U.S. patent application number 15/035950 was filed with the patent office on 2016-09-08 for systemic delivery of myostatin short interfering nucleic acids (sina) conjugated to a lipophilic moiety.
This patent application is currently assigned to SIRNA THERAPEUTICS, INC.. The applicant listed for this patent is Duncan BROWN, Vasant JADHAV, Laura SEPP-LORENZINO, SIRNA THERAPEUTICS, INC.. Invention is credited to Duncan Brown, Vasant Jadhav, Tayeba Khan, Laura Sepp-Lorenzino, Walter Richard Strapps, Marija Tadin-Strapps.
Application Number | 20160256570 15/035950 |
Document ID | / |
Family ID | 51999542 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160256570 |
Kind Code |
A1 |
Tadin-Strapps; Marija ; et
al. |
September 8, 2016 |
SYSTEMIC DELIVERY OF MYOSTATIN SHORT INTERFERING NUCLEIC ACIDS
(siNA) CONJUGATED TO A LIPOPHILIC MOIETY
Abstract
The present invention provides methods comprising the in vivo
delivery of small nucleic acid molecules capable of mediating RNA
interference and reducing the expression of myostatin, wherein the
small nucleic acid molecules are introduced to a subject by
systemic administration. Specifically, the invention relates to
methods comprising the in vivo delivery of short interfering
nucleic acid (siNA) molecules that target a myostatin gene
expressed by a subject, wherein the siNA molecule is conjugated to
a lipophilic moiety, such as cholesterol. The myostatin siNA
conjugates that are delivered as per the methods disclosed are
useful to modulate the in vivo expression of myostatin, increase
muscle mass and/or enhance muscle performance. Use of the disclosed
methods is further indicated for treating musculoskeletal diseases
or disorders and/or diseases or disorders that result in conditions
in which muscle is adversely affected.
Inventors: |
Tadin-Strapps; Marija;
(Boston, MA) ; Khan; Tayeba; (Lexington, MA)
; Strapps; Walter Richard; (Dedham, MA) ;
Sepp-Lorenzino; Laura; (Cambridge, MA) ; Jadhav;
Vasant; (Cambridge, MA) ; Brown; Duncan;
(Berkeley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEPP-LORENZINO; Laura
JADHAV; Vasant
BROWN; Duncan
SIRNA THERAPEUTICS, INC. |
Cambridge
Cambridge
Berkeley
Cambridge |
MA
MA
CA
MA |
US
US
US
US |
|
|
Assignee: |
SIRNA THERAPEUTICS, INC.
Cambridge
MA
|
Family ID: |
51999542 |
Appl. No.: |
15/035950 |
Filed: |
November 10, 2014 |
PCT Filed: |
November 10, 2014 |
PCT NO: |
PCT/US2014/064837 |
371 Date: |
May 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61902358 |
Nov 11, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 21/00 20180101;
C12N 15/1136 20130101; C12N 2310/14 20130101; A61K 48/0058
20130101; A61K 47/543 20170801; A61K 31/713 20130101; C12N
2310/3515 20130101; A61K 47/554 20170801; A61P 21/06 20180101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/113 20060101 C12N015/113; A61K 47/48 20060101
A61K047/48 |
Claims
1. A method of modulating in vivo expression of a myostatin gene in
a subject comprising introducing to said subject by systemic
administration an effective amount of a myostatin siNA conjugate,
or a pharmaceutical composition comprising said siNA conjugate,
wherein the siNA conjugate comprises an siNA molecule that targets
a myostatin gene expressed by said subject linked to a lipophilic
moiety, and wherein the siNA conjugate mediates RNA
interference.
2. A method according to claim 1, wherein the lipophilic moiety is
cholesterol.
3. A method according to claim 1, wherein the lipophilic moiety is
attached to a 3'-end of the siNA molecule.
4. A method according to claim 1, wherein the siNA molecule
comprises one or more chemically-modified nucleotides.
5. A method according to claim 1, wherein the siNA molecule is a
double-stranded molecule comprising an antisense strand and a sense
strand, wherein said antisense strand is complementary to said
sense strand.
6. A method according to claim 5, wherein the antisense strand and
the sense strand are each independently 15 to 30 nucleotides in
length.
7. A method according to claim 5, wherein the siNA molecule
comprises one or more 3'-overhanging nucleotides on one or both
strands.
8. A method according to claim 5, wherein the lipophilic moiety is
attached to either the 3'-end of the sense strand of the siNA
molecule, the 5'-end of the sense strand of the siNA molecule, or
the 3'-end of the antisense strand of the siNA molecule.
9. A method according to claim 1, wherein the siNA molecule
comprises a cap on a 3'-end of the molecule.
10. A method according to claim 1, wherein the subject is a
human.
11. A method according to claim 1, wherein the subject is
livestock.
12. A method of enhancing muscle mass in a subject comprising
reducing myostatin levels in said subject by introducing to said
subject by systemic administration an effective amount of a
myostatin siNA conjugate, or a pharmaceutical composition
comprising said siNA conjugate, wherein the siNA conjugate
comprises an siNA molecule that targets a myostatin gene expressed
by said subject linked to a lipophilic moiety, and wherein the siNA
conjugate mediates RNA interference.
13. A method of enhancing muscle performance mass in a subject
comprising reducing myostatin levels in said subject by introducing
to said subject by systemic administration an effective amount of a
myostatin siNA conjugate, or a pharmaceutical composition
comprising said siNA conjugate, wherein the siNA conjugate
comprises an siNA molecule that targets a myostatin gene expressed
by said subject linked to a lipophilic moiety, and wherein the siNA
conjugate mediates RNA interference.
14. A method of treating a musculoskeletal disease or disorder, or
a disease or disorder that results in conditions in which muscle is
adversely affected, in a subject comprising reducing myostatin
levels in said subject by introducing to said subject by systemic
administration an effective amount of a myostatin siNA conjugate,
or a pharmaceutical composition comprising said siNA conjugate,
wherein the siNA conjugate comprises an siNA molecule that targets
a myostatin gene expressed by said subject linked to a lipophilic
moiety, and wherein the siNA conjugate mediates RNA
interference.
15-18. (canceled)
19. A double-stranded short interfering nucleic acid (siNA)
molecule that inhibits the expression of myostatin, wherein: (a)
the siNA comprises a sense strand and an antisense strand; (b) each
strand is independently 15 to 30 nucleotides in length; and, (c)
the antisense strand comprises at least 15 nucleotides having
sequence complementary to any of: TABLE-US-00015 (SEQ ID NO: 1) 5'-
AUGGCAAAGAACAAAUAAU -3'; (SEQ ID NO: 2) 5'- GGCAAAGAACAAAUAAUAU
-3'; (SEQ ID NO: 3) 5'- ACUCCAGAAUAGAAGCCAU -3'; or (SEQ ID NO: 4)
5'- UUUGGAAGAUGACGAUUAU -3'.
20. A double-stranded short interfering nucleic acid (siNA)
molecule that inhibits the expression of myostatin, wherein: (a)
the siNA comprises a sense strand and an antisense strand; (b) each
strand is independently 15 to 30 nucleotides in length; and (c) the
antisense strand comprises at least a 15 nucleotide sequence of:
TABLE-US-00016 (SEQ ID NO: 18) 5'- AUUAUUUGUUCUUUGCCAU -3'; (SEQ ID
NO: 19) 5'- AUAUUAUUUGUUCUUUGCC -3'; (SEQ ID NO: 20) 5'-
AUGGCUUCUAUUCUGGAGU -3'; or (SEQ ID NO: 21) 5'- AUAAUCGUCAUCUUCCAAA
-3';
and wherein one or more of the nucleotides are optionally
chemically modified.
21. A double-stranded siNA molecule of claim 20, wherein the siNA
molecule comprises any of: TABLE-US-00017 (SEQ ID NO: 1) 5'-
AUGGCAAAGAACAAAUAAU -3' and (SEQ ID NO: 18) 5'- AUUAUUUGUUCUUUGCCAU
-3'; (SEQ ID NO: 2) 5'- GGCAAAGAACAAAUAAUAU -3' and (SEQ ID NO: 19)
5'- AUAUUAUUUGUUCUUUGCC -3'; (SEQ ID NO: 3) 5'- ACUCCAGAAUAGAAGCCAU
-3' and (SEQ ID NO: 20) 5'- AUGGCUUCUAUUCUGGAGU -3'; or (SEQ IN NO:
4) 5'- UUUGGAAGAUGACGAUUAU -3' and (SEQ ID NO: 21) 5'-
AUAAUCGUCAUCUUCCAAA -3'.
22. The double-stranded siNA molecule of claim 19, wherein the siNA
molecule is linked to a lipophilic moiety.
23. The double-stranded siNA molecule of claim 22, wherein the
lipophilic moiety is cholesterol.
24. The double-stranded siNA molecule of claim 23, wherein the
lipophilic moiety is attached to a 3'-end of the siNA molecule.
25. The double-stranded siNA molecule of claim 23, wherein the
lipophilic moiety is attached to a 5'-end of the siNA molecule.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/902,358, filed Nov. 11, 2013, the entire
contents of which are incorporated herein by reference.
REFERENCE TO SEQUENCE LISTING
[0002] A sequence listing text file is submitted via EFS-Web in
compliance with 37 CFR .sctn.1.52(e)(5) concurrently with the
specification. The sequence listing has the file name "A2038-7219WO
Sequence Listing", was created on Nov. 10, 2014, and is 24,773
bytes in size. The sequence listing is part of the specification
and is incorporated in its entirety by reference herein.
BACKGROUND OF THE INVENTION
[0003] RNA interference (RNAi) is an evolutionarily conserved
cellular mechanism of post-transcriptional gene silencing found in
fungi, plants and animals that uses small RNA molecules to inhibit
gene expression in a sequence-specific manner. RNAi is controlled
by the RNA-induced silencing complex (RISC) that is initiated by
short double-stranded RNA molecules in a cell's cytoplasm. The
short double-stranded RNA interacts with Argonaute 2 (Ago2), the
catalytic component of RISC, which cleaves target mRNA that is
complementary to the bound RNA. One of the two RNA strands, known
as the guide strand, binds the Ago2 protein and directs gene
silencing, while the other strand, known as the passenger strand,
is degraded during RISC activation. See, for example, Zamore and
Haley, 2005, Science, 309:1519-1524; Vaughn and Martienssen, 2005,
Science, 309:1525-1526; Zamore et al., 2000, Cell, 101:25-33; Bass,
2001, Nature, 411:428-429; and, Elbashir et al., 2001, Nature,
411:494-498. Single-stranded short interfering RNA has also been
shown to bind Ago2 and support cleavage activity (see, e.g., Lima
et al., 2012, Cell 150:883-894).
[0004] The RNAi machinery can be harnessed to destroy any mRNA of a
known sequence. This allows for suppression (knockdown) of any gene
from which it was generated, consequently preventing the synthesis
of the target protein. Modulation of gene expression through an
RNAi mechanism can be used to modulate therapeutically relevant
biochemical pathways, including ones which are not accessible
through traditional small molecule control.
[0005] Chemical modification of nucleotides incorporated into RNAi
molecules leads to improved physical and biological properties,
such as nuclease stability (see, e.g., Damha et al., 2008, Drug
Discovery Today, 13:842-855), reduced immune stimulation (see,
e.g., Sioud, 2006, TRENDS in Molecular Medicine, 12:167-176),
enhanced binding (see, e.g., Koller, E. et al., 2006, Nucleic Acid
Research, 34:4467-4476), and enhanced lipophilic character to
improve cellular uptake and delivery to the cytoplasm. Thus,
chemical modifications have the potential to increase potency of
RNA compounds, allowing lower doses of administration, reducing the
potential for toxicity, and decreasing overall cost of therapy.
[0006] In recent years, advances in oligonucleotide design and
chemical modification types/patterns have resulted in molecules
with increased resistance to nuclease-mediated degradation,
improved pharmokinetics, increased gene specificity and reduced
immunostimulatory responses (Lares, M. R. et al. 2010, Trends
Biotechnol. 58:570-9). Despite these major advances, siRNA delivery
to a diverse range of tissues remains a major obstacle in vivo.
While siRNA delivery in vivo has been achieved in eye, lung, brain,
tumor, and muscle by localized delivery (by intraocular,
intranasal, intrathecal, intratumoral, and intramuscular
injections, respectively), this delivery method is only suitable
for target validation studies due to its invasive nature and has
limited relevance as a clinical therapy (Golzio, M. et al., 2005,
Gene Ther. 12:246-51; Liang, Y. et al., 2010, PLoS One 5:e12860;
Reich, S. J. et al., 2003, Mol. Vis. 9:210-6; Tan, P. H. et al.,
2005, Gene Ther. 12:59-66; Zhang, X. et al., 2004, J. Biol. Chem.
279:10677-84). A good systemic delivery system is essential to
reach certain tissues of interest. Numerous studies have
demonstrated systemic and targeted systemic siRNA delivery in vivo
through a variety of methods, including cationic lipid and
polymers, cholesterol conjugates, cell-penetrating peptides,
recombinant viral vectors, small molecule carriers, antibody-linked
siRNA and targeting ligands (Frank-Kamenetsky, M. et al., 2008,
Proc. Natl. Acad. Sci. USA 105:11915-20; Khoury, M. et al., 2006,
Arthritis Rheum. 54:1867-77; Kim, B. et al., 2004, Am. J. Pathol.
165:2177-85; Kondo, E. et al., 2012, Nat. Commun. 3:951; Morrissey,
D. V. et al., 2005, Nat. Biotechnol. 23:1002-7; Schiffelers, R. M.
et al., 2004, Nucleic Acids Res. 32:e149; Song, E. et al., 2005,
Nat. Biotechnol. 23:709-17; Wolfrum, C. S. et al., 2007, Nat.
Biotechnol. 25:1149-57). However, systemic siRNA delivery has
remained limited to particular tissues, such as liver, tumors,
spleen and jejunum (Abrams, M. T. et al., 2010, Mol. Ther.
18:171-80; Chien, P. Y. et al., 2005, Cancer Gene Ther. 12:321-8;
Liang, Y. et al., supra; Sorensen, D. R. et al., 2003, J. Mol.
Biol. 327:761-6; Tadin-Strapps, M. et al., 2011, J. Lipid Res.
52:1084-97; Wolfrum, C. et al., supra).
[0007] Myostatin is an inhibitor of skeletal muscle differentiation
and growth. During development it is an inhibitor of myogenesis,
while during adulthood its major role is in negatively regulating
satellite cell activation and self-renewal. Myostatin is a member
of the TGF-.beta. family and acts as a catabolic stimulus through
the ActRIIB receptor to induce SMAD2/3/FOXO/NF-.kappa.B signaling
and muscle fiber atrophy (Sartori, R. G. et al., 2009, Am. J.
Physiol. Cell Physiol. 296:C1248-57; Stitt, T. N. et al., 2004,
Mol. Cell 14:395-403). Myostatin knockout mice, as well as other
mouse models of myostatin inhibition, display increased muscle
mass/strength and an attenuated/reversal of a muscle atrophy
phenotype in different muscle disease models (Akpan, I. et al.,
2009, Int. J. Obes. (Lond) 33:1265-73; Heineke, J. et al., 2010,
Circulation 121:419-25; Lin, J. et al., 2002, Biochem. Biophys.
Res. Commun. 291:701-6; Zhang, L. 2011, Faseb J. 25:1653-63; Zhou,
X. et al., 2010, Cell 142:531-43). Small-interfering RNAs targeting
myostatin may have numerous therapeutic applications in the
multitude of existing muscle disorders, which range from muscular
dystrophy, muscular atrophy in cachexia-inducing diseases, such as
cancer, heart disease, chronic obstructive pulmonary disease,
sarcopenia, chronic kidney disease, and metabolic diseases, and
also in insulin-resistant disorders (Asp, M. L. et al., 2010, Int.
J. Cancer 126:756-63; Bailey, J. L. et al., 2006, J. Am. Soc.
Nephrol. 17:1388-94; Engelen, M. P. et al., 1994, Eur. Respir. J.
7:1793-7; Ruegg, M. A. et al., 2011, Annu. Rev. Pharmacol. Toxicol.
51:373-95).
[0008] To date there has been limited success in siRNA or antisense
oligonucleotide (ASO) delivery systemically to muscle, with most
reports highlighting muscle targeting by local injection (Gebski,
B. L. et al., 2003, Hum. Mol. Genet. 12:1801-11; Guess, M. G. et
al., 2013, Skelet. Muscle 3:19; Laws, N. et al., 2008, J. Appl.
Physiol. 105:662-8; Tang, Y. et al., 2012, Mol. Pharmacol.
82:322-32). Several studies have used electroporation additively
with intramuscular (IM) injections to improve the transfer of
siRNAs or plasmid vectors into muscle cells (Eefting, D. et al.,
2007, Hum. Gene Ther. 18:861-9; Golzio, M. et al., 2005, supra;
Kishida, T. et al., 2004, J. Gene Med. 6:105-10). However, IM
injections have a long-standing history for causing pain, local
muscle damage and inflammation, which also minimizes their
usefulness for therapeutic applications (McMahon, J. M. et al.,
1998, Gene Ther. 5:1283-90). As an improvement to IM delivery, a
model of "local" venous delivery muscle system was developed, which
involves the use of a tourniquet to transiently isolate the
injection solution in the muscle of the limb, in order to deliver a
"high pressure" hydrodynamic injection of a luciferase pDNA vector
to muscle in rats, dogs and monkeys (Hagstrom, J. E. et al., 2004,
Mol. Ther. 10:386-98). Although it showed successful delivery into
multiple muscle groups in the limb and the ability for multiple
dosing, delivery efficiency was low and it is still an invasive
technique that requires a high degree of injection skill.
[0009] In recent years, the use of the carrier polymer,
atelocollagen, has been used for delivery of nucleic acids (siRNA,
ASOs and plasmids) and negatively-charged proteins. Recent studies
shows both local and systemic delivery of an atelocollagen/siRNA
complex to muscle in a model of Duchenne muscular dystrophy (DMD)
(Kawakami, E. et al., 2013, PLoS One 8:e64719; Kawakami, E. et al.,
2011, Dev. Growth Differ. 53:48-54; Kinouchi, N. et al., 2008, Gene
Ther. 15:1126-30).
[0010] There continues to be a need to develop therapies that can
easily and non-invasively deliver nucleic acids to the muscle,
which could have the potential for use in the future treatment of a
variety of muscle disorders, such as muscular atrophic diseases,
muscular dystrophy, and type II diabetes.
SUMMARY OF THE INVENTION
[0011] The present invention provides methods for delivering to a
subject small nucleic acid molecules capable of mediating RNA
interference and reducing the expression of myostatin. The small
nucleic acid molecules of the invention are more specifically
referred to herein as short interfering nucleic acid (siNA)
molecules. The siNA molecules that are delivered as per the methods
disclosed target a myostatin gene and are conjugated to a
lipophilic moiety, such as cholesterol (i.e., myostatin siNA
conjugates). Once delivered to their site of action (e.g., muscle
cells that express myostatin), the myostatin siNA conjugates act to
inhibit or down regulate myostatin gene expression by causing
destruction of a myostatin gene. By reducing the expression of a
myostatin gene and, in turn, reducing the level of myostatin
protein, the methods of the invention have the potential of
enhancing muscle mass and/or function. Thus, use of the disclosed
methods is indicated, for example, for treating musculoskeletal
diseases/disorders and diseases/disorders that result in conditions
in which muscle is adversely affected, such as neurodegenerative
diseases/disorders, sarcopenia, cachexia, obesity, Type-II
diabetes, HIV/AIDS and cancer. The methods of the invention are
also useful, for example, for enhancing muscle mass and/or function
in livestock including, but not limited to, cattle, pigs and
fowl.
[0012] An embodiment of the present invention relates to methods of
delivering to the muscle of a subject a short interfering nucleic
acid (siNA) molecule, or pharmaceutical compositions thereof, that
targets a myostatin gene comprising the step of systemically
administering to said subject a conjugate of said siNA, wherein
said conjugate comprises the siNA molecule linked to a lipophilic
moiety (e.g., cholesterol). Thus, the present invention relates to
methods of delivering to the muscle of a subject myostatin siNA
conjugates, or pharmaceutical compositions thereof, via systemic
administration. The myostatin siNA conjugates that are systemically
administered to the subject are delivered to muscle that expresses
myostatin. Once delivered to the muscle, the myostatin siNA
conjugate reduces myostatin expression by an RNA interference
mechanism. Thus, the present invention relates to methods of
delivering to the muscle of a subject a myostatin siNA conjugate,
or a pharmaceutical composition thereof, comprising systemically
administering the myostatin siNA conjugate to said subject in an
amount effective to modulate (e.g., inhibit or down-regulate)
myostatin expression in said muscle, wherein said myostatin siNA
conjugate comprises an siNA molecule that targets a myostatin gene
expressed by said subject linked to a lipophilic moiety (e.g.,
cholesterol), and wherein said myostatin siNA conjugate mediates
RNA interference.
[0013] An embodiment of the present invention relates to methods of
modulating (e.g., inhibiting or down-regulating) in vivo expression
of a myostatin gene in a subject comprising introducing to said
subject an effective amount of a myostatin siNA conjugate, or a
pharmaceutical composition thereof, by systemic administration,
wherein the siNA conjugate comprises a siNA molecule that targets a
myostatin gene expressed by said subject linked to a lipophilic
moiety (e.g., cholesterol), and wherein the siNA conjugate mediates
RNA interference. Another embodiment relates to methods of
modulating (e.g., inhibiting or down-regulating) in vivo expression
of a myostatin gene in a subject comprising delivering to the
muscle of said subject an effective amount of a myostatin siNA
conjugate, or a pharmaceutical composition thereof, by introducing
the siNA conjugate to said subject by systemic administration,
wherein the siNA conjugate comprises a siNA molecule that targets a
myostatin gene expressed by said subject linked to a lipophilic
moiety (e.g., cholesterol), and wherein the siNA conjugate mediates
RNA interference. Thus, the present invention relates to methods of
modulating in vivo expression of a myostatin gene in a subject
comprising systemically administering an effective amount of an
siNA conjugate, or a pharmaceutical composition thereof, to said
subject, wherein the siNA conjugate comprises an siNA molecule that
targets a myostatin gene expressed by said subject linked to a
lipophilic moiety, and wherein said siNA conjugate mediates RNA
interference. The myostatin siNA conjugates that are systemically
administered to the subject are delivered to muscle that expresses
myostatin and, by an RNA interference mechanism, inhibits or
down-regulates the expression of a myostatin gene in the
muscle.
[0014] A further aspect of the invention includes myostatin siNA
conjugates for use to modulate in vivo expression of a myostatin
gene expressed by a subject (i.e., a human or animal). Another
embodiment relates to the use of a conjugate comprising an siNA
molecule that targets a myostatin gene and a lipophilic moiety
(e.g., cholesterol), for the manufacture of a medicament for
modulating in vivo expression of a myostatin gene expressed by a
subject, which comprises systemic administration of said conjugate,
or a pharmaceutical composition thereof, to said subject.
[0015] Another embodiment of the present invention provides methods
for enhancing muscle mass in a subject comprising reducing
myostatin levels in said subject by introducing to said subject an
effective amount of a myostatin siNA conjugate, or a pharmaceutical
composition thereof, by systemic administration, wherein the siNA
conjugate comprises an siNA molecule that targets a myostatin gene
expressed by said subject linked to a lipophilic moiety (e.g.,
cholesterol), and wherein the siNA conjugate mediates RNA
interference. Another embodiment relates to methods for enhancing
muscle mass in a subject comprising reducing myostatin levels in
said subject by delivering to the muscle of said subject an
effective amount of a myostatin siNA conjugate, or a pharmaceutical
composition thereof, by introducing the siNA conjugate to said
subject by systemic administration, wherein the siNA conjugate
comprises an siNA molecule that targets a myostatin gene expressed
by said subject linked to a lipophilic moiety (e.g., cholesterol),
and wherein the siNA conjugate mediates RNA interference. The
phrase "reducing myostatin levels," as used herein, refers to
either reducing expression of a myostatin gene or reducing
myostatin protein levels. The myostatin siNA conjugates that are
systemically administered to the subject are delivered to muscle
that expresses myostatin and, by an RNA interference mechanism,
inhibits or down-regulates the expression of a myostatin gene in
the muscle. The decrease in myostatin expression results in an
increase in the muscle mass of the subject. The terms "muscle
enhancement" and "enhancing muscle" are intended to be
interchangeable herein and include, but are not limited to,
inducement of hyperplasia (increased muscle fiber number),
inducement of hypertrophy (increased muscle fiber diameter) or
both. The increase can be in type 1 and/or type 2 muscle fibers.
This aspect of the invention further relates to methods of
regenerating injured musculoskeletal tissue in a subject in need
thereof by systemically delivering myostatin siNA conjugates, or
pharmaceutical compositions thereof, described herein.
[0016] A further aspect of the invention includes myostatin siNA
conjugates for use to enhance muscle mass and/or to regenerate
injured musculoskeletal tissue in a subject. Another embodiment
relates to the use of a conjugate comprising an siNA molecule that
targets a myostatin gene linked to a lipophilic moiety (e.g.,
cholesterol), for the manufacture of a medicament for enhancing
muscle mass and/or regenerating injured musculoskeletal tissue in a
subject, which comprises systemic administration of said conjugate,
or a pharmaceutical composition thereof, to said subject.
[0017] Another embodiment of the present invention provides methods
for enhancing muscle performance in a subject comprising reducing
myostatin levels in said subject by introducing to said subject an
effective amount of a myostatin siNA conjugate, or a pharmaceutical
composition thereof, by systemic administration, wherein the siNA
conjugate comprises an siNA molecule that targets a myostatin gene
expressed by said subject linked to a lipophilic moiety (e.g.,
cholesterol), and wherein the siNA conjugate and mediates RNA
interference. Another embodiment relates to methods for enhancing
muscle performance in a subject comprising reducing myostatin
levels in said subject by delivering to the muscle of said subject
an effective amount of a myostatin siNA conjugate, or a
pharmaceutical composition thereof, by introducing the siNA
conjugate to said subject by systemic administration, wherein the
siNA conjugate comprises an siNA molecule that targets a myostatin
gene expressed by said subject linked to a lipophilic moiety (e.g.,
cholesterol), and wherein the siNA conjugate mediates RNA
interference. The myostatin siNA conjugates that are systemically
administered to the subject are delivered to muscle that expresses
myostatin and, by an RNA interference mechanism, inhibits or
down-regulates the expression of a myostatin gene in the muscle.
The decrease in myostatin expression results in an increase muscle
performance in the subject. "Enhanced muscle performance" includes,
but is not limited to, one or more of decreased atrophy, increased
muscle endurance and increased overall muscle strength (e.g.,
increased contractile force). A further aspect of the invention
includes myostatin siNA conjugates for use to enhance muscle
performance in a subject.
[0018] Another embodiment of the invention relates to methods of
treating musculoskeletal diseases or disorders and/or diseases or
disorders that result in conditions in which muscle is adversely
affected (e.g., muscle weakness, muscle atrophy) in a subject in
need thereof comprising the step of reducing myostatin levels in
said subject by introducing to said subject an effective amount of
a myostatin siNA conjugate, or a pharmaceutical composition
thereof, by systemic administration, wherein the siNA conjugate
comprises an siNA molecule that targets a myostatin gene expressed
by said subject linked to a lipophilic moiety (e.g., cholesterol),
and wherein the siNA conjugate mediates RNA interference. A further
embodiment of the invention relates to methods of treating
musculoskeletal diseases or disorders and/or diseases or disorders
that result in conditions in which muscle is adversely affected
(e.g., muscle weakness, muscle atrophy) in a subject in need
thereof comprising the step of reducing myostatin levels in said
subject by delivering to the muscle of said subject an effective
amount of a myostatin siNA conjugate, or a pharmaceutical
composition thereof, by systemic administration, wherein the siNA
conjugate comprises an siNA molecule that targets a myostatin gene
expressed by said subject linked to a lipophilic moiety (e.g.,
cholesterol), and wherein the siNA conjugate mediates RNA
interference. The myostatin siNA conjugates that are systemically
administered to the subject are delivered to muscle that expresses
myostatin and, by an RNA interference mechanism, inhibits or
down-regulates the expression of a myostatin gene in the muscle.
The decrease in myostatin expression results in an increased muscle
mass and/or enhanced muscle performance in the subject.
[0019] A further aspect of the invention includes myostatin siNA
conjugates for use to treat musculoskeletal diseases or disorders
and/or diseases or disorders that result in conditions in which
muscle is adversely affected in a subject. Another embodiment
relates to the use of a conjugate comprising an siNA molecule that
targets a myostatin gene linked to a lipophilic moiety (e.g.,
cholesterol), for the manufacture of a medicament for treating
musculoskeletal diseases or disorders and/or diseases or disorders
that result in conditions in which muscle is adversely affected in
a subject, which comprises systemic administration of said
conjugate, or a pharmaceutical composition thereof, to said
subject.
[0020] The methods of the present invention can be performed on a
subject to which nucleic acid molecules can be systemically
administered. The term "subject" as used herein is intended to
include human and non-human animals. Non-human animals include all
vertebrates, for example, mammals and non-mammals, such as
non-human primates, sheep, dogs, cats, cows, horses, chickens,
amphibians, and reptiles. In one embodiment, the methods of the
present invention are performed on a mammal. In another embodiment,
the methods of the present invention are performed on livestock. In
another embodiment, the methods of the present invention are
performed on humans. In a further embodiment, the human is
diagnosed with musculoskeletal disease. The term "subject" is also
intended to include an embryo, including a chicken embryo contained
within an egg.
[0021] An embodiment of the present invention relates to a
conjugate comprising an siNA molecule that targets a myostatin gene
and a lipophilic moiety (e.g., cholesterol). The myostatin siNA
conjugates of the invention may be used in a method of treatment of
a subject by therapy, which comprises systemic administration of
said conjugate, or a pharmaceutical composition thereof, to said
subject. A further embodiment relates to the use of a conjugate
comprising an siNA molecule that targets a myostatin gene and a
lipophilic moiety (e.g., cholesterol), for the manufacture of a
medicament for treating a subject, which comprises systemic
administration of said conjugate, or a pharmaceutical composition
thereof, to said subject.
[0022] An embodiment of the present invention relates to a
conjugate comprising an siNA molecule that targets a myostatin gene
and a lipophilic moiety (e.g., cholesterol), for use in a method of
treatment of a subject by therapy, wherein the conjugate is
formulated for systemic administration. A further embodiment
relates to the use of a conjugate comprising an siNA molecule that
targets a myostatin gene and a lipophilic moiety (e.g.,
cholesterol), for the manufacture of a medicament for treating a
subject, wherein the conjugate is formulated for systemic
administration.
[0023] The myostatin siNA conjugates of the present invention that
are delivered by the disclosed methods comprise a myostatin siNA
molecule linked to a lipophilic moiety. The myostatin siNA
conjugates delivered by the methods of the present invention are
not formulated with lipid formulations that form liposomes. While
not wishing to be bound by a particular theory, it is believed the
attachment of a lipophilic moiety to the myostatin siNA molecule
increases the lipophilicity of the siNA molecule, enhancing the
entry of the siNA molecule into muscle cells. Examples of
lipophilic moieties that can be linked to the myostatin siNA
molecule include, but are not limited to cholesterol, oleic acid,
stearic acid, palmitic acid, myristic acid, linoleic acid, oleyl,
retinyl, cholesteryl residues, cholic acid, adamantane acetic acid,
1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine. A preferred lipophilic moiety is
cholesterol.
[0024] The lipophilic moiety is attached to the myostatin siNA
molecule through linkage to a terminus of the siNA molecule (e.g.,
the 3' or 5' end of the sense strand of the siNA molecule) or
through linkage to an internal nucleotide of the siNA molecule. In
one embodiment, the lipophilic moiety is attached to the 3' end of
the passenger strand (sense strand) of a double-stranded myostatin
siNA molecule. In one embodiment, the lipophilic moiety is attached
to the 5' end of the passenger strand of a double-stranded
myostatin siNA molecule. In a further embodiment, the lipophilic
moiety is attached to the 3' end of the guide strand (antisense
strand) of a myostatin siNA molecule. In a further embodiment, a
myostatin siNA conjugate contains more than one attached lipophilic
moiety (e.g., a lipophilic moiety attached to both the 3' and the
5' end of the passenger strand; a lipophilic moiety attached to the
3' end of the guide strand and the 5' end of the passenger strand).
In this aspect of the invention, the lipophilic moieties can be the
same or different.
[0025] The present invention further provides siNA molecules useful
for modulating the expression of myostatin genes and to which a
lipophilic moiety can be attached to form the myostatin siNA
conjugates described herein. The siNA portion of the myostatin siNA
conjugates that are delivered by the methods of the present
invention can be single- or double-stranded small interfering
nucleic acid molecules and can take different oligonucleotide
forms, including but not limited to short interfering RNA (siRNA),
double-stranded RNA (dsRNA) and short hairpin RNA (shRNA)
molecules. In one embodiment, the myostatin siNA molecules are
double-stranded siNA molecules comprising a sense and an antisense
strand. The antisense strand comprises a sequence that is
complementary to a portion of a myostatin target RNA sequence, and
the sense strand is complementary to at least part of the antisense
strand. The double-stranded myostatin siNA molecules delivered by
the methods of the present invention can be symmetric or
asymmetric. In another aspect, the myostatin siNA molecules are
single-stranded siNA molecules, wherein the single oligonucleotide
strand (the antisense strand) comprises a sequence that is
complementary to at least part of a myostatin target RNA sequence.
The siNA portion of the myostatin siNA conjugates that are
delivered by the methods of the present invention inhibit myostatin
gene expression in a subject via an RNA interference (RNAi)
mechanism.
[0026] The myostatin siNA conjugates described herein are directed
to a myostatin gene that can be derived from any of a number of
animal species, including but not limited humans, cattle, swine,
fowl and rodent. In one embodiment, the myostatin gene is a human
myostatin RNA. In another embodiment, the myostatin gene is a
cattle myostatin RNA. In another embodiment, the myostatin gene is
a swine myostatin RNA. In a further embodiment, the myostatin gene
is a fowl myostatin RNA (e.g., chicken, turkey). In a further
embodiment, the myostatin gene is a rodent myostatin RNA (e.g.,
mouse).
[0027] In certain embodiments, the siNA molecules of the siNA
portion of the myostatin siNA conjugates that are delivered by the
methods of the present invention comprise an antisense strand
having at least 15 nucleotides with sequence complementarity to a
myostatin gene sequence. In other embodiments, the antisense strand
of an siNA molecule delivered by the methods of the present
invention is about 15 to 30 nucleotides in length. In further
embodiments, a double-stranded siNA molecule delivered by the
methods of the present invention comprises a sense strand and an
antisense strand, wherein each strand is independently about 15 to
30 nucleotides in length.
[0028] In one embodiment, the siNA portion of the siNA conjugates
of the invention are double-stranded siNA molecules that modulate
the expression of a myostatin gene, wherein the siNA molecule
comprises a sense strand and an antisense strand, wherein each
strand is independently 15 to 30 nucleotides in length, and wherein
the antisense strand comprises at least 15 nucleotides having
sequence complementary to any of:
TABLE-US-00001 (SEQ ID NO: 1) 5'- AUGGCAAAGAACAAAUAAU -3'; (SEQ ID
NO: 2) 5'- GGCAAAGAACAAAUAAUAU -3'; (SEQ ID NO: 3) 5'-
ACUCCAGAAUAGAAGCCAU -3'; or (SEQ ID NO: 4) 5'- UUUGGAAGAUGACGAUUAU
-3'.
In one embodiment, the "at least 15 nucleotides" are 15 contiguous
nucleotides.
[0029] In some embodiments, the antisense strand of the siNA
molecule portion of the myostatin siNA conjugates of the invention
comprises at least 15 nucleotides having sequence identity to any
of:
TABLE-US-00002 (SEQ ID NO: 18) 5'- AUUAUUUGUUCUUUGCCAU -3'; (SEQ ID
NO: 19) 5'- AUAUUAUUUGUUCUUUGCC -3'; (SEQ ID NO: 20) 5'-
AUGGCUUCUAUUCUGGAGU -3'; or (SEQ ID NO: 21) 5'- AUAAUCGUCAUCUUCCAAA
-3'.
In one embodiment, the "at least 15 nucleotides" are 15 contiguous
nucleotides. Thus, the antisense strand of the siNA molecule
comprises at least a 15 nucleotide sequence of any of SEQ ID NOs:
18-21.
[0030] In some embodiments, the sense strand of the siNA molecule
portion of the myostatin siNA conjugates of the invention comprises
at least 15 nucleotides having sequence identity to any of:
TABLE-US-00003 (SEQ ID NO: 1) 5'- AUGGCAAAGAACAAAUAAU -3'; (SEQ ID
NO: 2) 5'- GGCAAAGAACAAAUAAUAU -3'; (SEQ ID NO: 3) 5'-
ACUCCAGAAUAGAAGCCAU -3'; or (SEQ ID NO: 4) 5'- UUUGGAAGAUGACGAUUAU
-3'.
In one embodiment, the "at least 15 nucleotides" are 15 contiguous
nucleotides. Thus, the sense strand of the siNA molecule comprises
at least a 15 nucleotide sequence of any of SEQ ID NOs: 1-4.
[0031] In some embodiments, the siNA molecule portion of the
myostatin siNA conjugates of the invention comprises at least a 15
nucleotide sequence of both SEQ ID NO: 1 and 18; or both SEQ ID NO:
2 and 19; or both SEQ ID NO: 3 and 20; or both SEQ ID NO: 4 and 21.
In another embodiment, the siNA molecule portion of the myostatin
siNA conjugates comprises any of the following double-stranded
molecules:
TABLE-US-00004 (SEQ ID NO: 1) 5'- AUGGCAAAGAACAAAUAAU -3' and (SEQ
ID NO: 18) 5'- AUUAUUUGUUCUUUGCCAU -3'; (SEQ ID NO: 2) 5'-
GGCAAAGAACAAAUAAUAU -3' and (SEQ ID NO: 19) 5'- AUAUUAUUUGUUCUUUGCC
-3'; (SEQ ID NO: 3) 5'- ACUCCAGAAUAGAAGCCAU -3' and (SEQ ID NO: 20)
5'- AUGGCUUCUAUUCUGGAGU -3'; or (SEQ IN NO: 4) 5'-
UUUGGAAGAUGACGAUUAU -3' and (SEQ ID NO: 21) 5'- AUAAUCGUCAUCUUCCAAA
-3'.
[0032] In some embodiments of the invention, the siNA molecule is
linked to a lipophlic moiety. In anther embodiment, the lipophilic
moiety is cholesterol. In another embodiment, the lipophilic moiety
is attached to 3' end of the siNA molecule. In another embodiment,
the lipophilic moiety is attached to 5' end of the siNA molecule.
In another embodiment, a lipophilic moiety is attached to each of
the 3' and the 5' ends of the siNA molecule.
[0033] In some embodiments of the invention, all of the nucleotides
of siNA molecule portion of the myostatin siNA conjugates of the
invention are unmodified. In other embodiments, the siNA molecules
delivered by the methods of the present invention further comprise
one or more nucleotides in either one or both strands of the
molecule that are chemically-modified. Modifications include
nucleic acid sugar modifications, base modifications, backbone
(internucleoside linkage) modifications, non-nucleotide
modifications, and/or any combination thereof. In certain
instances, purine and pyrimidine nucleotides are differentially
modified. For example, purine and pyrimidine nucleotides can be
differentially modified at the 2'-sugar position (i.e., at least
one purine has a different modification from at least one
pyrimidine in the same or different strand at the 2'-sugar
position). In certain instances the purines are unmodified in one
or both strands, while the pyrimidines in one or both strands are
modified. In certain other instances, the pyrimidines are
unmodified in one or both strands, while the purines in one or both
strands are modified. In some instances, at least one modified
nucleotide is a 2'-deoxy-2'-fluoro nucleotide, a 2'-deoxy
nucleotide, or a 2'-O-alkyl nucleotide. In some instances, at least
5 or more of the pyrimidine nucleotides in one or both strands are
either all 2'-deoxy-2'-fluoro or all 2'-O-methyl pyrimidine
nucleotides. In some instances, at least 5 or more of the purine
nucleotides in one or both strands are either all
2'-deoxy-2'-fluoro or all 2'-O-methyl purine nucleotides. In
certain instances, wherein the siNA molecules comprise one or more
modifications as described herein, the nucleotides at positions 1,
2, and 3 at the 5' end of the guide (antisense) strand are
unmodified. In certain embodiments, the siNA molecules delivered by
the methods of the present invention comprise one or more modified
internucleoside linking groups. In certain embodiments, each
internucleoside linking group is, independently, a phosphodiester
or phosphorothioate linking group.
[0034] In certain embodiments, the siNA molecule portion of the
myostatin siNA conjugates of the invention have 3' overhangs of
one, two, three or four nucleotide(s) on one or both of the
strands. In other embodiments, the double-stranded siNA molecules
lack overhangs (i.e., have blunt ends). Preferably, the siNA
molecule has 3' overhangs of two nucleotides on both the sense and
antisense strands. The overhangs can be modified or unmodified.
Examples of modified nucleotides in the overhangs include, but are
not limited to, 2'-O-alkyl nucleotides, 2'-deoxy-2'-fluoro
nucleotides, locked nucleic acid (LNA) nucleotides, or 2'-deoxy
nucleotides. The overhanging nucleotides in the antisense strand
can comprise nucleotides that are complementary to nucleotides in
the myostatin target sequence. Likewise, the overhangs in the sense
strand can comprise nucleotides that are present in the myostatin
target sequence. In certain instances, the siNA molecules have two
3' overhanging nucleotides on the antisense strand that are
2'-O-alkyl (e.g., 2'-O-methyl) nucleotides and two 3' overhanging
nucleotides on the sense strand that are 2'-deoxy nucleotides. In
other instances, the siNA molecules have two 3' overhanging
nucleotides that are 2'-O-alkyl (e.g., 2'-O-methyl) nucleotides on
both the antisense strand and the sense strand. In certain
embodiments, the 2'-O-alkyl nucleotides are 2'-O-methyl uridine
nucleotides. In certain instances, the 3' overhangs also comprise
one or more phosphorothioate linkages between nucleotides of the
overhang.
[0035] In some embodiments, the siNA molecule portion of the
myostatin siNA conjugates of the invention have one or more
terminal caps (also referred to herein as "caps"). A cap may be
present at the 3'-terminus (3'-cap) of the antisense strand (guide
strand), at the 5'-terminus (5'-cap) of the sense strand (passenger
strand), and/or at 3'-terminus (3'-cap) of the sense strand
(passenger strand). The lipophilic moiety may be attached to the
same terminus of the siNA molecule that contains a terminal
cap.
[0036] In some embodiments, the siNA molecule portion of the
myostatin siNA conjugates of the invention are phosphorylated at
the 5' end of the antisense strand. The phosphate group can be a
phosphate, a diphosphate or a triphosphate.
[0037] In certain embodiments of this aspect of the invention, the
siNA portion of the myotstatin siNA conjugates of the invention are
double-stranded siNA molecules wherein the antisense and/or sense
strand comprises at least one nucleotide sequence selected from SEQ
ID NOs: 5-12, provided in Table 3. In a further embodiment, the
siNA portion of the myostatin siNA conjugates of the invention
comprises any of the following double-stranded molecules: SEQ ID
NO: 5 and 6; SEQ ID NO: 7 and 8; SEQ ID NO: 9 and 10; or SEQ ID NO:
11 and 12.
[0038] The present invention further provides compositions
comprising the myostatin siNA conjugates described herein with,
optionally, a pharmaceutically acceptable carrier or diluent. The
methods of the present invention include delivery of compositions
comprising the myostatin siNA conjugates described herein with a
pharmaceutically acceptable carrier or diluent, wherein said
compositions are formulated for systemic administration.
[0039] These and other aspects of the invention will be apparent
upon reference to the following Detailed Description and attached
figures. Moreover, it is contemplated that any method or
composition described herein can be implemented with respect to any
other method or composition described herein and that different
embodiments may be combined.
[0040] Additionally, patents, patent applications and other
documents are cited throughout the specification to describe and
more specifically set forth various aspects of this invention. Each
of these references cited herein is hereby incorporated by
reference in its entirety, including the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1: In vivo screen of Mstn-cholesterol conjugates. Four
Mstn-cholesterol siRNAs (.cndot.), PBS (.smallcircle.), and Placebo
5 non-targeting control (.cndot.) were screened in CD-1 mice (n=5)
at a 15 mpk dose by i.v. injection. (A) Mstn mRNA expression was
determined based on .DELTA..DELTA.Ct calculations, relative to PBS,
in gastrocnemius muscle 3 days post-injection. (B) Mstn protein
levels were measured in serum 3 days after dosing. ***, P<0.001
(by one-way ANOVA).
[0042] FIG. 2: In vivo dose titration and duration of myostatin
mRNA knockdown (KD) by Mstn-cholesterol siRNA.
Mstn:1169-cholesterol ("Mstn-chol") siRNA was tested in CD-1 mice
(n=5) at 5, 15, and 50 mpk, in addition to PBS and Placebo 5
non-targeting controls (50 mpk) by i.v. injection. Mstn mRNA
expression was determined based on .DELTA..DELTA.Ct calculations,
relative to PBS, in gastrocnemius (A), tricep muscles (B), and EDL
(C), at day 3, 7, and 21 post-injection. (D) Serum Mstn protein
levels were measured at indicated time points. *, P<0.05; **,
P<0.01; ***, P<0.001 (by one-way ANOVA).
[0043] FIG. 3: Long-term myostatin knockdown (KD) leads to increase
in muscle size. Mstn:1169-cholesterol siRNA (50 mpk) was injected
intravenously into CD-1 mice (n=12), in addition to PBS and Placebo
5 non-targeting controls. (A) Mstn mRNA levels, determined based on
.DELTA..DELTA.Ct calculations, relative to PBS, in gastrocnemius,
EDL, quadriceps, triceps and spinotrapezius muscles at day 21
post-injection. (B) Serum Mstn protein levels, determined for the
duration of the study. (C) Leg muscle size (maximum cross-sectional
area), monitored for the duration of the study and quantitated from
a series of 10 micro-CT images using a custom MATLAB and Definiens
Developer XD software algorithm. (D) Gastrocnemius muscle weight of
rested leg and exercised leg (used in situ muscle function assay)
at day 21. (E) Mean fiber cross-sectional area of gastrocnemius
muscle. (F) Mean total number of muscle fibers in gastrocnemius
muscle. (G) Size frequency distribution of muscle fibers in
gastrocnemius muscle. (H) Body weight measurements, determined for
the duration of the study. (I) Body composition analysis by qNMR
(EchoMRI) at day 20. *, P<0.05; **, P<0.01; ***, P<0.001
****, P<0.0001 (by one-way ANOVA (a-b) or two-way ANOVA
(c-h))
[0044] FIG. 4: Example of a muscle fatigue curve. Fatigue curves
exhibit three stages of muscle fatigue: early fatigue, late fatigue
and a non-fatigable stage. "Early fatigue" is represented by
F.sub.max-F.sub.0, typically representative of type IIb fibers,
which use creatine phosphate as an energy source. This stage is
followed by "late fatigue" (F.sub.0-F.sub.min), typically
representative of type IIa/x fibers, which use glycogen as an
energy source. The final stage of the fatigue curve is the
"non-fatigable" stage (F.sub.min), which is indicative of type I
fibers, which use fatty acids as an energy source.
[0045] FIG. 5: Long-term myostatin knockdown leads to changes in
muscle function. Mstn:1169 siRNA-cholesterol conjugate or Placebo
50-cholesterol non-targeting siRNA control (50 mpk) were injected
intravenously into CD-1 mice (n=12). Muscle fatigue curves, force
(A) or specific force (B), were generated from in situ muscle
function assay performed on day 21/day 22 (n=9 total). Function
parameters calculated from the indicated fatigue curve are plotted
below each curve in (A) and (B). Filled in bars indicate a
statistical significant change in the specified parameter between
Mstn siRNA-cholesterol conjugate and Placebo 5-cholesterol
treatment. *, P<0.05; **, P<0.01; ***, P<0.001 ****,
P<0.0001 (by one-way ANOVA (a-b) or two-way ANOVA (c-h))
[0046] FIG. 6: Heart weight measurements normalized to different
parameters for Placebo 5-chol and Mstn:1169 siRNA-conjugate.
Mstn:1169 siRNA-conjugate (50 mpk) was injected intravenously into
CD-1 mice (n=12), in addition to PBS and Placebo 5 cholesterol
conjugate non-targeting control. Hearts were weighed and normalized
to a variety of parameters (i.e., tibia length, body weight, lean
mass, and muscle cross sectional area (CSA)) after 21 days of Mstn
knockdown to assess signs of cardiac hypertrophy. Filled in bars
indicate a statistical significant change in the specified
parameter between Mstn siRNA-cholesterol conjugate and Placebo
5-cholesterol treatment. *, P<0.05; ****, P<0.0001 (by
one-way ANOVA (a-b) or two-way ANOVA (c-h))
[0047] FIG. 7: Ctnnb1-chol conjugates. (A) In vivo comparison of
efficacy of Ctnnb1-chol conjugates in WT, LDLR-/-, and ApoE-/-
mice. Ctnnb1-chol siRNA was screened in CD-1 mice (n=3 or 4) at a
14 mpk dose by i.v. injection. Ctnnb1 mRNA expression was
determined based on .DELTA..DELTA.Ct calculations, relative to PBS,
in gastrocnemius muscle 3 days post-injection. **, P<0.01; ***,
P<0.001 (by one-way ANOVA) (B) Ctnnb1-chol siRNAs with a single
cholesterol attached at either the 3' or 5' end of the passenger
strand, were screened in CD-1 mice (n=5) at a 15 mpk dose by i.v.
injection. Ctnnb1 mRNA expression was compared to PBS and Placebo 5
non-targeting control (3' chol). mRNA expression was determined
based on .DELTA..DELTA.Ct calculations, relative to PBS, in
gastrocnemius muscle 3 days post-injection. ***, P<0.001 (by
one-way ANOVA).
DETAILED DESCRIPTION OF THE INVENTION
A. Terms and Definitions
[0048] The following terminology and definitions apply as used in
the present application.
[0049] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a cell" includes a combination of two or more cells,
and the like.
[0050] Any concentration range, percentage range, ratio range or
integer range is to be understood to include the value of any
integer within the recited range, and when appropriate, fractions
thereof (such as one tenth and one hundredth of an integer), unless
otherwise indicated.
[0051] "About" or "approximately," as used herein, in reference to
a number are generally taken to include numbers that fall within a
range of 5% in either direction (greater than or less than) of the
number unless otherwise stated or otherwise evident from the
context (except where such number would exceed 100% of a possible
value). Where ranges are stated, the endpoints are included within
the range unless otherwise stated or otherwise evident from the
context.
[0052] The phrases "2'-modified nucleotide," "2'-substituted
nucleotide" or a nucleotide having a modification at the
"2'-position" of the sugar moiety, as used herein, generally refer
to nucleotides comprising a substituent at the 2' carbon position
of the sugar component that is other than H or OH. 2'-modified
nucleotides include, but are not limited to, bicyclic nucleotides
wherein the bridge connecting two carbon atoms of the sugar ring
connects the 2' carbon and another carbon of the sugar ring; and
nucleotides with non-bridging 2'substituents, such as allyl, amino,
azido, thio, O-allyl, OC.sub.1-10 alkyl, --OCF3,
O--(CH.sub.2).sub.2--O--CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-10 alkyl. 2'-modified nucleotides may further comprise
other modifications, for example at other positions of the sugar
and/or at the nucleobase. The phrases "3'-modified nucleotide,"
"3'-substituted nucleotide" or a nucleotide having a modification
at the "3'-position" of the sugar moiety generally refers to a
nucleotide comprising a modification, including a substituent, at
the 3' carbon position of the sugar component.
[0053] The term "abasic" as used herein refers to its meaning as is
generally accepted in the art. The term generally refers to sugar
moieties lacking a nucleobase or having a hydrogen atom (H) or
other non-nucleobase chemical groups in place of a nucleobase at
the 1' position of the sugar moiety, see for example Adamic et al.,
U.S. Pat. No. 5,998,203. In one embodiment, an siNA molecule of the
invention may contain an abasic moiety, wherein the abasic moiety
is ribose, deoxyribose, or dideoxyribose sugar.
[0054] The term "acyclic nucleotide" as used herein refers to its
meaning as is generally accepted in the art. The term generally
refers to any nucleotide having an acyclic ribose sugar, for
example where any of the ribose carbon/carbon or carbon/oxygen
bonds are independently or in combination absent from the
nucleotide.
[0055] If no number of carbon atoms is specified, the term "alkyl"
refers to a saturated aliphatic hydrocarbon group, branched or
straight-chain, containing from 1 to 10 carbon atoms. An alkyl
group can have a specific number of carbon atoms. For example,
C.sub.1-C.sub.10, as in "C.sub.1-C.sub.10 alkyl" or "C.sub.1-10
alkyl," is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8,
9 or 10 carbons in a linear or branched arrangement. For example,
"C.sub.1-C.sub.10 alkyl" specifically includes methyl, ethyl,
n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl,
heptyl, octyl, nonyl, decyl, and so on. The term "cycloalkyl" means
a monocyclic saturated aliphatic hydrocarbon group having the
specified number of carbon atoms. For example, "cycloalkyl"
includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl,
2-ethyl-cyclopentyl, cyclohexyl, and so on.
[0056] The term "antisense region" as used herein refers to its
meaning as is generally accepted in the art. With reference to the
siNA molecules delivered by the methods of the present invention,
the term refers to a nucleotide sequence of an siNA molecule having
complementarity to a myostatin RNA. In addition, the antisense
region of an siNA molecule comprises a nucleic acid sequence having
complementarity to a sense region of the siNA molecule. In one
embodiment, the antisense region of an siNA molecule is referred to
as the antisense strand or guide strand.
[0057] The term "biodegradable" as used herein refers to its
meaning as is generally accepted in the art. The term generally
refers to degradation in a biological system, for example,
enzymatic degradation or chemical degradation.
[0058] The term "biological system" as used herein refers to its
meaning as is generally accepted in the art. The term generally
refers to material, in a purified or unpurified form, from
biological sources including, but not limited to, human or animal,
wherein the system comprises the components required for RNAi
activity. Thus, the phrase includes, for example, a cell, tissue,
subject, or organism, or extract thereof. The term also includes
reconstituted material from a biological source.
[0059] The term "blunt end" as used herein refers to its meaning as
is generally accepted in the art. With reference to nucleic acid
molecules of the invention, the term refers to termini of a
double-stranded siNA molecule having no overhanging nucleotides. An
siNA duplex molecule of the invention can comprise blunt ends at
one or both termini of the duplex, such as termini located at the
5'-end of the antisense strand, the 5'-end of the sense strand, or
both termini of the duplex.
[0060] The term "cap" (also referred to herein as "terminal cap")
as used herein refers to its meaning as is generally accepted in
the art. With reference to exemplary nucleic acid molecules of the
invention, the term refers to a moiety, which can be a
chemically-modified nucleotide or a non-nucleotide, incorporated at
one or more termini of the nucleic acid molecules of the invention.
These terminal modifications may protect the nucleic acid molecule
from exonuclease degradation and may help in delivery and/or
localization of the nucleic acid molecule within a cell. The cap
can be present at a 5'-terminus (5'-cap) or 3'-terminus (3'-cap) of
a strand of the nucleic acid molecules of the invention, or can be
present on both termini. For example, a cap can be present at the
5'-end, 3'-end and/or 5' and 3'-ends of the sense strand of a
nucleic acid molecule of the invention. Additionally, a cap can be
present at the 3'-end of the antisense strand of a nucleic acid
molecule of the invention. In non-limiting examples, a 5'-cap
includes, but is not limited to, LNA; glyceryl; inverted deoxy
abasic residue (moiety); 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide;
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide; 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety. Non-limiting
examples of a 3'-cap include, but are not limited to, LNA;
glyceryl; inverted deoxy abasic residue (moiety); 4',5'-methylene
nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio
nucleotide; carbocyclic nucleotide; 5'-amino-alkyl phosphate;
1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate;
phosphorothioate and/or phosphorodithioate; bridging or
non-bridging methylphosphonate; and 5'-mercapto moieties (for more
details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein). In one embodiment, siNA
molecules of the present invention contain a vinyl phosphate 5'
terminal cap, wherein carbon 5 of the sugar ring contains the
following substituent (.dbd.CH)--P(.dbd.O)(OH).sub.2.
[0061] The term "cell" as used herein refers to its meaning as is
generally accepted in the art. The term is used herein in its usual
biological sense, and does not refer to an entire multicellular
organism, e.g., specifically does not refer to a human being. The
cell can be present in an organism, e.g., birds, plants and
mammals, such as humans, cows, sheep, apes, monkeys, swine, dogs,
and cats. The cell can be of somatic or germ line origin,
totipotent or pluripotent, dividing or non-dividing. The cell can
also be derived from or can comprise a gamete or embryo, a stem
cell, or a fully differentiated cell. The cell can be a muscle
cell.
[0062] The phrases "chemically-modified nucleotide," "modified
nucleotide" or, when used in reference to nucleotides within the
myostatin siNA molecules described herein, "chemical modification,"
refer to a nucleotide that contains a modification in the chemical
structure of the heterocyclic base moiety, sugar and/or phosphate
of the unmodified (or natural) nucleotide as is generally known in
the art (i.e., at least one modification compared to a naturally
occurring RNA or DNA nucleotide). In certain embodiments, the terms
can refer to certain forms of RNA that are naturally occurring in
certain biological systems, for example 2'-O-methyl modifications
or inosine modifications. A modified nucleotide includes abasic
nucleotides. Modified nucleotides include nucleotides with a
modified sugar ring or sugar surrogate. Modified heterocyclic base
moieties include without limitation, universal bases, hydrophobic
bases, promiscuous bases, size-expanded bases, and fluorinated
bases as defined herein. Certain of these nucleobases are
particularly useful for increasing the binding affinity of the siNA
molecules as provided herein. These include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. A modified internucleoside linkage refers to
any internucleoside linkage other than a naturally occurring
internucleoside linkage. Non-limiting examples of modified
nucleotides are described herein and in U.S. application Ser. No.
12/064,014 (published as US 20090176725).
[0063] The terms "complementarity" or "complementary" as used
herein refers to its meaning as is generally accepted in the art.
The terms generally refer to the formation or existence of hydrogen
bond(s) between one nucleic acid sequence and another nucleic acid
sequence by either traditional Watson-Crick or other
non-traditional types of bonding as described herein. In reference
to the nucleic molecules delivered by the methods of the present
invention, the binding free energy for a nucleic acid molecule with
its complementary sequence is sufficient to allow the relevant
function of the nucleic acid to proceed, e.g., RNAi activity.
Determination of binding free energies for nucleic acid molecules
is well known in the art (see, e.g., Turner et al., 1987, CSH Symp.
Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.
Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.
109:3783-3785). Perfect complementary means that all the contiguous
residues of a nucleic acid sequence will hydrogen bond with the
same number of contiguous residues in a second nucleic acid
sequence. Partial complementarity can include various mismatches or
non-based paired nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more mismatches, non-nucleotide linkers, or non-based paired
nucleotides) within the nucleic acid molecule, which can result in
bulges, loops, or overhangs between the sense strand or sense
region and the antisense strand or antisense region of a nucleic
acid molecule or between the antisense strand or antisense region
of a nucleic acid molecule and a corresponding target nucleic acid
molecule. Such partial complementarity can be represented by a %
complementarity that is determined by the number of non-base paired
nucleotides, e.g., about 50%, 60%, 70%, 80%, 90% etc. depending on
the total number of nucleotides involved. Such partial
complementarity is permitted to the extent that the nucleic acid
molecule (e.g., siNA) maintains its function, for example the
ability to mediate sequence specific RNAi.
[0064] The terms "composition" or "formulation" as used herein
refer to their generally accepted meaning in the art. These terms
generally refer to a composition or formulation, such as in a
pharmaceutically acceptable carrier or diluent, in a form suitable
for administration, e.g., systemic administration, into a cell or
subject, including, for example, a human. Suitable forms, in part,
depend upon the use or the route of entry, for example oral,
transdermal, inhalation, or by injection. Such forms should not
prevent the composition or formulation from reaching a target cell
(i.e., a cell to which the negatively charged nucleic acid is
desirable for delivery). For example, compositions injected into
the blood stream should be soluble. Other factors are known in the
art, and include considerations such as toxicity and forms that
prevent the composition or formulation from exerting its effect. As
used herein, pharmaceutical formulations include formulations for
human and veterinary use. A "pharmaceutically acceptable
composition" or "pharmaceutically acceptable formulation" can refer
to a composition or formulation that allows for the effective
distribution of the nucleic acid molecules of the instant invention
to the physical location most suitable for their desired
activity.
[0065] The term "conjugate" refers to an atom or group of atoms
bound to an siNA molecule delivered by the methods of the
invention. In general, conjugate groups modify one or more
properties of the molecule to which they are attached, including,
but not limited to pharmacodynamics, pharmacokinetic, binding,
absorption, cellular distribution, cellular uptake, charge and
clearance. Conjugate groups are routinely used in the chemical arts
and are linked directly or via an optional linking moiety or
linking group to the parent compound, such as an siNA molecule. The
siNA conjugates used in the methods of the present invention
comprise an siNA molecule that targets myostatin RNA linked to a
lipophilic moiety, such as cholesterol. In certain embodiments, the
lipophilic moiety is attached to a 3' or 5' terminal nucleotide or
to an internal nucleotide of a myostatin siNA molecule. As used
herein, "conjugate linking group" refers to any atom or group of
atoms used to attach the lipophilic moiety to a myostatin siNA
molecule. Linking groups or bifunctional linking moieties such as
those known in the art are amenable to the present invention.
[0066] The terms "detecting" or "measuring," as used herein in
connection with an activity, response or effect, indicate that a
test for detecting or measuring such activity, response, or effect
is performed. Such detection and/or measuring may include values of
zero. Thus, if a test for detection or measuring results in a
finding of no activity (activity of zero), the step of detecting or
measuring the activity has nevertheless been performed.
[0067] The phrase "effective amount" as used herein refers to its
meaning as is generally accepted in the art. The term generally
refers to the amount of a molecule, compound or composition that
will elicit the intended biological response of a cell, tissue,
system, animal or human that is be sought by the researcher,
veterinarian, medical doctor or other clinician (e.g., reduction in
myostatin protein levels, as measured in muscle tissue or serum). A
"therapeutically effective amount" generally refers to the amount
of a molecule, compound or composition that will elicit the medical
response if a given clinical treatment is considered effective when
there is at least a 25% reduction in a measurable parameter
associated with a disease or disorder, a therapeutically effective
amount of a drug for the treatment of that disease or disorder is
that amount necessary to effect at least a 25% reduction in that
parameter.
[0068] The term "expression" as used herein refers to its meaning
as is generally accepted in the art. The term generally refers to
the process by which a gene ultimately results in a protein.
Expression includes, but is not limited to, transcription,
splicing, post-transcriptional modification and translation.
[0069] The term "gene" as used herein refers to its meaning as is
generally accepted in the art. The term generally refers to a
nucleic acid (e.g., DNA or RNA) sequence that comprises partial
length or entire length coding sequences necessary for the
production of a polypeptide. A gene can also include the UTR or
non-coding region of the nucleic acid sequence. A gene can also
encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as
small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA
(snRNA), short interfering RNA (siRNA), small nucleolar RNA
(snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor
RNAs thereof. Such non-coding RNAs can serve as target nucleic acid
molecules for siNA mediated RNA interference in modulating the
activity of fRNA or ncRNA involved in functional or regulatory
cellular processes. Aberrant fRNA or ncRNA activity leading to
disease can therefore be modulated by delivery of
myostatin-directed siNA molecules by the methods of the invention.
siNA molecules targeting fRNA and ncRNA can also be used to
manipulate or alter the genotype or phenotype of a subject,
organism or cell, by intervening in cellular processes such as
genetic imprinting, transcription, translation, or nucleic acid
processing (e.g., transamination, methylation etc.). The term
"gene" can be used when referencing a gene to which an siNA
molecule, delivered by the methods of present invention, is either
directly (i.e., the siNA molecule comprises an antisense strand
having partial or complete complementarity to the gene) or
indirectly (i.e., the siNA molecule comprises an antisense strand
having partial or complete complementarity to a gene in the
expression or activity pathway of the gene) targeted.
[0070] The term "heteroaryl," as used herein, represents a stable
monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein
at least one ring is aromatic and contains from 1 to 4 heteroatoms
selected from the group consisting of O, N and S. Heteroaryl groups
within the scope of this definition include but are not limited to:
acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl,
indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl,
benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl,
isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl,
indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl,
pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.
"Heteroaryl" is also understood to include the N-oxide derivative
of any nitrogen-containing heteroaryl. In cases where the
heteroaryl substituent is bicyclic and one ring is non-aromatic or
contains no heteroatoms, it is understood that attachment is via
the aromatic ring or via the heteroatom containing ring,
respectively.
[0071] The term "heterocycle," as used herein, is intended to mean
a 3- to 10-membered aromatic or nonaromatic heterocycle containing
from 1 to 4 heteroatoms selected from the group consisting of O, N
and S, and includes bicyclic groups. "Heterocycle" includes the
above mentioned heteroaryls, as well as dihydro and tetrahydro
analogs thereof. Further examples of "heterocycle" include, but are
not limited to the following: azetidinyl, benzoimidazolyl,
benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl,
benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl,
furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl,
isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl,
naphthpyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline,
oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline,
oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,
pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,
quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl,
tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,
tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl,
triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl,
piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl,
thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,
dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,
dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,
dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,
dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,
dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,
dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,
dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,
dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl,
and tetrahydrothienyl, and N-oxides thereof. Attachment of a
heterocyclyl substituent can occur via a carbon atom or via a
heteroatom.
[0072] The terms "including" (and any form thereof, such as
"includes" and "include"), "comprising" (and any form thereof, such
as "has" or "have") or "containing" (and any form thereof
("contains" or "contain") are inclusive and open-ended and do not
exclude additional, unrecited elements or method steps.
[0073] The terms "inhibit," "down-regulate," or "reduce" as used
herein refer to their meanings as generally accepted in the art.
With reference to the siNA molecules delivered by the methods of
the present invention, the term generally refers to reduction in
the expression of a gene, or in the level of RNA molecules encoding
one or more proteins or protein subunits, or in the activity of one
or more proteins or protein subunits, below that observed in the
absence of the siNA molecules. Down-regulation can be associated
with post-transcriptional silencing, such as RNAi mediated
cleavage.
[0074] The terms "intermittent" or "intermittently" as used herein
refer to their meaning as generally accepted in the art. The terms
generally refer to periodic stopping and starting at either regular
or irregular intervals.
[0075] The terms "internucleoside linkage," "internucleoside
linker," "internucleoside linking group," "internucleotide
linkage," "internucleotide linker" or "internucleotide linking
group" are used herein interchangeably and refer to any linker or
linkage between two nucleoside (i.e., a heterocyclic base moiety
and a sugar moiety) units, as is known in the art, including, for
example, but not as limitation, phosphate, analogs of phosphate,
phosphonate, guanidium, hydroxylamine, hydroxylhydrazinyl, amide,
carbamate, alkyl, and substituted alkyl linkages. Internucleoside
linkages constitute the backbone of a nucleic acid molecule. In one
aspect, a nucleotide of an siNA molecule may be linked to a
consecutive nucleotide through a linkage between the 3'-carbon of
the sugar of the first nucleotide and the sugar moiety of the
second nucleotide (herein referred to as a 3' internucleoside
linkage). A 3'-5' internucleoside linkage, as used herein, refers
to an internucleoside linkage that links two consecutive nucleoside
units, wherein the linkage is between the 3' carbon of the sugar
moiety of the first nucleoside and the 5' carbon of the sugar
moiety of the second nucleoside. In another aspect, a nucleotide of
an siNA molecule may be linked to a consecutive nucleotide through
a linkage between the 2'-carbon of the sugar of the first
nucleotide and the sugar moiety of the second nucleotide (herein
referred to as a 2' internucleoside linkage). A 2'-5'
internucleoside linkage, as used herein, refers to an
internucleoside linkage that links two consecutive nucleoside
units, wherein the linkage is between the 2' carbon of the sugar
moiety of the first nucleoside and the 5' carbon of the sugar
moiety of the second nucleoside.
[0076] The term "linker" or "spacer," as used herein, refers to
their meaning as generally accepted in the art. Generally, they
refer to any molecule that links or joins components. In the case
of the present invention, a linker or spacer may be used to join a
myostatin siNA molecule to a lipophilic molecule to form a
myostatin siNA conjugate. The linker can be a nucleic acid or a
non-nucleic acid-based linker. The term "biodegradable linker"
refers to an optional linker molecule designed to connect the siNA
molecule to the lipophilic moiety and which is susceptible to
degradation in a biological system.
[0077] The term "livestock," in reference to animals, refers to
domesticated animals, semi-domesticated animals or captive wild
animals that are raised in an agricultural setting to produce
commodities such as food, fiber and labor. Livestock animals
include cattle, swine, fowl (e.g., chicken, turkey), sheep, bison,
goats and the like.
[0078] The phrase "metered dose inhaler" or "MDI" refers to a unit
comprising a can, a secured cap covering the can, and a formulation
metering valve situated in the cap. MDI systems include a suitable
channeling device. Suitable channeling devices comprise for
example, a valve actuator and a cylindrical or cone-like passage
through which medicament can be delivered from the filled canister
via the metering valve to the nose or mouth of a patient such as a
mouthpiece actuator.
[0079] The term "microRNA" or "miRNA" as used herein refers to its
meaning as is generally accepted in the art. The term generally
refers to a small non-coding RNA that regulates the expression of
target messenger RNAs either by mRNA cleavage, translational
repression/inhibition or heterochromatic silencing (see for example
Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116,
281-297; Cullen, 2004, Virus Research, 102, 3-9; He et al., 2004,
Nat. Rev. Genet., 5, 522-531; Ying et al., 2004, Gene, 342, 25-28;
and Sethupathy et al., 2006, RNA, 12:192-197). The phenomenon of
RNA interference includes the endogenously induced gene silencing
effects of miRNAs. As used herein, "microRNA mimetic" refers to an
siNA molecule having a sequence that is at least partially
identical to that of a microRNA. In certain embodiments, a microRNA
mimetic comprises the microRNA seed region of a microRNA. In
certain embodiments, a microRNA mimetic modulates translation of
more than one target nucleic acid.
[0080] The term "modulate" or "modulation" as used herein refers to
its meaning as is generally accepted in the art. With reference to
nucleic acid molecules delivered by the methods of the present
invention, the term refers to when the expression of a gene, or the
level of one or more RNA molecules (coding or non-coding), or the
activity of one or more RNA molecules or proteins or protein
subunits, is up-regulated or down-regulated, such that expression
level or activity is greater than or less than that observed in the
absence of the molecule that effects modulation. For example, the
term "modulate" in some embodiments can refer to inhibition and, in
other embodiments, can refer to potentiation or up-regulation,
e.g., of gene expression.
[0081] The phrases "muscle cell" or "muscle tissue" as used herein
refers to their meaning as is generally accepted in the art. They
refer to a cell or group of cells derived from muscle of any kind
(for example, skeletal muscle and smooth muscle, e.g. from the
digestive tract, urinary bladder, blood vessels or cardiac tissue).
Such muscle cells may be differentiated or undifferentiated, such
as myoblasts, myocytes, myotubes, cardiomyocytes and
cardiomyoblasts.
[0082] The phrase "myostatin siNA molecule" or "myostatin siNA" as
used herein refers to a siNA molecule that targets a myostatin
gene. The phrase "myostatin siNA conjugate" as used herein refers
to a siNA molecule that targets a myostatin gene and is linked to a
lipophilic moiety.
[0083] The phrase "non-base paired" refers to nucleotides that are
not base paired between the sense strand or sense region and the
antisense strand or antisense region of a double-stranded siNA
molecule. Non-base paired nucleotides can include, for example, but
not as limitation, mismatches, overhangs, and single stranded
loops.
[0084] The term "non-nucleotide" refers to any group or compound
that can be incorporated into a polynucleotide chain in the place
of one or more nucleotide units, such as for example but not
limitation, abasic moieties or alkyl chains. The group or compound
is "abasic" in that it does not contain a commonly recognized
nucleotide base, such as adenosine, guanine, cytosine, uracil or
thymine and, therefore, lacks a nucleobase at the 1'-position.
[0085] The term "nucleobase" is used herein to refer to the
heterocyclic base portion of a nucleotide. Nucleobases may be
naturally occurring or may be modified. In certain embodiments, a
nucleobase may comprise any atom or group of atoms capable of
hydrogen bonding to a base of another nucleic acid.
[0086] The term "nucleotide" is used as is generally recognized in
the art. Nucleotides generally comprise a heterocyclic base moiety
(i.e., a nucleobase), a sugar, and an internucleoside linkage,
e.g., a phosphate. The base can be a natural base (standard), a
modified base, or a base analog, as are well known in the art. Such
bases are generally located at the 1' position of a nucleotide
sugar moiety. Additionally, the nucleotides can be unmodified or
modified at the sugar, internucleoside linkage, and/or base moiety,
(also referred to interchangeably as nucleotide analogs, modified
nucleotides, non-natural nucleotides, non-standard nucleotides and
others; see, for example, U.S. application Ser. No. 12/064,014
(published as US 20090176725)). A naturally occurring
internucleoside linkage refers to a 3' to 5' phosphodiester linkage
(also referred to herein as a 3'-5' phosphodiester linkage).
[0087] The term "overhang" as used herein refers to its meaning as
is generally accepted in the art. With reference to exemplary
double-stranded nucleic acid molecules delivered by the methods of
the present invention, the term generally refers to the terminal
portion of a nucleotide sequence that is not base-paired between
the two strands of a double-stranded nucleic acid molecule.
Overhangs, when present, are typically at the 3'-end of one or both
strands in an siNA duplex.
[0088] The phrase "pharmaceutically acceptable carrier or diluent"
as used herein refers to its meaning as it generally accepted in
the art. The phrase generally refers to any substance suitable for
use in administering to a subject, such as an animal. In certain
embodiments, a pharmaceutically acceptable carrier or diluent is
sterile saline. In certain embodiments, such sterile saline is
pharmaceutical grade saline.
[0089] The term "phosphorothioate" refers to an internucleoside
phosphate linkage comprising one or more sulfur atoms in place of
an oxygen atom. Hence, the term phosphorothioate refers to both
phosphorothioate and phosphorodithioate internucleoside
linkages.
[0090] The term "ribonucleotide" as used herein refers to its
meaning as is generally accepted in the art. The term generally
refers to a nucleotide with a hydroxyl group at the 2' position of
a .beta.-D-ribofuranose moiety.
[0091] The term "RNA" as used herein refers to its generally
accepted meaning in the art. Generally, the term RNA refers to a
molecule comprising at least one ribofuranoside moiety. The term
can include double-stranded RNA, single-stranded RNA, isolated RNA
such as partially purified RNA, essentially pure RNA, synthetic
RNA, recombinantly produced RNA, as well as altered RNA that
differs from naturally occurring RNA by the addition, deletion,
substitution and/or alteration of one or more nucleotides. Such
alterations can include addition of non-nucleotide material, such
as to the end(s) of an siNA molecule or internally, for example at
one or more nucleotides of the RNA. Nucleotides in the nucleic acid
molecules of the instant invention can also comprise non-standard
nucleotides, such as non-naturally occurring nucleotides or
chemically synthesized nucleotides or deoxynucleotides. These
altered RNAs can be referred to as analogs or analogs of
naturally-occurring RNA.
[0092] The phrase "RNA interference" or term "RNAi" refer to the
biological process generally known in the art of inhibiting or down
regulating gene expression in a cell, typically by causing
destruction of specific target RNA and mediated by
sequence-specific nucleic acid molecules (e.g., short interfering
nucleic acid molecule), see for example Zamore and Haley, 2005,
Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science,
309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001,
Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498;
and Kreutzer et al., International PCT Publication No. WO 00/44895;
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646; Fire, International PCT Publication No. WO 99/32619;
Plaetinck et al., International PCT Publication No. WO 00/01846;
Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO
99/07409; and Li et al., International PCT Publication No. WO
00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,
2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297,
2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;
Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al.,
2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,
1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).
Additionally, the term RNAi is meant to be equivalent to other
terms used to describe sequence specific RNA interference, such as
post transcriptional gene silencing, translational inhibition,
transcriptional inhibition, or epigenetics. For example, siNA
molecules of the invention can be used to epigenetically silence
genes at either the post-transcriptional level or the
pre-transcriptional level. In a non-limiting example, epigenetic
modulation of gene expression by siNA molecules delivered by the
methods of the present invention can result from siNA mediated
modification of chromatin structure or methylation patterns to
alter gene expression (see, for example, Verdel et al., 2004,
Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303,
669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,
2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297,
2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).
Modulation of gene expression by siNA molecules delivered by the
methods of the present invention can result from siNA mediated
cleavage of RNA (either coding or non-coding RNA) via RISC.
[0093] The phrase "sense region" as used herein refers to its
meaning as is generally accepted in the art. With reference to siNA
molecules described herein, the term refers to a nucleotide
sequence of an siNA molecule having complementarity to an antisense
region of the siNA molecule. In addition, the sense region of a
siNA molecule can comprise a nucleic acid sequence having homology
or sequence identity with a target nucleic acid sequence. In one
embodiment, the sense region of the siNA molecule is also referred
to as the sense strand or passenger strand.
[0094] The phrases "short interfering nucleic acid," "siNA," "siNA
molecule," "short interfering RNA," "siRNA," "short interfering
nucleic acid molecule," "short interfering oligonucleotide
molecule," or "chemically modified short interfering nucleic acid
molecule" refer to any nucleic acid molecule capable of inhibiting
or down regulating gene expression or viral replication by
mediating RNA interference ("RNAi") in a sequence-specific manner.
These terms can refer to both individual nucleic acid molecules, a
plurality of such nucleic acid molecules, or pools of such nucleic
acid molecules. The siNA can be a symmetric or asymmetric
double-stranded nucleic acid molecule comprising self-complementary
sense and antisense strands or regions, wherein the antisense
strand/region comprises a nucleotide sequence that is complementary
to a nucleotide sequence in a target nucleic acid molecule or a
portion thereof, and the sense strand/region comprises a nucleotide
sequence corresponding to the target nucleic acid sequence or a
portion thereof. A symmetric duplex refers to an siNA molecule
comprising sense and antisense regions each comprising the same
number of nucleotides. An asymmetric duplex refers to an siNA
molecule comprising an antisense region and a sense region that
comprises fewer nucleotides than the antisense region, to the
extent that the sense region has enough complementary nucleotides
to base pair with the antisense region to form a duplex. For
example, an asymmetric double-stranded siNA molecule can comprise
an antisense region having length sufficient to mediate RNAi in a
cell or in vitro system, e.g. about 15 to about 30, and a sense
region having about 3 to about 25 nucleotides that are
complementary to the antisense region. As an example, an asymmetric
double-stranded hairpin siNA molecule can also comprise a loop
region comprising about 4 to about 12 nucleotides. The loop portion
of an asymmetric hairpin siNA molecule can comprise nucleotides,
non-nucleotides, linker molecules, or conjugate molecules as
described herein. An siNA molecule can also comprise a
single-stranded polynucleotide having a nucleotide sequence
complementary to a portion of a nucleotide sequence in a target
nucleic acid molecule (for example, where such siNA molecule does
not require the presence within the siNA molecule of a nucleotide
sequence corresponding to the target nucleic acid sequence or a
portion thereof). A single-stranded siNA molecule is an RNAi
molecule, functioning through an RNAi mechanism.
[0095] The term "subject" as used herein refers to its meaning as
is generally accepted in the art. As used herein, term generally
refers to an organism to which the siNA conjugates as described and
compositions thereof can be administered. The term "subject" is
intended to include human and non-human animals. Non-human animals
include all vertebrates, e.g. mammals and non-mammals, such as
non-human primates, sheep, dogs, cats, cows, horses, chickens,
amphibians, rabbits, hamsters, guinea pigs, livestock and reptiles.
A subject can be an organism that has been previously identified as
a suitable candidate for administration of the siNA conjugates as
per the methods of the invention. For example, a subject can be a
mammal, such as a human, diagnosed with a musculoskeletal disease,
wherein it is believed that treatment with the siNA conjugates
described herein has potential of resulting in a positive clinical
outcome. The term "subject" is also intended to include an embryo,
including a chicken embryo contained within an egg.
[0096] The term "sugar moiety" means a natural or modified sugar
ring or sugar surrogate.
[0097] The term "sugar surrogate" generally refers to a structure
that is capable of replacing the furanose ring of a naturally
occurring nucleotide. In certain embodiments, sugar surrogates are
non-furanose (or 4'-substituted furanose) rings or ring systems or
open systems. Such structures include simple changes relative to
the natural furanose ring, such as a 6-membered ring or may be more
complicated as is the case with the non-ring system used in peptide
nucleic acid. Sugar surrogates includes without limitation
morpholinos, cyclohexenyls and cyclohexitols. In most nucleotides
having a sugar surrogate group, the heterocyclic base moiety is
generally maintained to permit hybridization.
[0098] The phrase "systemic administration" as used herein refers
to its meaning as is generally accepted in the art. The term
generally refers to methods or techniques of administering a
molecule, drug, agent or compound in a manner resulting in in vivo
systemic absorption or accumulation of drugs in the blood stream
followed by distribution throughout the entire body. Systemic
administration includes in ovo administration.
[0099] The term "target" cellular protein, peptide, or polypeptide,
or polynucleotide or nucleic acid (such as "target DNA," "target
RNA," "target nucleic acid"), as used herein, refers to a protein
or nucleic acid, respectively, of which an siNA molecule may be
capable of inhibiting or down regulating the expression. In certain
embodiments, target RNA is mRNA, pre-mRNA, non-coding RNA,
pri-microRNA, pre-microRNA, mature microRNA, promoter-directed RNA,
or natural antisense transcripts. As used herein, "target mRNA"
refers to a pre-selected RNA molecule that encodes a protein. As
used herein, "target pre-mRNA" refers to a pre-selected RNA
transcript that has not been fully processed into mRNA. Notably,
pre-RNA includes one or more intron. As used herein, "target
microRNA" refers to a pre-selected non-coding RNA molecule about
18-30 nucleobases in length that modulates expression of one or
more proteins or to a precursor of such a non-coding molecule. As
used herein, "target non-coding RNA" refers to a pre-selected RNA
molecule that is not translated to generate a protein. Certain
non-coding RNA is involved in regulation of expression.
[0100] The phrases "target site," "target sequence" and "target
nucleic acid site" as used herein refer to their meanings as
generally accepted in the art. The term generally refers to a
sequence within a target nucleic acid (e.g., RNA) that is
"targeted," e.g., for cleavage mediated by an siNA molecule that
contains sequences within its antisense region that are
complementary to the target sequence.
[0101] The phrase "universal base" as used herein refers to its
meaning as is generally accepted in the art. The term universal
base generally refers to nucleotide base analogs that form base
pairs with each of the natural DNA/RNA bases with little or no
discrimination between them. Non-limiting examples of universal
bases include C-phenyl, C-naphthyl and other aromatic derivatives,
inosine, azole carboxamides, and nitroazole derivatives such as
3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as
known in the art (see, for example, Loakes, 2001, Nucleic Acids
Research, 29, 2437-2447).
[0102] The term "up-regulate" as used herein refers to its meaning
as is generally accepted in the art. With reference nucleic acid
molecules described herein, the term refers to an increase in
either the expression of a gene, or the level of RNA molecules or
equivalent RNA molecules encoding one or more proteins or protein
subunits, or the activity of one or more RNAs, proteins or protein
subunits, above that observed in the absence of the nucleic acid
molecules (e.g., siNA) of the invention. In certain instances,
up-regulation or promotion of gene expression with an siNA molecule
is above that level observed in the presence of an inactive or
attenuated molecule. In other instances, up-regulation or promotion
of gene expression with siNA molecules is above that level observed
in the presence of, for example, an siNA molecule with scrambled
sequence or with mismatches. In still other instances,
up-regulation or promotion of gene expression with a nucleic acid
molecule of the instant invention is greater in the presence of the
nucleic acid molecule than in its absence. In some instances,
up-regulation or promotion of gene expression is associated with
inhibition of RNA mediated gene silencing, such as RNAi mediated
cleavage or silencing of a coding or non-coding RNA target that
down-regulates, inhibits, or silences the expression of the gene of
interest to be up-regulated. The down-regulation of gene expression
can, for example, be induced by a coding RNA or its encoded
protein, such as through negative feedback or antagonistic effects.
The down-regulation of gene expression can, for example, be induced
by a non-coding RNA having regulatory control over a gene of
interest, for example by silencing expression of the gene via
translational inhibition, chromatin structure, methylation, RISC
mediated RNA cleavage, or translational inhibition. As such,
inhibition or down-regulation of targets that down-regulate,
suppress, or silence a gene of interest can be used to up-regulate
expression of the gene of interest toward therapeutic use.
[0103] The term "vector" as used herein refers to its meaning as is
generally accepted in the art. The term vector generally refers to
any nucleic acid- and/or viral-based expression system or technique
used to deliver one or more nucleic acid molecules.
B. Myostatin siNA Molecules
[0104] Myostatin is a known growth factor involved in regulation of
muscle growth. In particular, myostatin is a member of the
TGF-.beta. family of growth factors and is a potent negative
regulator of myogenesis. Knock-out mice for myostatin have greatly
increased muscle mass over their entire body, having approximately
30% greater body weight than normal mice, and exhibiting a 2 to 3
fold increase in individual muscle weights due to muscle fiber
hyperplasia and hypertrophy. Natural mutations in myostatin have
been identified as being responsible for the "double-muscled"
phenotype, such as the Belgian Blue and Piedmontese cattle breeds.
See McPherron, A. C. et al, 1997, Nature 387:83-92; McPherron, A.
C. et al., 1997, Proc. Natl. Acad. Sci. USA 94:12457-12461;
Kambadur, R. et al., 1997, Genome Res. 7:910-916; Grobet, L. et
al., 1997, Nat. Genet. 17:71-74).
[0105] The siNA molecules delivered by the methods of the present
invention are designed to target a myostatin gene. The siNA
molecules may be directed to a myostatin gene sequence derived from
an array of suitable animals, including for example human, cattle,
pigs, fowl or mouse. For example, a myostatin siNA molecule
delivered by the methods of the present invention may be designed
to target a myostatin mRNA as set forth in Table 1:
TABLE-US-00005 TABLE 1 NCBI GenBank Species Accession No. Bos
taurus GQ184147 Bos indicius AY794986 Home sapien AF104922 Sus
scrofa AY448008 Equus caballus AB033541 Gallus gallus AY448007
Meleagris gallopavo AF019625 Ovis aries AM992883 Capra hircus
GQ246167 Macaca fascicularis AY055750 Mus musculus NM_010834
[0106] The instant invention features single- or double-stranded
siNA molecules that target a myostatin gene, lipophilic conjugates
thereof, and methods of delivering and using the same in vivo,
wherein said delivered siNA molecules are capable of mediating RNA
interference. The antisense strand (or guide strand) of the siNA
portion of a myostatin siNA conjugate is complementary to a
myostatin target nucleic acid. The siNA molecule portion of the
conjugates can take different oligonucleotide forms, including but
not limited to short interfering RNA (siRNA), double-stranded RNA
(dsRNA), micro-RNA (miRNA) and short hairpin RNA (shRNA) molecules.
In certain embodiments, the siNA molecule is single-stranded. In
other embodiments, the siNA molecule is double-stranded molecules,
wherein said double-stranded molecule comprises an antisense strand
and a sense strand. The myostatin siNA molecules comprised within
the myostatin siNA conjugates modulate expression of a myostatin
target nucleic acid. In one embodiment, the siNA molecules inhibit
or reduce expression of a myostatin target nucleic acid. In one
aspect, the siNA molecule portion of the myostatin siNA conjugates
is single-stranded, wherein the single oligonucleotide strand
comprises a sequence that is complementary to at least a part of a
myostatin nucleic acid associated with myostatin gene expression.
For purposes of this disclosure, the single strand of a
single-stranded siNA molecule is referred to as the antisense
strand.
[0107] In another aspect, the siNA molecule portion of the
myostatin siNA conjugates is a double-stranded siNA molecule,
wherein a double-stranded siNA molecule comprises a sense and an
antisense oligonucleotide strand. The antisense strand comprises a
sequence that is complementary to at least a part of a myostatin
target nucleic acid associated with myostatin gene expression, and
the sense strand is complementary to the antisense strand. The
double-stranded siNA molecules can comprise two distinct and
separate strands that can be symmetric or asymmetric and are
complementary, i.e., two single-stranded oligonucleotides, or can
comprise one single-stranded oligonucleotide in which two
complementary portions, e.g., a sense region and an antisense
region (which, in this context, will be referred to herein as a
sense strand and an antisense strand, respectively), are
base-paired, and are covalently linked by one or more
single-stranded "hairpin" areas (i.e. loops) resulting in, for
example, a short-hairpin polynucleotide. The linker can be a
polynucleotide linker or a non-nucleotide linker. In some
embodiments, the linker is a non-nucleotide linker. In some
embodiments, a hairpin siNA molecule contains one or more loop
motifs, wherein at least one of the loop portions of the siNA
molecule is biodegradable. For example, a short hairpin siNA
molecule can be designed such that degradation of the loop portion
of the siNA molecule in vivo can generate a double-stranded siNA
molecule with 3'-terminal overhangs, such as 3'-terminal nucleotide
overhangs comprising 1, 2, 3 or 4 nucleotides.
[0108] The antisense strand of the siNA molecule portion of the
described conjugates is complementary to a portion of a myostatin
target nucleic acid sequence. In some embodiments, the target
nucleic acid is selected from a myostatin target mRNA, a myostatin
target pre-mRNA, a myostatin target microRNA, and a myostatin
target non-coding RNA. In certain embodiments, the antisense strand
of the siNA molecule comprises a region that is 100%
complementarity to a myostatin target nucleic acid sequence and
wherein the region of 100% complementarity is at least 10
nucleobases. In certain embodiments, the region of 100%
complementarity is at least 15 nucleobases. In certain embodiments,
the region of 100% complementarity is at least 20 nucleobases. In
certain embodiments, the region of 100% complementarity is at least
25 nucleobases. In certain embodiments, the region of 100%
complementarity is at least 30 nucleobases. In certain embodiments,
the antisense strand of the siNA molecule is at least 85%
complementary to a myostatin target nucleic acid sequence. In
certain embodiments, the antisense strand is at least 90%
complementary to a myostatin target nucleic acid sequence. In
certain embodiments, the antisense strand is at least 95%
complementary to a myostatin target nucleic acid sequence. In
certain embodiments, the antisense strand is at least 98%
complementary to a myostatin target nucleic acid sequence. In
certain embodiments, the antisense strand is 100% complementary to
a myostatin target nucleic acid sequence. The complementary
nucleotides may or may not be contiguous nucleotides. In one
embodiment, the complementary nucleotides are contiguous
nucleotides.
[0109] In certain embodiments, the siNA molecule portion of the
myostatin siNA conjugates that are administered in vivo as per the
methods of the present invention have between about 15 to about 30
(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30) nucleotides in the antisense strand that are
complementary to a nucleotide sequence of a myostatin target
nucleic acid. In certain embodiments, the siNA molecules of the
invention comprise an antisense strand having at least 15
nucleotides having sequence complementarity to a myostatin target
sequence. In certain embodiments, the siNA molecules of the
invention comprise an antisense strand having at least 18
nucleotides having sequence complementarity to a myostatin target
sequence. In certain embodiments, the siNA molecules of the
invention comprise an antisense strand having at least 19
nucleotides having sequence complementarity to a myostatin target
sequence. In certain embodiments, the siNA molecules of the
invention comprise an antisense strand having at least 20
nucleotides having sequence complementarity to a myostatin target
sequence. In certain embodiments, the siNA molecules of the
invention comprise an antisense strand having at least 21
nucleotides having sequence complementarity to a myostatin target
sequence. In certain embodiments of this aspect of the invention,
the complementary nucleotides are contiguous nucleotides.
[0110] In some embodiments, a double-stranded siNA molecule
comprised with the myostatin siNA conjugates described herein has
perfect complementarity between the sense strand or sense region
and the antisense strand or antisense region of the siNA molecule,
with the exception of any overhanging region.
[0111] In yet other embodiments, a double-stranded siNA molecule
has partial complementarity (i.e., less than 100% complementarity)
between the sense strand or sense region and the antisense strand
or antisense region of the siNA molecule. Thus, in some
embodiments, the double-stranded nucleic acid molecules have
between about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in one
strand (e.g., sense strand) that are complementary to the
nucleotides of the other strand (e.g., antisense strand). In
certain embodiments, the double-stranded siNA molecules have 17
nucleotides in the sense region that are complementary to
nucleotides of the antisense region of the molecule. In certain
embodiments, the double-stranded siNA molecules have 18 nucleotides
in the sense region that are complementary to nucleotides of the
antisense region of the molecule. In certain embodiments, the
double-stranded siNA molecules have 19 nucleotides in the sense
region that are complementary to nucleotides of the antisense
region of the molecule. In certain embodiments, the double-stranded
siNA molecules of the invention have 20 nucleotides in the sense
region that are complementary to nucleotides of the antisense
region of the molecule. In certain embodiments of this aspect of
the invention, the complementary nucleotides between the strands
are contiguous nucleotides.
[0112] For siNA molecules that are symmetric, each strand, the
sense (passenger) strand and antisense (guide) strand, are
independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in
length. Generally, each strand of a symmetric siNA molecule is
about 19-24 (e.g., about 19, 20, 21, 22, 23 or 24) nucleotides in
length. In certain embodiments, each strand of a symmetric siNA
molecule is 19 nucleotides in length. In certain embodiments, each
strand of a symmetric siNA molecule is 20 nucleotides in length. In
certain embodiments, each strand of a symmetric siNA molecule is 21
nucleotides in length. In certain embodiments, each strand of a
symmetric siNA molecule is 22 nucleotides in length.
[0113] For siNA molecules that are asymmetric, the antisense strand
of the molecule is about 15 to about 30 (e.g., about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides
in length, wherein the sense region is about 3 to about 25 (e.g.,
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25) nucleotides in length. Generally, the
antisense strand of an asymmetric siNA molecule is about 19-24
(e.g., about 19, 20, 21, 22, 23 or 24) nucleotides in length. In
one embodiment, the sense strand of an asymmetric siNA molecule is
about 19-24 (e.g., about 19, 20, 21, 22, 23 or 24) nucleotides in
length.
[0114] In yet other embodiments, siNA molecules comprised with the
myostatin siNA conjugates described herein are hairpin siNA
molecules, wherein the siNA molecules are about 25 to about 70
(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 40,
45, 50, 55, 60, 65, or 70) nucleotides in length.
[0115] In certain embodiments, siNA molecules comprised with the
myostatin siNA conjugates described herein are microRNA mimetics,
having a nucleotide sequence comprising a nucleotide portion that
is fully or partially identical to a seed region of a
myostatin-related microRNA. In certain embodiments, the nucleotide
sequence of a microRNA mimetic has a nucleotide portion that is
100% identical to a seed region of a myostatin-related microRNA. In
certain embodiments, the nucleotide sequence of a microRNA mimetic
has a nucleotide portion that is at least 75% identical (e.g.,
about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) to a seed
region of a myostatin-related microRNA. In certain embodiments, the
nucleotide sequence of a myostatin-related microRNA mimetic has a
nucleotide portion that is 75% identical to a seed region of a
myostatin-related microRNA. In certain embodiments, the nucleotide
sequence of a microRNA mimetic has a nucleotide portion that is 80%
identical to a seed region of a myostatin-related microRNA. In
certain embodiments, the nucleotide sequence of a microRNA mimetic
has a nucleotide portion that is 90% identical to a seed region of
a myostatin-related microRNA. In certain embodiments, the
nucleotide sequence of a microRNA mimetic has a nucleotide portion
that is 95% identical to a seed region of a myostatin-related
microRNA.
[0116] In other embodiments, siNA molecules comprised within the
myostatin siNA conjugates described herein can contain one or more
nucleotide deletions, substitutions, mismatches and/or additions
(in reference to a myostatin target site sequence, or between
strands of a duplex siNA molecule); provided, however, that the
siNA molecule maintains its activity, for example, to mediate RNAi.
In a non-limiting example, the deletion, substitution, mismatch
and/or addition can result in a loop or bulge, or alternately a
wobble or other alternative (non Watson-Crick) base pair. Thus, in
some embodiments, for example, double-stranded nucleic acid siNA
molecules have one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides
in one strand or region (e.g., sense strand) that are mismatches or
non-base-paired with the other strand or region (e.g., antisense
strand). In certain embodiments, the siNA molecules contain no more
than 3 mismatches. If the antisense strand of an siNA molecule
contains mismatches to a myostatin target sequence, it is
preferable that the area of mismatch is not located in the center
of a contiguous region of complementarity.
[0117] In certain embodiments, the siNA molecules comprised with
the conjugates described herein comprise overhangs of about 1 to
about 4 (e.g., about 1, 2, 3 or 4) nucleotides. The nucleotides in
the overhangs can be the same or different nucleotides. In some
embodiments, the overhangs occur at the 3'-end (or the 3' terminus)
of one or both strands of double-stranded siNA molecules. For
example, a double-stranded siNA molecule can comprise a nucleotide
or non-nucleotide overhang at the 3'-end of the antisense
strand/region, at the 3'-end of the sense strand/region, or at the
3' ends of both the antisense strand/region and the sense
strand/region. Overhanging nucleotides can be modified or
unmodified.
[0118] In some embodiments, the nucleotides comprising the
overhanging portion of an siNA molecule comprise sequences based on
a myostatin target nucleic acid sequence in which the nucleotides
comprising the overhanging portion of the antisense strand/region
are complementary to nucleotides in the myostatin target
polynucleotide sequence and/or the nucleotides comprising the
overhanging portion of the sense strand/region comprise nucleotides
from the myostatin target polynucleotide sequence. Thus, in some
embodiments, the overhang comprises a two nucleotide overhang that
is complementary to a portion of the myostatin target
polynucleotide sequence. In other embodiments, however, the
overhang comprises a two nucleotide overhang that is not
complementary to a portion of the myostatin target nucleic acid
sequence. In certain embodiments, the overhang comprises a 3'-UU
overhang that is not complementary to a portion of the myostatin
target nucleic acid sequence. In other embodiments, the overhang
comprises a UU overhang at the 3' end of the antisense strand and a
TT overhang at the 3' end of the sense strand.
[0119] In any of the embodiments of the siNA molecules described
herein having 3'-end nucleotide overhangs, the overhangs are
optionally chemically-modified at one or more nucleic acid sugar,
base, or backbone positions. Representative, but not limiting
examples of modified nucleotides in the overhanging portion of a
double-stranded siNA molecule include the following: 2'-O-alkyl
(e.g., 2'-O-methyl), 2'-deoxy, 2'-deoxy-2'-fluoro,
2'-deoxy-2'-fluoroarabino (FANA), 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, universal
base, acyclic, or 5-C-methyl nucleotides. In more preferred
embodiments, the overhang nucleotides are each independently, a
2'-O-alkyl nucleotide, a 2'-O-methyl nucleotide, a
2'-deoxy-2-fluoro nucleotide, or a 2'-deoxy ribonucleotide. In some
instances the overhanging nucleotides are linked by one or more
phosphorothioate linkages.
[0120] In yet other embodiments, siNA molecules comprised within
the myostatin siNA conjugates described herein comprise duplex
nucleic acid molecules with blunt ends (i.e., without nucleotide
overhangs), where both termini of the molecule are blunt, or
alternatively, where one of the ends is blunt. In some embodiments,
the siNA molecules comprise one blunt end, for example wherein the
5'-end of the antisense strand and the 3'-end of the sense strand
do not have any overhanging nucleotides, or wherein the 3'-end of
the antisense strand and the 5'-end of the sense strand do not have
any overhanging nucleotides. In other embodiments, siNA molecules
comprise two blunt ends, for example wherein the 3'-end of the
antisense strand and the 5'-end of the sense strand, as well as the
5'-end of the antisense strand and 3'-end of the sense strand, do
not have any overhanging nucleotides.
[0121] In any of the embodiments or aspects of the siNA molecules
comprised within the myostatin siNA conjugates described herein,
the sense strand and/or the antisense strand can further have a
cap, such as described herein or as known in the art. A cap can be
present at the 3'-end of the antisense strand, the 5'-end of the
sense strand, and/or the 3'-end of the sense strand. In the case of
a hairpin siNA molecule, a cap can be present at the 3'-end of the
polynucleotide. In some embodiments, a cap is at one or both ends
of the sense strand of a double-stranded siNA molecule. In other
embodiments, a cap is at the 3'-end of antisense (guide) strand. In
other embodiments, a cap is at the 3'-end of the sense strand and
at the 5'-end of the sense strand. Representative but non-limiting
examples of such terminal caps include an inverted abasic
nucleotide and derivatives thereof, an inverted nucleotide moiety,
a glyceryl modification, an alkyl or cycloalkyl group, a
heterocycle or any other cap as is generally known in the art.
[0122] Any of the embodiments of the siNA molecules described
herein can have a 5' phosphate terminus. In some embodiments, the
siNA molecules lack terminal phosphates.
[0123] In certain embodiments, double-stranded siNA molecules
comprised within the myostatin siNA conjugates described herein
comprise about 3 to about 30 (e.g., about 3, 4, 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) base pairs. Generally, the duplex structure of siNA
molecules is between 15 and 30 base pairs, more generally between
18 and 25 base pairs, yet more generally between 19 and 24 base
pairs, and most generally between 19 and 21 base pairs in length.
In one embodiment, a double-stranded siNA molecule comprises 19
base pairs. In one embodiment, a double-stranded siNA molecule
comprises 20 base pairs. In one embodiment, a double-stranded siNA
molecule comprises 21 base pairs. The double-stranded siNA
molecules can be asymmetric or symmetric. In other embodiments of
this aspect of the invention, the siNA duplex molecules are hairpin
structures.
[0124] Any siNA molecule can comprise one or more
chemically-modified nucleotides. Modifications can be used to
improve in vitro or in vivo characteristics such as stability,
activity, toxicity, immune response (e.g., prevent stimulation of
an interferon response, an inflammatory or pro-inflammatory
cytokine response, or a Toll-like Receptor response), and/or
bioavailability. Various chemically modified siNA motifs disclosed
herein have the potential to maintain an RNAi activity that is
substantially similar to either unmodified or minimally-modified
active siRNA (see for example Elbashir et al., 2001, EMBO J.,
20:6877-6888) while, at the same time, providing nuclease
resistance and pharmacokinetic properties suitable for use in
therapeutic applications.
[0125] In certain embodiments of the siNA molecules comprised with
the myostatin siNA conjugates used by the methods of the present
invention, any (e.g., one, more or all) nucleotides present in the
antisense and/or sense strand may be modified nucleotides (e.g.,
wherein one nucleotide is modified, some nucleotides (i.e., a
plurality or more than one) are modified, or all nucleotides of the
molecule are modified nucleotides). Modifications include sugar
modifications, base modifications, backbone (internucleoside
linkage) modifications, non-nucleotide modifications, and/or any
combination thereof.
[0126] Non-limiting examples of chemical modifications that are
suitable for use in the siNA molecule portion of the conjugates
described herein are disclosed in U.S. Pat. No. 8,202,979 and U.S.
patent application Ser. Nos. 10/981,966 and 12/064,014 (published
as US 20050266422 and US 20090176725, respectively), and in
references cited therein, and include sugar, base, and backbone
modifications, non-nucleotide modifications, and/or any combination
thereof. These U.S. patents and applications are incorporated
hereby as references for the purpose of describing chemical
modifications that are suitable for use with the siNA
molecules.
[0127] The chemical modifications of nucleotides present within a
single siNA molecule can be the same or different. In some
embodiments, at least one strand of an siNA molecule has at least
one chemical modification. In other embodiments, each strand has at
least one chemical modification, which can be the same or
different, such as sugar, base, or backbone (i.e., internucleotide
linkage) modifications. In other embodiments, siNA molecules
contain at least 2, 3, 4, 5, or more different chemical
modifications.
[0128] In some embodiments, the siNA molecules comprised within the
myostatin siNA conjugates administered as per the methods of the
present invention are partially modified (e.g., about 1, 2, 3, 4,
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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, or 59 nucleotides
are modified) with chemical modifications. In some embodiments, an
siNA molecule comprises at least about 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, or 60 nucleotides that are modified nucleotides, excluding
the 5' modified nucleotide of the antisense strand. In other
embodiments, the siNA molecules are completely modified (100%
modified) with chemical modifications, i.e., the siNA molecule does
not contain any ribonucleotides. In some embodiments, one or more
of the nucleotides in the sense strand of the siNA molecules are
modified. In the same or other embodiments, one or more of the
nucleotides in the antisense strand of the siNA molecules are
modified, excluding the 5' modified nucleotide of the antisense
strand. In some embodiments, one or more (e.g., 1, 2, 3, 4, 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) of the nucleotide positions
independently in either one or both strands of an siNA molecule are
modified.
[0129] Modified nucleotides contained within the siNA molecules
include those with modifications at the 2'-carbon of a sugar moiety
and/or the 3'-carbon of a sugar moiety of a nucleotide. In certain
specific embodiments of the invention, at least one modified
nucleotide is a 2'-deoxy-2-fluoro nucleotide, a 2'-deoxy
nucleotide, a 2'-O-alkyl (e.g., 2'-O-methyl) nucleotide, a
2'-methoxyethoxy or a locked nucleic acid (LNA) nucleotide, as is
generally recognized in the art.
[0130] In yet other embodiment of the invention, at least one
nucleotide has a ribo-like, Northern or A form helix configuration
(see e.g., Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag ed., 1984). Non-limiting examples of nucleotides
having a Northern configuration include locked nucleic acid (LNA)
nucleotides (e.g., 2'-0, 4'-C-methylene-(D-ribofuranosyl)
nucleotides); 2'-methoxyethoxy (MOE) nucleotides;
2'-methyl-thio-ethyl nucleotides; 2'-deoxy-2'-fluoro nucleotides;
2'-deoxy-2'-chloro nucleotides; 2'-azido nucleotides;
2'-O-trifluoromethyl nucleotides; 2'-O-ethyl-trifluoromethoxy
nucleotides; 2'-O-difluoromethoxy-ethoxy nucleotides; 4'-thio
nucleotides; and 2'-O-methyl nucleotides. In various embodiments, a
majority (e.g., greater than 50%) of the pyrimidine nucleotides
present in a double-stranded siNA molecule comprises a sugar
modification. In some of the same and/or other embodiments, a
majority (e.g., greater than 50%) of the purine nucleotides present
in a double-stranded siNA molecule comprises a sugar
modification.
[0131] In certain instances, purine and pyrimidine nucleotides of
an siNA molecule are differentially modified. In one example,
purine and pyrimidine nucleotides can be differentially modified at
the 2'-carbon of the sugar moiety (i.e., at least one purine has a
different modification from at least one pyrimidine in the same or
different strand at the 2'-carbon of the sugar moiety). In certain
embodiments, the purines are unmodified in one or both strands,
while the pyrimidines in one or both strands are modified. In
certain other instances, the pyrimidines are unmodified in one or
both strands, while the purines in one or both strands are
modified. In certain instances, wherein the siNA molecules comprise
one or more modifications as described herein, the nucleotides at
positions 2 and 3 at the 5' end of the antisense (guide) strand are
unmodified.
[0132] In some embodiments of the siNA molecules, the pyrimidine
nucleotides in the antisense strand are 2'-O-methyl or
2'-deoxy-2'-fluoro pyrimidine nucleotides, and the purine
nucleotides present in the antisense strand are 2'-O-methyl
nucleotides or 2'-deoxy nucleotides. In certain embodiments, all of
the pyrimidine nucleotides in a complementary region of an
antisense strand of an siNA molecule are 2'-deoxy-2'-fluoro
pyrimidine nucleotides. In certain embodiments, all of the purines
in the complementary region on the antisense strand are 2'-O-methyl
purine nucleotides.
[0133] In other embodiments of the siNA molecules, the pyrimidine
nucleotides in the sense strand are 2'-deoxy-2'-fluoro pyrimidine
nucleotides, and the purine nucleotides present in the sense strand
are 2'-O-methyl or 2'-deoxy purine nucleotides. In certain
embodiments of the invention, all the pyrimidine nucleotides in the
complementary region on the sense strand are 2'-deoxy-2'-fluoro
pyrimidine nucleotides. In certain embodiments, all the purine
nucleotides in the complementary region on the sense strand are
2'-deoxy purine nucleotides.
[0134] In certain embodiments, all of the pyrimidine nucleotides in
the complementary regions on the sense strand are
2'-deoxy-2'-fluoro pyrimidine nucleotides; all of the pyrimidine
nucleotides in the complementary region of the antisense strand are
2'-deoxy-2'-fluoro pyrimidine nucleotides; all the purine
nucleotides in the complementary region on the sense strand are
2'-deoxy purine nucleotides and all of the purines in the
complementary region on the antisense strand are 2'-O-methyl purine
nucleotides.
[0135] In some embodiments, at least 5 or more of the pyrimidine
nucleotides in one or both strands of an siNA molecule are
2'-deoxy-2'-fluoro pyrimidine nucleotides. In some embodiments, at
least 5 or more of the pyrimidine nucleotides in one or both
strands are 2'-O-methyl pyrimidine nucleotides. In some
embodiments, at least 5 or more of the purine nucleotides in one or
both strands are 2'-deoxy-2'-fluoro purine nucleotides In some
embodiments, at least 5 or more of the purine nucleotides in one or
both strands are 2'-O-methyl purine nucleotides.
[0136] In certain embodiments, the siNA molecules comprise one or
more modified internucleoside linking group. A modified
internucleoside linking group is a linking group other than a
phosphodiester 3'-5' internucleoside linking group, including but
not limited to 2' internucleoside linking groups (e.g.,
phosphodiester and phosphorothioate 2'-5' internucleoside
linkages). In certain embodiments, each internucleoside linking
group is, independently, a 2' or 3' phosphodiester or
phosphorothioate internucleoside linking group. In certain
embodiments, the 5'-most internucleoside linking group on either or
both strands of an siNA molecule is a phosphorothioate linking
group. In certain embodiments, the siNA molecules comprise from 3
to 12 contiguous phosphorothioate linking groups, wherein the
phosphorothioate linking groups are either 2' or 3' internucleoside
linking groups. In certain embodiments, the siNA molecules comprise
from 6 to 8 contiguous phosphorothioate linking groups, wherein the
phosphorothioate linking groups are either 2' or 3' internucleoside
linking groups. In certain embodiments, the 3' end of the antisense
and/or sense strand of the siNA molecules comprises a
phosphorothioate linking groups. In certain embodiments, the siNA
molecules comprise from 6 to 8 contiguous phosphorothioate linking
groups at the 3' end of the antisense and/or sense strand, wherein
the phosphorothioate linking groups are either 2' or 3'
internucleoside linking groups.
[0137] Any of the above described modifications, or combinations
thereof, including those in the references cited, can be applied to
any of the siNA molecules comprised within the myostatin siNA
conjugates that are administered as per the methods and uses of the
present invention.
[0138] The myostatin siNA molecules can be obtained using a number
of techniques known to those of skill in the art. For example the
siNA molecules can be chemically synthesized using protocols known
in the art (for example, as described in: Caruthers et al., 1992,
Methods in Enzymology 211, 3-19; Thompson et al., International PCT
Publication No. WO 99/54459; Wincott et al., 1995, Nucleic Acids
Res. 23, 2677-2684; Wincott et al., 1997, Methods Mol. Bio., 74,
59; Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45; Brennan,
U.S. Pat. No. 6,001,311; Usman et al., 1987, J. Am. Chem. Soc.,
109, 7845; and Scaringe et al., 1990, Nucleic Acids Res., 18,
5433). The syntheses of oligonucleotides described in the art makes
use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end and phosphoramidites at the 3'- or
2'-end.
[0139] In certain embodiments, the siNA molecules are synthesized,
deprotected, and analyzed according to methods described in, for
example, U.S. Pat. Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751,
6,989,442, and 7,205,399. In a non-limiting synthesis example,
small scale syntheses are conducted on a 394 Applied Biosystems,
Inc. synthesizer using a 0.2 .mu.mol scale protocol with a 2.5 min
coupling step for 2'-O-methylated nucleotides and a 45 second
coupling step for 2'-deoxy nucleotides or 2'-deoxy-2'-fluoro
nucleotides.
[0140] Alternatively, the siNA molecules can be synthesized
separately and joined together post-synthetically, for example, by
ligation (e.g., Moore et al., 1992, Science 256, 9923; Draper et
al., International PCT Publication No. WO 93/23569; Shabarova et
al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997,
Nucleosides & Nucleotides, 16, 951; and Bellon et al., 1997,
Bioconjugate Chem. 8, 204), or by hybridization following synthesis
and/or deprotection.
C. Myostatin siNA Conjugates
[0141] The present invention provides myostatin siNA molecules
capable of mediating RNA interference and reducing the in vivo
expression of myostatin and methods for delivering the same to a
subject, wherein the siNA molecules that are delivered as per the
methods disclosed herein are linked to a lipophilic moiety, such as
cholesterol. The lipophilic moiety-linked myostatin siNA molecules
are referred to herein as myostatin siNA conjugates. In one
embodiment, the myostatin siNA conjugates delivered by the methods
of the present invention are not formulated within lipid
formulations that form liposomes (e.g., a lipid nanoparticle).
While not wishing to be bound by any particular theory, it is
believed the attachment of a lipophilic moiety increases the
lipophilicity of the myostatin siNA molecule, enhancing the entry
of the siNA molecule into muscle cells.
[0142] Examples of lipophilic moieties that can be linked to a
myostatin siNA molecule to form a myostatin siNA conjugate include,
but are not limited to cholesterol, oleic acid, stearic acid,
palmitic acid, myristic acid, linoleic acid, oleyl, retinyl,
cholesteryl residues, cholic acid, adamantane acetic acid, 1-pyrene
butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol,
geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,
1,3-propanediol, heptadecyl group, O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In a
preferred embodiment, the linked lipophilic moiety is
cholesterol.
[0143] In certain embodiments, the lipophilic moiety is attached
directly to the siNA molecule. In these embodiments, the lipophilic
moiety is still considered, for the purposes of the present
invention, to be "linked" or "conjugated" to the siNA molecule. In
certain embodiments, the lipophilic moiety is attached to the siNA
molecule by means of a conventional linker or spacer molecule. The
linker or spacer can be a nucleic acid or non-nucleic acid
linker/spacer. A number of linker molecules are commercially
available. Suitable linkers include, but are not limited to,
straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers. Although a linker or spacer molecule
generally has no specific biological activity other than to join
the molecules being combined, or to preserve some minimum distance
or other spatial relationship between them, the constituent amino
acids of a peptide spacer may be selected to influence some
property of the molecule such as the folding, net charge, or
hydrophobicity.
[0144] The linker/spacer can be a nucleic acid linker that is
biodegradable. The stability of a nucleic acid-based biodegradable
linker molecule can be modulated by using various chemistries, for
example combinations of ribonucleotides, deoxyribonucleotides, and
chemically-modified nucleotides, such as 2'-O-methyl, 2'-fluoro,
2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl, and other 2'-modified
or base modified nucleotides. A biodegradable nucleic acid linker
molecule can be a dimer, trimer, tetramer or longer nucleic acid
molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides
in length, or can comprise a single nucleotide with a
phosphorus-based linkage, for example, a phosphoramidite or
phosphodiester linkage. A biodegradable nucleic acid linker
molecule can also comprise nucleic acid backbone, nucleic acid
sugar, or nucleic acid base modifications.
[0145] The lipophilic moiety is attached to a myostatin siNA
molecule through attachment to either a terminus of the siNA
molecule (e.g., the 3' or 5' end of an oligonucleotide strand of
the siNA molecule) or through linkage to an internal nucleotide of
the siNA molecule. In one embodiment, the lipophilic moiety is
attached to the 3' end of the passenger strand (sense strand) of a
double-stranded myostatin siNA molecule. In one embodiment, the
lipophilic moiety is attached to the 5' end of the passenger strand
of a double-stranded myostatin siNA molecule. In a further
embodiment, the lipophilic moiety is attached to the 3' end of the
guide strand (antisense strand) of a myostatin siNA molecule. In a
further embodiment, the lipophilic moiety is attached to the 5' end
of the guide strand (antisense strand) of a myostatin siNA
molecule. In a further embodiment, a myostatin siNA conjugate
contains more than one attached lipophilic moiety (e.g., a
lipophilic moiety attached to both the 3' and the 5' end of the
passenger strand; a lipophilic moiety attached to the 3' end of the
guide strand and the 5' end of the passenger strand). In this
aspect of the invention, the lipophilic moieties can be the same or
different.
[0146] In certain embodiments, a myostatin siNA conjugate is
prepared by chemically conjugating all or a portion of a myostatin
siNA molecule to the lipophilic group. Means of chemically
conjugating molecules are well known to those of skill in the art.
Such means will vary according to the structure of the moiety to be
attached, but will be readily ascertainable to those of skill in
the art.
[0147] The present invention further provides myostatin siNA
conjugates in kit form. The kit may comprise a container. In one
embodiment, the kit contains one or more myostatin siNA conjugate
with instructions for systemic administration. The kits may
comprise a myostatin siNA conjugate within a pharmaceutically
acceptable carrier or diluent. The kits may further comprise
excipients.
D. Uses
[0148] The methods for systemically administering myostatin siNA
conjugates described herein are useful to modulate and or regulate
(e.g., inhibit, down-regulate) the in vivo expression and/or
activity of a myostatin target nucleic acid (e.g., a myostatin
target gene) by an RNAi interference mechanism (e.g., by degrading
a myostatin mRNA). Modulation of the in vivo expression of a
myostatin target nucleic acid results increased muscle mass and/or
enhanced muscle performance. The methods may be further useful in
therapeutic regimens to treat one or more musculoskeletal disease
states. In one embodiment, inhibition of a disease may be evaluated
by directly measuring the progress of the disease in a subject. It
may also be inferred through observing a change or reversal in a
condition associated with the disease. The methods of the present
invention have the further potential of being used as a
prophylaxis. Thus, use of the myostatin siNA conjugates and
pharmaceutical compositions described herein have the potential of
ameliorating, treating, preventing, and/or curing diseases states
associated with regulation of myostatin gene expression. The
myostatin siNA conjugates further have the potential for use in
cosmetic applications and/or for veterinary purposes to increase
muscle mass and/or enhance muscle performance.
[0149] In an embodiment of the invention, the subject to which a
myostatin siNA conjugate described herein is systemically
administered is suffering from a musculoskeletal disease or
disorder. In one embodiment, a musculoskeletal disease or disorder
includes a condition that causes or results in muscle atrophy.
Muscle atrophy can result from treatment with a glucocorticoid such
as cortisol, dexamethasone, betamethasone, prednisone,
methylprednisolone or prednisolone. Muscle atrophy can also be a
result of denervation due to nerve trauma or a result of
degenerative, metabolic or inflammatory neuropathy. For example,
muscle atrophy can be a result of an adult motor neuron disease,
Guillian-Barre syndrome, infantile spinal muscular atrophy,
amyotrophic lateral sclerosis, juvenile spinal muscular atrophy,
autoimmune motor neuropathy with multifocal conductor block,
paralysis due to stroke or spinal cord injury, skeletal
immobilization due to trauma, prolonged bed rest, voluntary
inactivity, involuntary inactivity, and metabolic stress or
nutritional insufficiency. Muscle atrophy can be a result of
myopathy, including for example myotonia; a congenital myopathy,
including nemalene myopathy, multi/minicore myopathy and myotubular
(centronuclear) myopathy; mitochondrial myopathy; familial periodic
paralysis; inflammatory myopathy; metabolic myopathy, such as
caused by a glycogen or lipid storage disease; dermatomyositis;
polymyositis; inclusion body myositis; myositis ossificans;
rhabdomyolysis and myoglobinurias. Myopathy may be caused by a
muscular dystrophy syndrome, such as Duchenne muscular dystrophy
(DMD), Becker muscular dystrophy (also known as benign
pseudohypertrophic muscular dystrophy), myotonic dystrophy,
scapulohumeral and fascioscapulohumeral muscular dystrophy,
Emery-Dreifuss muscular dystrophy, oculopharyngeal muscular
dystrophy, limb girdle muscular dystrophy, Fukuyama congenital
muscular dystrophy, or hereditary distal myopathy.
[0150] Further examples musculoskeletal disease or disorder or
conditions that result in musculoskeletal disease or disorder
include sarcopenia, skin atrophy, muscle wasting, brain atrophy,
atherosclerosis, arteriosclerosis, pulmonary emphysema,
osteoporosis, osteoarthritis, immunologic incompetence, high blood
pressure, dementia, Huntington's disease, Alzheimer's disease,
cataracts, age-related macular degeneration, cancer, stroke,
frailty, memory loss, impaired kidney function, metabolic disorders
(including Type-II diabetes, metabolic syndrome, hyperglycemia,
obesity, thyroid gland disorder), cachexia (including cachexia
associated with a rheumatoid arthritis and cachexia associated with
cancer), acute and/or chronic renal disease or failure, liver
diseases (examples such as fibrosis, cirrhosis), cancer (including
rhabdomyosarcoma, prostate cancer, breast cancer, hepatocellular
carcinoma, and gastrointestinal cancer), Parkinson's Disease;
anemia, exposure to environmental toxins or drugs, HIV/AIDS,
fasting, benign congenital hypotonia, central core disease, burn
injury, chronic obstructive pulmonary disease, sepsis, congestive
heart failure, aging or an age-related condition, and space travel
or time spent in a zero gravity environment.
[0151] The myostatin siNA conjugates and pharmaceutical
formulations thereof can be administered to a subject alone or used
in combination with one or more other therapies, including known
therapeutic agents, treatments, or procedures to prevent or treat
musculoskeletal diseases, disorders, conditions, and traits. A
combination can conveniently be presented for use in the form of a
pharmaceutical composition, wherein the pharmaceutical composition
comprises a combination that includes a myostatin siNA conjugate, a
pharmaceutically acceptable diluent or carrier, and one or more
additional therapeutic agents. Alternatively, the individual
components of such combinations can be administered either
sequentially or simultaneously in separate or combined
pharmaceutical formulations.
[0152] Combinations of the methods of the invention with standard
medical treatments (e.g., corticosteroids for muscular dystrophies)
are specifically contemplated, as are combinations with novel
therapies. For example, for treatment of genetic muscular
dystrophies, methods of the invention may be combined with
follistatin administration, followed by simultaneous or concomitant
treatment to correct the genetic disorder. Correcting a genetic
disorder may involve, for example, replacing sarcoglycans in
sarcoglycan deficiency, correcting or replacing dystrophin in
disorders such as Duchenne's Muscular Dystrophy, treating ALS
patients with IGF-1 or mutant SOD1 interference strategies. Given
that in a disorder contemplated for treatment by the methods of the
present invention, a significant amount of muscle may be lost, the
rescue of muscle will provide a substrate (preserved or regenerated
muscle) for subsequent gene correction. In this respect, it may be
conceivable to inhibit myostatin to enhance muscle, increase muscle
size, and then provide the secondary treatment. Such secondary
treatments for muscular dystrophy may be IGF-1, exon-skipping,
calpain inhibition, dystrophin upregulation, and dystroglycan
expression. Myostatin inhibition in concert with muscle precursor
cells (satellite cells, stem cells) may allow more of these cells
to be incorporated into the tissue.
E. Pharmaceutical Compositions
[0153] The myostatin siNA conjugates of the invention are
preferably formulated as pharmaceutical compositions prior to
systemically administering to a subject, according to techniques
known in the art. Pharmaceutical compositions are characterized as
being at least sterile and pyrogen-free. Methods for preparing
pharmaceutical compositions are within the skill in the art for
example as described in Remington's Pharmaceutical Science,
17.sup.th ed., Mack Publishing Company, Easton, Pa. (1985).
[0154] Pharmaceutical compositions of the myostatin siNA conjugates
further comprise conventional pharmaceutical excipients and/or
additives. Suitable pharmaceutical excipients include
preservatives, flavoring agents, stabilizers, antioxidants,
osmolality adjusting agents, buffers, and pH adjusting agents.
Suitable additives include physiologically biocompatible buffers
(e.g., trimethylamine hydrochloride), addition of chelants (such
as, e.g., DTPA or DTPA-bisamide) or calcium chelate complexes (e.g.
calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of
calcium or sodium salts (e.g., calcium chloride, calcium ascorbate,
calcium gluconate or calcium lactate). In addition, antioxidants
and suspending agents can be used.
[0155] Compositions intended for oral use may be prepared according
to methods known in the art for the manufacture of pharmaceutical
compositions, especially methods known in the art for the
manufacture of pharmaceutical compositions comprising
oligonucleotides. For example, oral delivery of siRNA and antisense
oligonucleotides has been achieved through encapsulating siRNA
within biodegradable particles that protect them from degradation
and target them to M cells in intestinal Peyer's patches (see
Akhtar, S., 2009, J. Drug Target. 17:491-495). Oral composition can
contain one or more such sweetening agents, flavoring agents,
coloring agents or preservative agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets
contain the active ingredient in admixture with non-toxic
pharmaceutically acceptable excipients that are suitable for the
manufacture of tablets. These excipients can be, e.g., inert
diluents (such as calcium carbonate, sodium carbonate, lactose,
calcium phosphate or sodium phosphate), granulating and
disintegrating agents (e.g., corn starch, or alginic acid), binding
agents (e.g., starch, gelatin or acacia), and lubricating agents
(e.g., magnesium stearate, stearic acid or talc). The tablets can
be uncoated or they can be coated by known techniques. In some
cases such coatings can be prepared by known techniques to delay
disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monosterate or
glyceryl distearate can be employed.
[0156] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, e.g., calcium carbonate, calcium phosphate or
kaolin, or as soft gelatin capsules wherein the active ingredient
is mixed with water or an oil medium, e.g., peanut oil, liquid
paraffin or olive oil.
[0157] Aqueous suspensions contain the active materials in a
mixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, e.g., sodium
carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, e.g., lecithin, or condensation
products of an alkylene oxide with fatty acids, e.g.,
polyoxyethylene stearate; or condensation products of ethylene
oxide with long chain aliphatic alcohols, e.g.,
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, e.g., polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, e.g. ethyl, or n-propyl p-hydroxybenzoate, one or
more coloring agents, one or more flavoring agents, and one or more
sweetening agents, such as sucrose or saccharin.
[0158] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, e.g. arachis oil, olive oil, sesame
oil or coconut oil, or in a mineral oil such as liquid paraffin.
The oily suspensions can contain a thickening agent, e.g. beeswax,
hard paraffin or cetyl alcohol. Sweetening agents and flavoring
agents can be added to provide palatable oral preparations. These
compositions can be preserved by the addition of an anti-oxidant
such as ascorbic acid
[0159] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, e.g. gum acacia
or gum tragacanth, naturally-occurring phosphatides, e.g. soy bean,
lecithin, and esters or partial esters derived from fatty acids and
hexitol, anhydrides, for example sorbitan monooleate, and
condensation products of the said partial esters with ethylene
oxide, e.g. polyoxyethylene sorbitan monooleate. The emulsions can
also contain sweetening and flavoring agents.
[0160] Syrups and elixirs can be formulated with sweetening agents,
e.g. glycerol, propylene glycol, sorbitol, glucose or sucrose. Such
formulations can also contain a demulcent, a preservative, and
flavoring and coloring agents. The pharmaceutical compositions can
be in the form of a sterile injectable aqueous or oleaginous
suspension. This suspension can be formulated according to the
known art using those suitable dispersing or wetting agents and
suspending agents that have been mentioned above. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0161] The myostatin siNA conjugates can take the form of
suppositories, e.g., for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient that is solid at ordinary temperatures but
liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene glycols.
[0162] Myostatin siNA conjugates described herein can be formulated
in a sterile medium for intravenous administration. The molecule,
depending on the vehicle and concentration used, can either be
suspended or dissolved in the vehicle. Advantageously, adjuvants
such as local anesthetics, preservatives and buffering agents can
be dissolved in the vehicle.
[0163] In other embodiments, myostatin siNA conjugate formulations
provided herein for use in pulmonary delivery further comprise one
or more surfactants. Suitable surfactants or surfactant components
for enhancing the uptake of the compositions of the invention
include synthetic and natural as well as full and truncated forms
of surfactant protein A, surfactant protein B, surfactant protein
C, surfactant protein D and surfactant Protein E, di-saturated
phosphatidylcholine (other than dipalmitoyl),
dipalmitoylphosphatidylcholine, phosphatidylcholine,
phosphatidylglycerol, phosphatidylinositol,
phosphatidylethanolamine, phosphatidylserine; phosphatidic acid,
ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine,
palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone,
dolichols, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone
phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone,
palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline,
choline, and/or choline phosphate; as well as natural and
artificial lamellar bodies which are the natural carrier vehicles
for the components of surfactant, omega-3 fatty acids, polyenic
acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block
copolymers of ethylene or propylene oxides, polyoxypropylene,
monomeric and polymeric, polyoxyethylene, monomeric and polymeric,
poly (vinyl amine) with dextran and/or alkanoyl side chains, Brij
35, Triton X-100 and synthetic surfactants ALEC, Exosurf, Survan
and Atovaquone, among others. These surfactants can be used either
as single or part of a multiple component surfactant in a
formulation, or as covalently bound additions to the 5' and/or 3'
ends of the nucleic acid component of a pharmaceutical composition
herein.
[0164] In one embodiment, myostatin siNA conjugates can be
formulated for administration via pulmonary delivery, such as by
inhalation of an aerosol or spray dried formulation administered by
an inhalation device or nebulizer, providing rapid local uptake of
the nucleic acid molecules into relevant pulmonary tissues. Solid
particulate compositions containing respirable dry particles of
micronized nucleic acid compositions can be prepared by grinding
dried or lyophilized nucleic acid compositions, and then passing
the micronized composition through, for example, a 400 mesh screen
to break up or separate out large agglomerates. A solid particulate
composition comprising the siNA conjugate compositions can
optionally contain a dispersant which serves to facilitate the
formation of an aerosol as well as other therapeutic compounds. A
suitable dispersant is lactose, which can be blended with the
nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio
by weight.
[0165] Spray compositions comprising siNA conjugates described
herein can, for example, be formulated as aqueous solutions or
suspensions or as aerosols delivered from pressurized packs, such
as a metered dose inhaler, with the use of a suitable liquefied
propellant. In one embodiment, aerosol compositions suitable for
inhalation can be either a suspension or a solution and generally
contain an siNA conjugate and a suitable propellant such as a
fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures
thereof, particularly hydrofluoroalkanes, especially
1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or a
mixture thereof. The aerosol composition can optionally contain
additional formulation excipients well known in the art such as
surfactants. Non-limiting examples include oleic acid, lecithin or
an oligolactic acid or derivative such as those described in
WO94/21229 and WO98/34596 and co-solvents for example ethanol. In
one embodiment, a pharmaceutical aerosol formulation comprises an
siNA conjugate and a fluorocarbon or hydrogen-containing
chlorofluorocarbon or mixtures thereof as propellant, optionally in
combination with a surfactant and/or a co-solvent.
[0166] The aerosol formulations can be buffered by the addition of
suitable buffering agents.
[0167] Aerosol formulations can include optional additives
including preservatives if the formulation is not prepared sterile.
Non-limiting examples include, methyl hydroxybenzoate,
anti-oxidants, flavorings, volatile oils, buffering agents and
emulsifiers and other formulation surfactants. In one embodiment,
fluorocarbon or perfluorocarbon carriers are used to reduce
degradation and provide safer biocompatible non-liquid particulate
suspension compositions. In another embodiment, a device comprising
a nebulizer delivers an siNA conjugate composition comprising
fluorochemicals that are bacteriostatic thereby decreasing the
potential for microbial growth in compatible devices.
[0168] Capsules and cartridges comprising the myostatin siNA
conjugate compositions for use in an inhaler or insufflator, of for
example gelatin, can be formulated containing a powder mix for
inhalation of a compound of the invention and a suitable powder
base such as lactose or starch. In one embodiment, each capsule or
cartridge contains an siNA conjugate and one or more excipients. In
another embodiment, an siNA conjugate can be presented without
excipients such as lactose.
[0169] The siNA conjugates can also be formulated as a fluid
formulation for delivery from a fluid dispenser, such as those
described and illustrated in WO05/044354.
F. Administration
[0170] The myostatin siNA conjugates and pharmaceutical
compositions thereof are introduced into a subject by any of a
variety of forms of systemic administration. For the purposes of
the present invention, systemic administration include pulmonary
(inhalation, nebulization etc.), intravenous, subcutaneous,
catheterization, nasopharyngeal, or oral/gastrointestinal
administration as is generally known in the art. Further
non-limiting examples of administration methods of the invention
include buccal, sublingual, parenteral (i.e., intravenously,
intraperitoneally, subcutaneously, or intramuscularly), local
rectal administration or other local administration resulting in
absorption or accumulation of the myostatin siNA conjugates in the
blood stream followed by distribution throughout the entire body.
In one embodiment, the myostatin siNA conjugates and pharmaceutical
compositions thereof can be administered by insufflation and
inhalation. In one embodiment, the myostatin siNA conjugates and
pharmaceutical compositions thereof are administered intravenously
or intraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.
5,286,634). In another embodiment, the myostatin siNA conjugates
and pharmaceutical compositions thereof are administered
subcutaneously. In a further embodiment, the myostatin siNA
conjugates and pharmaceutical compositions thereof are administered
in ovo to an avian embryo while contained in the egg. The siNA
conjugates may be administered to any suitable compartment of the
egg (e.g., allantois fluid, yolk sac, amnion, air cell or into the
embryo).
[0171] For therapeutic applications, a pharmaceutically effective
dose of the myostatin siNA conjugates or pharmaceutical
compositions is systemically administered to a subject. A
pharmaceutically effective dose is that dose required to prevent,
inhibit the occurrence, or treat (alleviate a symptom to some
extent, preferably all of the symptoms) a disease state. One
skilled in the art can readily determine a therapeutically
effective dose of a myostatin siNA conjugate to be systemically
administered to a given subject, e.g., by taking into account
factors, such as the size and weight of the subject, the extent of
the disease progression or penetration, the age, health, and sex of
the subject, and the route of systemic administration. Generally,
an amount between 0.1 .mu.g/kg and 100 mg/kg body weight/day of
active ingredients is administered dependent upon potency of the
negatively charged polymer. Optimal dosing schedules can be
calculated from measurements of drug accumulation in the body of
the patient. The myostatin siNA conjugates can be administered in a
single dose or in multiple doses.
[0172] In one embodiment, the siNA conjugates described herein are
systemically delivered to a subject at a dose of between about 0.1
to about 500 mg/kg (mpk). In another embodiment, the siNA
conjugates are delivered at a dose of between about 1 to about 200
mpk. In another embodiment, the siNA conjugates are delivered at a
dose of between about 1 to about 100 mpk. In another embodiment,
the siNA conjugates are delivered at a dose of between about 5 to
about 60 mpk. In another embodiment, the siNA conjugates are
delivered at a dose of between about 10 to about 50 mpk.
[0173] The myostatin siNA conjugates can be administered once
monthly, once weekly, once daily (QD), or divided into multiple
monthly, weekly, or daily doses, such as, for example, twice daily
(BID), three times daily (TID), once every two weeks. Thus,
administration can be accomplished via single or divided doses.
Persons of ordinary skill in the art can easily estimate repetition
rates for dosing based on measured residence times and
concentrations of the drug in bodily fluids or tissues.
[0174] In addition, the administration can be continuous, e.g.,
every day, or intermittently. For example, intermittent
administration of a myostatin siNA conjugate may be administration
one to six days per week, administration in cycles (e.g., daily
administration for two to eight consecutive weeks, then a rest
period with no administration for up to one week), or
administration on alternate days.
[0175] Aerosol compositions can be administered into the
respiratory system as a formulation that includes particles of
respirable size, e.g. particles of a size sufficiently small to
pass through the nose, mouth and larynx upon inhalation and through
the bronchi and alveoli of the lungs. In general, respirable
particles range from about 0.5 to 10 microns in size. In one
embodiment, the particulate range can be from 1 to 5 microns. In
another embodiment, the particulate range can be from 2 to 3
microns. Particles of non-respirable size which are included in the
aerosol tend to deposit in the throat and be swallowed, and the
quantity of non-respirable particles in the aerosol is thus
minimized. For nasal administration, a particle size in the range
of 10-500 um is preferred to ensure retention in the nasal
cavity.
EXAMPLES
[0176] Examples provided are intended to assist in a further
understanding of the invention. Particular materials employed,
species and conditions are intended to be illustrative of the
invention and not limiting of the reasonable scope thereof. Certain
starting materials and reagents are either commercially available
or known in the chemical scientific or patent literature.
Example 1
Mtsn siRNA
[0177] siRNA Synthesis--
[0178] siRNAs were synthesized by methods similar to those
previously described (Wincott, F. et al., 1995, Nucleic Acids Res.
23:2677-84). For each oligonucleotide duplex, the individual,
complementary sense and antisense strands were first synthesized on
solid support, such as on controlled pore glass, using commercially
available automated oligosynthesizers. The solid support was
obtained pre-loaded with the first (3') nucleotide unit of the
desired sequence and placed in an appropriate column for the
oligosynthesizer. The first nucleotide was linked to the solid
support via a succinate linkage and contained a suitable acid
sensitive protecting group (trityl, dimethoxytrityl) on the
5'-terminal hydroxyl group. The solid-phase oligosynthesis employed
synthetic procedures that are generally known in the art.
Elongation of the desired oligomeric sequence went through a cycle
of four steps: 1) Acidic deprotection of the 5'-trityl protecting
group; 2) Coupling of the next nucleotide unit as the 5'-trityl (or
dimethoxytrityl) protected phosphoramidite in the presence of an
activating agent, such as S-ethyl-tetrazole; 3) Oxidation of the
P(III) phosphite triester to the P(V) phosphate triester by an
oxidizing agent, such as iodine; and 4) Capping any remaining
unreacted alcohol groups through esterification with an acylating
agent, such as acetic anhydride. The phosphoramidites used were
either derived from naturally occurring nucleotide units or from
chemical modified versions of these nucleotides. Oligonucleotide
synthesis cycles were continued until the last (5') nucleotide unit
was installed onto the extended oligomer. After the final cycle,
the 5'-trityl protecting group may or may not be removed from the
oligonucleotide while it remains on the solid support. In some
instances, the 5'-terminal trityl was first removed by treatment
with an acidic solution.
[0179] After deprotection, the solid support was treated with an
appropriate base, such as aqueous methylamine, in order to cleave
the oligonucleotide from the support, remove the cyanoethyl
protecting groups on the phosphates and deprotect the acyl
protecting groups on the nucleotide bases. After cleavage of each
strand from the solid support, each strand was purified
chromatographically with a reversed phase (C18) or anion exchange
(SAX) resin. Typically, the oligonucleotide was eluted from SAX
resin with a gradient of an inorganic salt, such as sodium
chloride. Salt was removed from the purified samples by dialysis or
tangential flow filtration.
[0180] Each purified oligonucleotide was analyzed for purity by
appropriate methods, including reversed phase HPLC, SAX HPLC, and
capillary gel electrophoresis. The identity of the oligonucleotide
was confirmed by mass spectrometry, using an ionization technique
such as ESI or MALDI. The yields of each oligonucleotide were
assessed by UV (260 nm) with a theoretically derived extinction
coefficient.
[0181] The corresponding sense and antisense strands were annealed
by mixing an equimolar amount of each material. The appropriate
amounts of each strand were approximated by UV (260 nm)
measurements and theoretical extinction coefficients. After the
annealing process, the extent of duplex formation and the presence
of any excess single strand material were assessed by an
appropriate chromatographic method, such as RP-HPLC or SAX. When
appropriate, the sample was adjusted with additional amounts of one
of the two strands in order to completely anneal the remaining
excess single strand. The final duplex material was lyophilized
prior to delivery for further biochemical or biological
testing.
[0182] Cells and Reagents--
[0183] Mouse hepatoma Hepa 1-6 cell line was obtained from the
American Type Tissue Collection (Cat # CRL-1830). Cells were grown
in Dulbecco's Modified Eagle Medium, High Glucose with Glutamax.TM.
(Invitrogen Cat #10566024) adjusted to 1 mM sodium pyruvate
(Invitrogen Cat #11360070 and supplemented with 10% fetal bovine
serum. Streptomycin and penicillin were added to the media at 100
.mu.g/mL and 100 U/mL, respectively. Cells were cultured at
37.degree. C. in the presence of 5% CO.sub.2.
[0184] Generation of Luciferase Reporter Constructs--
[0185] siRNAs were screened using the psiCHECK2 dual luciferase
reporter system. The luciferase reporter plasmids used were derived
from psiCHECK2 vector (Promega, Cat# C8021). Through de novo
synthesis, the full length transcript of mouse myostatin (NCBI
GenBank RefSeq NM_010834) was cloned into the XhoI/NotI sites of
the vector.
[0186] In Vitro Screening of siRNAs--
[0187] Hepa1-6 cells were seeded in 96-well plates at a density of
10,000 cells per well and incubated at 37.degree. C. After 24
hours, the cells were co-transfected with siRNAs and the MSTN
luciferase reporter plasmid using Lipofectamine 2000 reagent (Life
Technologies, Cat#11668019). Primary screens were performed by
co-transfecting siRNAs with the MSTN plasmid at final
concentrations of 10 nM and 0.6 ng/.mu.L, respectively. The cells
were incubated at 37.degree. C. and culture medium was replaced
with fresh media 24 hours post-transfection. After an additional 24
hour incubation, reporter Renilla luciferase and control firefly
luciferase activities were measured using Dual-Glo Luciferase Assay
System (Promega, Cat # E2940). Renilla luciferase activity of each
well was divided by firefly luciferase activity from the same cell
to normalize for different transfection efficiencies across
different wells. The normalized luciferase activities produced by
the Mstn siRNAs were further divided by the normalized luciferase
activity generated by non-targeting control siNA to calculate the
percent knockdown (% KD) of reporter expression. All calculations
of IC50s were performed using R.2.9.2 software. The data were
analyzed using the sigmoidal dose-response (variable slope)
equation for simple ligand binding.
[0188] Results--
[0189] Four unique, unconjugated siRNAs that target mouse Mstn mRNA
sequence and a non-targeting control siRNA were synthesized. The
target nucleotide sequences of the four Mstn siRNAs are set forth
in Table 2a ("target sequence").
TABLE-US-00006 TABLE 2a Mstn target sequences (5' to 3'), noting
the assigned mouse target site number (column 2) and the sequence
identification number (SEQ ID NO.) (column 3). Target Site Target
Sequence (mouse) SEQ ID NO: AUGGCAAAGAACAAAUAAU 1167 1
GGCAAAGAACAAAUAAUAU 1169 2 ACUCCAGAAUAGAAGCCAU 255 3
UUUGGAAGAUGACGAUUAU 421 4
TABLE-US-00007 TABLE 2b Various myostatin-related siNA sense
(passenger) and antisense (guide) sequences (5' to 3')
corresponding to the selected target site sequences in Table 2a.
Antisense sequences are readily identified as being complementary
to the sense sequence shown. The SEQ ID NOs listed in column 2
correspond to the sense sequences listed in column 3. The SEQ ID
NOs listed in column 5 correspond to the antisense sequences listed
in column 4. SEQ SEQ Target Site ID Sense (Target) Sequence
Antisense Sequence ID (mouse) NO: (5' to 3') (5' to 3') NO: 1167 1
AUGGCAAAGAACAAAUAAU AUUAUUUGUUCUUUGCCAU 18 1169 2
GGCAAAGAACAAAUAAUAU AUAUUAUUUGUUCUUUGCC 19 255 3
ACUCCAGAAUAGAAGCCAU AUGGCUUCUAUUCUGGAGU 20 421 4
UUUGGAAGAUGACGAUUAU AUAAUCGUCAUCUUCCAAA 21
[0190] The nucleotide sequences of the Mstn siRNAs and the
non-targeting control siRNA are indicated in Table 3. The
double-stranded siRNA molecules within Table 3 contain a sense
strand (also known as the passenger strand) and an antisense strand
(also known as the guide strand), wherein each strand is comprised
of 21 nucleotides (position 1 (5') to position 21 (3')) and
contains both internal and terminal chemically-modified
nucleotides. The name of each siRNA molecule is provided in column
1 and corresponds to the mouse Mstn mRNA region to which the
molecule is targeted. Column 2 of Table 3, "Strand", indicates
whether the particular sequence in the indicated row is the sense
(S) or antisense (A/S) strand of the duplex. Column 3 of Table 3,
"5-position 1 nuc," describes nucleotide position 1 of the sense
and antisense strands of the indicated siRNA molecules, each
comprising of a nucleotide with a 5' cap moiety. The chemical
structure of each 5'-position 1 nucleotides is provided in Table
6a, infra. The nucleotide sequence spanning positions 2-20 for each
of the sense and antisense strands of the siRNA molecules is
described in column 4 of Table 3, wherein the individual
nucleotides are separated by a semicolon. The chemical structure of
each nucleotide indicated within column 4 is provided for in Table
6b, infra. The 5th column of Table 3, "Nuc position 21-3',"
represents the 3' most nucleotide of the sense or antisense strand
of the siRNA, each represented by "omeU-iB" or "omeUSup" (for
structures, see Table 6c, infra). The SEQ ID NO: for each strand of
the siRNA molecules of Table 3 (positions 1-21) is provided in
column 6. Each siRNA molecule in Table 3 has 3' overhangs
consisting of 2 nucleotides at both ends of the molecule.
TABLE-US-00008 TABLE 3 Mstn siRNA sequences 5'- Nuc SEQ siRNA
position Nucleotide sequence - position 2 to position ID name
Strand 1 nuc position 20 21-3' NO: Mstn: S 6amiL- omeU; fluG; omeG;
fluC; omeA; fluA; omeA; omeU-iB 5 1167 iB-fluA fluG; omeA; fluA;
omeC; fluA; omeA; fluA; omeU; fluA; omeA; fluU; omeUs A/S vinylP-
fluU; omeU; fluA; omeU; fluU; omeU; fluG; omeUSup 6 moeT omeU;
fluU; omeC; fluU; omeU; fluU; omeG; fluC; omeC; fluA; omeU; omeUs
Mstn: S 6amiL- omeG; fluC; omeA; fluA; omeA; fluG; omeU-iB 7 1169
iB-fluG omeA; fluA; omeC; fluA; omeA; fluA; omeU; fluA; omeA; fluU;
omeA; fluU; omeUs A/S vinylP- fluU; omeA; fluU; omeU; fluA; omeU;
fluU; omeUSup 8 moeT omeU; fluG; omeU; fluU; omeC; fluU; omeU;
fluU; omeG; fluC; omeC; omeUs Mstn: S 6amiL- omeC; fluU; omeC;
fluC; omeA; fluG; omeU-iB 9 255 iB-fluA omeA; fluA; omeU; fluA;
omeG; fluA; omeA; fluG; omeC; fluC; omeA; fluU; omeUs A/S vinylP-
fluU; omeG; fluG; omeC; fluU; omeU; fluC; omeUSup 10 moeT omeU;
fluA; omeU; fluU; omeC; fluU; omeG; fluG; omeA; fluG; omeU; omeUs
Mstn: S 6 amiL- omeU; fluU; omeG; fluG; omeA; fluA; omeU-iB 11 421
iB-fluU omeG; fluA; omeU; fluG; omeA; fluC; omeG; fluA; omeU; fluU;
omeA; fluU; omeUs A/S vinylP- fluU; omeA; fluA; omeU; fluC; omeG;
fluU; omeUSup 12 moeT omeC; fluA; omeU; fluC; omeU; fluU; omeC;
fluC; omeA; fluA; omeA; omeUs Place- S 6 amiL- omeU; fluC; omeG;
fluC; omeC; fluU; omeU-iB 13 bo: 5 iB-fluG omeU; fluA; omeU; fluA;
omeU; fluC; omeG; fluG; omeU; fluC; omeG; fluA; omeUs A/S vinylP-
fluC; omeG; fluA; omeC; fluC; omeG; fluA; omeUSup 14 moeT omeU;
fluA; omeU; fluA; omeA; fluG; omeG; fluC; omeG; fluA; omeC;
omeUs
[0191] Each siRNA (10 nM) was co-transfected along with a Mstn
luciferase reporter plasmid (0.6 ng/.mu.l) into cells, and
luciferase activity was measured after 48 hours as a reflection of
mRNA knockdown (KD). The four siRNAs show a minimum of 90% Mstn
mRNA knockdown and 1050 values less than 0.016 nM (see Table
4).
TABLE-US-00009 TABLE 4 Mstn siRNA in vitro knockdown activity siRNA
Max mRNA KD (%) IC50 (nM) Mstn: 1167 91 0.007 Mstn: 1169 90 0.005
Mstn: 255 94 0.004 Mstn: 421 95 0.016
Example 2
Mtsn siRNA Cholesterol Conjugates
[0192] Cholesterol Conjugation--
[0193] A single cholesterol entity was attached to the 3' end of
the sense strand of each siRNA molecule described in Table 3. For
the preparation of oligonucleotides with a cholesterol unit on the
3'-terminus, deoxycytidylyl-deoxyguanosine (CpG) (see Table 6d,
infra, for structure) with a preloaded cholesterol succinate, which
also contained a dimethoxytrityl (DMT) protected primary alcohol,
was used for synthesis of the corresponding oligonucleotide
sequence (see Example 1). Typically, the final 5'-DMT protecting
group was removed during oligosynthesis. The oligonucleotide was
then cleaved from the CpG by treatment with a basic solution, such
as aqueous methylamine, and purified by chromatography with a
reversed phase resin, such as C18. This purified oligo was annealed
to its corresponding complementary strand to prepare the desired
oligonucleotide duplex.
[0194] Animals--
[0195] Female CD-1 mice were obtained from Charles River and were
between 8-9 weeks old at time of study. Mice were maintained on a
12-hour light and dark cycle with al libitum access to water and
standard chow diet (no. 5001; LabDiet). Control and experimental
cholesterol-conjugated siRNAs were administered to mice by tail
vein injections at indicated dosages. All animal studies were
conducted at Merck Research Laboratories in an AAALAC-accredited
West Point, Pa. animal facility using protocols approved by the
Institutional Animal Care and Use Committee (IACUC).
[0196] Quantitative Real-Time PCR Analysis--
[0197] Mice were sacrificed and tissues were homogenized in Trizol
(Invitrogen), extracted in 1-bromo-2-chloropropane (Acros
Organics), and total RNA was isolated using the MagMax RNA
isolation method (Ambion). RNA (125 ng) was reverse transcribed
using the High Capacity cDNA Reverse Transcription kit (Applied
Biosystems, Cat #4368813). Taqman qPCR analysis was performed using
an ABI 7900 Real-Time PCR System using TaqMan Fast Advanced Master
Mix (Applied Biosystems, Cat#4444555). All Taqman probes and
primers were purchased from Applied Biosystems as pre-validated
sets: mouse PPIB Assay ID Mm00478295_ml; mouse Mstn Assay ID
Mm01254559_ml. Taqman data analysis was performed on an ABI 7900
Real-Time PCR System, as described previously (Tadin-Strapps, M. et
al., 2011, J. Lipid Res. 52:1084-1097).
[0198] Serum Analysis of Myostatin Protein--
[0199] Blood was collected by tail vein collection, and serum was
collected at the specified time points and analyzed using the
GDF-8/Myostatin Quantikine ELISA kit (R&D, Cat # DGDF80).
Briefly, serum samples were activated as described in manufacturer
protocol, with the exception that the final activated serum sample
had an additional 1:2 dilution in calibrator diluent before
assaying.
[0200] Results--
[0201] In vivo efficacy studies in CD-1 mice were performed using
the four Mstn siRNA molecules of Table 3, each of which is
conjugated to a single cholesterol moiety at the 3' most nucleotide
of the sense (passenger) strand. Mice were injected intravenously
with 15 mpk siRNA, and Mstn mRNA knockdown was assessed in the
gastrocnemius muscle after 72 hours (FIGS. 1A and 1B). Reduction of
Mstn expression was seen, especially with Mstn:1169 and Mstn:1167
cholesterol conjugates. The Mstn:1169 cholesterol conjugate was
selected for use in follow-up siRNA optimization and functional
studies.
[0202] In order to examine the potency and efficacy of the
Mstn:1169-cholesterol conjugate, mice were treated with a single
injection of the conjugate at 5, 15, and 50 mpk. Mstn mRNA and
circulating protein levels were measured at various time points
(FIG. 2). Mstn:1169-cholesterol treatment caused a dose-dependent
knockdown in Mstn mRNA in gastrocnemius (FIG. 2A), tricep (FIG. 2B)
and EDL (FIG. 2C) muscles, demonstrating greater than 8-fold
difference in potency between the 5 mpk and 50 mpk doses. Maximum
knockdown (KD) was observed with the 50 mpk dose at day 7, showing
90-95% mRNA knockdown in all three muscles and 75% knockdown of
circulating protein levels (FIG. 2D). Mstn gene silencing with 50
mpk siRNA was between 88-95% KD in gastrocnemius, triceps and
spinotrapezius muscles (data not shown), which are mixed fiber type
muscles, as well as in the EDL muscle, a predominantly type II
fiber muscle. Mstn mRNA knockdown effects could not be determined
in the soleus muscle due to low Mstn mRNA levels in this muscle
(data not shown). Mstn siRNA also displays an extended duration of
silencing, maintaining 90-95% mRNA KD and 65% serum Mstn protein
levels for 21 days after 50 mpk dosing. There was no evidence of
liver toxicity and muscle damage at all doses examined, as
determined by monitoring serum ALT/AST and muscle creatine
phosphokinase, respectively (data not shown).
Example 3
Prolonged Mstn Knockdown Increases Muscle Size and Alters Muscle
Fatigue Profile
[0203] Body Composition--
[0204] Animal body composition was measured by quantitative
magnetic resonance spectroscopy using an EchoMRI instrument (Echo
Medical Systems, Houston, Tex.), in order to determine lean/fat
mass. Measurements were made 20 days after initiation of siRNA
dosing.
[0205] Micro-CT Imaging and Data Analysis--
[0206] Using the LaTheta micro-CT (LaTheta LCT-100A.TM., Aloka,
Echo Medical Systems, Houston, Tex.), a stack of 10 slices was
scanned between the knee and fibula-tibia junction. Images were
analyzed as previously described (Weber, H. et al., 2012, J. Appl.
Physiol. 112:2087-98) to find the largest cross sectional area
(CSA) of whole muscle in the lower leg. Mice were scanned at day-1
with respect to siRNA dosing, and also scanned at day 3, 7, 14 and
21.
[0207] In Situ Muscle Function Assay--
[0208] A custom build in situ assay system, as previously described
(see Weber, H. et al., supra), was performed 21 days after siRNA
dosing. Briefly, the Achilles tendon of an anesthetized animal was
connected to the lever of a combined servomotor/force transducer
unit. Electric impulses were delivered via the sciatic nerve to
stimulate the muscles of the lower leg to contract, while the
resulting force was recorded. Tetanic stimulation trains of 50 ms
length, containing 4 mA square pulses of 0.1 ms duration at 100 Hz,
were repeated at a frequency of 0.8 Hz for 300 s. A constant
baseline tension of 0.1 N was reestablished between stimulations.
After completion of the in situ assay, the hind limb muscles were
collected for weighing and histology.
[0209] Several parameters representing the fatigue envelopes were
extracted from a double sigmoidal curve fit, including maximum
force (F.sub.max), intermediate force (F.sub.o), minimum force
(F.sub.min), early fatigue force (F.sub.max-F.sub.o), late fatigue
force (F.sub.o-F.sub.min), maximal slope of early fatigue (S.sub.1)
and late fatigue (S.sub.2) and time constants of early
(.tau..sub.1) and late fatigue (.tau..sub.2) forces.
[0210] Measurement of Tibia Bone Lengths--
[0211] After sacrificing mice, each hindlimb was stored in 70%
ethanol overnight. Next day, legs were stripped of muscle and
tissue and length was measured using a digital caliper.
[0212] Quantitation of Myofiber Size and Number--
[0213] In order to detect muscle fiber cell size, laminin staining
was performed on gastrocnemius muscle 21 days-post siRNA/control
treatment. A transverse cross-section made through the widest
region of the gastrocnemius muscle was fixed in 10% formalin
overnight. Muscle was paraffin-embedded and sectioned (5 .mu.m) by
standard methods. Sections underwent heat-induced antigen retrieval
using citrate buffer (EZAR1, BioGenx #HJ521-XAK) at 103.degree. C.
for 10 minutes using a BioGenex EZ-Retriever Microwave. After PBS
washes, sections were incubated at room temperature for 1 hour with
a polyclonal Rabbit anti-Laminin antibody at a 1:100 dilution
(Abcam, cat # ab11575). A goat anti-rabbit A555 immunofluorescent
secondary antibody was applied after further PBS washes (Invitrogen
#A21429), and the slides were mounted using ProlongGold with Dapi
(Invitrogen #P36935). Images were captured on a BX-63 Olympus
Microscope using a Hamamatsu ORCA-R2 camera and cellSens Dimension
1.7 software (Olympus). Muscle fiber area and total number of
fibers were quantitated on the muscle cross-section using Definiens
software. Regions out of focus were eliminated from analysis prior
to the segmentation of the image. A multi-resolution algorithm was
used to extract the muscle fiber and the endomysiums in the
remaining muscle section. Fiber area was determined from 12 mice
per group, with 1200-7000 cell counts/mouse. Mean fiber size and
size frequency distribution of muscle fibers is shown.
[0214] Results--
[0215] Since Mstn is known to have a major inhibitory role in
muscle growth, the effects of Mstn silencing on muscle mass and
function was examined. In order to achieve maximum Mstn knockdown,
mice were treated with a single i.v. 50 mpk dose of the
Mstn:1169-siRNA-cholesterol conjugate (see Examples 1 and 2, supra)
or controls. After 21 days, mice treated with the Mstn siRNA
conjugate showed 85-90% Mstn mRNA knockdown in gastrocnemius, EDL,
quadriceps, triceps and spinotrapezius muscles (FIG. 3A). In
addition, circulating levels of Mstn protein were sustained at
>65% reduction through day 21 (FIG. 3B). Muscle size was
monitored in both hind limbs of mice by micro-CT at 3, 7, 14 and 20
days after dosing. Maximum muscle cross-sectional area (CSA) for
each leg, representative of plantarflexor muscle group, was
determined by quantitating a series of 10 slices using a custom
MATLAB and Definiens Architect XD software algorithm. Quantitation
results indicate a significant increase in muscle size by systemic
Mstn siRNA-cholesterol treatment as early as 3 days after
initiation of dosing and up to a 20% increase in leg muscle size by
day 21 (FIG. 3C). This data is supported by gastrocnemius muscle
weights at day 21/22, which also show .about.20% increase in the
weights of both rested and exercised gastrocnemius muscle by
Mstn-chol treatment in comparison to control mice (FIG. 3D) Laminin
immunofluorescent staining of a cross-section of gastrocnemius
muscle showed that the average fiber cross-sectional area was
increased, while the total number of fibers was unaffected by
Mstn-chol treatment (FIG. 3E, mean fiber area; FIG. 3F, mean fiber
number; FIG. 3G, size frequency distribution). In addition to the
significant increase in muscle size observed in mice treated with
the Mstn siRNA-cholesterol conjugate, body weight measurements also
indicate approximately 10% increase by day 20 (FIG. 311). Body
composition analysis, as determined by quantitative NMR, reveals
that this increase in body weight is attributed to an increase in
lean mass (FIG. 3I).
[0216] In order to assess potential changes to the strength and
fiber type composition of skeletal muscle as a result of
siRNA-mediated gene silencing of Mstn, muscle fatigue response to
exercise was assayed in an in situ muscle function assay where
repeated isometric contractions are induced by electrostimulation.
Since skeletal muscle consists of different fiber types, which
exhibit different contractile properties and differential energy
source usage, changes in muscle performance can be determined by
changes in several functional parameters in muscle fatigue curves,
as described previously (Weber, H. et al., 2012, supra). Briefly,
fatigue curves exhibit three stages of muscle fatigue: early
fatigue, late fatigue and a non-fatigable stage (FIG. 4). "Early
fatigue" is represented by F.sub.max-F.sub.0, indicative of type
IIb fibers, which are strong, fast, fatigue very quickly and use
creatine phosphate as an energy source. This stage is followed by
"late fatigue" (F.sub.0-F.sub.min), primarily indicative of type
IIa/x fibers, which are strong, fast, more fatigue-resistant and
use glycogen as an energy source. The final stage of the fatigue
curve is the "non-fatigable" stage (F.sub.min), which is marked
type I fibers, which are weak, slow, non-fatigable and use fatty
acids as an energy source.
[0217] Mstn knockdown results in increased F.sub.max and F.sub.0,
but since F.sub.max-F.sub.0 remains unchanged, the "early fatigue"
stage is unaffected (FIG. 5A). While there is no significant change
in F.sub.min, F.sub.0-F.sub.min is increased, suggesting an
increase in "late fatigue" indicative of increased contractile
force. This data suggests that Mstn knockdown results in a change
in type IIa/x fibers, due to a potential increase in the size,
quantity and/or strength of the fibers. In addition, there is no
change in any of the parameters associated with the timing of the
fatigue stages (.tau..sub.1, .tau..sub.2, S.sub.1, S.sub.2),
suggesting that fuel availability and usage are unaffected.
[0218] In order to determine changes to muscle quality, "specific
force" was calculated by normalizing muscle contractile force to
cross-sectional area (CSA) (FIG. 5B). There was no significant
difference in specific force or any additional functional parameter
in response to Mstn knockdown, suggesting that there is no effect
on muscle quality.
[0219] In addition to skeletal muscle, heart has also been reported
to express Mstn, although to a lesser degree (Sharma M. et al.,
1999, J. Cell Physiol. 180:1-9). Therefore, the heart was examined
for potential Mstn knockdown. Quantitative PCR indicates very low
Mstn mRNA expression in the heart (Ct values, 32-35, data not
shown) and, therefore, mRNA knockdown could not be determined.
There are many reports of changes in heart size in rodent Mstn
knockout models or in response to Mstn inhibition by small molecule
or neutralizing antibodies, although observations are varied, with
some reports of cardiac hypertrophy, and other reports of unchanged
heart size (Artaza, J. N. et al., 2007, J. Endocrinol. 194:63-76;
Morissette, M. R., 2006, Circ. Res. 99:15-24; Rodgers, B. D. et
al., 2009, J. Physiol. 587:4873-86; Whittemore, L. A. et al., 2003,
Biochem. Biophys. Res. Commun. 300:965-71). In order to access
signs of cardiac hypertrophy after 21 days of Mstn knockdown,
hearts were weighed and normalized to a variety of parameters (FIG.
6). Mstn knockdown resulted in a significant increase in heart
weight. When heart weight is normalized to tibia length, a
parameter anticipated to remain unchanged, mice treated with Mstn
siRNA-cholesterol continue to display a significant increase in
heart weight. However, if heart weight is normalized to body weight
(BW) or lean mass, cardiac hypertrophy is no longer observed,
suggesting that the increase in heart size is compensatory to the
increased BW resulting from Mstn knockdown.
Example 4
Mechanism of Uptake of Cholesterol Conjugates in Muscle
[0220] siRNA Synthesis--
[0221] siRNA was synthesized as described in Example 1, supra. The
sequences of two Ctnnb1 siRNAs used in this Example are indicated
in Table 4 (5'-3' direction). A non-targeting control siRNA was
also used (see "Placebo 5" from Examples 1 and 2, supra). The
content of each column is the same as those provided in Table 3,
supra.
TABLE-US-00010 TABLE 5 Ctnnb1 siRNA sequences 5'- Nuc SEQ siRNA
position Nucleotide sequence -position 2 to position ID name Strand
1 nuc position 20 21-3' NO: Ctnnb1: S 6amiL- omeU; fluG; omeU;
omeU; fluG; fluG; fluA; omeU- 15 1797 iB- omeU; omeU; fluG; fluA;
omeU; omeU; iBSup [3' Chol] omeC omeC; fluG; omeA; fluA; fluA;
omeUs A/S vinylP- fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeUSup
16 moeT omeC; fluA; omeA; fluU; omeC; fluC; omeA; fluA; omeC; fluA;
omeG; omeUs Ctnnb1: S iB- omeU; fluG; omeU; omeU; fluG; fluG; fluA;
omeU- 17 1797 omeC omeU; omeU; fluG; fluA; omeU; omeU; iBSup [5'
Chol] omeC; fluG; fluA; fluA; fluA; omeUs A/S vinylP- fluU; omeU;
fluC; omeG; fluA; omeA; fluU; omeUSup 16 moeT omeC; fluA; omeA;
fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs
[0222] Animals--
[0223] Female C57BL/6J wild-type LDL receptor -/- and ApoE -/- mice
were obtained from Jackson Laboratory (Stock #002207, 002052,
respectively) and were between 25-27 weeks old at time of study.
Female CD-1 mice were obtained from Charles River and were between
8-9 weeks old at time of study. All mice were maintained on a
12-hour light and dark cycle with al libitum access to water and
standard chow diet (no. 5001; LabDiet). Control and experimental
cholesterol-conjugated siRNAs were administered to mice by tail
vein injections at indicated dosages. Blood was collected by
cardiac puncture at the time of harvest. Tissue samples were
collected at specific time points after dosing. All animal studies
were conducted at Merck Research Laboratories in an
AAALAC-accredited West Point, Pa. animal facility using protocols
approved by the Institutional Animal Care and Use Committee
(IACUC).
[0224] Results--
[0225] In order to examine the potential mechanism of uptake of
cholesterol conjugates in muscle and whether association of the
conjugate with lipoprotein particles is required for uptake, the
efficacy of Ctnnb1 siRNA molecules with a single cholesterol entity
attached to either the 3' or 5' end of the passenger strand was
examined in wildtype, LDL receptor (LDLR) -/- and ApoE -/- mice.
Mice were treated with a single i.v. injection of Ctnnb1:1797[3'
Chol] siRNA at 14 mpk and Ctnnb1 mRNA was measured in the
gastrocnemius muscle after 72 hours (FIG. 7A). A maximum of 60%
mRNA knockdown was observed in wt mice. There is a reduction in
mRNA KD in ApoE -/- mice (50% KD) and a trend toward reduced KD in
LDLR -/- mice. The data suggests that Ctnnb1 cholesterol conjugate
uptake is only partially mediated through the LDL receptor or via
any ApoE-containing lipoprotein in muscle.
[0226] In order to determine whether the position of the
cholesterol on the passenger strand is essential for cholesterol
conjugate efficacy, Ctnnb1-chol conjugates with cholesterol on
either the 3' or 5' end of the passenger strand were examined in
vivo (FIG. 7B). CD-1 mice were treated with 15 mpk of either
version of Ctnnb1-chol and compared to a PBS control and a Placebo
5-chol, which has cholesterol at the 3' position. Ctnnb1 mRNA
levels were examined in gastrocnemius muscle 72 hours after
injection. The data suggests that both positions can be used to
create highly efficacious cholesterol conjugates in muscle.
Example 5
Chemical Structures of the Chemically-Modified Nucleotides Used to
Generate the siRNA Molecules Exemplified Herein
TABLE-US-00011 [0227] TABLE 6a Structure of 5'-position 1
nucleotides contained within siRNA molecules in Tables 3 and 5.
5'-position 1 nuc Structure vinylPmoeT ##STR00001## 6amil-iB-fluX X
= B = Base U, G, C, A ##STR00002## 6amil-iB-omeX X = B = Base U, G,
C, A ##STR00003##
TABLE-US-00012 TABLE 6b Structure of internally-located nucleotides
(i.e., positions 2-20) contained within the siRNA molecules in
Tables 3 and 5. Internal nuc (positions 2-20) Structure omeX X = B
= Base U, G, C, A ##STR00004## omeXs X = B = Base U, G, C, A
##STR00005## fluX X = B = Base U, G, C, A ##STR00006## fluXs X = B
= Base U, G, C, A ##STR00007##
TABLE-US-00013 TABLE 6c Structure of nucleotide position 21-3'
nucleotides exemplified in Tables 3 and 5. Nuc position 21-3'
Structure omeU-Sup ##STR00008## omeU-iBSup ##STR00009##
TABLE-US-00014 TABLE 6d Structure of cholesterol CpG Name Structure
Choles- terol CpG ##STR00010##
Sequence CWU 1
1
21119RNAArtificial SequenceSynthetic 1auggcaaaga acaaauaau
19219RNAArtificial SequenceSynthetic 2ggcaaagaac aaauaauau
19319RNAArtificial SequenceSynthetic 3acuccagaau agaagccau
19419RNAArtificial SequenceSynthetic 4uuuggaagau gacgauuau
19521RNAArtificial SequenceSynthetic 5auggcaaaga acaaauaauu u
21621DNAArtificial SequenceSynthetic 6tuuauuuguu cuuugccauu u
21721RNAArtificial SequenceSynthetic 7ggcaaagaac aaauaauauu u
21821DNAArtificial SequenceSynthetic 8tuauuauuug uucuuugccu u
21921RNAArtificial SequenceSynthetic 9acuccagaau agaagccauu u
211021DNAArtificial SequenceSynthetic 10tuggcuucua uucuggaguu u
211121RNAArtificial SequenceSynthetic 11uuuggaagau gacgauuauu u
211221DNAArtificial SequenceSynthetic 12tuaaucguca ucuuccaaau u
211321RNAArtificial SequenceSynthetic 13gucgccuuau aucggucgau u
211421DNAArtificial SequenceSynthetic 14tcgaccgaua uaaggcgacu u
211521RNAArtificial SequenceSynthetic 15cuguuggauu gauucgaaau u
211621DNAArtificial SequenceSynthetic 16tuucgaauca auccaacagu u
211721RNAArtificial SequenceSynthetic 17cuguuggauu gauucgaaau u
211819RNAArtificial SequenceSynthetic 18auuauuuguu cuuugccau
191919RNAArtificial SequenceSynthetic 19auauuauuug uucuuugcc
192019RNAArtificial SequenceSynthetic 20auggcuucua uucuggagu
192119RNAArtificial SequenceSynthetic 21auaaucguca ucuuccaaa 19
* * * * *