequenceSynthetic
Polynucleotide 35tacatggagg agatgcagaa tg 223622DNAArtificial
SequenceSynthetic Polynucleotide 36acttgccacc tatgagcttc tc
223722DNAArtificial SequenceSynthetic Polynucleotide 37gagatcccag
cagagaaggt tt 223820DNAArtificial SequenceSynthetic Polynucleotide
38agtctcccct gacagcatga 203920DNAArtificial SequenceSynthetic
Polynucleotide 39cataaagtcc ttcccgctga 204020DNAArtificial
SequenceSynthetic Polynucleotide 40tctgtgatct cctgcacagc
204120DNAArtificial SequenceSynthetic Polynucleotide 41acctcatcat
ggcctcactt 204219DNAArtificial SequenceSynthetic Polynucleotide
42gttgatgacc ggcacactc 194320DNAArtificial SequenceSynthetic
Polynucleotide 43gagctgcaga tcgatgacaa 204420DNAArtificial
SequenceSynthetic Polynucleotide 44tactggcggt cgttgatgta
204520DNAArtificial SequenceSynthetic Polynucleotide 45cctctcagga
aaggccagta 204620DNAArtificial SequenceSynthetic Polynucleotide
46cacttgatcg ttcaggtcca 204720DNAArtificial SequenceSynthetic
Polynucleotide 47ggagaacagc ccatacctga 204820DNAArtificial
SequenceSynthetic Polynucleotide 48gcccatccaa tgataaccac
204920DNAArtificial SequenceSynthetic Polynucleotide 49gacaagcaca
gcgacgagta 205020DNAArtificial SequenceSynthetic Polynucleotide
50agctgctcca ccttcttctg 205118DNAArtificial SequenceSynthetic
Polynucleotide 51cagaccacca tgcacctg 185220DNAArtificial
SequenceSynthetic Polynucleotide 52ctcgttgctg ttcttgtcca
205320DNAArtificial SequenceSynthetic Polynucleotide 53gcacctgccc
ctactgtaaa 205420DNAArtificial SequenceSynthetic Polynucleotide
54gcgtttccca cagtatgacc 205520DNAArtificial SequenceSynthetic
Polynucleotide 55tggctaccct ctgtgacctc 205620DNAArtificial
SequenceSynthetic Polynucleotide 56cccctcttca tccaggacta
205720DNAArtificial SequenceSynthetic Polynucleotide 57atcctcgacc
acatttaccg 205820DNAArtificial SequenceSynthetic Polynucleotide
58tgccactaac tcctgtgcat 205920DNAArtificial SequenceSynthetic
Polynucleotide 59tatcagcacg agaggctgaa 206020DNAArtificial
SequenceSynthetic Polynucleotide 60tcaaaacggt ccctcagttc
206120DNAArtificial SequenceSynthetic Polynucleotide 61gcaaacaccc
agctgtcaaa 206220DNAArtificial SequenceSynthetic Polynucleotide
62gcacatcatc agcaagccag 206318DNAArtificial SequenceSynthetic
Polynucleotide 63gagtgggctg cagtgaca 186420DNAArtificial
SequenceSynthetic Polynucleotide 64gcacgtactg tcggaagtca
206522RNAArtificial SequenceSynthetic Polynucleotide 65uucccuuugu
cauccuaugc cu 226622RNAArtificial SequenceSynthetic Polynucleotide
66ccucuucccc uugucucucc ag 226721DNAArtificial SequenceSynthetic
Polynucleotide 67uucuccgaac gugucacgut t 216823RNAHomo sapiens
68aagcaguguu ucuacuugca cua 236922RNAArtificial SequenceSynthetic
Polynucleotide 69cugagcaaug uuaaaagggc au 227023RNAArtificial
SequenceSynthetic Polynucleotide 70aagcaguguu ucuacuugcu caa 23
* * * * *
References