U.S. patent application number 14/367939 was filed with the patent office on 2014-12-11 for combination therapy for treating hearing and balance disorders.
This patent application is currently assigned to QUARK PHARMACEUTICALS, INC.. The applicant listed for this patent is QUARK PHARMACEUTICALS, INC.. Invention is credited to Svetlana Adamsky, Elena Feinstein.
Application Number | 20140364484 14/367939 |
Document ID | / |
Family ID | 48781880 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140364484 |
Kind Code |
A1 |
Adamsky; Svetlana ; et
al. |
December 11, 2014 |
COMBINATION THERAPY FOR TREATING HEARING AND BALANCE DISORDERS
Abstract
The present application relates to combinations of inhibitors
directed at down-regulation of genes associated with hearing loss
including HES1, HES5, HEY2, CDKN1B and NOTCH1, exhibiting a
beneficial effect and useful in treating or attenuating hearing
loss, treating balance impairment, promoting the replacement,
regeneration, or protection of otic (sensory) hair cells of the
inner ear, and or effecting hearing restoration/regeneration.
Inventors: |
Adamsky; Svetlana; (Gedera,
IL) ; Feinstein; Elena; (Rehovot, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
QUARK PHARMACEUTICALS, INC. |
Fremont |
CA |
US |
|
|
Assignee: |
QUARK PHARMACEUTICALS, INC.
Fremont
CA
|
Family ID: |
48781880 |
Appl. No.: |
14/367939 |
Filed: |
January 10, 2013 |
PCT Filed: |
January 10, 2013 |
PCT NO: |
PCT/US2013/020918 |
371 Date: |
June 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61585672 |
Jan 12, 2012 |
|
|
|
Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 2310/14 20130101; C12N 15/113 20130101; A61P 27/16 20180101;
C12N 2320/31 20130101 |
Class at
Publication: |
514/44.A |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2012 |
US |
PCT/US2012/049616 |
Claims
1. A combination of a HES1 inhibitor, a HES5 inhibitor and a HEY2
inhibitor, for simultaneous, separate or sequential use in a
subject for preventing, treating, or delaying of progression of a
hearing disorder, a hearing loss, and/or a balance impairment, or
for preventing the loss of otic (sensory) hair cells of the inner
ear in the subject.
2. A method of preventing, treating, or delaying of progression of
a hearing disorder, a hearing loss, and/or a balance impairment, or
of preventing the loss of otic (sensory) hair cells of the inner
ear in a subject, comprising use of a HES1 inhibitor, a HES5
inhibitor and a HEY2 inhibitor.
3. The combination or method of claim 1 or 2, wherein the HES1
inhibitor, the HES5 inhibitor and the HEY2 inhibitor are to be
administered substantially simultaneously or sequentially.
4. A composition comprising a combination of a HES1 inhibitor or a
pharmaceutically acceptable salt or prodrug thereof, a HES5
inhibitor or a pharmaceutically acceptable salt or prodrug thereof
and a HEY2 inhibitor or a pharmaceutically acceptable salt or
prodrug thereof; and a pharmaceutically acceptable carrier.
5. The combination, method, or composition of any one of claims 1
to 4, wherein each inhibitor is independently selected from the
group consisting of a small organic molecule; a protein, an
antibody or fragments thereof, a peptide, a peptidomimetic and a
nucleic acid molecule.
6. The combination, method, or composition of claim 5, wherein each
inhibitor comprises a nucleic acid molecule, or a pharmaceutically
acceptable salt thereof.
7. The combination, method or composition of claim 6, wherein a
first nucleic acid molecule is a double-stranded oligonucleotide
that binds a nucleotide sequence encoding a HES1 gene, a second
nucleic acid molecule is a double-stranded oligonucleotide that
binds a nucleotide sequence encoding a HES5 gene, and a third
nucleic acid molecule is a double-stranded oligonucleotide that
binds a nucleotide sequence encoding a HEY2 gene.
8. The combination, method or composition of claim 7, wherein each
of the double-stranded oligonucleotides comprises a dsRNA molecule
having a sense strand and an antisense strand.
9. The combination, method or composition of claim 7 or 8, wherein
the nucleic acid molecules are linked or wherein the nucleic acid
molecules are annealed in RNAistar formation.
10. The combination, method or composition of any one of claims 7
to 9, wherein: a first double-stranded oligonucleotide comprises a
sense strand and an antisense strand, wherein: (a) each strand is
independently 18 to 49 nucleotides in length; (b) a 18 to 49
nucleotide sequence of the antisense strand is complementary to a
sequence of an mRNA encoding HES1; and (c) a 18 to 49 nucleotide
sequence of the sense strand is complementary to the antisense
strand; a second double-stranded oligonucleotide comprises a sense
strand and an antisense strand, wherein: (a) each strand is
independently 18 to 49 nucleotides in length; (b) a 18 to 49
nucleotide sequence of the antisense strand is complementary to a
sequence of an mRNA encoding HES5; and (c) a 18 to 49 nucleotide
sequence of the sense strand is complementary to the antisense
strand; and a third double-stranded oligonucleotide comprises a
sense strand and an antisense strand, wherein: (a) each strand is
independently 18 to 49 nucleotides in length; (b) a 18 to 49
nucleotide sequence of the antisense strand is complementary to a
sequence of an mRNA encoding HEY2; and (c) a 18 to 49 nucleotide
sequence of the sense strand is complementary to the antisense
strand.
11. The combination, method or composition of any one of claims
1-10, wherein the inhibitors are co-administered to the subject in
the same formulation.
12. The combination or method of any one of claims 1-3 and 5-10,
wherein the inhibitors are co-administered to the subject in
different formulations.
13. The combination, method or composition of any one of claims
1-10, wherein the inhibitors are co-administered to the subject by
the same route.
14. The combination or method of any one of claim 1-3 or 5-10,
wherein the inhibitors are co-administered to the subject by
different routes.
15. The combination, method or composition of claim 13, wherein the
inhibitors are co-administered to the subject by transtympanic
injection.
16. The combination, method or composition of claim 13, wherein the
inhibitors are co-administered topically.
17. The combination, method or composition of claim 16, wherein the
topical administration comprises application of ear drops to the
eardrum.
18. The combination or method of any one of claims 1-3, wherein the
administering is substantially simultaneous.
19. The combination or method of any one of claims 1-3, wherein the
administering is sequential.
20. The combination, method or composition of any one of claims 7
to 19, wherein at least one double-stranded oligonucleotide
independently comprises a structure (A1): TABLE-US-00013 (A1)
(antisense strand) 5' (N)x - Z 3' (sense strand) 3' Z'-(N')y - z''
5'
wherein each of N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond; wherein
each of Z and Z' is independently present or absent, but if present
is independently 1-5 consecutive nucleotides, 1-5 consecutive
non-nucleotide moieties or a combination thereof covalently
attached at the 3' terminus of the strand in which it is present.
wherein z'' may be present or absent, but if present is a capping
moiety covalently attached at the 5' terminus of (N')y; wherein
each of x and y is independently an integer between 18 and 40;
wherein the sequence of (N')y is complementary to the sequence of
(N)x; and wherein (N)x comprises an antisense sequence
complementary to an mRNA selected from an mRNA encoding HES1, an
mRNA encoding HES5, and an mRNA encoding HEY2.
21. The combination, method or composition of claim 20, wherein in
the double-stranded oligonucleotide x=y=19.
22. The combination, method or composition of any one of claims 7
to 19, wherein at least one double-stranded oligonucleotide
independently comprises a structure (A2): TABLE-US-00014 (A2)
(antisense strand) 5' N.sup.1-(N)x - Z 3' (sense strand) 3'
Z'-N.sup.2-(N')y - z'' 5'
wherein each of N.sup.2, N and N' is independently an unmodified or
modified ribonucleotide, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the adjacent N or N' by a covalent bond; wherein
each of x and y is independently an integer between 17 and 39;
wherein N.sup.2 is covalently bound to (N')y; wherein N.sup.1 is
covalently bound to (N)x and is mismatched to the target mRNA
selected from HES1 mRNA, HES5 mRNA and HEY2 mRNA or is a
complementary deoxyribonucleotide moiety to the mRNA selected from
HES1 mRNA, HES5 mRNA and HEY2 mRNA; wherein N.sup.1 is a moiety
selected from the group consisting of natural or modified: uridine,
deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or
deoxyadenosine, an abasic ribose moiety and an abasic deoxyribose
moiety; wherein z'' may be present or absent, but if present is a
capping moiety covalently attached at the 5' terminus of N.sup.2--
(N')y; and wherein each of Z and Z' is independently present or
absent, but if present is independently 1-5 consecutive
nucleotides, 1-5 consecutive non-nucleotide moieties or a
combination thereof covalently attached at the 3' terminus of the
strand in which it is present; wherein the sequence of (N')y has
complementarity to the sequence of (N)x; and wherein the sequence
of (N)x has complementarity to a consecutive sequence selected from
a sequence in HES1 mRNA (SEQ ID NO:1); HES5 mRNA (SEQ ID NO:2), and
HEY2 mRNA (SEQ ID NO:10).
23. The combination, method or composition of claim 22, wherein in
the double-stranded oligonucleotide x=y=18.
24. A commercial package comprising the combination or composition
of any one of claims 1 or 3 to 23.
25. The commercial package of claim 24, wherein the package
includes a label or package insert that provides certain
information about methods of using the combination or composition
of any one of claims 1 or 3 to 23.
26. The commercial package of claim 25, wherein the label or
package insert includes dosing information.
27. The commercial package of claim 26, wherein the label or
package insert includes indications for use.
28. The commercial package of any one of claims 25 to 27, wherein
the label or package insert indicates that the combination of any
one of claims 1 or 3 to 15 is suitable for use in therapy.
29. The commercial package of any one of claims 25 to 27, wherein
the label or package insert indicates that the combination or
composition of any one of claims 1 or 3 to 15 is suitable for use
in preventing, treating, or delaying of progression of a hearing
disorder, a hearing loss, and/or a balance impairment, or for
preventing the loss of otic (sensory) hair cells of the inner ear
in a subject.
30. A combination of a CDKN1B inhibitor, a NOTCH1 inhibitor and a
HEY2 inhibitor, for simultaneous, separate or sequential use in
preventing, treating, or delaying of progression of a hearing
disorder, a hearing loss, and/or a balance impairment, or for
preventing the loss of otic (sensory) hair cells of the inner ear
in a subject.
31. A method of preventing, treating, or delaying of progression of
a hearing disorder, a hearing loss, and/or a balance impairment, or
of preventing the loss of otic (sensory) hair cells of the inner
ear in a subject, comprising use of a CDKN1B inhibitor, a NOTCH1
inhibitor and a HEY2 inhibitor.
32. The combination or method of claim 30 or 31, wherein the CDKN1B
inhibitor, the NOTCH1 inhibitor and the HEY2 inhibitor are to be
administered substantially simultaneously or sequentially.
33. A composition comprising at least one CDKN1B inhibitor or a
pharmaceutically acceptable salt or prodrug thereof, at least one
NOTCH1 inhibitor or a pharmaceutically acceptable salt or prodrug
thereof; and at least one HEY2 inhibitor or a pharmaceutically
acceptable salt or prodrug thereof; and a pharmaceutically
acceptable carrier.
34. The combination, method, or composition of any one of claims 30
to 33, wherein each inhibitor is independently selected from the
group consisting of a small organic molecule; a protein, an
antibody or fragments thereof, a peptide, a peptidomimetic and a
nucleic acid molecule.
35. The combination, method, or composition of claim 34, wherein
each inhibitor comprises a nucleic acid molecule or a
pharmaceutically acceptable salt thereof.
36. The combination, method, or composition of claim 35, wherein a
first nucleic acid molecule is a double-stranded oligonucleotide
that binds a nucleotide sequence encoding a CDKN1B gene, a second
nucleic acid molecule is a double-stranded oligonucleotide that
binds a nucleotide sequence encoding a NOTCH1 gene, and a third
nucleic acid molecule is a double-stranded oligonucleotide that
binds a nucleotide sequence encoding a HEY2 gene.
37. The combination, method, or composition of claim 36, wherein
each of the double-stranded oligonucleotides comprises a dsRNA
molecule having a sense strand and an antisense strand.
38. The combination, method, or composition of claim 36 or 37,
wherein the nucleic acid molecules are linked; or wherein the
nucleic acid molecules are annealed in RNAistar formation.
39. The combination, method, or composition of any one of claims
35-38, wherein: a first double-stranded oligonucleotide comprises a
sense strand and an antisense strand, wherein; (a) each strand is
independently 18 to 49 nucleotides in length; (b) a 18 to 49
nucleotide sequence of the antisense strand is complementary to a
sequence of an mRNA encoding CDKN1B; and (c) a 18 to 49 nucleotide
sequence of the sense strand is complementary to the antisense
strand; a second double-stranded oligonucleotide comprises a sense
strand and an antisense strand, wherein; (a) each strand is
independently 18 to 49 nucleotides in length; (b) a 18 to 49
nucleotide sequence of the antisense strand is complementary to a
sequence of an mRNA encoding NOTCH1; and (c) a 18 to 49 nucleotide
sequence of the sense strand is complementary to the antisense
strand; and a third double-stranded oligonucleotide comprises a
sense strand and an antisense strand, wherein; (a) each strand is
independently 18 to 49 nucleotides in length; (b) a 18 to 49
nucleotide sequence of the antisense strand is complementary to a
sequence of an mRNA encoding HEY2; and (c) a 18 to 49 nucleotide
sequence of the sense strand is complementary to the antisense
strand.
40. The combination, method or composition of any one of claims
30-39, wherein the inhibitors are co-administered to the subject in
the same formulation.
41. The combination or method of any one of claims 30-32 and 34-39,
wherein the inhibitors are co-administered to the subject in
different formulations.
42. The combination, method or composition of any one of claims
30-39, wherein the inhibitors are co-administered to the subject by
the same route.
43. The combination or method of any one of claims 30-32 and 34-39,
wherein the inhibitors are co-administered to the subject by
different routes.
44. The combination or method of claim 42, wherein the inhibitors
are co-administered to the subject by transtympanic injection.
45. The combination or method of claim 42, wherein the inhibitors
are co-administered topically.
46. The combination or method of claim 45, wherein the topical
administration comprises application of ear drops to the
eardrum.
47. The combination or method of any one of claims 30-32, wherein
the administering is substantially simultaneous.
48. The combination or method of any one of claims 30-32, wherein
the administering is sequential.
49. The combination, method, or composition of any one of claims 36
to 48, wherein at least one double-stranded oligonucleotide
independently comprises a structure (A1): TABLE-US-00015 (A1)
(antisense strand) 5' (N)x - Z 3' (scnsc strand) 3' Z'-(N')y - z''
5'
wherein each of N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond; wherein
each of Z and Z' is independently present or absent, but if present
is independently 1-5 consecutive nucleotides, 1-5 consecutive
non-nucleotide moieties or a combination thereof covalently
attached at the 3' terminus of the strand in which it is present.
wherein z'' may be present or absent, but if present is a capping
moiety covalently attached at the 5' terminus of (N')y; wherein
each of x and y is independently an integer between 18 and 40;
wherein the sequence of (N')y is complementary to the sequence of
(N)x; and wherein (N)x comprises an antisense sequence to an mRNA
selected from an mRNA encoding CDKN1B, an mRNA encoding NOTCH1, and
an mRNA encoding HEY2.
50. The combination, method, or composition of claim 49, wherein in
the double-stranded oligonucleotide x=y=19.
51. The combination, method, or composition of any one of claims 36
to 48, wherein at least one double-stranded oligonucleotide
independently comprises a structure (A2): TABLE-US-00016 (A2)
(antisense strand) 5' N.sup.1-(N)x - Z 3' (sense strand) 3'
Z'-N.sup.2-(N')y - z'' 5'
wherein each of N.sup.2, N and N' is independently an unmodified or
modified ribonucleotide, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the adjacent N or N' by a covalent bond; wherein
each of x and y is independently an integer between 17 and 39;
wherein N.sup.2 is covalently bound to (N')y; wherein N.sup.1 is
covalently bound to (N)x and is mismatched to the target mRNA
selected from CDKN1B mRNA, NOTCH1 mRNA and HEY2 mRNA or is a
complementary deoxyribonucleotide moiety to the mRNA selected from
CDKN1B mRNA, NOTCH1 mRNA and HEY2 mRNA; wherein N.sup.1 is a moiety
selected from the group consisting of natural or modified: uridine,
deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or
deoxyadenosine, an abasic ribose moiety and an abasic deoxyribose
moiety; wherein z'' may be present or absent, but if present is a
capping moiety covalently attached at the 5' terminus of
N.sup.2--(N')y; and wherein each of Z and Z' is independently
present or absent, but if present is independently 1-5 consecutive
nucleotides, 1-5 consecutive non-nucleotide moieties or a
combination thereof covalently attached at the 3' terminus of the
strand in which it is present; wherein the sequence of (N')y has
complementarity to the sequence of (N)x; and wherein the sequence
of (N)x has complementarity to a consecutive sequence selected from
a sequence in CDKN1B mRNA (SEQ ID NO:7); NOTCH1 mRNA (SEQ ID
NO:11), and HEY2 mRNA (SEQ ID NO:10)
52. The combination, method or composition of claim 51, wherein in
the double-stranded oligonucleotide x=y=18.
53. A commercial package comprising the composition or the
combination of any one of claims 30 to 32 or 34 to 52.
54. The commercial package of claim 53, wherein the label or
package insert indicates that the composition or the combination of
any one of claims 30 to 32 or 34 to 52 is suitable for use in
therapy.
55. The commercial package of claim 53 or 54, wherein the label or
package insert indicates that the composition or the combination of
any one of claims 30 to 32 or 34 to 52 is suitable for use in
preventing, treating, or delaying of progression of a hearing
disorder, a hearing loss, and/or a balance impairment, or for
preventing the loss of otic (sensory) hair cells of the inner ear
in a subject.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/585,672 filed Jan. 12, 2012 entitled
"Compounds, Compositions and Methods For Treating Hearing Loss" and
of PCT Application No. PCT/US12/49616 filed Aug. 3, 2012 entitled
"Double-Stranded Oligonucleotide Compounds and Methods of Use
Thereof for Treating Hearing and Balance Disorders", which are
incorporated herein by reference in their entirety and for all
purposes.
SEQUENCE LISTING
[0002] This application incorporates-by-reference nucleotide and/or
amino acid sequences which are present in the file named
"237-PCT2.ST25.txt", which is 5.044 megabytes in size, and which
was created on Jan. 9, 2013 in the IBM-PC machine format, having an
operating system compatibility with MS-Windows, and is submitted
herewith.
FIELD OF THE INVENTION
[0003] The present disclosure relates to combination therapy,
including compositions and methods useful for treating hearing
loss, treating balance impairment, promoting the replacement,
regeneration, or protection of otic hair (sensory) cells of the
inner ear, or effecting hearing restoration/regeneration.
BACKGROUND OF THE INVENTION
[0004] PCT application No. PCT/US12/49616 to the assignee of the
present application relates to double stranded RNA compounds,
pharmaceutical compositions comprising same and methods of use
thereof for the down-regulation of target genes associated with
hearing loss and balance impairment, including HES1, HES5, HEY1,
HEY2, ID1, ID2, ID3, CDKN1B, and NOTCH1, the inhibition of which is
useful for treating hearing loss, treating balance impairment,
promoting the replacement, regeneration, or protection of otic hair
(sensory) cells of the inner ear, or effecting hearing
restoration/regeneration.
SUMMARY OF THE INVENTION
[0005] It has now been found that certain tripartite combinations
of inhibitors directed at down-regulation of certain target genes
associated with hearing and balance disorders are beneficial in
treating or attenuating hearing loss, treating balance impairment,
promoting the replacement, regeneration, or protection of otic
(sensory) hair cells of the inner ear, and/or effecting hearing
restoration/regeneration. In particular, the combination includes a
first agent targeting HES1, a second agent targeting HES5 and a
third agent targeting HEY2, or includes a first agent targeting
CDKN1B, a second agent targeting NOTCH1 and a third agent targeting
HEY2.
[0006] In one aspect, provided herein is a combination of a HES1
inhibitor, a HES5 inhibitor and a HEY2 inhibitor for use in
therapy. In some embodiments the therapy comprises preventing,
treating, or delaying of progression of a hearing disorder, a
hearing loss, and/or a balance impairment, or for preventing the
loss of otic (sensory) hair cells of the inner ear in a subject. In
another aspect provided herein is a composition comprising a
combination of a HES1 inhibitor, or a pharmaceutically acceptable
salt or prodrug thereof, a HES5 inhibitor, or a pharmaceutically
acceptable salt or prodrug thereof and a HEY2 inhibitor, or a
pharmaceutically acceptable salt or prodrug thereof; and a
pharmaceutically acceptable carrier. In yet another aspect provided
herein is a method of preventing, treating, or delaying of
progression of a hearing disorder, a hearing loss, and/or a balance
impairment, or of preventing the loss of otic (sensory) hair cells
of the inner ear in a subject, comprising administering to the
subject a HES1 inhibitor, a HES5 inhibitor and a HEY2 inhibitor. In
another aspect provided herein is a product, a kit or a commercial
package comprising a HES1 inhibitor, a HES5 inhibitor and a HEY2
inhibitor or a composition thereof as disclosed hereinabove.
[0007] In yet another aspect provided herein is a combination of a
CDKN1B inhibitor, a NOTCH1 inhibitor, and a HEY2 inhibitor for use
in for use in therapy. In some embodiments the therapy comprises
preventing, treating, or delaying of progression of a hearing
disorder, a hearing loss, and/or a balance impairment, or for
preventing the loss of otic (sensory) hair cells of the inner ear
in a subject. In another aspect provided herein is a composition
comprising at least one CDKN1B inhibitor, or a pharmaceutically
acceptable salt or prodrug thereof, at least one NOTCH1 inhibitor,
or a pharmaceutically acceptable salt or prodrug thereof; and at
least one HEY2 inhibitor, or a pharmaceutically acceptable salt or
prodrug thereof; and a pharmaceutically acceptable carrier. In yet
another aspect provided herein is a method of preventing, treating,
or delaying of progression of a hearing disorder, a hearing loss,
and/or a balance impairment, or of preventing the loss of otic
(sensory) hair cells of the inner ear in a subject, comprising
administering to the subject a CDKN1B inhibitor, a NOTCH1 inhibitor
and a HEY2 inhibitor In another aspect provided herein is a
product, a kit or a commercial package comprising a CDKN1B
inhibitor, a NOTCH1 inhibitor and a HEY2 inhibitor or a composition
thereof as disclosed hereinabove.
[0008] In various aspects and embodiments of the combinations and
methods provided herein a therapeutically effective amount of each
of the inhibitors is administered substantially simultaneously,
separately or sequentially and in any order, and the components are
administered separately or as a fixed combination (for example in a
single dosage form). In some embodiments of the combinations and
methods provided herein, each of the inhibitors is administered
substantially simultaneously or sequentially and in any order. In
other embodiments of the combinations and methods provided herein,
a therapeutically effective amount of two of the inhibitors is
administered simultaneously or substantially simultaneously and the
third inhibitor is administered separately.
[0009] In one embodiment, provided herein is a combination of a
HES1 inhibitor, a HES5 inhibitor and a HEY2 inhibitor, for
substantially simultaneous or sequential use in preventing,
treating, or delaying of progression of a hearing disorder, a
hearing loss, and/or a balance impairment, or for preventing the
loss of otic (sensory) hair cells of the inner ear in a
subject.
[0010] In another embodiment, provided herein is a combination of a
CDKN1B inhibitor, a NOTCH1 inhibitor and a HEY2 inhibitor, for
substantially simultaneous or sequential use in preventing,
treating, or delaying of progression of a hearing disorder, a
hearing loss, and/or a balance impairment, or for preventing the
loss of otic (sensory) hair cells of the inner ear in a
subject.
[0011] In one embodiment provided are methods for treating,
including preventing, the incidence or severity of hearing loss in
a subject in which expression of HES1, HES5, and HEY2 genes is
associated with the etiology or progression of the hearing
disorder/hearing loss.
[0012] In another embodiment, provided are methods for treating,
including preventing, the incidence or severity of balance
impairment in a subject in which expression of HES1, HES5, and HEY2
genes is associated with the etiology or progression of the balance
impairment.
[0013] In yet another embodiment, provided are methods for
treating, including preventing, the incidence or severity of loss
of otic (sensory) hair cells of the inner ear in a subject, in
which expression of HES1, HES5, and HEY2, genes is associated with
the etiology or progression of the otic (sensory) hair cell
loss.
[0014] In one embodiment, provided are methods for treating,
including preventing, the incidence or severity of hearing loss in
a subject in which expression of CDKN1B, NOTCH1 and HEY2 genes is
associated with the etiology or progression of the hearing
disorder/hearing loss.
[0015] In another embodiment, provided are methods for treating,
including preventing, the incidence or severity of balance
impairment in a subject in which expression of CDKN1B, NOTCH1 and
HEY2 genes is associated with the etiology or progression of the
balance impairment.
[0016] In yet another embodiment, provided are methods for
treating, including preventing, the incidence or severity of loss
of otic (sensory) hair cells of the inner ear of a subject, in
which expression of CDKN1B, NOTCH1 and HEY2, genes is associated
with the etiology or progression of the otic (sensory) hair cell
loss.
[0017] In one embodiment provided herein is a method of preventing,
treating or delaying progression of a hearing disorder, a hearing
loss, and/or a balance impairment, or of preventing the loss of
otic (sensory) hair cells of the inner ear in a subject, comprising
administering to the subject a HES1 inhibitor, a HES5 inhibitor and
a HEY2 inhibitor.
[0018] In another embodiment provided herein is a method of
preventing, treating, or delaying of progression of a hearing
disorder, a hearing loss, and/or a balance impairment, or of
preventing the loss of otic (sensory) hair cells of the inner ear
in a subject, comprising administering to the subject a CDKN1B
inhibitor, a NOTCH1 inhibitor and a HEY2 inhibitor.
[0019] In some embodiments the subject is a mammal. In a preferred
embodiment the subject is a human subject.
[0020] Further provided is a method of preventing degeneration of
the auditory nerve in a subject comprising administering to the
subject a combination disclosed herein.
[0021] Such methods involve administering to a mammal in need of
such treatment a combination or a composition comprising
prophylactically or therapeutically effective amount of a HES1
inhibitor, a HES5 inhibitor and a HEY2 inhibitor. In another
embodiment, such methods involve administering to a mammal in need
of such treatment a combination or a composition comprising
prophylactically or therapeutically effective amount of a CDKN1B
inhibitor, a NOTCH1 inhibitor and a HEY2 inhibitor.
[0022] In various embodiments, each inhibitor (the HES1 inhibitor,
the HES5 inhibitor, the HEY2 inhibitor, the CDKN1B inhibitor, and
the NOTCH1 inhibitor) is independently selected from the group
consisting of a small organic molecule; a protein, an antibody or a
fragment thereof, a peptide, a peptidomimetic and a nucleic acid
molecule.
[0023] In preferred embodiments, each inhibitor comprises a
therapeutic nucleic acid molecule, or a pharmaceutically acceptable
salt thereof. In some embodiments, the nucleic acid compound is
applied directly to the round window membrane of the cochlea or
administered by transtympanic injection or via a transtympanic
device including a canula or an implant. Methods include sustained
delivery and controlled delivery for local or systemic delivery
including delivery of inhibitors using, for example, a pump, a slow
or sustained release composition or an implant comprising a drug
depot.
[0024] In preferred embodiments of the compositions, combinations,
methods, commercial packages and kits provided herein, each
inhibitor comprises a separate nucleic acid molecule, or a
pharmaceutically acceptable salt thereof. In some embodiments of
the compositions, combinations, methods, commercial packages and
kits provided herein, the nucleic acid molecules are linked one to
the other. In some embodiments of the compositions, combinations,
methods, commercial packages and kits provided herein, the nucleic
acid molecules are annealed and covalently linked in a multi-arm
formation (RNAistar).
[0025] In preferred embodiments of the compositions, combinations,
methods, commercial packages and kits provided herein, each
inhibitor comprises a nucleic acid compound. Preferably, the
nucleic acid compound is applied directly to the round window
membrane of the cochlea or administered by transtympanic injection
or via a transtympanic device including a canula.
[0026] Thus, the compositions, combinations, methods, commercial
packages and kits provided herein preferably involve use of nucleic
acid molecules (for example, short interfering nucleic acid (siNA),
short interfering RNA (siRNA), double-stranded RNA (dsRNA),
micro-RNA (miRNA) or short hairpin RNA (shRNA)) that bind a
nucleotide sequence (such as an mRNA sequence) or portion thereof,
encoding HES1, HES5, HEY2, CDKN1B, or NOTCH1, for example, the mRNA
coding sequence (SEQ ID NOS:1, 2, 10, 7, and 11) for human HES1,
HES5, HEY2, CDKN1B, or NOTCH1 mRNA, respectively, encoding one or
more proteins or protein subunits exemplified by SEQ ID NOS:12, 13,
21, 18 and 22, respectively. In certain preferred embodiments, the
compositions, combinations, methods, commercial packages and kits
disclosed herein down-regulate or inhibit expression of HES1, HES5
and HEY2, or CDKN1B, NOTCH1, and HEY2 genes. In various embodiments
each nucleic acid molecule is selected from the group consisting of
unmodified or chemically modified dsRNA compound such as a
chemically modified siRNA or shRNA that down-regulates HES1, HES5,
HEY2, CDKN1B or NOTCH1, expression.
[0027] In some preferred embodiments the HES1 inhibitor is a
synthetic, chemically modified double stranded RNA (dsRNA) compound
that down-regulates HES1 expression. In certain preferred
embodiments, "HES1" refers to human HES1 gene. In some preferred
embodiments the HES5 inhibitor is a synthetic, chemically modified
double stranded RNA (dsRNA) compound that down-regulates HES5
expression. In certain preferred embodiments, "HES5" refers to
human HES5 gene. In some preferred embodiments the HEY2 inhibitor
is a synthetic, chemically modified double stranded RNA (dsRNA)
compound that down-regulates HEY2 expression. In certain preferred
embodiments, "HEY2" refers to human HEY2 gene. In some preferred
embodiments the CDKN1B inhibitor is a synthetic, chemically
modified double stranded RNA (dsRNA) compound that down-regulates
CDKN1B expression. In certain preferred embodiments, "CDKN1B"
refers to human CDKN1B gene. In some preferred embodiments the
NOTCH1 inhibitor is a synthetic, chemically modified double
stranded RNA (dsRNA) compound that down-regulates NOTCH1
expression. In certain preferred embodiments, "NOTCH1" refers to
human NOTCH1 gene.
[0028] In some preferred embodiments of the compositions,
combinations, methods, commercial packages and kits provided
herein, the first nucleic acid molecule is a double-stranded
oligonucleotide that binds a nucleotide sequence encoding a HES1
gene, the second nucleic acid molecule is a double-stranded
oligonucleotide that binds a nucleotide sequence encoding a HES5
gene, and the third nucleic acid molecule is a double-stranded
oligonucleotide that binds a nucleotide sequence encoding a HEY2
gene.
[0029] In some preferred embodiments of the compositions,
combinations, methods, commercial packages and kits provided
herein, the first nucleic acid molecule is a double-stranded
oligonucleotide that binds a nucleotide sequence encoding a CDKN1B
gene, the second nucleic acid molecule is a double-stranded
oligonucleotide that binds a nucleotide sequence encoding a NOTCH1
gene, and the third nucleic acid molecule is a double-stranded
oligonucleotide that binds a nucleotide sequence encoding a HEY2
gene.
[0030] In various preferred embodiments of the compositions,
combinations, methods, commercial packages and kits provided
herein, each of the double-stranded oligonucleotides (for example
double-stranded RNA (dsRNA)) comprises a sense strand and an
antisense strand.
[0031] In some preferred embodiments of the compositions,
combinations, methods, commercial packages and kits provided
herein, each of the double-stranded oligonucleotides comprises a
sense strand and an antisense strand, wherein (a) each strand is
independently 18 to 49 nucleotides in length; (b) a 18 to 49
nucleotide sequence of the antisense strand is complementary to a
sequence of an mRNA encoding a target gene; and (c) a 18 to 49
nucleotide sequence of the sense strand is complementary to the
antisense strand. In various embodiment the mRNA encoding a target
gene is selected from mammalian HES1 (SEQ ID NO:1), HES5 (SEQ ID
NO:2), HEY2 (SEQ ID NO:10), CDKN1B (SEQ ID NO:7), or NOTCH1 (e.g.,
SEQ ID NO:11 or portion thereof; and the sense strand and the
antisense strand comprise sequence pairs set forth in any of SEQ ID
NOS:23-1495 or 26667-26706 (HES1), SEQ ID NOS:1496-2703 or
26707-26732 (HES5), SEQ ID NOS:13004-16621 or 26779-26788 (HEY2),
SEQ ID NOS:7444-10533 or 26867-26900 (CDKN1B) or SEQ ID
NOS:16622-26666 or 26901-26912 (NOTCH1).
[0032] In some preferred embodiments of the compositions,
combinations, methods, commercial packages and kits provided
herein, a first double-stranded oligonucleotide comprises a sense
strand and an antisense strand, wherein;
(a) each strand is independently 18 to 49 nucleotides in length;
(b) a 18 to 49 nucleotide sequence of the antisense strand is
complementary to a sequence of an mRNA encoding HES1; and (c) a 18
to 49 nucleotide sequence of the sense strand is complementary to
the antisense strand;
[0033] a second double-stranded oligonucleotide comprises a sense
strand and an antisense strand, wherein;
(a) each strand is independently 18 to 49 nucleotides in length;
(b) a 18 to 49 nucleotide sequence of the antisense strand is
complementary to a sequence of an mRNA encoding HES5; and (c) a 18
to 49 nucleotide sequence of the sense strand is complementary to
the antisense strand; and
[0034] a third double-stranded oligonucleotide comprises a sense
strand and an antisense strand, wherein;
(a) each strand is independently 18 to 49 nucleotides in length;
(b) a 18 to 49 nucleotide sequence of the antisense strand is
complementary to a sequence of an mRNA encoding HEY2; and (c) a 18
to 49 nucleotide sequence of the sense strand is complementary to
the antisense strand.
[0035] In other preferred embodiments of the compositions,
combinations, methods, commercial packages and kits provided
herein, a first double-stranded oligonucleotide is a dsRNA molecule
comprising a sense strand and an antisense strand, wherein;
(a) each strand is independently 18 to 49 nucleotides in length;
(b) a 18 to 49 nucleotide sequence of the antisense strand is
complementary to a sequence of an mRNA encoding CDKN1B; and (c) a
18 to 49 nucleotide sequence of the sense strand is complementary
to the antisense strand; a second double-stranded oligonucleotide
comprises a sense strand and an antisense strand, wherein; (a) each
strand is independently 18 to 49 nucleotides in length; (b) a 18 to
49 nucleotide sequence of the antisense strand is complementary to
a sequence of an mRNA encoding NOTCH1; and (c) a 18 to 49
nucleotide sequence of the sense strand is complementary to the
antisense strand; and a third double-stranded oligonucleotide
comprises a sense strand and an antisense strand, wherein; (a) each
strand is independently 18 to 49 nucleotides in length; (b) a 18 to
49 nucleotide sequence of the antisense strand is complementary to
a sequence of an mRNA encoding HEY2; and (c) a 18 to 49 nucleotide
sequence of the sense strand is complementary to the antisense
strand.
[0036] In some embodiments the dsRNA molecule has a structure set
forth as Structure (A1) or Structure (A2), disclosed herein.
[0037] In particular embodiments, compositions, combinations,
methods, commercial packages and kits provided herein are useful in
the treatment of an ear (otic, aural) condition or pathology,
particularly pathologies involving death of otic (sensory) hair
cells if the inner ear, are provided herein.
[0038] In another aspect provided is use of a combination of a HES1
inhibitor, a HES5 inhibitor and a HEY2 inhibitor, for the
preparation of a medicament for the treatment of a disease or
disorder of the inner ear or of the middle ear. In another aspect
provided is use of a combination of a CDKN1B inhibitor, a NOTCH1
inhibitor and a HEY2 inhibitor, for the preparation of a medicament
for the treatment of a disease or disorder of the inner ear or of
the middle ear.
[0039] In particular embodiments, provided herein are combinations,
compositions and methods of use thereof in the treatment of
auditory and vestibular diseases, disorders, injuries and
conditions including, without being limited to ototoxin induced
hearing loss, age-related hearing loss, a hearing impairment due to
end-organ lesions involving inner ear hair cells, e.g., acoustic
trauma, viral endolymphatic labyrinthitis, Meniere's disease;
tinnitus which may be intermittent or continuous, wherein there is
diagnosed a sensorineural loss; hearing loss due to bacterial or
viral infection, such as in herpes zoster oticus; purulent
labyrinthitis arising from acute otitis media, purulent meningitis,
chronic otitis media, sudden deafness including that of viral
origin, e.g., viral endolymphatic labyrinthitis caused by viruses
including mumps, measles, influenza, chicken pox, mononucleosis and
adenoviruses; congenital hearing loss such as that caused by
rubella, anoxia during birth, bleeding into the inner ear due to
trauma during delivery, ototoxic drugs administered to the mother,
erythroblastosis fetalis, and hereditary conditions including
Waardenburg's syndrome and Hurler's syndrome.
[0040] In another aspect provided herein is a commercial package or
a kit comprising any of the composition or the combination
disclosed herein. In some embodiments, the commercial package or
kit, includes a label or package insert that provides certain
information about how the composition or combination disclosed
herein may be used. In some embodiments of the commercial package
or kit, the label or package insert includes dosing information. In
some embodiments of the commercial package or kit, the label or
package insert includes indications for use. In some embodiments of
the commercial package or kit, the label or package insert
indicates that the composition or the combination is suitable for
use in therapy. In some embodiments of the commercial package or
kit, wherein the label or package insert indicates that the
composition or the combination is suitable for use in preventing,
treating, or delaying of progression of a hearing disorder, a
hearing loss, and/or a balance impairment, or for preventing the
loss of otic (sensory) hair cells of the inner ear in a
subject.
[0041] The preferred methods, materials, and examples that will now
be described are illustrative only and are not intended to be
limiting; materials and methods similar or equivalent to those
described herein can be used in practice or testing of the
invention. Other features and advantages of the invention will be
apparent from the following figures, detailed description, and from
the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIGS. 1A-1E show the ABR response obtained in this study at
Day 0, after 3 weeks, after 5 weeks, after 7 weeks and after 9
weeks.
[0043] FIG. 1A shows the ABR response obtained in this study at Day
0, after 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks
for 1 KHz stimulus.
[0044] FIG. 1B shows the ABR response obtained in this study at Day
0, after 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks
for 4 KHz stimulus.
[0045] FIG. 1C shows the ABR response obtained in this study at Day
0, after 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks
for 8 KHz stimulus.
[0046] FIG. 1D shows the ABR response obtained in this study at Day
0, after 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks
for 16 KHz stimulus.
[0047] FIG. 1E shows the ABR response obtained in this study at Day
0, after 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks
for 32 KHz stimulus.
[0048] FIG. 1F provides the legend for FIGS. 1A-1E.
DETAILED DESCRIPTION OF THE INVENTION
[0049] Provided herein are compositions and combinations which
down-regulate expression of certain genes associated with hearing
loss and their use in treating a subject suffering from hearing
loss and/or balance impairment, for promoting the replacement,
regeneration, or protection of otic (sensory) hair cells of the
inner ear, and or effecting hearing restoration/regeneration. In
preferred embodiments the methods comprise partial or full hearing
regeneration Inhibition of expression of a combination of the HES1,
HES5 and HEY2 genes, or of a combination of the CDKN1B, NOTCH1 and
HEY2 genes was shown to be beneficial in regeneration of hearing.
The present application relates in particular to use of therapeutic
agents, for example double-stranded oligonucleotide molecules,
including dsRNA/small interfering RNA (siRNA) compounds which
inhibit expression of HES1, HES5, CDKN1B, HEY2 and NOTCH1 and to
the use of these dsRNA molecules in the treatment of hearing loss.
Sense strands and complementary antisense strands useful in
generating the compositions and the combinations of dsRNA molecules
as provided herein are set forth in SEQ ID NOS:23-1495 or
26667-26706 (HES1), SEQ ID NOS:1496-2703 or 26707-26732 (HES5), SEQ
ID NOS:13004-16621 or 26779-26788 (HEY2), SEQ ID NOS:7444-10533 or
26867-26900 (CDKN1B) or SEQ ID NOS:16622-26666 or 26901-26912
(NOTCH1). Certain currently preferred sense strand and antisense
strand pairs are set forth in tables I-V infra.
[0050] Provided herein are methods, combinations and compositions
for inhibiting expression of HES1, HES5 and HEY2 genes, or for
inhibiting expression of CDKN1B, NOTCH1 and HEY2 genes in vivo. In
general, the method includes administering oligoribonucleotide
combination/composition, such as combination/composition of dsRNA
molecules, including small interfering RNAs (i.e., dsRNAs), that
are targeted to the target mRNAs, and hybridize to, or interact
with, the mRNAs under biological conditions (within the cell), or a
nucleic acid material that can produce siRNAs in a cell, in an
amount sufficient to down-regulate expression of a target gene by
an RNA interference mechanism. Details of target genes are
presented in Table A, hereinbelow.
TABLE-US-00001 TABLE A Target genes Gene No. abbrev Full name and
gi and accession numbers 1 HES1 hairy and enhancer of split 1,
(Drosophila) Alternative Names: bHLHb39; FLJ20408; HES-1; HHL; HRY
gi|8400709|ref|NM_005524.2|(SEQ ID NO: 1) 2 HES5 hairy and enhancer
of split 5 (Drosophila) Alternative Names: bHLHb
38gi|145301612|ref|NM_001010926.2|(SEQ ID NO: 2) 3 ID1 inhibitor of
DNA binding 1, dominant negative HLH protein. Alternative Names:
bHLHb24; ID gi|31317298|ref|NM_002165.2|transcript v.1 (SEQ ID NO:
3) gi|31317296|ref|NM_181353.1|transcript v.2 (SEQ ID NO: 4) 4 ID2
inhibitor of DNA binding 2, dominant negative HLH protein
Alternative Names: bHLHb26; GIG8; ID2A; ID2H; MGC26389
gi|33946335|ref|NM_002166.4|(SEQ ID NO: 5) 5 ID3 inhibitor of DNA
binding 3, dominant negative HLH protein. Alternative Names:
bHLHb25; HEIR-1 gi|156119620|ref|NM_002167.3|(SEQ ID NO: 6) 6
CDKN1B cyclin-dependent kinase inhibitor 1B (p27, Kip1) Alternative
Names: CDKN4; KIP1; MEN1B; MEN4; P27KIP1
gi|17978497|ref|NM_004064.2|(SEQ ID NO: 7) 7 HEY1 HEY1 -
hairy/enhancer-of-split related with YRPW motif 1
gi|105990527|ref|NM_012258.3|transcript v.1 (SEQ ID NO: 8)
gi|105990525|ref|NM_001040708.1|transcript v.2 (SEQ ID NO: 9) 8
HEY2 HEY2 - hairy/enhancer-of-split related with YRPW motif 2
gi|105990529|ref|NM_012259.2|(SEQ ID NO: 10) 9 NOTCH1 NOTCH1 Notch
homolog 1, translocation-associated (Drosophila)
gi|148833507|ref|NM_017617.3|Homo sapiens mRNA (SEQ ID NO: 11)
[0051] Preferred targets are HES1 (mRNA SEQ ID NO:1; polypeptide
SEQ ID NO:12), HES5 (mRNA SEQ ID NO:2; polypeptide SEQ ID NO:13),
HEY2 (mRNA SEQ ID NO:10; polypeptide SEQ ID NO:21); CDKN1B (mRNA
SEQ ID NO:7; polypeptide SEQ ID NO:18) and NOTCH1 (mRNA SEQ ID
NO:11; polypeptide SEQ ID NO:22).
[0052] In various embodiments, provided is the use of
compositions/combinations of double stranded RNAs, (dsRNAs)
including chemically modified small interfering RNAs (siRNAs), in
the treatment of various diseases and medical conditions.
Particular diseases and conditions to be treated are related to
hearing loss and/or balance loss.
[0053] Preferred sense and antisense nucleic acid sequences useful
in generating dsRNA for use in the combinations, compositions and
methods as provided herein were prioritized based on their score
according to a proprietary algorithm as the best sequences for
targeting the human gene expression. SEQ ID NOS:23-693 and
26691-26706 (HES1); SEQ ID NOS:1496-2029 and 26725-26732 (HES5);
SEQ ID NOS:7444-9007 and 26887-26900 (CDKN1B); SEQ ID
NOS:13004-14801 and 26785-26788 (HEY2); SEQ ID NOS:16622-18643 and
26922-26912 (NOTCH1) set forth 19-mer oligomers. SEQ ID
NOS:694-1495 (HES1); SEQ ID NOS:2030-2703 (HES5); SEQ ID
NOS:9008-10533 (CDKN1B); SEQ ID NOS:14802-16389 (HEY2); SEQ ID
NOS:18644-26666 (NOTCH1) set forth 18-mer oligomers useful in
generating dsRNA molecules according to Structure A2, as described
hereinbelow.
DEFINITIONS
[0054] For convenience certain terms employed in the specification,
examples and claims are described herein.
[0055] It is to be noted that, as used herein, the singular forms
"a", "an" and "the" include plural forms unless the content clearly
dictates otherwise.
[0056] Where aspects or embodiments of the invention are described
in terms of Markush groups or other grouping of alternatives, those
skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the group.
[0057] An "inhibitor" is a compound, which is capable of reducing
(partially or fully) the expression of a gene or the activity of
the product of such gene to an extent sufficient to achieve a
desired biological or physiological effect. The term "inhibitor" as
used herein refers to one or more of a small organic molecule, a
protein, an antibody or fragments thereof, a peptide, a
peptidomimetic and a nucleic acid molecule, including siRNA, shRNA,
synthetic shRNA; miRNA, antisense RNA and DNA and ribozymes.
[0058] A "dsRNA molecule" or "dsRNA inhibitor" is a compound which
is capable of down-regulating or reducing the expression of a gene
or the activity of the product of such gene to an extent sufficient
to achieve a desired biological or physiological effect and
includes one or more of a siRNA, shRNA, synthetic shRNA; miRNA
Inhibition may also be referred to as down-regulation or, for RNAi,
silencing.
[0059] The term "inhibit" as used herein refers to reducing the
expression of a gene or the activity of the product of such gene to
an extent sufficient to achieve a desired biological or
physiological effect. Inhibition is either complete or partial.
[0060] As used herein, the term "inhibition" of a target gene means
attenuation, reduction or down regulation of gene expression
(transcription or translation) or polypeptide activity of a target
gene wherein the target gene is selected from a gene transcribed
into an mRNA set forth in any one of SEQ ID NOS:1, 2, 10, 7, or 11
or an SNP (single nucleotide polymorphism) or other variants
thereof. The gi number for the mRNA of each target gene is set
forth in Table A ("v" refers to transcript variant). The
polynucleotide sequence of the target mRNA sequence, or the target
gene having a mRNA sequence refer to the mRNA sequences set forth
in SEQ ID NO:1, 2, 10, 7 or 11, or any homologous sequences thereof
preferably having at least 70% identity, more preferably 80%
identity, even more preferably 90% or 95% identity to any one of
mRNA set forth in SEQ ID NO:1, 2, 10, 7 or 11. Therefore,
polynucleotide sequences derived from any one of SEQ ID NO:1, 2,
10, 7 or 11 which have undergone mutations, alterations or
modifications as described herein are encompassed in the present
invention. The terms "mRNA polynucleotide sequence", "mRNA
sequence" and "mRNA" are used interchangeably.
[0061] As used herein, the terms "polynucleotide" and "nucleic
acid" may be used interchangeably and refer to nucleotide sequences
comprising deoxyribonucleic acid (DNA), and ribonucleic acid (RNA).
The terms are to be understood to include, as equivalents, analogs
of either RNA or DNA made from nucleotide analogs. Throughout this
application, mRNA sequences are set forth as representing the
corresponding genes.
[0062] "Oligonucleotide" or "oligomer" refers to a
deoxyribonucleotide or ribonucleotide sequence from about 2 to
about 50 nucleotides. Each DNA or RNA nucleotide may be
independently natural or synthetic, and or modified or unmodified.
Modifications include changes to the sugar moiety, the base moiety
and or the linkages between nucleotides in the oligonucleotide. The
compounds according to various aspect and embodiments of the
present disclosure encompass molecules comprising
deoxyribonucleotides, ribonucleotides, modified
deoxyribonucleotides, modified ribonucleotides, unconventional
moieties and combinations thereof.
[0063] "Substantially complementary" refers to complementarity of
greater than about 84%, to another sequence. For example in a
duplex region consisting of 19 base pairs one mismatch results in
94.7% complementarity, two mismatches results in about 89.5%
complementarity and 3 mismatches results in about 84.2%
complementarity, rendering the duplex region substantially
complementary. Accordingly substantially identical refers to
identity of greater than about 84%, to another sequence.
[0064] "Nucleotide" is meant to encompass deoxyribonucleotides and
ribonucleotides, which may be natural or synthetic, and or modified
or unmodified. Modifications include changes to the sugar moiety,
the base moiety and or the linkages between ribonucleotides in the
oligoribonucleotide. As used herein, the term "ribonucleotide"
encompasses natural and synthetic, unmodified and modified
ribonucleotides. Modifications include changes to the sugar moiety,
to the base moiety and/or to the linkages between ribonucleotides
in the oligonucleotide.
[0065] The nucleotides can be selected from naturally occurring or
synthetic modified bases. Naturally occurring bases include
adenine, guanine, cytosine, thymine and uracil. Modified bases of
nucleotides include inosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo
uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo
uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol
adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other
8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol
guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other
substituted guanines, other aza and deaza adenines, other aza and
deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
In some embodiments one or more nucleotides in an oligomer is
substituted with inosine.
[0066] According to some embodiments the present disclosure
provides use of inhibitory oligonucleotide compounds comprising
unmodified and modified nucleotides and or unconventional moieties.
The compounds comprise at least one modified nucleotide selected
from the group consisting of a sugar modification, a base
modification and an internucleotide linkage modification and may
contain DNA, and modified nucleotides such as LNA (locked nucleic
acid), ENA (ethylene-bridged nucleic acid), PNA (peptide nucleic
acid), arabinoside, phosphonocarboxylate or phosphinocarboxylate
nucleotide (PACE nucleotide), mirror nucleotide, or nucleotides
with a 6 carbon sugar.
[0067] All analogs of, or modifications to, a
nucleotide/oligonucleotide are employed with the present
embodiments, provided that said analog or modification does not
substantially adversely affect the function of the
nucleotide/oligonucleotide. Acceptable modifications include
modifications of the sugar moiety, modifications of the base
moiety, modifications in the internucleotide linkages and
combinations thereof.
[0068] A sugar modification includes a modification on the 2'
moiety of the sugar residue and encompasses amino, fluoro, alkoxy
e.g. methoxy, alkyl, amino, fluoro, chloro, bromo, CN, CF,
imidazole, carboxylate, thioate, C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl or aralkyl, OCF.sub.3, OCN, O-,
S-, or N-alkyl; O-, S, or N-alkenyl; SOCH.sub.3; SO.sub.2CH.sub.3;
ONO.sub.2; NO.sub.2, N.sub.3; heterozycloalkyl; heterozycloalkaryl;
aminoalkylamino; polyalkylamino or substituted silyl, as, among
others, described in European patents EP 0 586 520 B1 or EP 0 618
925 B1.
[0069] In one embodiment a dsRNA molecule useful in methods,
compositions, combinations, commercial packages and kits provided
herein, comprises at least one ribonucleotide comprising a 2'
modification on the sugar moiety ("2' sugar modification"). In
certain embodiments the compound comprises 2'O-alkyl or 2'-fluoro
or 2'O-allyl or any other 2' modification, optionally on alternate
positions. Other stabilizing modifications are also possible (e.g.
terminal modifications). In some embodiments a preferred 2'O-alkyl
is 2'O-methyl (methoxy) sugar modification.
[0070] In some embodiments the backbone of the oligonucleotides is
modified and comprises phosphate-D-ribose entities but may also
contain thiophosphate-D-ribose entities, triester, thioate, 2'-5'
bridged backbone (also may be referred to as 5'-2'), PACE and the
like.
[0071] As used herein, the terms "non-pairing nucleotide analog"
means a nucleotide analog which comprises a non-base pairing moiety
including but not limited to: 6 des amino adenosine (Nebularine),
4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U,
N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC,
N3-Me dC. In some embodiments the non-base pairing nucleotide
analog is a ribonucleotide. In other embodiments it is a
deoxyribonucleotide. In addition, analogues of polynucleotides may
be prepared wherein the structure of one or more nucleotide is
fundamentally altered and better suited as therapeutic or
experimental reagents. An example of a nucleotide analogue is a
peptide nucleic acid (PNA) wherein the deoxyribose (or ribose)
phosphate backbone in DNA (or RNA) is replaced with a polyamide
backbone which is similar to that found in peptides. PNA analogues
have been shown to be resistant to enzymatic degradation and to
have extended stability in vivo and in vitro. Other modifications
that can be made to oligonucleotides include polymer backbones,
cyclic backbones, acyclic backbones, thiophosphate-D-ribose
backbones, triester backbones, thioate backbones, 2'-5' bridged
backbone, artificial nucleic acids, morpholino nucleic acids,
glycol nucleic acid (GNA), threose nucleic acid (TNA), arabinoside,
and mirror nucleoside (for example, beta-L-deoxyribonucleoside
instead of beta-D-deoxyribonucleoside). Examples of dsRNA molecules
comprising LNA nucleotides are disclosed in Elmen et al., (NAR
2005, 33(1):439-447). The compounds useful in accordance with the
present disclosure can be synthesized using one or more inverted
nucleotides, for example inverted thymidine or inverted adenine
(see, for example, Takei, et al., 2002, JBC 277(26):23800-06).
[0072] Other modifications include terminal modifications on the 5'
and/or 3' part of the oligonucleotides and are also known as
capping moieties. Such terminal modifications are selected from a
nucleotide, a modified nucleotide, a lipid, a peptide, a sugar and
inverted abasic moiety.
[0073] An "alkyl moiety or derivative thereof" refers to straight
chain or branched carbon moieties and moieties per se or further
comprising a functional group including alcohols, phosphodiester,
phosphorothioate, phosphonoacetate and also includes amines,
carboxylic acids, esters, amides aldehydes. "Hydrocarbon moiety"
and "alkyl moiety" are used interchangeably.
[0074] "Terminal functional group" includes halogen, alcohol,
amine, carboxylic, ester, amide, aldehyde, ketone, ether
groups.
[0075] Provided are methods, compositions and combinations for
inhibiting expression of target genes in vivo. In certain
embodiments, the methods include administering a composition or a
combination of oligoribonucleotides, in particular small
interfering RNAs (i.e. siRNAs) that target mRNAs transcribed from
target genes in an amount sufficient to down-regulate expression of
target genes by, e.g. an RNA interference mechanism. In particular,
the subject methods can be used to inhibit expression of target
genes for treatment of a disease. Provided herein are compositions
and combinations of dsRNA molecules directed to target genes
disclosed herein and useful as therapeutic agents to treat various
otic and vestibular system pathologies.
[0076] Provided are methods, combinations and compositions for
inhibiting expression of a hearing loss-associated gene in vivo. In
general, the methods includes administering combinations of
oligoribonucleotides, in particular double-stranded RNAs (such as,
for example, siRNAs), that target mRNAs, or pharmaceutical
compositions comprising them, in an amount sufficient to
down-regulate expression of target genes by, e.g. an RNA
interference mechanism. In particular, the subject methods can be
used to inhibit expression of a hearing loss-associated genes for
treatment of a disease or a disorder or a condition disclosed
herein.
[0077] Provided herein are methods, combinations and compositions
for inhibiting expression of HES1, HES5 and HEY2, in vivo. Provided
herein are methods and compositions for inhibiting expression of
HEY2, CDKN1B and NOTCH1, in vivo. In general, the methods includes
administering oligoribonucleotides, in particular double stranded
RNAs (i.e. dsRNAs) or a nucleic acid material that can produce
dsRNA in a cell, that target mRNAs transcribed from HES1, HES5 and
HEY2 genes or HEY2, CDKN1B and NOTCH1 genes in an amount sufficient
to down-regulate expression of the target genes e.g., by an RNA
interference mechanism.
dsRNA and RNA Interference
[0078] RNA interference (RNAi) is a phenomenon involving
double-stranded (ds) RNA-dependent gene-specific
posttranscriptional silencing. Initial attempts to study this
phenomenon and to manipulate mammalian cells experimentally were
frustrated by an active, non-specific antiviral defense mechanism
which was activated in response to long dsRNA molecules (Gil et
al., Apoptosis, 2000. 5:107-114). Later, it was discovered that
synthetic duplexes of 21 nucleotide RNAs could mediate gene
specific RNAi in mammalian cells, without stimulating the generic
antiviral defense mechanisms Elbashir et al. Nature 2001,
411:494-498 and Caplen et al. PNAS 2001, 98:9742-9747). As a
result, small interfering RNAs (siRNAs), which are short
double-stranded RNAs, have been widely used to inhibit gene
expression and understand gene function.
[0079] RNA interference (RNAi) is mediated by small interfering
RNAs (siRNAs) (Fire et al, Nature 1998, 391:806) or microRNAs
(miRNAs) (Ambros V. Nature 2004, 431:350-355); and Bartel D P.
Cell. 2004 116(2):281-97). The corresponding process is commonly
referred to as specific post-transcriptional gene silencing when
observed in plants and as quelling when observed in fungi.
[0080] A siRNA compound is a double-stranded RNA which
down-regulates or silences (i.e. fully or partially inhibits) the
expression of an endogenous or exogenous gene/mRNA. RNA
interference is based on the ability of certain dsRNA species to
enter a specific protein complex, where they are then targeted to
complementary cellular RNAs and specifically degrades them. Thus,
the RNA interference response features an endonuclease complex
containing a siRNA, commonly referred to as an RNA-induced
silencing complex (RISC), which mediates cleavage of
single-stranded RNA having a sequence complementary to the
antisense strand of the siRNA duplex. Cleavage of the target RNA
may take place in the middle of the region complementary to the
antisense strand of the siRNA duplex (Elbashir, et al., Genes Dev.,
2001, 15:188). In more detail, longer dsRNAs are digested into
short (17-29 bp) dsRNA fragments (also referred to as short
inhibitory RNAs or "siRNAs") by type III RNAses (DICER, DROSHA,
etc., (see Bernstein et al., Nature, 2001, 409:363-6 and Lee et
al., Nature, 2003, 425:415-9). The RISC protein complex recognizes
these fragments and complementary mRNA. The whole process is
culminated by endonuclease cleavage of target mRNA (McManus and
Sharp, Nature Rev Genet, 2002, 3:737-47; Paddison and Hannon, Curr
Opin Mol Ther. 2003, 5(3): 217-24). (For additional information on
these terms and proposed mechanisms, see for example, Bernstein, et
al., RNA. 2001, 7(11):1509-21; Nishikura, Cell. 2001, 107(4):415-8
and PCT Publication No. WO 01/36646).
[0081] The selection and synthesis of dsRNA compounds corresponding
to known genes has been widely reported; see for example Ui-Tei et
al., J Biomed Biotechnol. 2006; 65052; Chalk et al., BBRC. 2004,
319(1):264-74; Sioud and Leirdal, Met. Mol Biol.; 2004, 252:457-69;
Levenkova et al., Bioinform. 2004, 20(3):430-2; Ui-Tei et al., NAR
2004, 32(3):936-48. For examples of the use of, and production of,
modified siRNA see Braasch et al., Biochem., 2003, 42(26):7967-75;
Chiu et al., RNA, 2003, 9(9):1034-48; PCT publications WO
2004/015107 (atugen); WO 02/44321 (Tuschl et al), and U.S. Pat.
Nos. 5,898,031 and 6,107,094.
[0082] Several groups have described the development of DNA-based
vectors capable of generating siRNA within cells. The method
generally involves transcription of short hairpin RNAs that are
efficiently processed to form siRNAs within cells (Paddison et al.
PNAS USA 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002,
16:948-958; Sui et al. PNAS USA 2002, 8:5515-5520; and Brummelkamp
et al. Science 2002, 296:550-553). These reports describe methods
of generating siRNAs capable of specifically targeting numerous
endogenously and exogenously expressed genes.
[0083] Studies have revealed that siRNA can be effective in vivo in
mammals, including humans. Specifically, Bitko et al., showed that
specific siRNAs directed against the respiratory syncytial virus
(RSV) nucleocapsid N gene are effective in treating mice when
administered intranasally (Nat. Med. 2005, 11(1):50-55). For
reviews of therapeutic applications of siRNAs see for example Barik
(Mol. Med 2005, 83: 764-773) and Chakraborty (Current Drug Targets
2007 8(3):469-82). In addition, clinical studies with short siRNAs
that target the VEGFR1 receptor in order to treat age-related
macular degeneration (AMD) have been conducted in human patients
(Kaiser, Am J Ophthalmol. 2006 142(4):660-8). Further information
on the use of siRNA as therapeutic agents may be found in Durcan,
2008. Mol. Pharma. 5(4):559-566; Kim and Rossi, 2008. BioTechniques
44:613-616; Grimm and Kay, 2007, JCI, 117(12):3633-41.
[0084] A dsRNA useful with the combination therapy or compositions
is a duplex oligoribonucleotide in which the sense strand is
substantially complementary to an 18-40 consecutive nucleotide
segment of the mRNA polynucleotide sequence of a target gene, and
the antisense strand is substantially complementary to the sense
strand. In general, some deviation from the target mRNA sequence is
tolerated without compromising the siRNA activity (see e.g.
Czauderna et al., Nuc. Acids Res. 2003, 31(11):2705-2716). A siRNA
of the invention inhibits gene expression on a post-transcriptional
level with or without destroying the mRNA. Without being bound by
theory, siRNA may target the mRNA for specific cleavage and
degradation and/or may inhibit translation from the targeted
message.
[0085] In some embodiments the dsRNA is blunt ended, on one or both
ends. More specifically, the dsRNA may be blunt ended on the end
defined by the 5'-terminus of the first strand and the 3'-terminus
of the second strand, or the end defined by the 3'-terminus of the
first strand and the 5'-terminus of the second strand.
[0086] In other embodiments at least one of the two strands may
have an overhang of at least one nucleotide at the 5'-terminus; the
overhang may consist of at least one deoxyribonucleotide. At least
one of the strands may also optionally have an overhang of at least
one nucleotide at the 3'-terminus. The overhang may consist of from
about 1 to about 5 nucleotides.
[0087] The length of RNA duplex is from about 18 to about 49
ribonucleotides, preferably 19 to 23 ribonucleotides. Further, the
length of each strand (oligomer) may independently have a length
selected from the group consisting of about 18 to about 49 bases,
preferably 18 to 23 bases and more preferably 19, 20 or 21
ribonucleotides.
[0088] Additionally, in certain preferred embodiments the
complementarity between said first strand and the target nucleic
acid is perfect. In some embodiments, the strands are substantially
complementary, i.e. having one, two or up to three mismatches
between said first strand and the target nucleic acid.
[0089] Further, the 5'-terminus of the first strand of the siRNA
may be linked to the 3'-terminus of the second strand, or the
3'-terminus of the first strand may be linked to the 5'-terminus of
the second strand, said linkage being via a nucleic acid linker
typically having a length between 3-100 nucleotides, preferably
about 3 to about 10 nucleotides.
[0090] The dsRNAs compounds useful in methods, combinations and
compositions disclosed herein, possess structures and modifications
which impart one or more of increased activity, increased
stability, reduced toxicity, reduced off target effect, and/or
reduced immune response. The siRNA structures as disclosed herein,
are beneficially applied to double-stranded RNA useful in methods,
combinations and compositions disclosed herein for use in
preventing or attenuation target gene expression, in particular the
target genes discussed herein.
[0091] According to one aspect, the present disclosure provides use
of combinations or compositions of chemically modified
double-stranded oligonucleotides comprising at least one modified
nucleotide selected from the group consisting of a sugar
modification, a base modification and an internucleotide linkage
modification. Accordingly, the chemically modified double stranded
oligonucleotide compounds useful in the methods, compositions and
combinations provided herein, may contain modified nucleotides such
as DNA, LNA (locked nucleic acid), ENA (ethylene-bridged nucleic
acid), PNA (peptide nucleic acid), arabinoside, PACE, mirror
nucleoside, or nucleotides with a 6 carbon sugar. Examples of PACE
nucleotides and analogs are disclosed in U.S. Pat. Nos. 6,693,187
and 7,067,641 both incorporated herein by reference. The
oligonucleotide may further comprise 2'O-methyl or 2'-fluoro or
2'O-allyl or any other 2' modification, optionally on alternate
positions. Other stabilizing modifications, which do not
significantly reduce the activity are also possible (e.g. terminal
modifications). The backbone of the active part of the
oligonucleotides may comprise phosphate-D-ribose entities but may
also contain thiophosphate-D-ribose entities, triester, thioate,
2'-5' bridged backbone (also may be referred to as 5'-2'), PACE or
any other type of modification. Terminal modifications on the 5'
and/or 3' part of the oligonucleotides are also possible. Such
terminal modifications may be lipids, peptides, sugars, inverted
abasic moieties or other molecules.
Chemical Synthesis of Oligonucleotide Compounds
[0092] The oligonucleotide compounds for use in the methods,
compositions, combinations, commercial packages and kits disclosed
herein can be synthesized by any of the methods that are well-known
in the art for synthesis of ribonucleic (or deoxyribonucleic)
oligonucleotides. Such synthesis is, among others, described in
Beaucage and Iyer, Tetrahedron 1992; 48:2223-2311; Beaucage and
Iyer, Tetrahedron 1993; 49: 6123-6194 and Caruthers, et. al.,
Methods Enzymol. 1987; 154: 287-313; the synthesis of thioates is,
among others, described in Eckstein, Annu Rev. Biochem. 1985; 54:
367-402, the synthesis of RNA molecules is described in Sproat, in
Humana Press 2005 edited by Herdewijn P.; Kap. 2: 17-31 and
respective downstream processes are, among others, described in
Pingoud et. al., in IRL Press 1989 edited by Oliver R. W. A.; Kap.
7: 183-208.
[0093] Other synthetic procedures are known in the art e.g. the
procedures as described in Usman et al., 1987, J. Am. Chem. Soc.,
109, 7845; Scaringe et al., 1990, NAR., 18, 5433; Wincott et al.,
1995, NAR. 23, 2677-2684; and Wincott et al., 1997, Methods Mol.
Bio., 74, 59, and these procedures may make use of common nucleic
acid protecting and coupling groups, such as dimethoxytrityl at the
5'-end, and phosphoramidites at the 3'-end. The modified (e.g.
2'-O-methylated) nucleotides and unmodified nucleotides are
incorporated as desired.
[0094] The oligonucleotides useful in accordance with embodiments
provided herein can be synthesized separately and joined together
post-synthetically, for example, by ligation (Moore et al., 1992,
Science 256, 9923; Draper et al., International Patent Publication
No. WO 93/23569; Shabarova et al., 1991, NAR 19, 4247; Bellon et
al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al.,
1997, Bioconjugate Chem. 8, 204), or by hybridization following
synthesis and/or deprotection.
[0095] It is noted that a commercially available machine
(available, inter alia, from Applied Biosystems) can be used; the
oligonucleotides are prepared according to the sequences disclosed
herein. Overlapping pairs of chemically synthesized fragments can
be ligated using methods well known in the art (e.g., see U.S. Pat.
No. 6,121,426). The strands are synthesized separately and then are
annealed to each other in the tube. Then, the double-stranded
siRNAs are separated from the single-stranded oligonucleotides that
were not annealed (e.g. because of the excess of one of them) by
HPLC. In relation to the dsRNAs or dsRNA fragments useful in
accordance with embodiments of present disclosure, two or more such
sequences can be synthesized and linked together for use in the
methods, compositions and combinations disclosed herein.
[0096] The compounds for use in accordance with can also be
synthesized via tandem synthesis methodology, as described for
example in US Patent Publication No. US 2004/0019001 (McSwiggen),
wherein both siRNA strands are synthesized as a single contiguous
oligonucleotide fragment or strand separated by a cleavable linker
which is subsequently cleaved to provide separate siRNA fragments
or strands that hybridize and permit purification of the siRNA
duplex. The linker can be a polynucleotide linker or a
non-nucleotide linker.
[0097] The present disclosure provides for a pharmaceutical
composition comprising three dsRNA molecules for the treatment of
any of the diseases and conditions mentioned herein, whereby at
least two of the molecules may be physically mixed together in the
pharmaceutical composition in amounts which generate equal or
otherwise beneficial activity, or may be covalently or
non-covalently bound, or joined together by a nucleic acid linker
of a length ranging from 2-100, preferably 2-50 or 2-30
nucleotides. In one embodiment, the dsRNA molecules are comprised
of a double-stranded nucleic acid structure as described herein,
wherein the dsRNA molecules are selected from the oligonucleotides
described herein. Thus, the dsRNA molecules may be covalently or
non-covalently bound or joined by a linker to form a tandem or a
triplet dsRNA compound. Such tandem dsRNA molecules comprising two
siRNA sequences are typically of 38-150 nucleotides in length, more
preferably 38 or 40-60 nucleotides in length, and longer
accordingly if more than two siRNA sequences are included in the
tandem molecule. A longer tandem compound comprised of two or more
longer sequences which encode siRNA produced via internal cellular
processing, e.g., long dsRNAs, is also envisaged, as is a tandem
molecule encoding two or more shRNAs. Such tandem molecules are
also considered to be a part of the disclosure. A compound
comprising two (tandem) or more (RNAistar) dsRNA sequences
disclosed herein is envisaged. Examples of such "tandem" or "star"
molecules are provided in PCT patent publication no. WO
2007/091269, assigned to the assignee of the present application
and incorporated herein by reference in its entirety.
[0098] The dsRNA molecules that target HES1, HES5, and HEY2, or
HEY2, CDKN1B and NOTCH1 may be the main active components in a
pharmaceutical composition, Simultaneous inhibition of said
additional gene(s) will likely have an additive or synergistic
effect for treatment of the diseases disclosed herein.
[0099] Additionally, the dsRNA disclosed herein or any nucleic acid
molecule comprising or encoding such dsRNA can be linked or bound
(covalently or non-covalently) to antibodies (including aptamer
molecules) against cell surface internalizable molecules expressed
on the target cells, in order to achieve enhanced targeting for
treatment of the diseases disclosed herein. For example, anti-Fas
antibody (preferably a neutralizing antibody) may be combined
(covalently or non-covalently) with any dsRNA. In another example,
an aptamer which can act like a ligand/antibody may be combined
(covalently or non-covalently) with any dsRNA.
[0100] The nucleic acid molecules disclosed herein can be delivered
either directly or with viral or non-viral vectors. When delivered
directly, the sequences are generally rendered nuclease resistant.
Alternatively the sequences can be incorporated into expression
cassettes or constructs such that the sequence is expressed in the
cell as discussed herein below. Generally the construct contains
the proper regulatory sequence or promoter to allow the sequence to
be expressed in the targeted cell. Vectors optionally used for
delivery of the compounds of the present invention are commercially
available, and may be modified for the purpose of delivery of the
compounds of the present invention by methods known to one of skill
in the art.
dsRNA Useful for Combination Therapy
[0101] In particular embodiments of compositions, combinations,
methods, commercial packages and kits provided herein, the
double-stranded oligonucleotide (for example dsRNA) possess
modifications which may increase activity, increase stability,
minimize toxicity and/or affect delivery of the double-stranded
oligonucleotide to the middle and inner ear when compared to the
corresponding unmodified double-stranded oligonucleotide compound.
The double-stranded oligonucleotide molecules are designed to
down-regulate target gene expression and attenuate target gene
function. In certain embodiments the target gene is transcribed
into any one of the mRNA polynucleotides set forth in SEQ ID NOS:1,
2, 7, 10 and 11. In particular embodiments of compositions,
combinations, methods, commercial packages and kits provided
herein, the double-stranded oligonucleotide (for example dsRNA)
possess a sense strand sequence and an antisense strand sequence
selected from sense strand oligonucleotide and corresponding
antisense strand oligonucleotide set forth in SEQ ID NOS:23-1495 or
26667-26706 (HES1), SEQ ID NOS:1496-2703 or 26707-26732 (HES5), SEQ
ID NOS:13004-16621 or 26779-26788 (HEY2), SEQ ID NOS:7444-10533 or
26867-26900 (CDKN1B) or SEQ ID NOS:16622-26666 or 26901-26912
(NOTCH1) useful in generating the chemically modified
double-stranded oligonucleotide molecules.
[0102] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, in each
of a double-stranded oligonucleotide molecule (e.g., dsRNA
molecule) the antisense strand may be 18 to 49 nucleotides in
length (e.g., 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 or 49 nucleotides in length); or 18-35 nucleotides in length; or
18-30 nucleotides in length; or 18-25 nucleotides in length; or
18-23 nucleotides in length; or 19-21 nucleotides in length; or
25-30 nucleotides in length; or 26-28 nucleotides in length.
compositions, combinations, methods, commercial packages and kits,
as disclosed herein, in each of a double-stranded oligonucleotide
molecule (e.g., dsRNA molecule), the antisense strand is 19
nucleotides in length. Similarly the sense strand of a
double-stranded oligonucleotide molecule (e.g., dsRNA molecule) may
be 18 to 49 nucleotides in length (e.g., 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 or 49 nucleotides in length); or
18-35 nucleotides in length; or 18-30 nucleotides in length; or
18-25 nucleotides in length; or 18-23 nucleotides in length; or
19-21 nucleotides in length; or 25-30 nucleotides in length; or
26-28 nucleotides in length. In various preferred embodiments of
compositions, combinations, methods, commercial packages and kits,
as disclosed herein, in each double-stranded oligonucleotide (e.g.,
dsRNA molecule), the sense strand is 19 nucleotides in length and
the antisense strand is 19 nucleotides in length. In various
preferred embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, the duplex
region of the double-stranded oligonucleotide molecule (e.g., dsRNA
molecule) may be 18-49 nucleotides in length (e.g., about 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 or 49 nucleotides in
length), 18-35 nucleotides in length; or 18-30 nucleotides in
length; or 18-25 nucleotides in length; or 18-23 nucleotides in
length; or 18-21 nucleotides in length; or 25-30 nucleotides in
length; or 25-28 nucleotides in length. In various preferred
embodiments of compositions, combinations, methods, commercial
packages and kits, as disclosed herein, the duplex region of the
double-stranded oligonucleotide molecule is 19 nucleotides in
length.
[0103] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
sense strand and the antisense strand of the double-stranded
oligonucleotide (e.g., an dsRNA molecule) are separate
oligonucleotide strands. In some embodiments, the separate sense
strand and antisense strand form a double stranded structure, also
known as a duplex, via hydrogen bonding, for example, Watson-Crick
base pairing. In some embodiments one or more nucleotide pairs form
non-Watson-Crick base pairing. In some embodiments the sense strand
and the antisense strand are two separate strands that are
covalently linked to each other. In other embodiments, the sense
strand and the antisense strands are part of a single
oligonucleotide having both a sense and antisense region; in some
preferred embodiments the oligonucleotide has a hairpin
structure.
[0104] In certain embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide is symmetrical with regard to
overhangs, and has a blunt end on both ends. In other embodiments
the double-stranded oligonucleotide is a dsRNA molecule that is
symmetrical with regard to overhangs, and has a nucleotide or a
non-nucleotide or a combination of a nucleotide and non-nucleotide
overhang on both ends of the dsRNA molecule. In certain preferred
embodiments, the nucleic acid molecule is a dsRNA molecule that is
asymmetrical with regard to overhangs, and has a blunt end on one
end of the molecule and an overhang on the other end of the
molecule. In some embodiments an asymmetrical dsRNA molecule has a
3'-overhang on one side of a duplex occurring on the sense strand;
and a blunt end on the other side of the molecule occurring on both
the 5'-end of the sense strand and the 5'-end of the antisense
strand. In some embodiments an asymmetrical dsRNA molecule has a
5'-overhang on one side of a duplex occurring on the sense strand;
and a blunt end on the other side of the molecule occurring on both
the 3'-end of the sense strand and the 3'-end of the antisense
strand. In other embodiments an asymmetrical dsRNA molecule has a
3'-overhang on one side of a duplex occurring on the antisense
strand; and a blunt end on the other side of the molecule occurring
on both the 5'-end of the sense strand and the 5'-end of the
antisense strand. In some embodiments an asymmetrical dsRNA
molecule has a 5'-overhang on one side of a duplex occurring on the
antisense strand; and a blunt end on the other side of the molecule
occurring on both the 3'-end of the sense strand and the 3'-end of
the antisense strand. In some embodiments the overhangs are
nucleotide overhangs, in other embodiments the overhangs are
non-nucleotide overhangs. In some embodiments the overhangs are 5'
overhangs; in alternative embodiments the overhangs are 3'
overhangs.
[0105] In certain embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide has a hairpin structure (having the
sense strand and antisense strand on one oligonucleotide), with a
loop structure on one end and a blunt end on the other end. In some
embodiments, the double-stranded oligonucleotide has a hairpin
structure, with a loop structure on one end and an overhang end on
the other end; in certain embodiments, the overhang is a
3'-overhang; in certain embodiments the overhang is a 5'-overhang;
in certain embodiments the overhang is on the sense strand; in
certain embodiments the overhang is on the antisense strand.
[0106] In certain embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecule) may include
one or more modifications or modified nucleotides such as described
herein. For example, the double-stranded oligonucleotide (e.g.,
dsRNA molecule) may include a modified nucleotide having a modified
sugar; a modified nucleotide having a modified nucleobase; or a
modified nucleotide having a modified phosphate group, a modified
phosphodiester backbone and/or a modified terminal phosphate
group.
[0107] In certain embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecules) may have
one or more ribonucleotides that include a modified sugar moiety,
for example as described herein. A non-limiting example of a
modified sugar moiety is a 2'alkoxy modified sugar moiety. In some
preferred embodiments the nucleic acid comprises at least one
2'-O-methyl sugar modified ribonucleotide.
[0108] In certain embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecule) may have one
or more modified nucleobase(s), for example as described
herein.
[0109] In certain embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecule) may have one
or more modifications to the phosphodiester backbone, for example
as described herein.
[0110] In certain embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecule) may have one
or more modified phosphate group(s), for example as described
herein.
[0111] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecule) may include
an unmodified antisense strand and a sense strand having one or
more modifications. In some embodiments the double-stranded
oligonucleotide (e.g., dsRNA molecule) may include an unmodified
sense strand and an antisense strand having one or more
modifications. In preferred embodiments, the double-stranded
oligonucleotide (e.g., dsRNA molecule) may include one or more
modified nucleotides in the both the sense strand and the antisense
strand.
[0112] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecules) may include
a phosphate group at the 5' end of the sense and/or the antisense
strand (i.e. a 5'-terminal phosphate group). In some embodiments
the double-stranded oligonucleotide may include a phosphate group
at the 5' terminus of the antisense strand.
[0113] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecules) may include
a phosphate group at the 3' end of the sense and/or the antisense
strand (i.e. a 3'-terminal phosphate group). In some embodiments
the double-stranded oligonucleotide may include a phosphate group
at the 3' terminus of the antisense strand.
[0114] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecules) may include
a phosphate group at the 3' terminus of the antisense strand and
the sense strand.
[0115] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide (e.g., dsRNA molecules) the
antisense strand and the sense strand of the nucleic acid molecule
are non-phosphorylated at both the 3' terminus and at the 5'
terminus.
[0116] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, at
least one double-stranded oligonucleotide is independently having
the structure (A1):
TABLE-US-00002 (A1) (antisense strand) 5' (N)x - Z 3' (sense
strand) 3' Z'-(N')y - z'' 5'
[0117] wherein each N and N' is a ribonucleotide which may be
unmodified or modified, or an unconventional moiety; wherein each
of (N)x and (N')y is an oligonucleotide in which each consecutive N
or N' is joined to the next N or N' by a covalent bond;
wherein each of Z and Z' is independently present or absent, but if
present independently comprises 1-5 consecutive nucleotides, 1-5
consecutive non-nucleotide moieties or a combination thereof
covalently attached at the 3' terminus of the strand in which it is
present; wherein z'' may be present or absent, but if present is a
capping moiety covalently attached at the 5' terminus of (N')y;
each of x and y is independently an integer from 18 to 40; wherein
the sequence of (N')y is complementary to the sequence of (N)x; and
wherein (N)x comprises an antisense sequence to a consecutive
sequence in an mRNA selected from an mRNA encoding HES1, an mRNA
encoding HES5, an mRNA encoding HEY2, an mRNA encoding CDKN1B and
an mRNA encoding NOTCH1.
[0118] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, at
least one double-stranded oligonucleotide is independently having
the structure (A1), (N)x comprises an antisense sequence and (N')y
comprises a sense sequence set forth in any one of SEQ ID
NOS:23-693 and 26691-26706 (HES1); SEQ ID NOS:1496-2029 and
26725-26732 (HES5); SEQ ID NOS:7444-9007 and 26887-26900 (CDKN1B);
SEQ ID NOS:13004-14801 and 26785-26788 (HEY2); SEQ ID
NOS:16622-18643 and 26922-26912 (NOTCH1). In some embodiments
preferred (N)x and (N')y are set forth in any one of SEQ ID
NOS:26691-26706 (HES1); SEQ ID NOS:26725-26732 (HES5); SEQ ID
NOS:26887-26900 (CDKN1B); SEQ ID NOS:26785-26788 (HEY2); SEQ ID
NOS:26922-26912 (NOTCH1).
[0119] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, at
least one double-stranded oligonucleotide is independently having
the structure (A1) and the covalent bond joining each consecutive N
and/or N' is a phosphodiester bond.
[0120] In various embodiments of compositions, combinations,
methods, commercial packages and kits, as disclosed herein, at
least one double-stranded oligonucleotide is independently having
the structure (A1) x=y and each of x and y is 19, 20, 21, 22 or 23.
In preferred embodiments x=y=19.
[0121] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide comprise a DNA moiety or a mismatch
to the target at position 1 of the antisense strand (5' terminus).
In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2) set forth
below:
TABLE-US-00003 (A2) (antisense strand) 5' N1-(N)x - Z 3' (sense
strand) 3' Z'-N2-(N')y - z'' 5'
wherein each N1, N2, N and N' is independently an unmodified or
modified nucleotide, or an unconventional moiety; wherein each of
(N)x and (N')y is an oligonucleotide in which each consecutive N or
N' is joined to the adjacent N or N' by a covalent bond; wherein
each of x and y is independently an integer between 17 and 39;
wherein N2 is covalently bound to (N')y; wherein N1 is covalently
bound to (N)x and is mismatched to the target mRNA or is a
complementary DNA moiety to the target mRNA; wherein N1 is a moiety
selected from the group consisting of natural or modified: uridine,
deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or
deoxyadenosine, an abasic ribose moiety and an abasic deoxyribose
moiety; wherein z'' may be present or absent, but if present is a
capping moiety covalently attached at the 5' terminus of N2-(N')y;
wherein each of Z and Z' is independently present or absent, but if
present is independently 1-5 consecutive nucleotides, 1-5
consecutive non-nucleotide moieties or a combination thereof
covalently attached at the 3' terminus of the strand in which it is
present; and wherein the sequence of (N')y has complementarity to
the sequence of (N)x; and wherein the sequence of (N)x is an
antisense sequence to a consecutive sequence in HES1 mRNA (SEQ ID
NO:1); HES5 mRNA (SEQ ID NO:2), HEY2 mRNA (SEQ ID NO:10), CDKN1B
mRNA (SEQ ID NO:7) or NOTCH1 mRNA (SEQ ID NO:11).
[0122] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2) wherein the
sequence of (N')y is complementary to the sequence of (N)x; and
wherein the sequence of (N)x comprises an antisense sequence and
(N')y comprises a sense sequence set forth in any one of SEQ ID
NOS:694-1495 and 26667-26690 (HES1); SEQ ID NOS:2030-2703 and
26707-26724 (HES5); SEQ ID NOS:9008-10533 and 26867-26886 (CDKN1B);
SEQ ID NOS:14802-16389 and 26779-26784 (HEY2); SEQ ID
NOS:18644-26666 and 26901-26910 (NOTCH1). Preferred (N)x and (N')y
are set forth in any one of SEQ ID NOS:26667-26690 (HES1); SEQ ID
NOS:26707-26724 (HES5); SEQ ID NOS:26867-26886 (CDKN1B); SEQ ID
NOS:26779-26784 (HEY2); SEQ ID NOS:26901-26910 (NOTCH1). Molecules
covered by the description of Structure (A2) are also referred to
herein as "18+1" or "18+1 mer". In some embodiments, the N2-(N')y
and N1-(N)x useful in generating double-stranded oligonucleotides
having Structure (A2) are presented in Tables I-V, particularly the
sequences designated as "18+1" type. In certain preferred
embodiments (N)x and (N')y are selected from the sequence pairs
shown in Tables I-V.
[0123] In preferred In some embodiments of compositions,
combinations, methods, commercial packages and kits, as disclosed
herein, at least one double-stranded oligonucleotide has a
structure (A2), the sequence of (N')y is fully complementary to the
sequence of (N)x. In various embodiments sequence of N2-(N')y is
complementary to the sequence of N1-(N)x. In some embodiments (N)x
comprises an antisense that is fully complementary to about 17 to
about 39 consecutive nucleotides in a target mRNA set forth in any
one of SEQ ID NOS:1, 2, 10, 7 or 11. In other embodiments (N)x
comprises an antisense that is substantially complementary to about
17 to about 39 consecutive nucleotides in a target mRNA set forth
in SEQ ID NOS:1, 2, 10, 7 or 11. In some embodiments, at least one
double-stranded oligonucleotide has a structure (A2) and N1 and N2
form a Watson-Crick base pair. In other embodiments, at least one
double-stranded oligonucleotide has a structure (A2) and N1 and N2
form a non-Watson-Crick base pair. In some embodiments a base pair
is formed between a ribonucleotide and a deoxyribonucleotide.
[0124] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2), x=y=18,
x=y=19 or x=y=20. In preferred embodiments x=y=18. When x=18 in
N1-(N)x, N1 refers to position 1 and positions 2-19 are included in
(N).sub.18. When y=18 in N2-(N')y, N2 refers to position 19 and
positions 1-18 are included in (N').sub.18. In some embodiments at
least one double-stranded oligonucleotide has a structure (A2), N1
is covalently bound to (N)x and is mismatched to the target mRNA
set forth in SEQ ID NO:1, 2, 10, 7 or 11. In various embodiments at
least one double-stranded oligonucleotide has a structure (A2), N1
is covalently bound to (N)x and is a DNA moiety complementary to
the target mRNA set forth in SEQ ID NO:1, 2, 10, 7 or 11.
[0125] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2), a uridine in
position 1 of the antisense strand is substituted with an N1
selected from natural or modified: adenosine, deoxyadenosine,
uridine, deoxyuridine (dU), ribothymidine or deoxythymidine. In
various embodiments, at least one double-stranded oligonucleotide
has a structure (A2), N1 is selected from natural or modified:
adenosine, deoxyadenosine or deoxyuridine. For example, in some
embodiments a cytidine in position 1 is replaced with an adenine or
a uridine; a guanosine in position 1 is replaced with an adenine or
a uridine; or an adenine is replaced with a uridine.
[0126] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2), guanosine in
position 1 (N1) of the antisense strand is substituted with a
natural or modified: adenosine, deoxyadenosine, uridine,
deoxyuridine, ribothymidine or deoxythymidine. In various
embodiments N1 is selected from a natural or modified: adenosine,
deoxyadenosine, uridine or deoxyuridine.
[0127] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2), cytidine in
position 1 (N1) of the antisense strand is substituted with a
natural or modified: adenosine, deoxyadenosine, uridine,
deoxyuridine, ribothymidine or deoxythymidine. In various
embodiments N1 is selected from a natural or modified: adenosine,
deoxyadenosine, uridine or deoxyuridine.
[0128] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2), adenosine in
position 1 (N1) of the antisense strand is substituted with a
natural or modified: deoxyadenosine, deoxyuridine, ribothymidine or
deoxythymidine.
[0129] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has a structure (A2), N1 and N2
form a base pair between natural or modified: uridine or
deoxyuridine, and adenosine or deoxyadenosine. In other embodiments
N1 and N2 form a base pair between natural or modified:
deoxyuridine and adenosine.
[0130] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, the
double-stranded oligonucleotide molecules are also referred to as
"duplexes". In some embodiments at least one double-stranded
oligonucleotide has a structure (A2), and the double stranded
oligonucleotide is a chemically modified dsRNA.
[0131] In certain preferred embodiments of compositions,
combinations, methods, commercial packages and kits, as disclosed
herein, the double-stranded oligonucleotide molecules has Structure
(A2), and x=y=18. In some embodiments x=y=18 and (N)x consists of
an antisense oligonucleotide present in SEQ ID NOS:694-1495 and
26667-26690 (HES1); SEQ ID NOS:2030-2703 and 26707-26724 (HES5);
SEQ ID NOS:9008-10533 and 26867-26886 (CDKN1B); SEQ ID
NOS:14802-16389 and 26779-26784 (HEY2); SEQ ID NOS:18644-26666 and
26901-26910 (NOTCH1).
[0132] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has Structure (A2) and N1 is
selected from a natural uridine and a modified uridine. In some
embodiments, N1 is a natural uridine. In some embodiments, (N)x
comprises an antisense oligonucleotide and (N')y comprises a sense
oligonucleotide present in sequence pairs set forth in SEQ ID
NOS:694-1495 (HES1); SEQ ID NOS:2030-2703 (HES5); SEQ ID
NOS:9008-10533 (CDKN1B); SEQ ID NOS:14802-16389 (HEY2); SEQ ID
NOS:18644-26666 (NOTCH1).
[0133] In some embodiments of compositions, combinations, methods,
commercial packages and kits, as disclosed herein, at least one
double-stranded oligonucleotide has Structure (A2), x=y=18 and
N1-(N)x comprises an antisense oligonucleotide and N2-(N')y
comprises a sense oligonucleotide present in sequence pairs set
forth in SEQ ID 26667-26690 (HES1); SEQ ID NOS: 26707-26724 (HES5);
SEQ ID NOS: 26867-26886 (CDKN1B); SEQ ID NOS: 26779-26784 (HEY2);
SEQ ID NOS: 26901-26910 (NOTCH1).
[0134] In some embodiments, at least one double-stranded
oligonucleotide has Structure (A2), x=y=18 and N1 is selected from
a natural or modified uridine, a natural or modified adenine, and a
natural or modified thymidine.
[0135] In some embodiments at least one double-stranded
oligonucleotide has Structure (A2), wherein N1 is a 2'OMe
sugar-modified uridine or a 2'OMe sugar-modified adenosine. In
certain embodiments at least one double-stranded oligonucleotide
has Structure (A2), N2 is a 2'OMe sugar modified ribonucleotide or
deoxyribonucleotide.
[0136] In some embodiments of Structure (A1) and/or Structure (A2),
each N consists of an unmodified ribonucleotide. In some
embodiments of Structure (A1) and/or Structure (A2) each N'
consists of an unmodified ribonucleotide. In preferred embodiments
at least one of N and/or N' comprises a chemically modified
ribonucleotide, an unmodified deoxyribonucleotide, a chemically
modified deoxyribonucleotide or an unconventional moiety. In some
embodiments the unconventional moiety is selected from a mirror
nucleotide, an abasic ribose moiety and an abasic deoxyribose
moiety. In some embodiments the unconventional moiety is a mirror
nucleotide, preferably an L-DNA moiety. In some embodiments at
least one of N or N' comprises a 2'OMe sugar-modified
ribonucleotide.
[0137] In some embodiments of Structure (A1) and/or Structure (A2)
the sequence of (N')y is fully complementary to the sequence of
(N)x. In other embodiments of Structure (A1) and/or Structure (A2)
the sequence of (N')y is substantially complementary to the
sequence of (N)x.
[0138] In some embodiments of Structure (A1) and/or Structure (A2)
(N)x includes an antisense sequence that is fully complementary to
about 17 to about 39 consecutive nucleotides in a target mRNA set
forth in any one of SEQ ID NO:1, 2, 10, 7 OR 11. In other
embodiments of Structure A1 and/or Structure A2 (N)x includes an
antisense that is substantially complementary to about 17 to about
39 consecutive nucleotides in a target mRNA set forth in any one of
SEQ ID NO:1, 2, 10, 7 OR 11. In some embodiments of Structure (A1)
and/or Structure (A2), the dsRNA compound is blunt ended, for
example, wherein each of z'', Z and Z' is absent. In an alternative
embodiment, at least one of z'', Z or Z' is present.
[0139] In various embodiments Z and Z' independently include one or
more covalently linked modified and or unmodified nucleotides,
including deoxyribonucleotides and ribonucleotides, or one or more
unconventional moieties for example inverted abasic deoxyribose
moiety or abasic ribose moiety or a mirror nucleotide; one or more
non-nucleotide C3 moiety or a derivative thereof, non-nucleotide C4
moiety or a derivative thereof or non-nucleotide C5 moiety or a
derivative thereof, an non-nucleotide amino-C6 moiety or a
derivative thereof, as defined herein, and the like. In some
embodiments Z' is absent and Z is present and includes one or more
non-nucleotide C3 moieties. In some embodiments Z is absent and Z'
is present and includes one or more non-nucleotide C3 moieties. In
some embodiments each of Z and Z' independently comprises one or
more non-nucleotide C3 moieties or one or more non-nucleotide
amino-C6 moieties. In some embodiments z'' is present and is
selected from a mirror nucleotide, an abasic moiety and an inverted
abasic moiety. In some embodiments of Structures (A1) and/or (A2)
each of Z and Z' includes an abasic moiety, for example a
deoxyriboabasic moiety (referred to herein as "dAb") or riboabasic
moiety (referred to herein as "rAb"). In some embodiments each of Z
and/or Z' comprises two covalently linked abasic moieties and is
for example dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb, wherein each
moiety is covalently attached to an adjacent moiety, preferably via
a phospho-based bond. In some embodiments the phospho-based bond
includes a phosphorothioate, a phosphonoacetate or a phosphodiester
bond. In preferred embodiments the phospho-based bond is a
phosphodiester bond.
[0140] In some embodiments each of Z and/or Z' independently
includes an alkyl moiety, optionally propane [(CH2)3] moiety (C3)
or a derivative thereof including propanol (C3OH) and phospho
derivative of propanediol ("C3Pi"). In some embodiments each of Z
and/or Z' includes two alkyl moieties and in some examples is
C3Pi-C3OH. In the example of C3Pi-C3OH, the 3' terminus of the
antisense strand and/or the 3' terminus of the sense strand is
covalently attached to a C3 moiety via a phospho-based bond and the
C3 moiety is covalently bound to a C3OH moiety via a phospho-based
bond. In some embodiments the phospho-based bonds include a
phosphorothioate, a phosphonoacetate or a phosphodiester bond. In
preferred embodiments the phospho-based bond is a phosphodiester
bond.
[0141] In specific embodiments of Structures (A1) and (A2), Z
comprises C3Pi-C3OH. In specific embodiments of Structures (A1) and
(A2), Z' comprises C3Pi or C3OH. In some embodiments of Structures
(A1) and (A2), a double stranded nucleic acid molecule includes a
C3Pi-C3OH moiety covalently attached to the 3' terminus of the
antisense strand and a C3Pi or C3OH moiety covalently attached to
the 3' terminus of the sense strand.
[0142] In some embodiments of Structure (A1) and/or Structure (A2)
each N consists of an unmodified ribonucleotide. In some
embodiments of Structure (A1) and/or Structure (A2) each N'
consists of an unmodified ribonucleotide. In preferred embodiments,
at least one of N and/or N' is a chemically modified
ribonucleotide, an unmodified deoxyribonucleotide, a chemically
modified deoxyribonucleotide or an unconventional moiety.
[0143] In other embodiments a compound of Structure (A1) and/or
(A2) includes at least one ribonucleotide modified in its sugar
residue. In some embodiments the compound comprises a modification
at the 2' position of the sugar residue. In some embodiments the
modification in the 2' position comprises the presence of an amino,
a fluoro, an alkoxy or an alkyl moiety. In certain embodiments the
2' modification includes an alkoxy moiety. In preferred embodiments
the alkoxy moiety is a methoxy moiety (also referred to as
2'-O-methyl; 2'OMe; 2'OMe; 2'-OCH3). In some embodiments a nucleic
acid compound includes 2'OMe sugar modified alternating
ribonucleotides in one or both of the antisense strand and the
sense strand. In other embodiments a compound includes 2'OMe sugar
modified ribonucleotides in the antisense strand, (N)x or N1-(N)x,
only. In some embodiments, the 2'OMe sugar modified ribonucleotides
alternate with unmodified nucleotides. In certain embodiments the
middle ribonucleotide of the antisense strand; e.g. ribonucleotide
in position 10 in a 19-mer strand, is unmodified. In various
embodiments the nucleic acid compound includes at least 5
alternating 2'OMe sugar modified ribonucleotides and unmodified
ribonucleotides. In additional embodiments a compound of Structure
(A1) and/or (A2) includes modified ribonucleotides in alternating
positions wherein each ribonucleotide at the 5' terminus and at the
3' terminus of (N)x or N1-(N)x is modified in its sugar residue,
and each ribonucleotide at the 5' terminus and at the 3' terminus
of (N')y or N2-(N)y is unmodified in its sugar residue. In various
embodiments the ribonucleotides in alternating positions are
modified at the 2' position of the sugar residue.
[0144] In some embodiments the nucleic acid compound includes at
least 5 alternating 2'OMe sugar modified ribonucleotides and
unmodified ribonucleotides, for example at positions 1, 3, 5, 7 and
9 or at positions 11, 13, 15, 17, 19 (5'>3'). In some
embodiments, (N)x of Structure (A1) or N1-(N)x of Structure (A2)
includes 2'OMe sugar modified ribonucleotides in positions 2, 4, 6,
8, 11, 13, 15, 17 and 19. In some embodiments, (N)x of Structure
(A1) or N1-(N)x of Structure (A2) includes 2'OMe sugar modified
ribonucleotides in positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.
In some embodiments, (N)x of Structure (A1) or N1-(N)x of Structure
(A2) includes 2'OMe sugar modified ribonucleotides in one or more
pyrimidines.
[0145] In some embodiments of Structure (A1) and/or (A2), neither
of the sense strand nor the antisense strand is phosphorylated at
the 3' terminus and at the 5' terminus. In other embodiments one or
both of the sense strand and/or the antisense strand are
phosphorylated at the 3' termini. In other embodiments one or both
of the sense strand and/or the antisense strand are phosphorylated
at the 5' terminus.
[0146] In some embodiments the double stranded molecule disclosed
herein includes one or more of the following modifications:
[0147] N in at least one of positions 5, 6, 7, 8, or 9 from the 5'
terminus of the antisense strand is selected from a DNA, TNA, a
2'5' nucleotide or a mirror nucleotide;
[0148] N' in at least one of positions 9 or 10 from the 5' terminus
of the sense strand is selected from a TNA, 2'5' nucleotide and a
pseudoUridine;
[0149] N' in 4, 5, or 6 consecutive positions at the 3' terminus of
(N')y comprises a 2'5' ribonucleotide;
[0150] one or more pyrimidine ribonucleotides are 2' sugar modified
in the sense strand, the antisense strand or both the sense strand
and the antisense strand.
[0151] In some embodiments the double stranded molecule disclosed
herein includes a combination of the following modifications:
[0152] the antisense strand includes a DNA, TNA, a 2'5' nucleotide
or a mirror nucleotide in at least one of positions 5, 6, 7, 8, or
9 from the 5' terminus;
[0153] the sense strand includes at least one of a TNA, a 2'5'
nucleotide and a pseudoUridine in positions 9 or 10 from the 5'
terminus; and
[0154] one or more pyrimidine ribonucleotides are 2' modified in
the sense strand, the antisense strand or both the sense strand and
the antisense strand.
[0155] In some embodiments the double stranded molecule disclosed
herein includes a combination of the following modifications:
[0156] the antisense strand includes a DNA, 2'5' nucleotide or a
mirror nucleotide in at least one of positions 5, 6, 7, 8, or 9
from the 5' terminus;
[0157] the sense strand includes 4, 5, or 6 consecutive 2'5'
nucleotides at the 3' penultimate or 3' terminal positions; and
[0158] one or more pyrimidine ribonucleotides are 2' sugar modified
in the sense strand, the antisense strand or both the sense strand
and the antisense strand.
[0159] In some embodiments of Structure (A1) and/or (A2) (N)y
includes at least one unconventional moiety selected from a mirror
nucleotide, a 2'5' ribonucleotide and a TNA. In some embodiments
the unconventional moiety is a mirror nucleotide. In various
embodiments the mirror nucleotide is selected from an
L-ribonucleotide (L-RNA) and an L-deoxyribonucleotide (L-DNA). In
preferred embodiments the mirror nucleotide is L-DNA. In certain
embodiments the sense strand comprises an unconventional moiety in
position 9 or 10 (from the 5' terminus). In preferred embodiments
the sense strand includes an unconventional moiety in position 9
(from the 5' terminus). In some embodiments the sense strand is 19
nucleotides in length and comprises 4, 5, or 6 consecutive
unconventional moieties in positions 15 (from the 5' terminus). In
some embodiments the sense strand includes 4 consecutive 2'5'
ribonucleotides in positions 15, 16, 17, and 18. In some
embodiments the sense strand includes 5 consecutive 2'5'
ribonucleotides in positions 15, 16, 17, 18 and 19. In various
embodiments the sense strand further comprises Z'. In some
embodiments Z' includes a C3OH moiety or a C3Pi moiety.
[0160] In some embodiments of Structure (A1) and/or (A2) (N)y
comprises at least one unconventional moiety selected from a mirror
nucleotide and a nucleotide joined to an adjacent nucleotide by a
2'-5' internucleotide phosphate bond. In some embodiments the
unconventional moiety is a mirror nucleotide. In various
embodiments the mirror nucleotide is selected from an
L-ribonucleotide (L-RNA) and an L-deoxyribonucleotide (L-DNA). In
preferred embodiments the mirror nucleotide is L-DNA.
[0161] In some embodiments of Structure A1 (N')y comprises at least
one L-DNA moiety. In some embodiments x=y=19 and (N')y consists of
unmodified ribonucleotides at positions 1-17 and 19 and one L-DNA
at the 3' penultimate position (position 18). In other embodiments
x=y=19 and (N')y consists of unmodified ribonucleotides at position
1-16 and 19 and two consecutive L-DNA nucleotides at the 3'
penultimate position (positions 17 and 18). In various embodiments
the unconventional moiety is a nucleotide joined to an adjacent
nucleotide by a 2'-5' internucleotide phosphate linkage. According
to various embodiments (N')y comprises 2, 3, 4, 5, or 6 consecutive
ribonucleotides at the 3' terminus linked by 2'-5' internucleotide
linkages. In one embodiment, four consecutive ribonucleotides at
the 3' terminus of (N')y are joined by three 2'-5' phosphodiester
bonds. In one embodiment, five consecutive ribonucleotides at the
3' terminus of (N')y are joined by four 2'-5' phosphodiester bonds.
In some embodiments, wherein one or more of the 2'-5'
ribonucleotides form a 2'-5' phosphodiester bonds the nucleotide
further comprises a 3'-O-methyl (3'OMe) sugar modification. In some
embodiments the 3' terminal nucleotide of (N')y comprises a 3'OMe
sugar modification. In certain embodiments x=y=19 and (N')y
comprises two or more consecutive nucleotides at positions 15, 16,
17, 18 and 19 which are joined to an adjacent nucleotide by a 2'-5'
internucleotide bond. In various embodiments, the nucleotide
forming the 2'-5' internucleotide bond comprises a ribonucleotide.
In preferred embodiments the 2'-5' internucleotide bond is a
phosphosdiester internucleotide bond. In various embodiments the
nucleotide forming the 2'-5' internucleotide bond comprises a 3'
deoxyribose nucleotide or a 3' methoxy nucleotide. In various
embodiments, the ribonucleotide forming the 2'-5' internucleotide
bond comprises a 3' deoxyribose ribonucleotide or a 3' methoxy
ribonucleotide. In some embodiments x=y=19 and (N')y comprises
nucleotides joined to the adjacent nucleotide by a 2'-5'
internucleotide bond between positions 15-16, 16-17 and 17-18 or
between positions 16-17, 17-18 and 18-19. In some embodiments
x=y=19 and (N')y comprises nucleotides joined to the adjacent
nucleotide by a 2'-5' internucleotide bond between positions 16-17
and 17-18 or between positions 17-18 and 18-19 or between positions
15-16 and 17-18. In various embodiments, the nucleotides forming
the 2'-5' internucleotide bond comprise ribonucleotides. In various
embodiments, the nucleotides forming the 2'-5' internucleotide bond
are ribonucleotides. In other embodiments the pyrimidine
ribonucleotides (rU, rC) in (N')y are substituted with a
ribonucleotide joined to the adjacent ribonucleotide by a 2'-5'
internucleotide bond.
[0162] In some embodiments of Structure (A2), (N)y comprises at
least one L-DNA moiety. In some embodiments x=y=18 and N2-(N')y,
consists of unmodified ribonucleotides at positions 1-17 and 19 and
one L-DNA at the 3' penultimate position (position 18). In other
embodiments x=y=18 and N2-(N')y consists of unmodified
ribonucleotides at position 1-16 and 19 and two consecutive L-DNA
at the 3' penultimate position (positions 17 and 18). In various
embodiments the unconventional moiety is a nucleotide joined to an
adjacent nucleotide by a 2'-5' internucleotide phosphate linkage.
According to various embodiments N2-(N')y comprises 2, 3, 4, 5, or
6 consecutive ribonucleotides at the 3' terminus linked by 2'-5'
internucleotide linkages. In one embodiment, four consecutive
ribonucleotides at the 3' terminus of N2-(N')y are joined by three
2'-5' phosphodiester bonds, wherein one or more of the 2'-5'
ribonucleotides which form the 2'-5' phosphodiester bonds further
comprises a 3'-O-methyl (3'OMe) sugar modification. In some
embodiments the 3' terminal ribonucleotide of N2-(N')y comprises a
2'OMe sugar modification. In certain embodiments x=y=18 and
N2-(N')y comprises two or more consecutive nucleotides at positions
15, 16, 17, 18 and 19 joined to an adjacent nucleotide by a 2'-5'
internucleotide bond. In various embodiments the nucleotide forming
the 2'-5' internucleotide bond comprises a 3' deoxyribose
nucleotide or a 3' methoxy nucleotide. In various embodiments, the
ribonucleotide forming the 2'-5' internucleotide bond comprises a
3' deoxyribose ribonucleotide or a 3' methoxy ribonucleotide. In
some embodiments x=y=18 and N2-(N')y comprises nucleotides joined
to the adjacent nucleotide by a 2'-5' internucleotide bond between
positions 16-17 and 17-18 or between positions 17-18 and 18-19 or
between positions 15-16 and 17-18. In various embodiments, the
nucleotides forming the 2'-5' internucleotide bond comprise
ribonucleotides. In various embodiments, the nucleotides forming
the 2'-5' internucleotide bond are ribonucleotides. In other
embodiments a pyrimidine ribonucleotide (rU, rC) in (N')y comprises
a ribonucleotide joined to the adjacent ribonucleotide by a 2'-5'
internucleotide bond.
[0163] In further embodiments of Structures (A1) and/or (A2) (N')y
comprises 1-8 modified ribonucleotides wherein the modified
ribonucleotide is a deoxyribose (DNA) nucleotide. In certain
embodiments (N')y comprises 1, 2, 3, 4, 5, 6, 7, or up to 8 DNA
moieties.
[0164] In presently preferred embodiments the inhibitor provided
herein is a synthetic, chemically modified double-stranded
oligonucleotide (e.g. dsRNA) compound, selected form: a
double-stranded oligonucleotide that down-regulates HES1 expression
and includes an oligonucleotide pair selected from Table I; a
double-stranded oligonucleotide that down-regulates HES5 expression
and includes an oligonucleotide pair selected from Table II; a
double-stranded oligonucleotide that down-regulates HEY2 expression
and includes an oligonucleotide pair selected from Table III; a
double-stranded oligonucleotide that down-regulates CDKN1B
expression and includes an oligonucleotide pair selected from Table
IV; a double-stranded oligonucleotide that down-regulates NOTCH1
expression and includes an oligonucleotide pair selected from Table
V. Tables I-V are provided herein below.
TABLE-US-00004 TABLE I Selected HES1 dsRNA dsRNA SEQ ID SEQ ID Name
NO: Sense strand (5' > 3') NO: Antisense strand (5' > 3')
Type HES1_12 26667 GCCAGCUGAUAUAAUGGAA 26679 UUCCAUUAUAUCAGCUGGC 18
+ 1 HES1_13 26668 GCCAGUGUCAACACGACAA 26680 UUGUCGUGUUGACACUGGC 18
+ 1 HES1_14 26669 CAGCGAGUGCAUGAACGAA 26681 UUCGUUCAUGCACUCGCUG 18
+ 1 HES1_16 26670 GAACGAGGUGACCCGCUUA 26682 UAAGCGGGUCACCUCGUUC 18
+ 1 HES1_19 26671 CCAGUGUCAACACGACACA 26683 UGUGUCGUGUUGACACUGG 18
+ 1 HES1_20 26672 CGAGUGCAUGAACGAGGUA 26684 UACCUCGUUCAUGCACUCG 18
+ 1 HES1_21 26673 UGUCAACACGACACCGGAA 26685 UUCCGGUGUCGUGUUGACA 18
+ 1 HES1_22 26674 CAGUGUCAACACGACACCA 26686 UGGUGUCGUGUUGACACUG 18
+ 1 HES1_24 26675 GGCGGACUCCAUGUGGAGA 26687 UCUCCACAUGGAGUCCGCC 18
+ 1 HES1_28 26676 CGGAUAAACCAAAGACAGA 26688 UCUGUCUUUGGUUUAUCCG 18
+ 1 HES1_33 26677 AGUGCAUGAACGAGGUGAA 26689 UUCACCUCGUUCAUGCACU 18
+ 1 HES1_36 26678 CAGCGAGUGCAUGAACGAU 26690 AUCGUUCAUGCACUCGCUG 18
+ 1 HES1_10 26691 GUAUUAAGUGACUGACCAU 26699 AUGGUCAGUCACUUAAUAC 19
HES1_11 26692 GAAAACACUGAUUUUGGAU 26700 AUCCAAAAUCAGUGUUUUC 19
HES1_15 26693 ACUGCAUGACCCAGAUCAA 26701 UUGAUCUGGGUCAUGCAGU 19
HES1_17 26694 AGCCAGUGUCAACACGACA 26702 UGUCGUGUUGACACUGGCU 19
HES1_18 26695 GUGUCAACACGACACCGGA 26703 UCCGGUGUCGUGUUGACAC 19
HES1_26 26696 CAGUGAAGCACCUCCGGAA 26704 UUCCGGAGGUGCUUCACUG 19
HES1_27 26697 CAUGGAGAAAAGACGAAGA 26705 UCUUCGUCUUUUCUCCAUG 19
HES1_35 26698 CAGCUGAUAUAAUGGAGAA 26706 UUCUCCAUUAUAUCAGCUG 19
TABLE-US-00005 TABLE II Selected HES5 dsRNA dsRNA SEQ ID SEQ ID
Name NO: Sense strand (5' > 3') NO: Antisense strand (5' >
3') Type HES5_19 26707 GGAGUUCGCGCGGCACCAA 26716
UUGGUGCCGCGCGAACUCC 18 + 1 HES5_20 26708 GCGACACGCAGAUGAAGCA 26717
UGCUUCAUCUGCGUGUCGC 18 + 1 HES5_22 26709 CGGGCACAUUUGCCUUUUA 26718
UAAAAGGCAAAUGUGCCCG 18 + 1 HES5_23 26710 CGCCAGCGACACGCAGAUA 26719
UAUCUGCGUGUCGCUGGCG 18 + 1 HES5_24 26711 CCGACUGCGGAAGCCGGUA 26720
UACCGGCUUCCGCAGUCGG 18 + 1 HES5_26 26712 GCGCGGCACCAGCCCAACA 26721
UGUUGGGCUGGUGCCGCGC 18 + 1 HES5_27 26713 AACCGACUGCGGAAGCCGA 26722
UCGGCUUCCGCAGUCGGUU 18 + 1 HES5_28 26714 CGACUGCGGAAGCCGGUGA 26723
UCACCGGCUUCCGCAGUCG 18 + 1 HES5_29 26715 CGACACGCAGAUGAAGCUA 26724
UAGCUUCAUCUGCGUGUCG 18 + 1 HES5_10 26725 CUGUAGAGGACUUUCUUCA 26729
UGAAGAAAGUCCUCUACAG 19 HES5_21 26726 GCCAGCGACACGCAGAUGA 26730
UCAUCUGCGUGUCGCUGGC 19 HES5_25 26727 GCGACACGCAGAUGAAGCU 26731
AGCUUCAUCUGCGUGUCGC 19 HES5_8 26728 GGGUUCUAUGAUAUUUGUA 26732
UACAAAUAUCAUAGAACCC 19
TABLE-US-00006 TABLE III Selected HEY2 dsRNA dsRNA SEQ ID Sense
strand SEQ ID AntiSense strand Name NO: (5' > 3') NO: (5' >
3') Type HEY2_1 26779 GGGAGCGAGAACAAUUACA 26782 UGUAAUUGUUCUCGCUCCC
18 + 1 HEY2_2 26780 GGGUAAAGGCUACUUUGAA 26783 UUCAAAGUAGCCUUUACCC
18 + 1 HEY2_5 26781 GAAAAGGCGUCGGGAUCGA 26784 UCGAUCCCGACGCCUUUUC
18 + 1 HEY2_3 26785 GGGUAAAGGCUACUUUGAC 26787 GUCAAAGUAGCCUUUACCC
19 HEY2_4 26786 CCAUGGCCCACCACCAUCA 26788 UGAUGGUGGUGGGCCAUGG
19
TABLE-US-00007 TABLE IV Selected CDKN1B (p27) duplexes DsRNA SEQ ID
Sense strand SEQ ID AntiSense strand name NO: (5' > 3') NO: (5'
> 3') Type CDKN1B_29 26867 AGCCAAAGUGGCAUGUUUA 26877
UAAACAUGCCACUUUGGCU 18 + 1 CDKN1B_30 26868 GCAUACUGAGCCAAGUAUA
26878 UACAUCCUGGCUCUCCUGC 18 + 1 CDKN1B_31 26869
CAGCGCAAGUGGAAUUUCA 26879 UGAAAUUCCACUUGCGCUG 18 + 1 CDKN1B_33
26870 UGCAUACUGAGCCAAGUAA 26880 UAUGCCACUUUGGCUUGUA 18 + 1
CDKN1B_34 26871 GGAGCGGAUGGACGCCAGA 26881 UCUGACAUCCUGGCUCUCC 18 +
1 CDKN1B_35 26872 AGGGCAGCUUGCCCGAGUA 26882 UACUCGGGCAAGCUGCCCU 18
+ 1 CDKN1B_36 26873 GUACUACCUGUGUAUAUAG 26883 UUUGGCUCAGUAUGCAACC
18 + 1 CDKN1B_37 26874 UGCAUACUGAGCCAAGUAU 26884
UUACUUGGCUCAGUAUGCA 18 + 1 CDKN1B_38 26875 GAGUGUCUAACGGGAGCCA
26885 UCCGCUGACAUCCUGGCUC 18 + 1 CDKN1B_40 26876
GCGCAAGUGGAAUUUCGAA 26886 UUCGAAAUUCCACUUGCGC 18 + 1 CDKN1B_3 26887
CGCAUUUGGUGGACCCAAA 26894 UUUGGGUCCACCAAAUGCG 19 CDKN1B_4 26888
GCAAUUAGGUUUUUCCUUA 26895 UAAGGAAAAACCUAAUUGC 19 CDKN1B_10 26889
CAUUGUACUACCUGUGUAU 26896 AUACACAGGUAGUACAAUG 19 CDKN1B_11 26890
GGUUUUUCCUUAUUUGCUU 26897 AAGCAAAUAAGGAAAAACC 19 CDKN1B_18 26891
AGCGCAAGUGGAAUUUCGA 26898 UCGAAAUUCCACUUGCGCU 19 CDKN1B_28 26892
GGUUGCAUACUGAGCCAAA 26899 AUCCUGGCUCUCCUGCGCC 19 CDKN1B_32 26893
AGCCAAAGUGGCAUGUUUU 26900 AAAACAUGCCACUUUGGCU 19
TABLE-US-00008 TABLE V Selected NOTCH1 dsRNA SEQ ID Sense strand
SEQ ID Antisense strand Name NO: (5' > 3') NO: (5' > 3')
NOTCH1_1 26901 CCUUCUACUGCGAGUGUCA 26906 UGACACUCGCAGUAGAAGG 18 + 1
NOTCH1_2 26902 GCUACAACUGCGUGUGUGA 26907 UCACACACGCAGUUGUAGC 18 + 1
NOTCH1_3 26903 UCCUUCUACUGCGAGUGUA 26908 UACACUCGCAGUAGAAGGA 18 + 1
NOTCH1_4 26904 CUCCUUCUACUGCGAGUGA 26909 UCACUCGCAGUAGAAGGAG 18 + 1
NOTCH1_5 26905 CAGCGCAGAUGCCAACAUA 26910 UAUGUUGGCAUCUGCGCUG 18 + 1
NOTCH1_6 26911 ACAACUGCGUGUGUGUCAA 26912 UUGACACACACGCAGUUGU 19
[0165] In some embodiments of the combinations, compositions and
methods, the double-stranded oligonucleotide molecule includes a
sense strand and an antisense strand selected from the
oligonucleotide pairs set forth in Tables I-V. Unless otherwise
stated all positions along a sense strand or antisense strand are
counted from the 5' to the 3' (5'-3').
[0166] In some embodiments the double stranded oligonucleotide
includes a particular sense strand and a particular antisense
strand set forth in SEQ ID NOS:23-1495 or 26667-26706 (HES1), SEQ
ID NOS:1496-2703 or 26707-26732 (HES5), SEQ ID NOS:13004-16621 or
26779-26788 (HEY2), SEQ ID NOS:7444-10533 or 26867-26900 (CDKN1B)
or SEQ ID NOS:16622-26666 or 26901-26912 (NOTCH1).
[0167] In some embodiments the double stranded nucleic acid
molecule has the structure:
##STR00001##
wherein each "|" represents base pairing between the
ribonucleotides; wherein each X is any one of A, C, G, U and is
independently an unmodified or modified ribonucleotide, an
unmodified or modified deoxyribonucleotide or an unconventional
moiety; wherein each of Z and Z' is independently present or
absent, but if present is independently 1-5 consecutive nucleotides
or non-nucleotide moieties or a combination thereof covalently
attached at the 3' terminus of the strand in which it is present;
and wherein z'' may be present or absent, but if present is a
capping moiety covalently attached at the 5' terminus of the sense
strand.
[0168] In preferred embodiments the double-stranded oligonucleotide
molecule comprises modified ribonucleotides and unconventional
moieties.
Chemical Modifications
[0169] All analogs of, or modifications to, a
nucleotide/oligonucleotide may be employed with the present
embodiments, provided that said analogue or modification does not
substantially affect the function of the
nucleotide/oligonucleotide. The nucleotides can be selected from
naturally occurring or synthetic modified bases. Naturally
occurring bases include adenine, guanine, cytosine, thymine and
uracil. Modified bases of nucleotides are described herein.
[0170] In addition, analogues of polynucleotides can be prepared
wherein the structure of one or more nucleotide is fundamentally
altered and better suited as therapeutic or experimental reagents.
An example of a nucleotide analogue is a peptide nucleic acid (PNA)
wherein the deoxyribose (or ribose) phosphate backbone in DNA (or
RNA is replaced with a polyamide backbone which is similar to that
found in peptides. PNA analogues have been shown to be resistant to
enzymatic degradation and to have extended stability in vivo and in
vitro. Other modifications that can be made to oligonucleotides
include polymer backbones, cyclic backbones, acyclic backbones,
thiophosphate-D-ribose backbones, triester backbones, thioate
backbones, 2'-5' bridged backbone, artificial nucleic acids,
morpholino nucleic acids, locked nucleic acid (LNA), glycol nucleic
acid (GNA), threose nucleic acid (TNA), arabinoside, and mirror
nucleoside (for example, beta-L-deoxynucleoside instead of
beta-D-deoxynucleoside). Examples of dsRNA molecules comprising LNA
nucleotides are disclosed in Elmen et al., (NAR 2005,
33(1):439-447).
[0171] The nucleic acid compounds useful in methods, compositions
and combinations disclosed herein can be synthesized using one or
more inverted nucleotides, for example inverted thymidine or
inverted adenine (see, for example, Takei, et al., 2002, JBC
277(26):23800-06).
[0172] The term "unconventional moiety" as used herein refers to
abasic ribose moiety, an abasic deoxyribose moiety, a
deoxyribonucleotide, a modified deoxyribonucleotide, a mirror
nucleotide, a non-base pairing nucleotide analog and a nucleotide
joined to an adjacent nucleotide by a 2'-5' internucleotide
phosphate bond; C3, C4, C5 and C6 moieties; bridged nucleic acids
including LNA and ethylene bridged nucleic acids.
[0173] The term "capping moiety" as used herein includes abasic
ribose moiety, abasic deoxyribose moiety, modifications of abasic
ribose and abasic deoxyribose moieties including 2'O alkyl
modifications; inverted abasic ribose and abasic deoxyribose
moieties and modifications thereof; C6-imino-Pi; a mirror
nucleotide including L-DNA and L-RNA; 5'OMe nucleotide; and
nucleotide analogs including 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; 12-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; alpha-nucleotide;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted abasic moiety; 1,4-butanediol phosphate; 5'-amino;
and bridging or non bridging methylphosphonate and 5'-mercapto
moieties.
[0174] Abasic deoxyribose moiety includes for example abasic
deoxyribose-3'-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate;
1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic
deoxyribose moiety includes inverted deoxyriboabasic; 3',5'
inverted deoxyriboabasic 5'-phosphate.
[0175] A "mirror" nucleotide is a nucleotide with reversed
chirality to the naturally occurring or commonly employed
nucleotide, i.e., a mirror image (L-nucleotide) of the naturally
occurring (D-nucleotide). The nucleotide can be a ribonucleotide or
a deoxyribonucleotide and may further comprise at least one sugar,
base and/or backbone modification. U.S. Pat. No. 6,602,858
discloses nucleic acid catalysts comprising at least one
L-nucleotide substitution. Mirror nucleotide includes for example
L-DNA (L-deoxyriboadenosine-3'-phosphate (mirror dA);
L-deoxyribocytidine-3'-phosphate (mirror dC);
L-deoxyriboguanosine-3'-phosphate (mirror dG);
L-deoxyribothymidine-3'-phosphate (mirror image dT)) and L-RNA
(L-riboadenosine-3'-phosphate (mirror rA);
L-ribocytidine-3'-phosphate (mirror rC);
L-riboguanosine-3'-phosphate (mirror rG); L-ribouracil-3'-phosphate
(mirror dU).
[0176] In various embodiments of Structure A1 or Structure A2, Z
and Z' are absent. In other embodiments Z or Z' is present. In some
embodiments each of Z and/or Z' independently includes a C2, C3,
C4, C5 or C6 alkyl moiety, optionally a C3 [propane, --(CH2)3-]
moiety or a derivative thereof including propanol (C3-OH/C3OH),
propanediol, and phosphodiester derivative of propanediol ("C3Pi").
In preferred embodiments each of Z and/or Z' includes two
hydrocarbon moieties and in some examples is C3Pi-C3OH or
C3Pi-C3Pi. Each C3 is covalently conjugated to an adjacent C3 via a
covalent bond, preferably a phospho-based bond. In some embodiments
the phospho-based bond is a phosphorothioate, a phosphonoacetate or
a phosphodiester bond.
[0177] In specific embodiments, at least one double-stranded
oligonucleotide has Structure A1 x=y=19 and Z comprises at least
one C3 alkyl overhang. In specific embodiments, at least one
double-stranded oligonucleotide has Structure A2 x=y=18 and Z
comprises at least one C3 alkyl overhang. In some embodiments the
C3-C3 overhang is covalently attached to the 3' terminus of (N)x or
(N')y via a covalent linkage, preferably a phosphodiester linkage.
In some embodiments the linkage between a first C3 and a second C3
is a phosphodiester linkage. In some embodiments the 3'
non-nucleotide overhang is C3Pi-C3Pi. In some embodiments the 3'
non-nucleotide overhang is C3Pi-C3Ps. In some embodiments the 3'
non-nucleotide overhang is C3Pi-C3OH (OH is hydroxy). In some
embodiments the 3' non-nucleotide overhang is C3Pi-C3OH.
[0178] In various embodiments the alkyl moiety comprises an alkyl
derivative including a C3 alkyl, C4 alkyl, C5 alky or C6 alkyl
moiety comprising a terminal hydroxyl, a terminal amino, or
terminal phosphate group. In some embodiments the alkyl moiety is a
C3 alkyl or C3 alkyl derivative moiety. In some embodiments the C3
alkyl moiety comprises propanol, propylphosphate,
propylphosphorothioate or a combination thereof. The C3 alkyl
moiety is covalently linked to the 3' terminus of (N')y and/or the
3' terminus of (N)x via a phosphodiester bond. In some embodiments
the alkyl moiety comprises propanol, propyl phosphate or propyl
phosphorothioate. In some embodiments each of Z and Z' is
independently selected from propanol, propyl phosphate propyl
phosphorothioate, combinations thereof or multiples thereof in
particular 2 or 3 covalently linked propanol, propyl phosphate,
propyl phosphorothioate or combinations thereof. In some
embodiments each of Z and Z' is independently selected from propyl
phosphate, propyl phosphorothioate, propyl phospho-propanol; propyl
phospho-propyl phosphorothioate; propylphospho-propyl phosphate;
(propyl phosphate)3, (propyl phosphate)2-propanol, (propyl
phosphate)2-propyl phosphorothioate. Any propane or propanol
conjugated moiety can be included in Z or Z'.
[0179] Exemplary 3' terminal non-nucleotide moieties are as
follows:
##STR00002##
Indications
[0180] The molecules and compositions disclosed herein are useful
in the treatment of diseases and disorders of the ear, as well as
other diseases and conditions herein described.
The Human Ear
[0181] The human ear is comprised of three major structural
components: the outer, middle, and inner ears, which function
together to convert sound waves into nerve impulses that travel to
the brain, where they are perceived as sound. The inner ear also
helps to maintain balance.
[0182] The anatomy of the middle and the inner ear is well known to
those of ordinary skill in the art (see, e.g., Atlas of Sensory
Organs: Functional and Clinical Analysis, Andrs Csillag, Humana
Press (2005), pages 1-82, incorporated herein by reference). In
brief, the middle ear consists of the eardrum and a small
air-filled chamber containing a sequence of three tiny bones known
as the ossicles, which link the eardrum to the inner ear.
[0183] The inner ear (labyrinth) is a complex structure consisting
of the cochlea, which is the organ of hearing and the vestibular
system, the organ of balance. The vestibular system consists of the
saccule and the utricle, which determine position sense, and the
semicircular canals, which help maintain balance.
[0184] The cochlea houses the organ of Corti, which consists, in
part, of about 20,000 specialized sensory cells, called "inner ear
hair cells" or "hair cells". These cells have small hairline
projections (cilia) that extend into the cochlear fluid. Sound
vibrations transmitted from the ossicles in the middle ear to the
oval window in the inner ear cause the fluid and cilia to vibrate.
Hair cells in different parts of the cochlea vibrate in response to
different sound frequencies and convert the vibrations into nerve
impulses which are sent to the brain for processing and
interpretation. The inner ear hair cells (IHC) are surrounded by
inner ear support cells. Supporting cells underlie, at least
partially surround, and physically support sensory hair cells
within the inner ear. Representative examples of support cells
include inner rod (pillar cells), outer rod (pillar cells), inner
phalangeal cells, outer phalangeal cells (of Deiters), cells of
Held, cells of Hensen, cells of Claudius, cells of Boettcher,
interdental cells and auditory teeth (of Huschke).
[0185] The spiral ganglion is the group of nerve cells that send a
representation of sound from the cochlea to the brain. The cell
bodies of the spiral ganglion neurons are found in the spiral
structure of the cochlea and are part of the central nervous
system. Their dendrites make synaptic contact with the base of hair
cells, and their axons are bundled together to form the auditory
portion of the eighth cranial nerve (vestibulocochlear nerve).
Hearing Loss
[0186] Auditory hair cells are sensory receptors located in the
organ of Corti of the cochlea involved in detecting sound. The
cochlear hair cells come in two anatomically and functionally
distinct types: the outer and inner hair cells. Auditory hair cells
convert sound information into electrical signals that are sent via
nerve fibers to the brain and processed.
[0187] Vestibular hair cells, located in the vestibular organs of
the inner ear (utricle, saccule, ampullae), detect changes in head
position and convey this information to the brain to help maintain
balance posture and eye position.
[0188] In the absence of auditory hair cells, sound waves are not
converted into neural signals and hearing deficits ensue, for
example, decreased hearing sensitivity, i.e. sensorineural hearing
loss. In the absence of vestibular hair cells, balance deficits
ensue.
[0189] Despite the protective effect of the acoustic reflex, loud
noise can damage and destroy hair cells. Irreversible hair cell
death is elicited by metabolic or biochemical changes in the hair
cells that involve reactive oxygen species (ROS). Exposure to
certain drugs and continued exposure to loud noise, inter alia,
cause progressive damage, eventually resulting in ringing in the
ears (tinnitus) and or hearing loss.
[0190] Acquired hearing loss can be caused by several factors
including exposure to harmful noise levels, exposure to ototoxic
drugs such as cisplatin and aminoglycoside antibiotics and
aging.
[0191] U.S. Ser. No. 11/655,610 to the assignee of the present
invention relates to methods of treating hearing impairment by
inhibiting a pro-apoptotic gene in general and p53 in particular.
International Patent Publication No. WO 2005/119251 relates to
methods of treating deafness. International Patent Publication No.
WO/2005/055921 relates to foam compositions for treatment of ear
disorders. U.S. Pat. No. 7,087,581 relates to methods of treating
diseases and disorders of the inner ear. PCT Publication No. WO
2009/147684, assigned to the assignee of the present application,
and incorporated herein by reference in its entirety discloses
certain compounds and compositions for treating otic disorders and
diseases.
Ear Disorders
[0192] The present disclosure is directed, inter alia, to
compositions, combinations and methods useful in treating a patient
suffering from or at risk of various ear disorders. Ear disorders
include hearing loss induced for example by ototoxins, excessive
noise or ageing. Middle and inner ear disorders produce many of the
same symptoms, and a disorder of the middle ear may affect the
inner ear and vice versa.
[0193] In addition to hearing loss, ear disorders include
myringitis, an eardrum infection caused by a variety of viruses and
bacteria; temporal bone fracture for example due to a blow to the
head; auditory nerve tumors (acoustic neuroma, acoustic neurinoma,
vestibular schwannoma, eighth nerve tumor).
[0194] In various embodiments, the methods, combinations and
compositions disclosed herein are useful in treating various
conditions of hearing loss. Without being bound by theory, the
hearing loss may be due to apoptotic inner ear hair cell damage or
loss (Zhang et al., Neuroscience 2003. 120:191-205; Wang et al., J.
Neuroscience 23((24):8596-8607), wherein the damage or loss is
caused by infection, mechanical injury, loud sound (noise), aging
(presbycusis), or chemical-induced ototoxicity.
[0195] By "ototoxin" in the context disclosed herein is meant a
substance that through its chemical action injures, impairs or
inhibits the activity of the sound receptors component of the
nervous system related to hearing, which in turn impairs hearing
(and/or balance). In the context of the present invention,
ototoxicity includes a deleterious effect on the inner ear hair
cells. Ototoxic agents that cause hearing impairments include, but
are not limited to, neoplastic agents such as vincristine,
vinblastine, cisplatin and cisplatin-like compounds, taxol and
taxol-like compounds, dideoxy-compounds, e.g., dideoxyinosine;
alcohol; metals; industrial pollutants involved in occupational or
environmental exposure; contaminants of food or medicinals; and
over-doses of vitamins or therapeutic drugs, e.g., antibiotics such
as penicillin or chloramphenicol, and megadoses of vitamins A, D,
or B6, salicylates, quinines and loop diuretics. By "exposure to an
ototoxic agent" is meant that the ototoxic agent is made available
to, or comes into contact with, a mammal. Exposure to an ototoxic
agent can occur by direct administration, e.g., by ingestion or
administration of a food, medicinal, or therapeutic agent, e.g., a
chemotherapeutic agent, by accidental contamination, or by
environmental exposure, e g., aerial or aqueous exposure.
Typically, treatment is performed to prevent or reduce ototoxicity,
especially resulting from or expected to result from administration
of therapeutic drugs. Preferably a composition comprising a
therapeutically effective amount of a chemically modified siRNA
compound of the invention is given immediately after the exposure
to prevent or reduce the ototoxic effect. More preferably,
treatment is provided prophylactically, either by administration of
the pharmaceutical composition of the invention prior to or
concomitantly with the ototoxic pharmaceutical or the exposure to
the ototoxin. Incorporated herein by reference are chapters 196,
197, 198 and 199 of The Merck Manual of Diagnosis and Therapy, 14th
Edition, (1982), Merck Sharp & Dome Research Laboratories, N.J.
and corresponding chapters in the most recent 16th edition,
including Chapters 207 and 210) relating to description and
diagnosis of hearing and balance impairments.
[0196] Accordingly, in one aspect provided are methods,
combinations and pharmaceutical compositions for treating a mammal,
preferably human, to prevent, reduce, or treat a hearing
impairment, disorder or imbalance, preferably an ototoxin-induced
hearing condition, by administering to a mammal in need of such
treatment a combination or composition comprising inhibitors of
target genes as disclosed herein. Some embodiments are directed to
methods for treating a hearing disorder or impairment wherein the
ototoxicity results from administration of a therapeutically
effective amount of an ototoxic pharmaceutical drug. Typical
ototoxic drugs are chemotherapeutic agents, e.g. antineoplastic
agents, and antibiotics. Other possible candidates include
loop-diuretics, quinines or a quinine-like compound, PDE-5
inhibitors and salicylate or salicylate-like compounds.
[0197] Ototoxicity is a dose-limiting side effect of antibiotic
administration. From 4 to 15% of patients receiving 1 gram per day
for greater than 1 week develop measurable hearing loss, which
slowly becomes worse and can lead to complete permanent deafness if
treatment continues. Ototoxic aminoglycoside antibiotics include
but are not limited to neomycin, paromomycin, ribostamycin,
lividomycin, kanamycin, amikacin, tobramycin, viomycin, gentamicin,
sisomicin, netilmicin, streptomycin, dibekacin, fortimicin, and
dihydrostreptomycin, or combinations thereof. Particular
antibiotics include neomycin B, kanamycin A, kanamycin B,
gentamicin C1, gentamicin C1a, and gentamicin C2, and the like that
are known to have serious toxicity, particularly ototoxicity and
nephrotoxicity, which reduce the usefulness of such antimicrobial
agents (see Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 6th ed., A. Goodman Gilman et al., eds; Macmillan
Publishing Co., Inc., New York, pp. 1169-71 (1980)).
[0198] Ototoxicity is also a serious dose-limiting side-effect for
anti-cancer agents. Ototoxic neoplastic agents include but are not
limited to vincristine, vinblastine, cisplatin and cisplatin-like
compounds and taxol and taxol-like compounds. Cisplatin-like
compounds include carboplatin (Paraplatin.RTM.), tetraplatin,
oxaliplatin, aroplatin and transplatin inter alia and are platinum
based chemotherapeutics.
[0199] Diuretics with known ototoxic side-effect, particularly
"loop" diuretics include, without being limited to, furosemide,
ethacrylic acid, and mercurials.
[0200] Ototoxic quinines include but are not limited to synthetic
substitutes of quinine that are typically used in the treatment of
malaria. In some embodiments the hearing disorder is side-effect of
inhibitors of type 5 phosphodiesterase (PDE-5), including
sildenafil (Viagra.RTM.), vardenafil (Levitra.RTM.) and tadalafil
(Cialis).
[0201] Salicylates, such as aspirin, are the most commonly used
therapeutic drugs for their anti-inflammatory, analgesic,
anti-pyretic and anti-thrombotic effects. Unfortunately, they too
have ototoxic side effects. They often lead to tinnitus ("ringing
in the ears") and temporary hearing loss. Moreover, if the drug is
used at high doses for a prolonged time, the hearing impairment can
become persistent and irreversible.
[0202] In some embodiments of the methods provided herein, the
subject is a mammal suffering of infection and treated by
administration of an aminoglycoside antibiotic. The methods
disclosed herein improve the outcome of such treatment by reducing
or preventing ototoxin-induced hearing impairment associated with
the antibiotic.
[0203] The methods, combinations and pharmaceutical compositions
described herein are also effective in the treatment of acoustic
trauma or mechanical trauma, preferably acoustic or mechanical
trauma that leads to inner ear hair cell loss. With more severe
exposure, injury can proceed from a loss of adjacent supporting
cells to complete disruption of the organ of Corti. Death of the
sensory cell can lead to progressive Wallerian degeneration and
loss of primary auditory nerve fibers. The methods, combinations
and compositions provided herein are useful in treating acoustic
trauma caused by a single exposure to an extremely loud sound, or
following long-term exposure to everyday loud sounds above 85
decibels, for treating mechanical inner ear trauma, for example,
resulting from the insertion of an electronic device into the inner
ear or for preventing or minimizing the damage to inner ear hair
cells associated with the operation.
[0204] Another type of hearing loss is presbycusis, which is
hearing loss that gradually occurs in most individuals as they age.
About 30-35 percent of adults between the ages of 65 and 75 years
and 40-50 percent of people 75 and older experience hearing loss.
The methods, combinations and compositions disclosed herein are
useful in preventing, reducing or treating the incidence and/or
severity of inner ear disorders and hearing impairments associated
with presbycusis.
Acoustic Trauma
[0205] Acoustic trauma is a type of hearing loss that is caused by
prolonged exposure to loud noises. Without wishing to be bound to
theory, exposure to loud noise causes the hair cells on the cochlea
to become less sensitive. With more severe exposure, injury can
proceed from a loss of adjacent supporting cells to complete
disruption of the organ of Corti. Death of the sensory cell can
lead to progressive Wallerian degeneration and loss of primary
auditory nerve fibers. Disclosed herein are, inter alia,
combinations, pharmaceutical compositions and methods useful in
attenuating hearing loss due to acoustic trauma. In certain
embodiments of the methods, combinations and compositions, dsRNA
molecules that target HES1, HES5, and HEY2 are used for treating or
preventing acoustic trauma in a subject exposed to acoustic trauma.
In certain embodiments of the methods, combinations and
compositions, dsRNA molecules that target CDKN1B, NOTCH1 and HEY2
are used for treating or preventing acoustic trauma in a subject
exposed to acoustic trauma.
[0206] In certain embodiments, provided herein are methods of
treating a subject suffering from or at risk of an ear disorder
which comprises topically administering to the canal of the
subject's ear a pharmaceutical composition comprising inhibitors to
target genes associated with the disorder, such as dsRNA
inhibitors, in an amount effective to treat the subject, and a
pharmaceutically acceptable excipient or mixtures thereof, thereby
treating the subject. In certain embodiments, provided herein are
methods of treating a subject suffering from or at risk of an ear
disorder which comprises transtympanically administering to the
canal of the subject's ear a pharmaceutical composition comprising
inhibitors to target genes associated with the disorder, such as
oligonucleotide inhibitors, in an amount effective to treat the
subject, and a pharmaceutically acceptable excipient or mixtures
thereof, thereby treating the subject. In one embodiment, the
pharmaceutical composition is delivered via a posterior
semicircular canalostomy. In one embodiment, the pharmaceutical
composition is delivered as ear drops. In another embodiment the
pharmaceutical composition is delivered by a pump.
[0207] In some embodiments, the pharmaceutical composition is
applied to the ear canal when the subject's head is tilted to one
side and the treated ear is facing upward. In some embodiments, the
pharmaceutical composition is applied to the ear using a receptacle
for eardrops, for example using a dropper of for example, 10-100
microliter per drop, or a wick.
[0208] In some embodiments an ear disorder relates to
chemical-induced hearing loss; for example hearing loss induced by
inter alia cisplatin and its analogs; aminoglycoside antibiotics,
quinine and its analogs; salicylate and its analogs;
phosphodiesterase type 5 (PDE5) inhibitors or loop-diuretics. In
some embodiments the ear disorder refers to noise-induced hearing
loss. In other embodiments the ear disorder is age related hearing
loss.
[0209] Without being bound by theory, inhibition of HES1, HES5,
HEY2, CDKN1B or NOTCH1 results in regeneration of or protection of
otic hair (sensory) cells of the inner ear, optionally via an
increase in Atoh1 expression. The methods, compositions and
combinations as disclosed herein, are useful in treating,
ameliorating or preventing any disease, disorder or injury in which
promoting proliferation of supporting cells of the inner ear or of
outer hair cells or of inner hair cells in the cochlea is required.
In various embodiments the methods, compositions and combinations
provided herein are useful in treating hearing and balance
disorders, such as, without being limited to, ototoxin-induced
hearing loss, hearing loss associated with Meniere's disease, and
trauma-induced hearing loss, such as acoustic trauma and pressure
trauma, including blasts and surgical procedures in the inner
and/or middle ear.
Diseases and Disorders of the Vestibular System
[0210] In various embodiments the nucleic acid compounds and
pharmaceutical compositions disclosed herein are useful for
treating disorders and diseases affecting the vestibular system in
which expression of HES1, HES5, and HEY2, or CDKN1B, NOTCH1 and
HEY2 is detrimental, for example Meniere's Disease. The vestibular
sensory system in most mammals, including humans, contributes to
balance, and to a sense of spatial orientation and stability.
Together with the cochlea it constitutes the labyrinth of the inner
ear. The vestibular system comprises two components: the
semicircular canal system, which indicate rotational movements; and
the otoliths, which indicate linear accelerations.
[0211] The primary morbidity associated with Meniere's disease is
the debilitating nature of vertigo and the progressive hearing
loss. Current therapies have not been successful at preventing
progression of neuronal degeneration and associated hearing loss. A
therapeutic treatment, which would protect the neurons of the inner
ear including the vestibulocochlear nerve from damage and/or induce
regeneration of the vestibulocochlear nerve and thereby attenuate
or prevent hearing loss in Meniere's patients would be highly
desirable.
[0212] In certain aspects and embodiments, the combinations,
compositions, methods, commercial packages and kits provided herein
are useful in treating subjects at risk of or suffering from
Meniere's disease.
[0213] In conclusion, there are no effective modes of therapy for
the prevention and/or treatment of the conditions disclosed herein.
Treatments that are available suffer from, inter alia, the
drawbacks of severe side effects due to the lack of selective
targeting and there remains a need therefore to develop novel
compositions and methods of treatment for these purposes.
[0214] In various embodiments the combinations and pharmaceutical
compositions provided herein are useful in treating or preventing
various diseases, disorders and injury that affect the ear, such
as, without being limited to, the diseases, disorders and injury
that are disclosed herein below. Without being bound by theory, it
is believed that the combinations/compositions disclose herein
prevent death or various types of cells within the ear and/or
promote differentiation of supporting cells within the inner ear
into otic sensory cells.
Pharmaceutical Compositions
[0215] Provided are compositions, combinations and methods for
down-regulation of expression of HES1, HES5 and HEY2, or for
down-regulation of expression of CDKN1B, HEY2 and NOTCH1. In
certain embodiments, the compositions, combinations and methods use
small nucleic acid molecules, such as short interfering nucleic
acid (siNA), interfering RNA (RNAi), short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin
RNA (shRNA) molecules capable of mediating down-regulation of HES1,
HES5, HEY2, CDKN1B, or NOTCH1 gene expression or that mediate RNA
interference against HES1, HES5, HEY2, CDKN1B, or NOTCH1 gene
expression.
[0216] While it may be possible for the molecules disclosed herein
to be administered as the raw chemical, it is preferable to present
them as a pharmaceutical composition. Accordingly provided is a
pharmaceutical composition comprising one or more of the dsRNA
molecules disclosed herein or pharmacological salts thereof; and a
pharmaceutically acceptable carrier. The composition may comprise a
mixture of two or three different nucleic acid compounds.
[0217] Compositions, methods and kits provided herein may include
one or more nucleic acid molecules (e.g., dsRNA) and methods that
independently or in combination modulate (e.g., down-regulate) the
expression of HES1, HES5, HEY2, CDKN1B or NOTCH1 protein and/or
genes encoding HES1, HES5, HEY2, CDKN1B or NOTCH1 protein, proteins
and/or genes associated with the maintenance and/or development of
diseases, conditions or disorders associated with HES1, HES5, HEY2,
CDKN1B or NOTCH1, particularly disorders associated with the ear.
The description of the various aspects and embodiments is provided
with reference to exemplary genes HES1, HES5, HEY2, CDKN1B or
NOTCH1. However, the various aspects and embodiments are also
directed to other related genes, such as homolog genes and
transcript variants, and polymorphisms (e.g., single nucleotide
polymorphism, (SNPs)) associated with certain HES1, HES5, HEY2,
CDKN1B or NOTCH1 genes. As such, the various aspects and
embodiments are also directed to other genes that are involved in
HES1, HES5, HEY2, CDKN1B or NOTCH1 mediated pathways of signal
transduction or gene expression that are involved, for example, in
the maintenance or development of diseases, traits, or conditions
described herein. These additional genes can be analyzed for target
sites using the methods described for the HES1, HES5, HEY2, CDKN1B
or NOTCH1 gene herein. Thus, the down-regulation of other genes and
the effects of such modulation of the other genes can be performed,
determined, and measured as described herein.
[0218] Further provided is a pharmaceutical composition comprising
at least one compound of the invention covalently or non-covalently
bound to one or more compounds of the invention in an amount
effective to down regulate HES1, HES5, HEY2, CDKN1B, or NOTCH1
expression; and a pharmaceutically acceptable carrier. Further
provided are nucleic acid compounds which are processed
intracellularly by endogenous cellular complexes to produce one or
more oligoribonucleotides useful in accordance with the aspects and
embodiments described herein.
[0219] Further provided is a pharmaceutical composition comprising
a pharmaceutically acceptable carrier and one or more of the
compounds useful in methods disclosed herein in an amount effective
to inhibit expression in a cell of human HES1, HES5, HEY2, CDKN1B
or NOTCH1, the compound comprising a sequence which is
substantially complementary to a consecutive sequence selected from
a sequence in HES1 mRNA, HES5 mRNA, HEY2 mRNA, CDKN1B mRNA or
NOTCH1 mRNA.
[0220] Substantially complementary refers to complementarity of
greater than about 84%, to another sequence. For example in a
duplex region consisting of 19 base pairs one mismatch results in
94.7% complementarity, two mismatches results in about 89.5%
complementarity and 3 mismatches results in about 84.2%
complementarity, rendering the duplex region substantially
complementary. Accordingly substantially identical refers to
identity of greater than about 84%, to another sequence.
[0221] Additionally, provided herein are methods of preventing,
treating, or delaying of progression of a hearing disorder, a
hearing loss, and/or a balance impairment, or of preventing the
loss of otic (sensory) hair cells of the inner ear in a subject,
comprising inhibiting the expression of HES1, HES5, HEY2, CDKN1B or
NOTCH1 gene by at least 20%, by at least 30% by at least 40%,
preferably by 50%, 60% or 70%, more preferably by 75%, 80% or 90%
as compared to a control comprising contacting an mRNA transcript
of the respective gene with a HES1 inhibitor, a HES5 inhibitor, a
HEY2 inhibitor, a CDKN1B inhibitor or a NOTCH1 inhibitor,
[0222] In various embodiments as provided herein, the inhibitor is
an oligoribonucleotide compound. Compositions, combinations and
methods disclosed herein inhibit/down-regulate the HES1, HES5,
HEY2, CDKN1B, or NOTCH1 gene, whereby the
inhibition/down-regulation is selected from the group comprising
inhibition/down-regulation of gene function,
inhibition/down-regulation of polypeptide and
inhibition/down-regulation of mRNA expression.
[0223] In certain embodiments, compositions, combinations and
methods provided herein include a double-stranded short interfering
nucleic acid (siNA) compound that down-regulates expression of a
HES1, HES5, HEY2, CDKN1B or NOTCH1 gene (e.g., the mRNA coding
sequence for human HES1, HES5, HEY2, CDKN1B or NOTCH1 exemplified
by SEQ ID NO:1, 2, 10, 7 or 11, where the nucleic acid molecule
includes about 18 to about 49 base pairs.
[0224] In some embodiments, a nucleic acid disclosed herein may be
used to inhibit the expression of the HES1, HES5, HEY2, CDKN1B or
NOTCH1 gene or a HES1, HES5, HEY2, CDKN1B, or NOTCH1 gene family
where the genes or gene family sequences share sequence homology.
Such homologous sequences can be identified as is known in the art,
for example using sequence alignments. Nucleic acid molecules can
be designed to target such homologous sequences, for example using
perfectly complementary sequences or by incorporating non-canonical
base pairs, for example mismatches and/or wobble base pairs, that
can provide additional target sequences. In instances where
mismatches are identified, non-canonical base pairs (for example,
mismatches and/or wobble bases) can be used to generate nucleic
acid molecules that target more than one gene sequence. In a
non-limiting example, non-canonical base pairs such as UU and CC
base pairs are used to generate nucleic acid molecules that are
capable of targeting sequences for differing HES1, HES5, HEY2,
CDKN1B, or NOTCH1 targets that share sequence homology. As such,
one advantage of using dsRNAs disclosed herein is that a single
nucleic acid can be designed to include nucleic acid sequence that
is complementary to the nucleotide sequence that is conserved
between the homologous genes. In this approach, a single nucleic
acid can be used to inhibit expression of more than one gene
instead of using more than one nucleic acid molecule to target the
different genes.
[0225] Nucleic acid molecules may be used to target conserved
sequences corresponding to a gene family or gene families such as
HES1, HES5, HEY2, CDKN1B, or NOTCH1 family genes. As such, nucleic
acid molecules targeting multiple HES1, HES5, HEY2, CDKN1B, or
NOTCH1 targets can provide increased therapeutic effect. In
addition, nucleic acid can be used to characterize pathways of gene
function in a variety of applications. For example, nucleic acid
molecules can be used to inhibit the activity of target gene(s) in
a pathway to determine the function of uncharacterized gene(s) in
gene function analysis, mRNA function analysis, or translational
analysis. The nucleic acid molecules can be used to determine
potential target gene pathways involved in various diseases and
conditions toward pharmaceutical development. The nucleic acid
molecules can be used to understand pathways of gene expression
involved in, for example ear disorders.
[0226] In various embodiments of the compositions, combinations and
methods provided herein, nucleic acid compounds inhibit the HES1,
HES5, HEY2, CDKN1B, or NOTCH1 polypeptide, whereby the inhibition
is selected from the group comprising inhibition of function (which
may be examined by an enzymatic assay or a binding assay with a
known interactor of the native gene/polypeptide, inter alia),
down-regulation of protein or inhibition of protein (which may be
examined by Western blotting, ELISA or immuno-precipitation, inter
alia) and inhibition of mRNA expression (which may be examined by
Northern blotting, quantitative RT-PCR, in-situ hybridisation or
microarray hybridisation, inter alia).
[0227] In certain embodiments, the compositions, combinations and
methods provided herein include a nucleic acid molecule having RNAi
activity against HES1, HES5, HEY2, CDKN1B, or NOTCH1 RNA, where the
nucleic acid molecule includes a sequence complementary to any RNA
having HES1, HES5, HEY2, CDKN1B or NOTCH1 encoding sequence, such
as that sequence set forth in SEQ ID NO:1, 2, 10, 7 or 11. In
another embodiment, a nucleic acid molecule may have RNAi activity
against HES1, HES5, HEY2, CDKN1B, or NOTCH1 RNA, where the nucleic
acid molecule includes a sequence complementary to an RNA having
variant HES1, HES5, HEY2, CDKN1B or NOTCH1 encoding sequence, for
example mutations in HES1, HES5, HEY2, CDKN1B or NOTCH1 genes not
shown in SEQ ID NO:1, 2, 10, 7 or 11 but known in the art to be
associated with the onset and/or maintenance and/or development of
any of the disorders disclosed herein, for example a SNP. Chemical
modifications as described herein can be applied to any nucleic
acid construct disclosed herein. In another embodiment, a nucleic
acid molecule disclosed herein includes a nucleotide sequence that
can interact with nucleotide sequence of a HES1, HES5, HEY2, CDKN1B
or NOTCH1 gene and thereby mediate down-regulation or silencing of
HES1, HES5, HEY2, CDKN1B or NOTCH1 gene expression, for example,
wherein the nucleic acid molecule mediates regulation of HES1,
HES5, HEY2, CDKN1B or NOTCH1 gene expression by cellular processes
that modulate the chromatin structure or methylation patterns of
the gene and prevent transcription of the gene.
Delivery and Formulations
[0228] The inhibitors useful in accordance with the aspects and
embodiments disclosed herein (e.g. dsRNA molecules) may be
delivered to the ear of a subject by direct application of a
pharmaceutical composition to the outer ear; by transtympanic
injection, by a pump or by ear drops. In some embodiments the
pharmaceutical composition is applied to the ear canal. Delivery to
the ear may also be refereed to as aural or otic delivery
comprising, e.g. siRNA; a penetration enhancer and a
pharmaceutically acceptable vehicle.
[0229] In various embodiments, inhibitors, e.g. nucleic acid
molecules, as disclosed herein may be delivered to the target
tissue by direct application of the naked molecules prepared with a
carrier or a diluent.
[0230] The terms "naked nucleic acid" or "naked dsRNA" or "naked
siRNA" refers to nucleic acid molecules that are free from any
delivery vehicle that acts to assist, promote or facilitate entry
into the cell, including viral sequences, viral particles, liposome
formulations, lipofectin or precipitating agents and the like. For
example, dsRNA in PBS is "naked dsRNA".
[0231] In various embodiments, inhibitors, e.g. nucleic acid
molecules disclosed herein, may be delivered or administered
directly with a carrier or diluent that acts to assist, promote or
facilitate entry to the cell, including viral vectors, viral
particles, liposome formulations, lipofectin or precipitating
agents and the like.
[0232] A nucleic acid molecule may include a delivery vehicle,
including liposomes, for administration to a subject, carriers and
diluents and their salts, and/or can be present in pharmaceutically
acceptable formulations. In some embodiments, the dsRNA molecules
of disclosed herein are delivered in liposome formulations and
lipofectin formulations and the like and can be prepared by methods
well known to those skilled in the art. Such methods are described,
for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859,
which are herein incorporated by reference.
[0233] Delivery systems aimed specifically at the enhanced and
improved delivery of siRNA into mammalian cells have been
developed, (see, for example, Shen et al., FEBS Let. 2003,
539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et
al., Mol. Vision 2003, 9: 210-216; Sorensen et al., J. Mol. Biol.
2003. 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 and
Simeoni et al., NAR 2003, 31,11: 2717-2724). siRNA has recently
been successfully used for inhibition of gene expression in
primates (see for example, Tolentino et al., Retina 24(4):660).
[0234] Delivery of naked or formulated RNA molecules to the ear,
optionally the inner ear, is accomplished, inter alia, by
transtympanic injection or by administration of the desired
compounds formulated as an ear drop. Otic compositions comprising
dsRNA are disclosed in US Publication No. 20110142917, to the
assignee of the present application and incorporated herein by
reference in its entirety.
[0235] Polypeptides that facilitate introduction of nucleic acid
into a desired subject are known in the art, e.g. such as those
described in US. Application Publication No. 20070155658 (e.g., a
melamine derivative such as 2,4,6-Triguanidino Traizine and
2,4,6-Tramidosarcocyl Melamine, a polyarginine polypeptide, and a
polypeptide including alternating glutamine and asparagine
residues).
[0236] The pharmaceutically acceptable carriers, solvents,
diluents, excipients, adjuvants and vehicles as well as implant
carriers generally refer to inert, non-toxic solid or liquid
fillers, diluents or encapsulating material not reacting with the
active ingredients of the invention and they include liposomes and
microspheres. Examples of delivery systems useful in the present
invention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;
4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;
4,439,196; and 4,475,196. Many other such implants, delivery
systems, and modules are well known to those skilled in the
art.
[0237] In a particular embodiment, the administration comprises
transtympanic administration. In another embodiment the
administration comprises topical or local administration. The
compounds are administered as eardrops, ear cream, ear ointment, a
solution, a foam, a mousse or any of the above in combination with
a delivery device. Implants of the compounds are also useful.
Liquid forms are prepared as drops or for continuous application.
The liquid compositions include aqueous solutions, with and without
organic co-solvents, aqueous or oil suspensions, emulsions with
edible oils, as well as similar pharmaceutical vehicles. These
compositions may also be injected transtympanically. Eardrops may
also be referred to as otic drops or aural drops. In a preferred
embodiment, the ear drops remain in the ear canal for about 30 min
in order to prevent leakage of the drops out of the canal. It is
thus preferable that the subject receiving the drops keep his head
on the side with the treated ear facing upward to prevent leakage
of the drop out of the canal.
[0238] Methods for the delivery of nucleic acid molecules are
described in Akhtar et al., Trends Cell Bio., 2: 139 (1992);
Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.
Akhtar, (1995), Maurer et al., Mol. Membr. Biol., 16: 129-140
(1999); Hofland and Huang, Handb. Exp. Pharmacol., 137: 165-192
(1999); and Lee et al., ACS Symp. Ser., 752: 184-192 (2000); U.S.
Pat. Nos. 6,395,713; 6,235,310; 5,225,182; 5,169,383; 5,167,616;
4,959217; 4,925,678; 4,487,603; and 4,486,194 and Sullivan et al.,
PCT WO 94/02595; PCT WO 00/03683 and PCT WO 02/08754; and U.S.
Patent Application Publication No. 2003077829. These protocols can
be utilized for the delivery of virtually any nucleic acid
molecule. Nucleic acid molecules can be administered to cells by a
variety of methods known to those of skill in the art, including,
but not restricted to, encapsulation in liposomes, by
iontophoresis, or by incorporation into other vehicles, such as
biodegradable polymers, hydrogels, cyclodextrins (see e.g.,
Gonzalez et al., Bioconjugate Chem., 10: 1068-1074 (1999); Wang et
al., International PCT publication Nos. WO 03/47518 and WO
03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA
microspheres (see for example U.S. Pat. No. 6,447,796 and U.S.
Application Publication No. 2002130430), biodegradable
nanocapsules, and bioadhesive microspheres, or by proteinaceous
vectors (O'Hare and Normand, International PCT Publication No. WO
00/53722). Alternatively, the nucleic acid/vehicle combination is
locally delivered by direct injection or by use of an infusion
pump. Direct injection of the nucleic acid molecules of the
invention, whether intravitreal, subcutaneous, transtympanic,
intramuscular, or intradermal, can take place using standard needle
and syringe methodologies, or by needle-free technologies such as
those described in Conry et al., Clin. Cancer Res., 5: 2330-2337
(1999) and Barry et al., International PCT Publication No. WO
99/31262. The molecules of the instant invention can be used as
pharmaceutical agents. Pharmaceutical agents prevent, modulate the
occurrence, or treat or alleviate a symptom to some extent
(preferably all of the symptoms) of a disease state in a subject.
In one specific embodiment of this invention topical and
transdermal formulations may be selected.
[0239] The pharmaceutical compositions and combinations disclosed
herein are administered and dosed in accordance with good medical
practice, taking into account the clinical condition of the
individual subject, the disease to be treated, the site and method
of administration, scheduling of administration, patient age, sex,
body weight and other factors known to medical practitioners.
[0240] In another embodiment, the administration comprises topical
or local administration such as via eardrops or ointment. In a
non-limiting example, dsRNA compounds that target HES1, HES5, HEY2,
CDKN1B, or NOTCH1 are useful in treating a subject suffering from
damage to the ear, wherein the dsRNA compounds are delivered to the
ear via topical delivery (e.g., ear drops or ointments). Nucleic
acid molecules may be complexed with cationic lipids, packaged
within liposomes, or otherwise delivered to target cells or
tissues. The nucleic acid or nucleic acid complexes can be locally
administered to relevant tissues ex vivo, or in vivo through direct
dermal application, transdermal application, or injection, with or
without their incorporation in biopolymers. Preferred
oligonucleotides useful in generating dsRNA molecules are disclosed
herein.
[0241] Delivery systems may include surface-modified liposomes
containing poly (ethylene glycol) lipids (PEG-modified, or
long-circulating liposomes or stealth liposomes). These
formulations offer a method for increasing the accumulation of
drugs in target tissues. This class of drug carriers resists
opsonization and elimination by the mononuclear phagocytic system
(MPS or RES), thereby enabling longer blood circulation times and
enhanced tissue exposure for the encapsulated drug (Lasic et al.
Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull.
1995, 43, 1005-1011).
[0242] Nucleic acid molecules may be formulated or complexed with
polyethylenimine (e.g., linear or branched PEI) and/or
polyethylenimine derivatives, including for example
polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine
(PEI-PEG-GAL) or
polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine
(PEI-PEG-triGAL) derivatives, grafted PEIs such as galactose PEI,
cholesterol PEI, antibody derivatized PEI, and polyethylene glycol
PEI (PEG-PEI) derivatives thereof (see for example Ogris et al.,
2001, AAPA Pharm Sci, 3, 1-11; Furgeson et al., 2003, Bioconjugate
Chem., 14, 840-847; Kunath et al., 2002, Pharmaceutical Research,
19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52;
Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson
et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al.,
1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al.,
1999, PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of
Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of
Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002,
PNAS USA, 99, 14640-14645; Sagara, U.S. Pat. No. 6,586,524 and US
Patent Application Publication No. 20030077829).
[0243] Nucleic acid molecules may be complexed with membrane
disruptive agents such as those described in U.S. Patent
Application Publication No. 20010007666. The membrane disruptive
agent or agents and the nucleic acid molecule may also be complexed
with a cationic lipid or helper lipid molecule, such as those
lipids described in U.S. Pat. No. 6,235,310.
[0244] Delivery systems may include, for example, aqueous and
nonaqueous gels, creams, multiple emulsions, microemulsions,
liposomes, ointments, aqueous and nonaqueous solutions, lotions,
aerosols, hydrocarbon bases and powders, and can contain excipients
such as solubilizers, permeation enhancers (e.g., fatty acids,
fatty acid esters, fatty alcohols and amino acids), and hydrophilic
polymers (e.g., polycarbophil and polyvinylpyrolidone). In one
embodiment, the pharmaceutically acceptable carrier is a liposome
or a transdermal enhancer. Non-limiting examples of liposomes which
can be used with the compounds of this invention include the
following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the
cationic lipid
N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and
dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2)
Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid
and DOPE (Glen Research); (3) DOTAP
(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)
(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome
formulation of the polycationic lipid DOSPA, the neutral lipid DOPE
(GIBCO BRL) and Di-Alkylated Amino Acid (DiLA2).
[0245] Delivery systems may include patches, tablets,
suppositories, pessaries, gels, aqueous and nonaqueous solutions,
lotions and creams, and can contain excipients such as solubilizers
and enhancers (e.g., propylene glycol, bile salts and amino acids),
and other vehicles (e.g., polyethylene glycol, glycerol, fatty acid
esters and derivatives, and hydrophilic polymers such as
hydroxypropylmethylcellulose and hyaluronic acid).
[0246] Nucleic acid molecules may include a bioconjugate, for
example a nucleic acid conjugate as described in Vargeese et al.,
U.S. Ser. No. 10/427,160; U.S. Pat. No. 6,528,631; U.S. Pat. No.
6,335,434; U.S. Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S.
Pat. No. 5,214,136; U.S. Pat. No. 5,138,045.
[0247] Compositions, combinations, methods and kits disclosed
herein may include an expression vector that includes a nucleic
acid sequence encoding at least one nucleic acid molecule disclosed
herein in a manner that allows expression of the nucleic acid
molecule. Methods of introducing nucleic acid molecules or one or
more vectors capable of expressing the strands of dsRNA into the
environment of the cell will depend on the type of cell and the
make up of its environment. The nucleic acid molecule or the vector
construct may be directly introduced into the cell (i.e.,
intracellularly); or introduced extracellularly into a cavity,
interstitial space, into the circulation of an organism, introduced
orally, or may be introduced by bathing an organism or a cell in a
solution containing dsRNA. The cell is preferably a mammalian cell;
more preferably a human cell. The nucleic acid molecule of the
expression vector can include a sense region and an antisense
region. The antisense region can include a sequence complementary
to a RNA or DNA sequence encoding HES1, HES5, HEY2, CDKN1B, or
NOTCH1, and the sense region can include a sequence complementary
to the antisense region. The nucleic acid molecule can include two
distinct strands having complementary sense and antisense regions.
The nucleic acid molecule can include a single strand having
complementary sense and antisense regions.
[0248] Nucleic acid molecules that interact with target RNA
molecules and down-regulate gene encoding target RNA molecules
(e.g., HES1, HES5, HEY2, CDKN1B, or NOTCH1 mRNA, SEQ ID NO:1, 2,
10, 7 or 11) may be expressed from transcription units inserted
into DNA or RNA vectors. Recombinant vectors can be DNA plasmids or
viral vectors. Nucleic acid molecule expressing viral vectors can
be constructed based on, but not limited to, adeno-associated
virus, retrovirus, adenovirus, or alphavirus. The recombinant
vectors capable of expressing the nucleic acid molecules can be
delivered as described herein, and persist in target cells.
Alternatively, viral vectors can be used that provide for transient
expression of nucleic acid molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the nucleic
acid molecules bind and down-regulate gene function or expression,
e.g., via RNA interference (RNAi). Delivery of nucleic acid
molecule expressing vectors can be systemic, such as by intravenous
or intramuscular administration, by local administration, by
administration to target cells ex-planted from a subject followed
by reintroduction into the subject, or by any other means that
would allow for introduction into the desired target cell.
[0249] Expression vectors may include a nucleic acid sequence
encoding at least one nucleic acid molecule disclosed herein, in a
manner which allows expression of the nucleic acid molecule. For
example, the vector may contain sequence(s) encoding both strands
of a nucleic acid molecule that include a duplex. The vector can
also contain sequence(s) encoding a single nucleic acid molecule
that is self-complementary and thus forms a nucleic acid molecule.
Non-limiting examples of such expression vectors are described in
Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and
Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002,
Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature
Medicine, advance online publication doi:10.1038/nm725. Expression
vectors may also be included in a mammalian (e.g., human) cell
[0250] An expression vector may encode one or both strands of a
nucleic acid duplex, or a single self-complementary strand that
self hybridizes into a nucleic acid duplex. The nucleic acid
sequences encoding nucleic acid molecules can be operably linked in
a manner that allows expression of the nucleic acid molecule (see
for example Paul et al., 2002, Nature Biotechnology, 19, 505;
Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et
al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002,
Nature Medicine, advance online publication doi:
10.1038/nm725).
[0251] An expression vector may include one or more of the
following: a) a transcription initiation region (e.g., eukaryotic
pol I, II or III initiation region); b) a transcription termination
region (e.g., eukaryotic pol I, II or III termination region); c)
an intron and d) a nucleic acid sequence encoding at least one of
the nucleic acid molecules, wherein said sequence is operably
linked to the initiation region and the termination region in a
manner that allows expression and/or delivery of the nucleic acid
molecule. The vector can optionally include an open reading frame
(ORF) for a protein operably linked on the 5'-side or the 3'-side
of the sequence encoding the nucleic acid molecule; and/or an
intron (intervening sequences).
[0252] Transcription of the nucleic acid molecule sequences can be
driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA
polymerase II (pol II), or RNA polymerase III (pol III).
Transcripts from pol II or pol III promoters are expressed at high
levels in all cells; the levels of a given pol II promoter in a
given cell type depends on the nature of the gene regulatory
sequences (enhancers, silencers, etc.) present nearby. Prokaryotic
RNA polymerase promoters are also used, providing that the
prokaryotic RNA polymerase enzyme is expressed in the appropriate
cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87,
6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber
et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol.
Cell. Biol., 10, 4529-37). Several investigators have demonstrated
that nucleic acid molecules expressed from such promoters can
function in mammalian cells (e.g. Kashani-Sabet et al., 1992,
Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl.
Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res.,
20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90,
6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et
al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4; Thompson et
al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,
1993, Science, 262, 1566). More specifically, transcription units
such as the ones derived from genes encoding U6 small nuclear
(snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in
generating high concentrations of desired RNA molecules such as
siNA in cells (Thompson et al., supra; Couture and Stinchcomb,
1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830;
Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene
Ther., 4, 45; Beigelman et al., International PCT Publication No.
WO 96/18736). The above nucleic acid transcription units can be
incorporated into a variety of vectors for introduction into
mammalian cells, including but not restricted to, plasmid DNA
vectors, viral DNA vectors (such as adenovirus or adeno-associated
virus vectors), or viral RNA vectors (such as retroviral or
alphavirus vectors) (see Couture and Stinchcomb, 1996 supra).
[0253] Nucleic acid molecule may be expressed within cells from
eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science,
229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA
83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88,
10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15;
Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al.,
1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.
Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,
4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et
al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene
Therapy, 4, 45. Those skilled in the art realize that any nucleic
acid can be expressed in eukaryotic cells from the appropriate
DNA/RNA vector. The activity of such nucleic acids can be augmented
by their release from the primary transcript by a enzymatic nucleic
acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO
94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6;
Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et
al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994,
J. Biol. Chem., 269, 25856.
[0254] A viral construct packaged into a viral particle would
accomplish both efficient introduction of an expression construct
into the cell and transcription of dsRNA construct encoded by the
expression construct.
[0255] Methods for oral introduction include direct mixing of RNA
with food of the organism, as well as engineered approaches in
which a species that is used as food is engineered to express an
RNA, then fed to the organism to be affected. Physical methods may
be employed to introduce a nucleic acid molecule solution into the
cell. Physical methods of introducing nucleic acids include
injection of a solution containing the nucleic acid molecule,
bombardment by particles covered by the nucleic acid molecule,
soaking the cell or organism in a solution of the RNA, or
electroporation of cell membranes in the presence of the nucleic
acid molecule. In one embodiment provided herein is a cell
comprising a nucleic acid molecule disclosed herein.
[0256] Other methods known in the art for introducing nucleic acids
to cells may be used, such as chemical mediated transport, such as
calcium phosphate, and the like. Thus the nucleic acid molecules
may be introduced along with components that perform one or more of
the following activities: enhance RNA uptake by the cell, promote
annealing of the duplex strands, stabilize the annealed strands, or
other-wise increase inhibition/down-regulation of the target
gene.
[0257] Polymeric nanocapsules or microcapsules facilitate transport
and release of the encapsulated or bound dsRNA into the cell. They
include polymeric and monomeric materials, especially including
polybutylcyanoacrylate. A summary of materials and fabrication
methods has been published (see Kreuter, 1991). The polymeric
materials which are formed from monomeric and/or oligomeric
precursors in the polymerization/nanoparticle generation step, are
per se known from the prior art, as are the molecular weights and
molecular weight distribution of the polymeric material which a
person skilled in the field of manufacturing nanoparticles may
suitably select in accordance with the usual skill.
[0258] Nucleic acid molecules may be formulated as a microemulsion.
A microemulsion is a system of water, oil and amphiphile which is a
single optically isotropic and thermodynamically stable liquid
solution. Typically microemulsions are prepared by first dispersing
an oil in an aqueous surfactant solution and then adding a
sufficient amount of a 4th component, generally an intermediate
chain-length alcohol to form a transparent system.
[0259] Surfactants that may be used in the preparation of
microemulsions include, but are not limited to, ionic surfactants,
non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers,
polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310),
tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310),
hexaglycerol pentaoleate (PO500), decaglycerol monocaprate
(MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate
(SO750), decaglycerol decaoleate (DA0750), alone or in combination
with cosurfactants. The cosurfactant, usually a short-chain alcohol
such as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules.
[0260] Delivery formulations can include water soluble degradable
crosslinked polymers that include one or more degradable
crosslinking lipid moiety, one or more PEI moiety, and/or one or
more mPEG (methyl ether derivative of PEG (methoxypoly (ethylene
glycol)).
Dosages
[0261] The useful dosage to be administered and the particular mode
of administration will vary depending upon such factors as the cell
type, or for in vivo use, the age, weight and the particular
subject and region thereof to be treated, the particular nucleic
acid and delivery method used, the therapeutic or diagnostic use
contemplated, and the form of the formulation, for example,
suspension, emulsion, micelle or liposome, as will be readily
apparent to those skilled in the art. Typically, dosage is
administered at lower levels and increased until the desired effect
is achieved.
[0262] The "therapeutically effective dose" for purposes herein is
thus determined by such considerations as are known in the art. The
dose must be effective to achieve improvement including but not
limited to improved survival rate or more rapid recovery, or
improvement or elimination of symptoms and other indicators as are
selected as appropriate measures by those skilled in the art.
[0263] Suitable amounts of inhibitors, e.g. nucleic acid molecules,
may be introduced and these amounts can be empirically determined
using standard methods. Effective concentrations of individual
nucleic acid molecule species in the environment of a cell may be
about 1 femtomolar, about 50 femtomolar, 100 femtomolar, 1
picomolar, 1.5 picomolar, 2.5 picomolar, 5 picomolar, 10 picomolar,
25 picomolar, 50 picomolar, 100 picomolar, 500 picomolar, 1
nanomolar, 2.5 nanomolar, 5 nanomolar, 10 nanomolar, 25 nanomolar,
50 nanomolar, 100 nanomolar, 500 nanomolar, 1 micromolar, 2.5
micromolar, 5 micromolar, 10 micromolar, 100 micromolar or
more.
[0264] In general, the active dose of nucleic acid compound for
humans is in the range of from 1 ng/kg to about 20-100 milligrams
per kilogram (mg/kg) body weight of the recipient per day,
preferably about 0.01 mg to about 2-10 mg/kg body weight of the
recipient per day, in a regimen of a single dose, a one dose per
day or twice or three or more times per day for a period of 1-4
weeks or longer. A suitable dosage unit of nucleic acid molecules
may be in the range of 0.001 to 0.25 milligrams per kilogram body
weight of the recipient per day, or in the range of 0.01 to 20
micrograms per kilogram body weight per day, or in the range of
0.01 to 10 micrograms per kilogram body weight per day, or in the
range of 0.10 to 5 micrograms per kilogram body weight per day, or
in the range of 0.1 to 2.5 micrograms per kilogram body weight per
day. Dosage may be from 0.01 ug to 1 g per kg of body weight (e.g.,
0.1 ug, 0.25 ug, 0.5 ug, 0.75 ug, 1 ug, 2.5 ug, 5 ug, 10 ug, 25 ug,
50 ug, 100 ug, 250 ug, 500 ug, 1 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50
mg, 100 mg, 250 mg, or 500 mg per kg of body weight).
[0265] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
subject per day). The amount of active ingredient that can be
combined with the carrier materials to produce a single dosage form
varies depends upon the host treated and the particular mode of
administration. Dosage unit forms generally contain between from
about 1 mg to about 500 mg of an active ingredient.
[0266] It is understood that the specific dose level for any
particular subject depends upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0267] Pharmaceutical compositions that include the inhibitors,
e.g. nucleic acid molecules, disclosed herein may be administered
once, once daily (QD), twice a day (bid), three times a day (tid),
four times a day (qid), at any interval and for any duration, or by
continuous application for any duration, as is medically
appropriate. The therapeutic agent may also be dosed in dosage
units containing two, three, four, five, six or more sub-doses
administered at appropriate intervals throughout the day. In that
case, the nucleic acid molecules contained in each sub-dose may be
correspondingly smaller in order to achieve the total daily dosage
unit. The dosage unit can also be compounded for a single dose over
several days, e.g., using a conventional sustained release
formulation which provides sustained and consistent release of the
dsRNA over a several day period. Sustained release formulations are
well known in the art. The dosage unit may contain a corresponding
multiple of the daily dose. The composition can be compounded in
such a way that the sum of the multiple units of a nucleic acid
together contain a sufficient dose. The combination of therapeutic
agents may be adminstered prior to, during or after exposure to the
insult (i.e. ototoxin, mechanical injury etc.)
Pharmaceutical Compositions, Kits, and Containers
[0268] Also provided are compositions, combinations, commercial
packages, kits, containers and formulations that include an
inhibitor, for example a nucleic acid molecule (e.g., an siNA
molecule), as provided herein for down-regulating expression of
HES1, HES5, HEY2, CDKN1B or NOTCH1 for administering or
distributing the nucleic acid molecule to a patient. A kit may
include at least one container and at least one label. Suitable
containers include, for example, bottles, vials, syringes, and test
tubes. The containers can be formed from a variety of materials
such as glass, metal or plastic. The container can hold amino acid
sequence(s), small molecule(s), nucleic acid sequence(s), cell
population(s) and/or antibody(s) and/or any other component
required for relevant laboratory, prognostic, diagnostic,
prophylactic and therapeutic purposes. Indications and/or
directions for such uses can be included on or with such container,
as can reagents and other compositions or tools used for these
purposes.
[0269] The container can alternatively hold a composition that is
effective for treating, diagnosis, prognosing or prophylaxing a
condition and can have a sterile access port (for example the
container can be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The active
agents in the composition can be a nucleic acid molecule capable of
specifically binding HES1, HES5, HEY2, CDKN1B or NOTCH1 mRNA and/or
down-regulating the function of HES1, HES5, HEY2, CDKN1B or
NOTCH1.
[0270] A kit may further include a second container that includes a
pharmaceutically-acceptable buffer, such as phosphate-buffered
saline, Ringer's solution and/or dextrose solution. It can further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, stirrers,
needles, syringes, and/or package inserts with indications and/or
instructions for use.
[0271] Federal law requires that the use of pharmaceutical
compositions in the therapy of humans be approved by an agency of
the Federal government. In the United States, enforcement is the
responsibility of the Food and Drug Administration, which issues
appropriate regulations for securing such approval, detailed in 21
U.S.C. .sctn.301-392. Similar approval is required by most foreign
countries. Regulations vary from country to country, but individual
procedures are well known to those in the art and the compositions,
combinations and methods provided herein preferably comply
accordingly.
[0272] The combinations disclosed herein can be used to treat
diseases, conditions or disorders associated with expression of
HES1, HES5, and HEY2, or CDKN1B, NOTCH1 and HEY2 such as disease,
injury, condition or pathology in the ear, vestibular sensory
system, and any other disease or conditions that are related to or
will respond to the levels of expression of HES1, HES5 and HEY2, or
CDKN1B, NOTCH1 and HEY2 in a cell or tissue, alone or in
combination with other therapies. As such, compositions,
combinations, commercial packages, kits and methods disclosed
herein may include packaging a nucleic acid molecule disclosed
herein that includes a label or package insert. The label may
include indications for use of the nucleic acid molecules such as
use for treatment or prevention of diseases, disorders, injuries
and conditions of the ear or vestibular system, including, without
being limited to, Meniere's disease, acoustic trauma, deafness,
hearing loss, presbycusis and any other disease or condition
disclosed herein. The label may include indications for use of the
nucleic acid molecules such as use for treatment or prevention of
attenuation of neuronal degeneration. Neuronal degeneration
includes for example degeneration of the auditory nerve, (also
known as the vestibulocochlear nerve or acoustic nerve and
responsible for transmitting sound and equilibrium information from
the inner ear to the brain); the hair cells of the inner ear that
transmit information to the brain via the auditory nerve, which
consists of the cochlear nerve, and the vestibular nerve, and
emerges from the medulla oblongata and enters the inner skull via
the internal acoustic meatus (or internal auditory meatus) in the
temporal bone, along with the facial nerve. The label may include
indications for use of the nucleic acid molecules such as use for
treatment or prevention of any other disease or conditions that are
related to or will respond to the levels of expression of HES1,
HES5 and HEY2, or expression of CDKN1B, NOTCH1 and HEY2, in a cell
or tissue, alone or in combination with other therapies. A label
may include an indication for use in reducing and/or
down-regulating expression of expression of HES1, HES5 and HEY2, or
CDKN1B, NOTCH1 and HEY2. A "package insert" is used to refer to
instructions customarily included in commercial packages of
therapeutic products, that contain information about the
indications, usage, dosage, administration, contraindications,
other therapeutic products to be combined with the packaged
product, and/or warnings concerning the use of such therapeutic
products, etc.
[0273] Those skilled in the art will recognize that other
treatments, drugs and therapies known in the art can be readily
combined with the nucleic acid molecules herein (e.g. dsNA
molecules) and are hence contemplated herein.
Methods of Treatment
[0274] In another aspect, the present invention relates to a method
for the treatment of a subject in need of treatment for a disease
or disorder associated with the abnormal expression of HES1, HES5,
HEY2, CDKN1B or NOTCH1, comprising administering to the subject an
amount of inhibitors which reduce or inhibit expression of HES1,
HES5 and HEY2 genes, or of inhibitor that reduce or inhibit
expression of CDKN1B, NOTCH1 and HEY2 genes.
[0275] In one embodiment, nucleic acid molecules may be used to
down-regulate or inhibit the expression of HES1, HES5, HEY2,
CDKN1B, or NOTCH1 and/or HES1, HES5, HEY2, CDKN1B, or NOTCH1
proteins arising from HES1, HES5, HEY2, CDKN1B, or NOTCH1 and/or
haplotype polymorphisms that are associated with a disease or
condition, (e.g., neurodegeneration). Analysis of HES1, HES5, HEY2,
CDKN1B, or genes, and/or protein or RNA levels can be used to
identify subjects with such polymorphisms or those subjects who are
at risk of developing traits, conditions, or diseases described
herein. These subjects are amenable to treatment, for example,
treatment with nucleic acid molecules disclosed herein and any
other composition useful in treating diseases related to HES1,
HES5, HEY2, CDKN1B, or NOTCH1 gene expression. As such, analysis of
HES1, HES5, HEY2, CDKN1B, or NOTCH1 gene and/or protein or RNA
levels can be used to determine treatment type and the course of
therapy in treating a subject. Monitoring of protein or RNA levels
can be used to predict treatment outcome and to determine the
efficacy of combinations and compositions that modulate the level
and/or activity of certain genes and/or proteins associated with a
trait, a condition, or a disease.
[0276] Provided herein are methods of inhibiting the expression
target genes selected from the group consisting of a gene
transcribed into mRNA set forth in any one of SEQ ID NOS:1, 2, 10,
7 or 11 by at least 40%, preferably by 50%, 60% or 70%, more
preferably by 75%, 80% or 90% as compared to a control, comprising
contacting an mRNA transcript of the target gene disclosed herein
with a combination or a compositions provided herein.
[0277] In one embodiment the oligoribonucleotide inhibits one or
more of the target genes disclosed herein, whereby the inhibition
is selected from the group comprising inhibition of gene function,
inhibition of polypeptide and inhibition of mRNA expression.
[0278] In one embodiment the compound inhibits the target
polypeptide, whereby the inhibition is selected from the group
comprising inhibition of function (which is examined by, for
example, an enzymatic assay or a binding assay with a known
interactor of the native gene/polypeptide, inter alia), inhibition
of protein (which is examined by, for example, Western blotting,
ELISA or immuno-precipitation, inter alia) and inhibition of mRNA
expression (which is examined by, for example, Northern blotting,
quantitative RT-PCR, in-situ hybridization or microarray
hybridization, inter alia).
[0279] In one embodiment the compound is down-regulating a
mammalian polypeptide, whereby the down-regulation is selected from
the group comprising down-regulation of function (which is examined
by, for example, an enzymatic assay or a binding assay with a known
interactor of the native gene/polypeptide, inter alia),
down-regulation of protein (which is examined by, for example,
Western blotting, ELISA or immuno-precipitation, inter alia) and
down-regulation of mRNA expression (which is examined by, for
example, Northern blotting, quantitative RT-PCR, in-situ
hybridization or microarray hybridization, inter alia).
[0280] In additional embodiments provided herein is a method of
treating a patient suffering from a disease accompanied by an
elevated level of a mammalian gene elected from the group
consisting of a gene transcribed into mRNA set forth in any one of
SEQ ID NOS:1, 2, 10, 7 or 11, the method comprising administering
to the patient a combination or composition as disclosed herein in
a therapeutically effective dose thereby treating the patient.
[0281] Methods, combinations and compositions which inhibit a
mammalian gene or polypeptide as disclosed herein are discussed
herein at length, and any of said molecules and/or compositions are
beneficially employed in the treatment of a patient suffering from
any of said conditions. Novel methods of treatment using known
compounds and compositions fall within the scope of the present
invention.
[0282] In various embodiments, the methods disclosed herein include
administering a therapeutically effective amount of compounds which
down-regulate expression of a hearing loss associated gene. By
"exposure to a toxic agent" is meant that the toxic agent is made
available to, or comes into contact with, a mammal. A toxic agent
can be toxic to the nervous system. Exposure to a toxic agent can
occur by direct administration, e.g., by ingestion or
administration of a food, medicinal, or therapeutic agent, e.g., a
chemotherapeutic agent, by accidental contamination, or by
environmental exposure, e g., aerial or aqueous exposure.
[0283] Further provided is a method of preventing degeneration of
the auditory nerve, also known as the vestibulocochlear nerve or
acoustic nerve, responsible for transmitting sound and equilibrium
information from the inner ear to the brain. The hair cells of the
inner ear transmit information to the brain via the auditory nerve,
which consists of the cochlear nerve, and the vestibular nerve, and
emerges from the medulla oblongata and enters the inner skull via
the internal acoustic meatus (or internal auditory meatus) in the
temporal bone, along with the facial nerve.
[0284] Further provided is a process of preparing a pharmaceutical
composition, which comprises:
providing one or more double stranded molecule disclosed herein;
and admixing said molecule with a pharmaceutically acceptable
carrier.
[0285] In a preferred embodiment, the molecule used in the
preparation of a pharmaceutical composition is admixed with a
carrier in a pharmaceutically effective dose. In a particular
embodiment the compound of the present invention is conjugated to a
steroid or to a lipid or to another suitable molecule e.g. to
cholesterol.
[0286] The nucleic acid molecules disclosed herein are able to
down-regulate the expression of HES1, HES5, HEY2, CDKN1B, or NOTCH1
in a sequence specific manner. The nucleic acid molecules may
include a sense strand and an antisense strand which include
contiguous nucleotides that are at least partially complementary
(antisense) to a portion of HES1, HES5, HEY2, CDKN1B, or NOTCH1
mRNA.
[0287] In some embodiments, dsRNA specific for HES1, HES5, HEY2,
CDKN1B or NOTCH1 can be used in conjunction with other therapeutic
agents and/or dsRNA specific for other molecular targets, such as,
without being limited to various proapoptotic genes.
[0288] A method for treating or preventing HES1, HES5, HEY2, CDKN1B
or NOTCH1 associated disease or condition in a subject or organism
may include contacting the subject or organism with a combination
or a composition as provided herein under conditions suitable to
down-regulate the expression of the gene in the subject or
organism.
[0289] A method for treating or preventing an ear disorder in a
subject or organism may include contacting the subject or organism
with a combination or a composition as provided herein under
conditions suitable to down-regulate the expression of the HES1,
HES5, HEY2, CDKN1B or NOTCH1 gene in the subject or organism.
[0290] In preferred embodiments the subject being treated is a
warm-blooded animal and, in particular, mammals including
human.
[0291] The methods disclosed herein comprise administering to the
subject a combination or a composition of inhibitory compounds
which down-regulate expression of HES1, HES5 and HEY2 or CDKN1B,
NOTCH1 and HEY2, in a therapeutically effective dose so as to
thereby treat the subject.
[0292] Methods, combinations and compositions which down-regulate
HES1, HES5, HEY2, CDKN1B or NOTCH1, in particular combination of a
HES1 inhibitor or a pharmaceutically acceptable salt or prodrug
thereof, a HES5 inhibitor or a pharmaceutically acceptable salt or
prodrug thereof and a HEY2 inhibitor or a pharmaceutically
acceptable salt or prodrug thereof, or combination of a CDKN1B
inhibitor or a pharmaceutically acceptable salt or prodrug thereof,
a NOTCH1 inhibitor or a pharmaceutically acceptable salt or prodrug
thereof and a HEY2 inhibitor or a pharmaceutically acceptable salt
or prodrug thereof, are discussed herein at length, and any of said
combinations and/or compositions may be beneficially employed in
the treatment of a subject suffering from any of said conditions.
Sense strand and antisense strand oligonucleotide sequences useful
in generating dsRNA are set forth herein. Preferred oligonucleotide
sequences useful in the preparation of dsRNA that down-regulate
expression of HES1 are set forth in SEQ ID NOS:26667-26690 and
26691-26706; of HES5 are set forth in SEQ ID NOS:26707-26724 and
26725-26732; of HEY2 are set forth in SEQ ID NOS:26779-26784 and
26785-26788; of CDKN1B are set forth in SEQ ID NOS:26867-26886 and
26887-26900 or NOTCH1 are set forth in SEQ ID NOS:26901-26910 and
26911-26912.
[0293] The methods disclosed herein comprise administering to the
subject a composition or a combination as disclosed herein, of
inhibitory compounds which down-regulate expression of the genes
set forth in SEQ ID NOS: 1, 2, 10, 7 or 11; and in particular
compositions or combinations of oligonucleotide compounds in a
therapeutically effective dose so as to thereby treat the
subject.
[0294] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent a disorder or reduce the symptoms of a disorder, such as
hearing disorder or impairment (or balance impairment), to prevent
or reduce cell death associated with a hearing loss-associated
disease as listed herein, to promote regeneration of otic (sensory)
cells or to promote differentiation of supporting cells in the
inner ear into otic (sensory) cells. Those in need of treatment
include those already experiencing the disease or condition, those
prone to having the disease or condition, and those in which the
disease or condition is to be prevented. The compositions or
combinations disclosed herein are administered before, during or
subsequent to the onset of the disease or condition.
[0295] Without being bound by theory, the hearing impairment may be
due to apoptotic inner ear hair cell damage or loss, wherein the
damage or loss is caused by infection, mechanical injury, loud
sound, aging, or, in particular, chemical-induced ototoxicity.
Ototoxins include therapeutic drugs including antineoplastic
agents, salicylates, quinines, and aminoglycoside antibiotics,
contaminants in foods or medicinals, and environmental or
industrial pollutants. Typically, treatment is performed to prevent
or reduce ototoxicity, especially resulting from or expected to
result from administration of therapeutic drugs. Preferably a
therapeutically effective composition is given immediately after
the exposure to prevent or reduce the ototoxic effect. More
preferably, treatment is provided prophylactically, either by
administration of the composition prior to or concomitantly with
the ototoxic pharmaceutical or the exposure to the ototoxin.
[0296] Hearing impairments relevant to the present disclosure may
be due to end-organ lesions involving inner ear hair cells, e.g.,
acoustic trauma, viral endolymphatic labyrinthitis, Meniere's
disease. Hearing impairments include tinnitus, which is a
perception of sound in the absence of an acoustic stimulus, and may
be intermittent or continuous, wherein there is diagnosed a
sensorineural loss. Hearing loss may be due to bacterial or viral
infection, such as in herpes zoster oticus, purulent labyrinthitis
arising from acute otitis media, purulent meningitis, Chronic
otitis media, sudden deafness including that of viral origin, e.g.,
viral endolymphatic labyrinthitis caused by viruses including
mumps, measles, influenza, chicken pox, mononucleosis and
adenoviruses. The hearing loss can be congenital, such as that
caused by rubella, anoxia during birth, bleeding into the inner ear
due to trauma during delivery, ototoxic drugs administered to the
mother, erythroblastosis fetalis, and hereditary conditions
including Waardenburg's syndrome and Hurler's syndrome.
[0297] The hearing loss can be noise-induced, generally due to a
noise greater than 85 decibels (db) that damages the inner ear. In
a particular aspect, the hearing loss is caused by an ototoxic drug
that effects the auditory portion of the inner ear, particularly
inner ear hair cells. Incorporated herein by reference are chapters
196, 197, 198 and 199 of The Merck Manual of Diagnosis and Therapy,
14th Edition, (1982), Merck Sharp & Dome Research Laboratories,
N.J. and corresponding chapters in the most recent 16th edition,
including Chapters 207 and 210) relating to description and
diagnosis of hearing and balance impairments.
[0298] In one embodiment, provided is a method for treating a
mammal having or prone to a hearing (or balance) impairment or
treating a mammal prophylactically in conditions where inhibition
of the genes of the invention is beneficial. The method would
prevent or reduce the occurrence or severity of a hearing (or
balance) impairment that would result from inner ear cell injury,
loss, or degeneration, in particular caused by an ototoxic agent.
In some embodiments the method includes administering a
therapeutically effective amount of a HES1 inhibitor, a HES5
inhibitor and a HEY2 inhibitor. In other embodiments the method
includes administering a therapeutically effective amount of a
CDKN1B inhibitor, a NOTCH1 inhibitor and a HEY2 inhibitor.
[0299] It is the object of the present disclosure to provide
methods, combinations and compositions for treating a mammal, to
prevent, reduce, or treat a hearing impairment, disorder or
imbalance, optionally an ototoxin-induced hearing condition, by
administering to a mammal in need of such treatment a composition
or a combination as disclosed herein. In some embodiments the
methods are for treating a hearing disorder or impairment wherein
the ototoxicity results from administration of a therapeutically
effective amount of an ototoxic pharmaceutical drug. Typical
ototoxic drugs are chemotherapeutic agents, e.g. antineoplastic
agents, and antibiotics. Other possible candidates include
loop-diuretics, quinines or a quinine-like compound, and salicylate
or salicylate-like compounds.
[0300] In some embodiments, the combinations and compositions
provided herein are co-administered with an ototoxin. For example,
an improved method is provided for treatment of infection of a
mammal by administration of an aminoglycoside antibiotic, the
improvement comprising administering a therapeutically effective
amount of a composition or a combination of inhibitors
(particularly dsRNAs) which down-regulate expression of HES1, HES5
and HEY2, or which down-regulate expression of CDKN1B, NOTCH1 and
HEY2 to the patient in need of such treatment to reduce or prevent
ototoxin-induced hearing impairment associated with the antibiotic.
The compounds which down-regulate expression of HES1, HES5, HEY2,
CDKN1B or NOTCH1, e.g. dsRNAs are preferably administered locally
within the inner ear.
[0301] In yet another embodiment an improved method for treatment
of cancer in a mammal by administration of a chemotherapeutic
compound is provided, wherein the improvement comprises
administering a therapeutically effective amount of a composition
or combination as disclosed herein to the patient in need of such
treatment to reduce or prevent ototoxin-induced hearing impairment
associated with the chemotherapeutic drug. The compositions or
combinations which reduce or prevent the ototoxin-induced hearing
impairment, e.g. compositions and combinations comprising dsRNA
molecules as disclosed herein, inter alia are preferably
administered directly to the cochlea as naked dsRNAs in a vehicle
such as PBS or other physiological solutions, but may alternatively
be administered with a delivery vehicle as described above.
[0302] In another embodiment the methods of treatment are applied
to treatment of hearing impairment resulting from the
administration of a chemotherapeutic agent in order to treat its
ototoxic side-effect.
[0303] In another embodiment the methods, compositions and
combinations are applied to hearing impairments resulting from the
administration of quinine and its synthetic substitutes, typically
used in the treatment of malaria, to treat its ototoxic
side-effect.
[0304] In some embodiments of combinations provided herein,
combination therapy is achieved by administering two or three
inhibitors (i.e. dsRNAs) each of which is formulated and
administered separately, or by administering the inhibitors in a
single formulation. Other combinations are also encompassed by
combination therapy. For example, two inhibitors can be formulated
together and administered in conjunction with a separate
formulation containing a third inhibitor. While the two or more
inhibitors in the combination therapy can be administered
simultaneously, they need not be. For example, administration of a
first inhibitor (or combination of inhibitors) can precede
administration of a second inhibitor (or combination of inhibitors)
by minutes, hours, days, or weeks. Thus, the two or more inhibitors
can be administered within minutes of each other or within one or
several hours of each other or within one or several days of each
other or within several weeks of each other. In some cases even
longer intervals are possible. The two or more inhibitors used in
combination therapy may or may not be present within the patient's
body at the same time. Combination therapy includes two or more
administrations of one or more of the inhibitors used in the
combination. For example, if dsRNA1 and dsRNA2 (i.e. wherein dsRNA1
targets gene 1 and dsRNA2 targets gene 2) are used in a
combination, one could administer them sequentially in any
combination one or more times, e.g., in the order dsRNA1-dsRNA2,
dsRNA2-dsRNA1, dsRNA1-dsRNA2-dsRNA1, dsRNA2-dsRNA1-dsRNA2,
dsRNA1-dsRNA1-dsRNA2, dsRNA1-dsRNA2-dsRNA2 etc.
[0305] The combinations as disclosed herein can be administered in
a form of a single pharmaceutical formulation, optionally together
with a pharmaceutically acceptable diluent or carrier. The
individual components of such a combination referred to as
inhibitors, can be administered either simultaneously,
concurrently, separately or sequentially, from the same or separate
pharmaceutical formulations.
[0306] In some embodiments, each inhibitor is administered by the
same route, either from the same or from different pharmaceutical
compositions. In other embodiments, using the same route of
administration for the first inhibitor, the second inhibitor and
the third inhibitor either is impossible or is not preferred.
Persons skilled in the art are aware of the best modes of
administration for each inhibitor, either alone or in a
combination.
[0307] As used herein, the term "substantially simultaneously" with
regard to administration of at least two inhibitors means that a
second inhibitor is administered within a time period of no greater
than about 5 minutes after administration of the first inhibitor,
preferably within a time period of about 1 minute, more preferably
within a time period of about 30 seconds, and most preferably, is
administered simultaneously with the first inhibitor from the same
or separate pharmaceutical formulations. Similarly, with regard to
administration of three inhibitors, a third inhibitor may be
administered within a time period of no greater than about 5
minutes after administration of the second inhibitor, preferably
within a time period of about 1 minute, more preferably within a
time period of about 30 seconds, and most preferably is
administered simultaneously with the second inhibitor from the same
or separate pharmaceutical formulations.
Hearing Regeneration
[0308] Sensory progenitor cells can develop as either hair cells or
supporting cells. Ablation studies indicate that removal of a hair
cell changes the fate of a surrounding cell from a supporting to a
hair cell. This response suggests that hair cells generate
inhibitory signals that prevent neighboring cells from developing
as hair cells. This type of interaction is consistent with the
effects of Notch-mediated lateral inhibition. Consistent with this
hypothesis, two Notch ligands, Jag2 and delta 1 (Dll1) are rapidly
upregulated in a subset of Atoh1-positive cells. The expression of
these ligands leads to activation of Notch 1 and the increased
transcription of two Notch pathway target genes, HES1 and HES5 in
cells that will develop as supporting cells. Deletion of any of the
genes in this pathway leads to an overproduction of hair cells,
suggesting that Notch signalling has a role in diverting progenitor
cells from the hair cell fate. The mechanism of this diversion has
been examined using cells in Kolliker's organ. First,
co-transfection of Kolliker's organ cells with Atoh1 and HES1 was
sufficient to inhibit hair cell formation, suggesting that Atoh1
transcription is a target of HES1 in the ear. Second, transient
activation of ATOH1 in patches of Kolliker's organ cells leads to
activation of the Notch signalling pathway within those cells and
to the inhibition of ATOH1 and hair cell fate in a subset of those
cells.
[0309] Zine et al. (J Neurosci. 2001 21(13):4712-20) demonstrate
that HES1 and HES5 activities are important for repressing the
commitment of progenitor cells to IHCs and OHCs fates,
respectively, likely by antagonizing Math1. This negative
regulation is critical for the correct number of hair cells to be
produced and for the establishment of the normal cochlear mosaic of
a single row of IHCs and three rows of OHCs. In the vestibular
system, HES1 and HES5 also act as negative regulators of hair cell
differentiation within the utricle and saccule epithelia. It is
possible that simultaneous down-regulation of both of HES1 and HES5
in the cochlea might be used to stimulate the replacement of lost
auditory hair cells. Such studies may have a significant
therapeutic value, because loss of auditory hair cells through
disease, trauma, and aging is a common cause of hearing loss and/or
deafness.
[0310] Details of certain indications in which the compounds
disclosed herein are useful as therapeutics are described
herein.
[0311] The aspects and embodiments provided herein have been
described in an illustrative manner, and it is to be understood
that the terminology used is intended to be in the nature of words
of description rather than of limitation.
[0312] Many modifications and variations are possible in light of
the above teachings. It is, therefore, to be understood that within
the scope of the appended claims, the invention can be practiced
otherwise than as specifically described.
[0313] Throughout this application, various publications, including
United States patents, are referenced by author and year and
patents by number. The disclosures of these publications and
patents and patent applications in their entireties are hereby
incorporated by reference into this application in order to more
fully describe the state of the art to which this invention
pertains.
[0314] The present disclosure is illustrated in detail below with
reference to examples, but is not to be construed as being limited
thereto.
[0315] Citation of any document herein is not intended as an
admission that such document is pertinent prior art, or considered
material to the patentability of any claim of the present
disclosure. Any statement as to content or a date of any document
is based on the information available to applicant at the time of
filing and does not constitute an admission as to the correctness
of such a statement.
EXAMPLES
[0316] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the claimed invention in any
way.
[0317] Standard molecular biology protocols known in the art not
specifically described herein are generally followed essentially as
in Sambrook et al., Molecular cloning: A laboratory manual, Cold
Springs Harbor Laboratory, New-York (1989, 1992), and in Ausubel et
al., Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md. (1988), and as in Ausubel et al., Current Protocols
in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989)
and as in Perbal, A Practical Guide to Molecular Cloning, John
Wiley & Sons, New York (1988), and as in Watson et al.,
Recombinant DNA, Scientific American Books, New York and in Birren
et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4
Cold Spring Harbor Laboratory Press, New York (1998) and
methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202;
4,801,531; 5,192,659 and 5,272,057 and incorporated herein by
reference. Polymerase chain reaction (PCR) was carried out as in
standard PCR Protocols: A Guide To Methods And Applications,
Academic Press, San Diego, Calif. (1990). In situ PCR in
combination with Flow Cytometry (FACS) can be used for detection of
cells containing specific DNA and mRNA sequences (Testoni et al.,
Blood 1996, 87:3822.) Methods of performing RT-PCR are well known
in the art.
Example 1
In Vitro Testing of dsRNA Molecules
[0318] About 1.5-2.times.10.sup.5 tested cells (HeLa cells and/or
293T cells for siRNA targeting human genes and NRK52 (normal rat
kidney proximal tubule cells) cells and/or NMuMG cells (mouse
mammary epithelial cell line) for siRNA targeting the rat/mouse
gene) were seeded per well in 6 wells plate (70-80% confluent).
[0319] 24 hours later, cells were transfected with dsRNA molecules
using the Lipofectamine.TM. 2000 reagent (Invitrogen) at final
concentrations of 5 nM or 20 nM. The cells were incubated at
37.degree. C. in a CO.sub.2 incubator for 72 h.
[0320] As positive control for transfection, PTEN-Cy3 labeled dsRNA
molecules were used. GFP dsRNA molecules were used as negative
control for siRNA activity. At 72 h after transfection, cells were
harvested and RNA was extracted from cells. Transfection efficiency
was tested by fluorescent microscopy. The percent of inhibition of
gene expression using specific preferred siRNA structures was
determined using qPCR analysis of a target gene in cells expressing
the endogenous gene.
Body Fluid/Cell Stability Assay
[0321] The modified compounds disclosed herein are tested for
duplex stability in human, rat or mouse plasma or human, rat or
mouse serum (to test in model system), or CSF (cerebrospinal fluid;
human, mouse or rat) or human cell extract, as follows:
[0322] For example: dsRNA molecules at final concentration of 7 uM
are incubated at 37.degree. C. in 100% human serum (Sigma Cat#
H4522). (siRNA stock 100 uM diluted in human serum 1:14.29 or human
tissue extract from various tissue types.). Five ul (5 ul) are
added to 15 ul 1.5.times.TBE-loading buffer at different time
points (for example 0, 30 min, 1 h, 3 h, 6 h, 8 h, 10 h, 16 h and
24 h). Samples are immediately frozen in liquid nitrogen and are
kept at -20.degree. C.
[0323] Each sample is loaded onto a non-denaturing 20% acrylamide
gel, prepared according to methods known in the art. The oligos are
visualized with ethidium bromide under UV light.
Exonuclease Stability Assay
[0324] To study the stabilization effect of 3' non-nucleotide
moieties on a nucleic acid molecule the sense strand, the antisense
strand and the annealed dsRNA duplex are incubated in cytosolic
extracts prepared from different cell types.
[0325] Extract: HCT116 cytosolic extract (12 mg/ml).
[0326] Extract buffer: 25 mM Hepes pH-7.3 at 37.degree. C.; 8 mM
MgCl.sub.2; 150 mM NaCl with 1 mM DTT was added fresh immediately
before use.
[0327] Method: 3.5 ml of test dsRNA (100 mM), were mixed with 46.5
ml contain 120 mg of HCT116 cytosolic extract. The 46.5 ml consists
of 12 ml of HCT116 extract, and 34.5 ml of the extract buffer
supplemented with DTT and protease inhibitors cocktail/100
(Calbiochem, setIII-539134). The final concentration of the siRNA
in the incubation tube is 7 mM. The sample is incubated at
37.degree. C., and at the indicated time point 5 ml are moved to
fresh tube, mixed with 15 ml of 1XTBE-50% Glycerol loading buffer,
and snap frozen in Liquid N.sub.2. The final concentration of the
siRNA in the loading buffer is 1.75 mM (21 ng siRNA/ml). For
analyses by native PAGE and EtBr staining 50 ng are loaded per
lane. For Northern analyses 1 ng of tested siRNA are loaded per
lane.
Innate Immune Response to dsRNA Molecules:
[0328] Fresh human blood (at RT) is mixed at 1:1 ratio with sterile
0.9% NaCl at RT, and gently loaded (1:2 ratio) on Ficoll
(Lymphoprep, Axis-Shield cat#1114547). Samples are centrifuged at
RT (22.degree. C., 800 g) in a swinging centrifuge for 30 minutes,
washed with RPMI1640 medium and centrifuged (RT, 250 g) for 10
minutes. Cells are counted and seeded at final concentration of
1.5.times.10.sup.6 cell/ml in growth medium (RPMI1640+10% FBS+2 mM
L-glutamine+1% Pen-Strep) and incubated for 1 hour at 37.degree. C.
before dsRNA treatment. Cells are exposed to the test dsRNAs at
different concentrations using the Lipofectamine.TM. 2000 reagent
(Invitrogen) according to manufacturer's instructions and incubated
at 37.degree. C. in a 5% CO.sub.2 incubator for 24 hours.
[0329] As a positive control for IFN response, cells are treated
with either poly(I:C), a synthetic analog of double strand RNA
(dsRNA) which is a TLR3 ligand (InvivoGen Cat# tlrl-pic) at final
concentrations of 0.25-5.0 .mu.g/mL or to Thiazolaquinolone
(CLO75), a TLR 7/8 ligand (InvivoGen Cat# tlrl-c75) at final
concentrations of 0.075-2 .mu.g/mL. Cell treated with
Lipofectamine.TM. 2000 reagent are used as negative (reference)
control for IFN response.
[0330] At about 24 hours following incubation, cells are collected
and supernatant is transferred to new tubes. Samples are frozen
immediately in liquid nitrogen and secretion of IL-6 and
TNF-.alpha. cytokines was tested using IL-6, DuoSet ELISA kit
(R&D System DY2060), and TNF-.alpha., DuoSet ELISA kit (R&D
System DY210), according to manufacturer's instructions. RNA is
extracted from the cell pellets and mRNA levels of human genes
IFIT1 (interferon-induced protein with tetratricopeptide repeats 1)
and MX1 (myxovirus (influenza virus) resistance 1,
interferon-inducible protein p78) were measured by qPCR. Measured
mRNA quantities are normalized to the mRNA quantity of the
reference gene peptidylprolyl isomerase A (cyclophilin A; CycloA).
Induction of IFN-signaling is evaluated by comparing the quantity
of mRNA from IFIT1 and MX1 genes from treated cells, relative to
their quantities non-treated cells. The qPCR results are those that
passed QC standards, i.e. the value of the standard curve slope was
in the interval [-4, -3], R2>0.99, no primer dimers. Results
that do not pass the QC requirements are disqualified from
analysis.
[0331] In general, the dsRNAs having specific sequences that were
selected for in vitro testing were specific for human and a second
species such as rat or rabbit genes. The dsRNA were tested for
activity to Human (Hu), mouse (Ms), rat (Rt), chinchilla (Chn) and
or guinea-pig (GP) target gene. For example, activity in chinchilla
was tested by cloning the chinchilla target gene (i.e. CDKN1B) and
expressing in a 293 or HeLa cell line. Similar results are obtained
using siRNAs having these RNA sequences and modified as described
herein.
[0332] PCT/US12/49616 discloses chemically modified dsRNA nucleic
acid molecules, and is incorporated herein by reference in its
entirety.
Example 2
Generation of Sequences for Active dsRNA Molecules to the Target
Genes and Production of the siRNAs
[0333] Using proprietary algorithms and the known sequence of the
mRNA of the target genes disclosed herein, the sequences of many
potential dsRNA, i.e. siRNAs were generated.
[0334] Specifically, SEQ ID NOS:23-381 provide human 19 mer
oligonucleotides; SEQ ID NOS:382-693 provide best 19-mer
human-cross species oligonucleotides; SEQ ID NOS:694-1367 provide
human 18 mer oligonucleotides; and SEQ ID NO:16-1495 provide best
18-mer human-cross species oligonucleotides useful in generating
dsRNA to down-regulate HES1 expression; Table I includes certain
preferred 19 mer oligonucleotides based on Structure A1, set forth
in SEQ ID NOS: 26,667-26,690 and based on Structure A2, set forth
in SEQ ID NOS:26,691-26,706 useful in generating dsRNA to
down-regulate HES1 expression.
[0335] SEQ ID NOS:1496-1759 provide human 19 mer oligonucleotides;
SEQ ID NOS:1760-2029 provide best 19-mer human-cross species
oligonucleotides; SEQ ID NOS:2030-2575 provide human 18 mer
oligonucleotides; and SEQ ID NOS:2576-2703 provide best 18-mer
human-cross species oligonucleotides useful in generating dsRNA to
down-regulate HES5 expression; Table II includes certain preferred
19 mer oligonucleotides based on Structure A1, set forth in SEQ ID
NOS: 26,707-26,724 and based on Structure A2, set forth in SEQ ID
NOS:26,725-26,732 useful in generating dsRNA to down-regulate HES5
expression.
[0336] SEQ ID NOS:13004-14077 provide human 19 mer
oligonucleotides; SEQ ID NOS:14078-14801 provide best 19-mer
human-cross species oligonucleotides; SEQ ID NOS:14802-16389
provide best human 18 mer oligonucleotides; and SEQ ID
NOS:16390-16621 provide best 18-mer human-cross species
oligonucleotides useful in generating dsRNA to down-regulate HEY2
expression; Table III includes certain preferred 19 mer
oligonucleotides based on Structure A1, set forth in SEQ ID NOS:
26,779-26,784 and based on Structure A2, set forth in SEQ ID
NOS:26,785-26,788 useful in generating dsRNA to down-regulate HEY2
expression.
[0337] SEQ ID NOS:7444-8185 provide human 19 mer oligonucleotides;
SEQ ID NOS:8186-9007 provide best human-cross species
oligonucleotides; SEQ ID NOS:9008-10233 provide human 18 mer
oligonucleotides; and SEQ ID NOS:10234-10533 provide best 18-mer
human-cross species oligonucleotides useful in generating dsRNA to
down-regulate CDKN1B expression; Table IV includes certain
preferred 19 mer oligonucleotides based on Structure A1, set forth
in SEQ ID NOS:26,867-26,886 and based on Structure A2, set forth in
SEQ ID NOS:26,887-26,900 useful in generating dsRNA to
down-regulate CDKN1B expression.
[0338] SEQ ID NOS:16622-18469 provide human 19 mer
oligonucleotides; SEQ ID NOS:18470-18643 provide best human-cross
species oligonucleotides; SEQ ID NOS:18644-26211 provide human 18
mer oligonucleotides; and SEQ ID NOS:26212-26666 provide best
18-mer human-cross species oligonucleotides useful in generating
dsRNA to down-regulate NOTCH1 expression; Table V includes certain
preferred 19 mer oligonucleotides based on Structure A1, set forth
in SEQ ID NOS:26,901-26,910 and based on Structure A2, set forth in
SEQ ID NOS: 26,911-26,912 useful in generating dsRNA to
down-regulate NOTCH1 expression.
[0339] The oligonucleotide sequences prioritized based on their
score in the proprietary algorithm as the best predicted sequences
for targeting the human gene expression.
[0340] "18+1" refers to a molecule that is 19 nucleotides in length
and includes a mismatch to the mRNA target at position 1 of the
antisense strand, according to Structure A2. In preferred
embodiments the sense strand is fully complementary to the
antisense strand. In some embodiments the sense strand is
mismatched to the antisense strand in 1, 2, or 3 positions.
Example 3
On-Target and Off-Target Testing of Double Stranded RNA
Molecules
[0341] The psiCHECK.TM. system enables evaluation of the guide
strand (GS) (antisense) and the passenger strand (PS) (sense
strand) to elicit targeted (on-target) and off-targeted effects, by
monitoring the changes in expression levels of their target
sequences. Four psiCHECK.TM.-2-based (Promega) constructs were
prepared for the evaluation of target activity and potential
off-target activity of each test molecule GS and PS strands. In
each of the constructs one copy or three copies of either the full
target or the seed-target sequence, of test molecule PS or GS, was
cloned into the multiple cloning site located downstream of the
Renilla luciferase translational stop codon in the 3'-UTR
region.
Example 4
The Effect of Combination Treatment on Carboplatin-Induced Hair
Cell Death in the Cochlea of Chinchilla
[0342] Eight Chinchillas are pre-treated by direct administration
of a composition or a combination of HES1 dsRNA, HES5 dsRNA and
HEY2 dsRNA, in saline or by a composition or a combination of
CDKN1B dsRNA, NOTCH1 dsRNA and HEY2 dsRNA in saline (to the left
ear of each animal. Saline is administered to the right ear of each
animal as placebo. Two days following the administration of the
composition or the combination, the animals are treated with
carboplatin (75 mg/kg iP). After sacrifice of the chinchillas (two
weeks post carboplatin treatment) the % of dead cells of inner hair
cells (IRC) and outer hair cells (ONC) is calculated in the left
ear (composition/combination treated) and in the right ear (saline
treated). Since the effect of the siRNA is similar across dose, the
data is pooled from the 3 doses. As was previously shown,
carboplatin preferentially damages the inner hair cells in the
chinchilla at the 75 mg/kg dose while the outer hair cells remain
intact. The compositions/combinations provided herein reduce
ototoxin-induced (e.g. carboplatin-induced) inner hair cells loss
in the cochlea.
Example 5
The Effect of Combination Treatment on Acoustic-Induced Hair Cell
Death in the Cochlea of Chinchilla
[0343] The activity of the compositions/combinations of the present
invention in an acoustic trauma model is studied in chinchilla. A
group of 7 animals undergo acoustic trauma by exposing them to an
octave band of noise centered at 4 kHz for 2.5 h at 105 dB. The
left ear of the noise-exposed chinchillas is pre-treated (48 h
before the acoustic trauma) with a composition/combination of
dsRNAs as disclosed herein, in saline; the right ear is pre-treated
with vehicle (saline). The compound action potential (CAP) is a
convenient and reliable electrophysiological method for measuring
the neural activity transmitted from the cochlea. The CAP is
recorded by placing an electrode near the base of the cochlea in
order to detect the local field potential that is generated when a
sound stimulus, such as click or tone burst, is abruptly turned on.
The functional status of each ear is assessed at about 2.5 weeks
after the acoustic trauma. Specifically, the mean threshold of the
compound action potential recorded from the round window is
determined 2.5 weeks after the acoustic trauma in order to
determine if the thresholds in the composition/combination treated
ear were lower (better) than the untreated (saline) ear. In
addition, the amount of inner and outer hair cell loss is
determined in the composition/combination treated and the control
ear. The results indicate that the compositions/combinations
provided herein, reduce acoustic trauma-induced ONC loss in the
cochlea.
Example 6
The Effect of Combination Treatment on Cisplatin-Induced Hair Cell
Death in the Cochlea of Rats
[0344] Male Wistar Rats are tested for basal auditory brainstem
response (ABR) thresholds for signals of clicks, 8, 16 and 32 kHz
prior to cisplatin treatment. Following the basal auditory
brainstem response testing, cisplatin is administered as an
intraperitoneal infusion of 12 mg/kg over 30 minutes. Treated ears
receive the dsRNA compositions/combinations disclosed herein in PBS
(applied directly to the round window membrane). Control ears are
treated either with non-related GFP dsRNA or PBS. The
compositions/combinations are administered between 3-5 days prior
to cisplatin administration in order to permit protective effect on
the cochlea.
[0345] The auditory brainstem response (ABR) testing is repeated 3
days after cisplatin administration. The auditory brainstem
response thresholds are compared between pretreatment and post
treatment and the shift in thresholds is measured. Higher shift in
thresholds following cisplatin treatment is indicative for more
severe hair cells loss in the cochlea. After the repeat of auditory
brainstem response testing, animals are sacrificed and cochleae are
removed and processed for scanning electron microscopy (SEM) to
quantify outer hair cell (ONC) loss in the hook region (high
frequency region). The % outer hair cell loss is calculated by
dividing the number of missing or severely damaged cells by the
total number of outer hair cells in the field of the photograph.
The results indicate that compositions/combinations disclosed
herein provide a protective effect to the cochlea when administered
prior to ototoxin (e.g. cisplatin) administration.
Example 7
Additional Hearing Loss Models
A) Hearing Regeneration (Plasticity) Model in Guinea-Pig
[0346] Deafness is induced by systemically treating albino guinea
pigs with a single is injection of kanamycin (450-500 mg/kg)
followed by a single iv (jugular) injection of ethacrynic acid
(EA). This pharmacological deafening eliminates bilaterally all
hair cells approximately after 1-2 days and leaves the supporting
cells differentiated. Therapeutic composition/combination as
disclosed herein are applied to the middle ear by transtympanic
injection (TT) or into the external auditory canal or eardrum by
ear drops (ErD).
[0347] The efficacy of the compositions/combinations is examined as
follows:
[0348] 1) Cochleae/s are morphologically analyzed as whole-mounts
stained for myosin VIIa (hair cell marker) and phalloidin.
[0349] 2) BrdU incorporation is measured as an indicator of
proliferation rate of hair cells.
B) Noise Induced Acute Hearing Loss Model in Guinea Pig
[0350] Noise can cause hearing damage with temporary or permanent
sensorineural hearing loss (SNHL) and tinnitus. SNHL and tinnitus
can occur singular or in combination. In humans, noise induced
hearing loss (NIHL) is demonstrated by a threshold shift in the
pure tone audiogram, in recruitment, in pathological results of
supra-threshold hearing tests and in amplitude decline of
oto-acoustic emissions. Hearing damage is induced by exposure to
continuous noise or impulsive noise. In addition the possibility of
impulse noise traumata or explosion trauma should be taken into
consideration. Exposure to impulse noise can result in a more
severe lesion of the inner ear than exposure to continuous noise.
Important criteria for the development of noise damage are sound
pressure level (SPL), level increase velocity, exposure time, as
well as individual susceptibility ("the vulnerable inner ear").
Noise exposure usually leads to an elevation of threshold which may
be later resolved in part, such that the temporary component is
called "temporary threshold shift" (TTS). If there isn't complete
restitution in the recovery phase after TTS, this may result in
permanent inner ear damage (permanent threshold shift=PTS). Very
high sound intensity may lead to immediate cellular death and
mechanical rupture of structures in the inner ear and PTS.
[0351] In this model, a bilateral lesion is induced with noise
exposure; Guinea pigs are exposed to 117 dB SPL broadband noises
for 6 hours.
[0352] In a pilot study according to this model, the
compositions/combinations as disclosed herein are employed in this
model with beneficial result.
Example 8
The Effect of Combination Treatment on Noise-Induced Death of Otic
Sensory Cells of the Inner Ear
[0353] Model system: Exposure of guinea pigs to one-octave-band
noise centered at 6 kHz, at 130 dB SPL for 2 hours (Futon et al,
NeuroReport 19:277-281, 2008)
[0354] Experimental Groups
[0355] Adult Hartley albino guinea pigs (age 3 months), with normal
Preyer's reflex, are exposed to noise and randomized to the
following groups: noise control group without treatment (n=6),
noise control animals with vehicle (n=6), animals treated with the
composition/combination provided herein at a dose of 1 .mu.g (n=6);
animals treated with the compositions/combinations provided herein
at a dose of 5 .mu.g (n=6); animals treated with the
compositions/combinations provided herein at a dose of 10 .mu.g
(n=6); animals treated with the compositions/combinations provided
herein at a dose of 50 .mu.g (n=6); 4 groups of noise control
animals with control dsRNA compound which down-regulates expression
of EGFP gene (each n=6), at a dose of 1 .mu.g, 5 .mu.g, 10 .mu.g
and 50 .mu.g respectively.
[0356] Treatment is performed 1 h before noise exposure and once
daily for 3 days thereafter.
[0357] The following exemplary vehicles are used in this
experiment: PBS, artificial perilymph solution.
[0358] The compositions/combinations as provided herein are
formulated for administration in the vehicle of the experiment.
[0359] Vehicle, the compositions/combinations as provided herein or
dsRNA control compound, is injected intraperitoneally or by bolus
injection. All animals are sacrificed after functional evaluation
with a lethal dose of anesthetic: three animals for each group at
day 1 for immunolabeling and the remaining animals at day 21, of
which three are further processed for scanning electron microscopy
(SEM).
Noise Exposure
[0360] Acoustic trauma is induced by a continuous pure tone of 6
kHz generated by a waveform generator (for example: Generator
LAG-120B, Leader Electronics Corp, Yokohama, Japan), and amplified
by an audio amplifier (for example: A-207R, Pioneer Electronics,
Long Beach, Calif., USA). All animals, under anesthetic, are
exposed for 40 min to a 6 kHz, 120 db SPL (Sound Pressure Level)
sound presented in an open field (for example: dome tweeter
TW340.times.0, Audax, Chateau de Loir, France).
Electrophysiological Measurements of Auditory Function
[0361] Auditory brainstem responses (ARB) are measured before noise
exposure and 1 h, 3 days, 7 days and 21 days after noise exposure.
Animals are mildly anaesthetized and placed in a soundproof room.
Three electrodes are subcutaneously inserted into the right mastoid
(active), vertex (reference) and left mastoid (ground). A
computer-controlled data acquisition system, for example TDT System
3 (Tucker-Davis Technologies, Alachu, Fla., USA) data acquisition
system with real-time digital signal processing is used to record
the ABR and to generate the auditory stimulus. Tone bursts of pure
tones ranging from 2 to 24 kHz (rise/fall time, 1 ms; total
duration, 10 ms; repetition rate, 20/s) is presented monaurally in
an open field. Responses are filtered (0.3-3 kHz), digitized and
averaged across 500 discrete samples at each frequency-level
combination.
Morphological Studies: Scanning Electron Microscopy
[0362] SEM analysis is performed, e.g. as described in Sergi B, et
al. Protective properties of idebenone in noise-induced hearing
loss in the guinea pig. NeuroReport 2006; 17:857-861. Briefly, the
cochlea (n=3) of three animals for each group is perfused with 2.5%
glutaraldehyde in 0.1 M phosphate buffer and post-fixed overnight
and then incubated for 2 h in 2% osmium tetroxide cacodylate
buffer. After micro-dissection, the cochlea is dehydrated with
increasing concentrations of ethanol from 30 to 100% and dried in
the critical point and finally coated with gold. Each specimen is
viewed and photographed by means of, e.g. a Zeiss Supra 50 Field
Emission SEM apparatus (Carl Zeiss Inc., Gottingen, Germany).
Quantitative EM observations of the surface morphology of the organ
of Corti are performed by determining the number of hair cells in
20 segments (1 mm length of basilar membrane each). A hair cell is
counted as missing if the stereociliary bundle is absent or the
stereocillia of the bunch are completely fused. Results of hair
cell counts are expressed as the percentage of remaining hair cells
in each row of inner hair cells and outer hair cells over the
entire length of cochlea.
Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End
Labeling Assay
[0363] The cochlea (n=3) of three animals for each group are
stained by using TUNEL (terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling) assay (for example,
Molecular Probes, Inc., Carslbad, Calif., USA) as described in B
Sergi et al. Protective properties of idebenone in noise-induced
hearing loss in the guinea pig. NeuroReport (2006) 17:857-861.
Briefly, the cochlea are fixed with 10% formaldehyde in 0.1 M
phosphate-buffered saline (PBS), pH 7.3. After micro-dissection,
surface preparations of the organ of Corti are incubated in
ice-cold 70% (v/v) ethanol overnight and then in freshly prepared
DNA labeling solution containing 10 .mu.l of reaction buffer, 0.75
.mu.l of TdT enzyme, 8.0 .mu.l of BrdUTP and 31.25 .mu.l of
dH.sub.2O for 16 h at room temperature. The tissues are then
stained with Alexa Fluor 488 dye-labelled anti BrdU
antibody--contained in the TUNEL assay kit (e.g., Molecular Probes
Inc., Carlsbad, Calif., USA) (5 .mu.l of antibody plus 95 .mu.l of
the Corti are double stained with propidium iodide (5 .mu.g/ml in
10 mM PBS) for 20 min at room temperature. After rinsing in PBS,
the organs of Corti are mounted on slides containing an anti-fade
medium (for example, Prolong Gold, Molecular Probes, Inc.).
Specimens are observed using confocal laser scanning microscopy
(e.g., Leica TCS-SP2, Leica Inc., Wetzlar, Germany).
Results
[0364] The results obtained in this model indicate that the
compositions/combinations provided herein: (a) attenuated
noise-induced threshold shift; and (b) decreased noise-induced
outer hair cell loss; provided protection against noise-induced
hearing loss (NIHL).
Example 9
Therapeutic Activity of Combination Treatments in Hair Cell
Regeneration in Rat Model of Ototoxic Hearing Loss
[0365] Model system. 40 ul of cocktail Kanamycin (200 mg/ml) and
ethacrynic acid (20 mg/ml) in PBS (pH 8) were administered by
transtympanic injection. 6 groups of rats (N=5, 4 or 3) were
used.
[0366] In groups 1-4, animals (Wistar and Norway Brown young adult
male rats (180-220 g)) were bilaterally deafened using a combined
treatment with transtympanic administration of kanamycin (KM) and
ethacrynic acid (EA) cocktail, as described above. Animals in group
5 had one ear deafened as above while receiving PBS in the
contralateral ear. Animals in group 6 remained undeafened. dsRNA to
EGFP was used as control siRNA. Test or control siRNAs were
delivered to groups 1-4 as assigned below. Groups 5 and 6 did not
receive combination/control siRNA treatment.
[0367] The following groups were performed (total n=25):
[0368] Group 1 (5 rats): KM+EA. Left ear: control, Right ear:
combination of HES1 dsRNA+HEY2 dsRNA+HES5 dsRNA;
[0369] Group 2 (5 rats): KM+EA. Left ear: control, Right ear:
combination of HES1 dsRNA+HEY2 dsRNA+EGFP dsRNA);
[0370] Group 3 (5 rats): KM+EA. Left ear: control, Right ear:
combination of NOTCH1 dsRNA+CDKN1B dsRNA+HEY2 dsRNA;
[0371] Group 4 (4 rats): KM+EA. Left ear: control, Right ear: EGFP
dsRNA (control siRNA);
[0372] Group 5 (3 rats): KM+EA. Left ear: control, Right ear:
vehicle;
[0373] Group 6 (3 rats): Normal control. Left ear: un-operated.
Right ear: sham-operated (surgical procedure)
[0374] Details of dsRNA Combinations Administration:
[0375] control siRNA or dsRNA combinations were administered by
combined GelFoam and pump administration, as follows.
[0376] On day 4 after KM+EA administration, control siRNA or dsRNA
combinations were administered to the anesthetized animals via
surgical procedure of opening the bulla and applying a 3 .mu.l
volume of control siRNA or combinations of dsRNAs (see Table B for
bolus dsRNA doses) on a small piece of GelFoam placed on the round
window membrane. Next, the catheter of a 2006 Alzet miniosmotic
pump was placed on a GelFoam in the adequate orientation and
secured to the bulla. The catheter was then connected to the pump
filled with 200 .mu.l volume of control siRNA or dsRNA combinations
(see Table B for pump dsRNA doses), followed by pump implantation
between the 2 scapulae. Control siRNA or dsRNA combinations were
applied continuously over a period of 6 weeks via 2006 Alzet
miniosmotic pump.
TABLE-US-00009 TABLE B dsRNA doses GelFoam dose, .mu.g of Dose in a
6 weeks each (in total volume pump, .mu.g of each Group dsRNA/dsRNA
of 3 .mu.l/foam piece/ (in total volume No. combination ear) of 200
.mu.l/ear) 1 HES1 + HEY2 + HES5 30 .mu.g + 30 .mu.g + 30 .mu.g 30
.mu.g + 30 .mu.g + 30 .mu.g 2 HES1 + HEY2 + EGFP 30 .mu.g + 30
.mu.g + 30 .mu.g 30 .mu.g + 30 .mu.g + 30 .mu.g 3 NOTCH1 + CDKN1B +
HEY2 30 .mu.g + 30 .mu.g + 30 .mu.g 30 .mu.g + 30 .mu.g + 30 .mu.g
4 EGFP (control siRNA) 90 .mu.g 90 .mu.g
[0377] Details of dsRNA compounds: Table C hereinbelow provides
details of chemically modified dsRNA compounds that were used in
this model system.
TABLE-US-00010 TABLE C dsRNA type/ Name Sense 5 -> 3 Antisense 5
-> 3 NOTCH1/ zidB; rG; rC; rU; rA; rC; rA; rA; mU; rC; rA; rC;
rA; rC; rA2p; rC; NOTCH1_2_S2085 rC; rU; rG; rC; rG; rU; rG; rU2p;
rG; rC; rA; rG; rU; rU; rG; rU; rG2p; rU2p; rG2p; rA2p; zc3p mA;
rG; rC; zc3p; zc3p CDKN1B/ zc6Np; rG; mC; rA; rA; rU; mU; rA; 5'p;
mU; rA; rA; rG; rG; rA; mA; CDKN1B_4_S2102 rG; rG; rU; rU; rU; rU;
rU; mC; rC; rA; rA; rA; rC; rC; mU; rA; rA; rU; rU; mU; rA; zc3p
mU; rG; rC; zc3p; zc3p$ HEY2/ zidB; rG; rG; rG; mU; rA; rA; rA; mU;
rU; mC; rA; rA; rA; rG2p; mU; HEY2_2_S1970 rG; rG; rC; mU; rA; mC;
rU; rU; mU; rA; rG; mC; rC; mU; rU; mU; rA; rG; rA; rA; zc3p mC;
rC; mC; zc3p; zc3p HES1/ zidB; rC; rA; rG; rC; rG; rA; rG; rA; rU;
mC; rG; rU; rU; rC; rA; mU; HES1_36_S2086 rU; rG; rC; rA; rU; rG;
rA; rA2p; rG; mC; rA; rC; rU; mC; rG; rC; rC2p; rG2p; rA2p; rU2p;
zc3p rU; rG; zc3p; zc3p EGFP/ rG; mG; rC; mU; rA; mC; rG; mU; rC;
mG; rG; mU; rG; mC; rG; mC; rU; mC; EGFP_5_S763 mC; rA; mG; rG; mA;
rG; mC; rG; mU; rC; mU; rG; mG; rA; mC; rG; mU; rA; mC; rC$ rA; mG;
rC; mC$ HES5/ rG; mG; rG; mU; rU; mC; rU; mA; rU; mU; rA; mC; rA;
mA; rA; mU; rA; mU; HES5_8_S500 mG; rA; mU; rA; mU; rU; mU; rG; mU;
rC; mA; rU; mA; rG; mA; rA; mC; rA rC; mC
[0378] Table D hereinbelow provides a legend of the modified
ribonucleotides/unconventional moieties utilized in the dsRNA
compounds in Table C.
TABLE-US-00011 TABLE D Legend Code Modification Nuc c6Np Amino
modifier C6 (Glen Research) iB inverted deoxyabasic mA
2'-O-methyladenosine-3'-phosphate mA$ 2'-O-methyladenosine (no
phosphate) mC 2'-O-methylcytidine-3'-phosphate mC$
2'-O-methylcytidine (no 3'-phosphate) mG
2'-O-methylguanosine-3'-phosphate mG$ 2'-O-methylguanosine (no
phosphate) mU 2'-O-methyluridine-3'-phosphate mU$
2'-O-methyluridine (no phosphate) rA riboadenosine-3'-phosphate rA$
riboadenosine (no phosphate) rA2p riboadenosine-2'-phosphate rC
ribocytidine-3'-phosphate rC$ ribocytidine (no phosphate) rC2p
ribocytidine-2'-phosphate rG riboguanosine-3'-phosphate rC2p
riboguanosine-2'-phosphate rU ribouridine-3'-phosphate rU$
ribouridine (no phosphate) rU2p ribouridine-2'-phosphate p
5'-phosphate z Prefix for Capping moiety zc3p C3Pi covalently
attached zc3p$ C3OH covalently attached $ No terminal phosphate
[0379] Schedule of ABR/DPOAE measurements: For all animals outlined
in Table B above, damage baseline ABR/DPOAE was measured on days 3
or 4 after KM+EA administration, but prior to surgery/dsRNA
combinations/dsRNA control administration on day 4. For ABR and
DPOAE recording: the rat was anesthetized with a Ketamine (40
mg/kg)-Xylazine (5 mg/kg) cocktail.
[0380] In addition, all the animals were tested for ABR/DPOAE on
weeks 3, 5, 7 and 9. In addition, all animals are tested for
ABR/DPOAE at week 11.
[0381] Euthanasia of the animals and histology studies: all
experimental animals were euthanized on week 11 after dsRNA
combinations/dsRNA control administration (after ABR/DPOAE tests on
week 11). Inner ear tissues were harvested and subjected to
histology studies, qPCR study for target genes and Atoh1 mRNA
levels; RACE analysis of the cleavage products, etc.
[0382] Results: FIGS. 1A-1E show the ABR response obtained in this
study at Day 0, after 3 weeks, after 5 weeks, after 7 weeks and
after 9 weeks. FIG. 1A shows the ABR response obtained in this
study at Day 0, after 3 weeks, after 5 weeks, after 7 weeks and
after 9 weeks for 1 KHz stimulus. FIG. 1B shows the ABR response
obtained in this study at Day 0, after 3 weeks, after 5 weeks,
after 7 weeks and after 9 weeks for 4 KHz stimulus. FIG. 1C shows
the ABR response obtained in this study at Day 0, after 3 weeks,
after 5 weeks, after 7 weeks and after 9 weeks for 8 KHz stimulus.
FIG. 1D shows the ABR response obtained in this study at Day 0,
after 3 weeks, after 5 weeks, after 7 weeks and after 9 weeks for
16 KHz stimulus. FIG. 1E shows the ABR response obtained in this
study at Day 0, after 3 weeks, after 5 weeks, after 7 weeks and
after 9 weeks for 32 KHz stimulus. FIG. 1F provides the legend for
FIGS. 1A-1E.
[0383] Table E hereinbelow provides explanation of the legend for
the test groups in FIGS. 1A-1E.
TABLE-US-00012 TABLE E Explanation of the legend for the test
groups in the Figures Legend Explanation "Contralateral" Normal
control. Left ear: un-operated. Right ear: sham-operated (surgical
procedure) "PBS + vehicle" Left ear: control, Right ear: PBS +
Vehicle, which is artificial perilymph KM + EA KM + EA. Left ear:
control, Right ear: vehicle (artificial perilymph) KM + EA +
vehicle KM + EA + vehicle (artificial perilymph). Left ear:
control, Right ear: vehicle KM + EA + combination of HES1 KM + EA.
Left ear: control, Right ear: dsRNA + HES5 dsRNA + HEY2 combination
of HES1 dsRNA + HES5 dsRNA dsRNA + HEY2 dsRNA KM + EA + combination
of HES1 KM + EA. Left ear: control, Right ear: dsRNA + HEY2 dsRNA +
EGFP combination of HES1 dsRNA + HEY2 dsRNA + dsRNA EGFP dsRNA KM +
EA + combination of NOTCH1 KM + EA. Left ear: control, Right ear:
dsRNA + CDKN1B dsRNA + HEY2 combination of NOTCH1 dsRNA + CDKN1B
dsRNA dsRNA + HEY2 dsRNA KM + EA + EGFP dsRNA KM + EA. Left ear:
control, Right ear: EGFP dsRNA - (control siRNA)
[0384] FIGS. 1A-1E show that combination of HES1 dsRNA+HES5
dsRNA+HEY2 dsRNA and combination of NOTCH1 dsRNA+CDKN1B dsRNA+HEY2
dsRNA were effective in significantly improving ABR response in
this model of ototoxin-induced hearing loss.
Example 10
Treatment of Disorders with Impaired Vestibular Function
[0385] An animal model useful for testing the combinations,
compositions and methods disclosed herein for improving vestibular
function may be found in Schlecker C, et al., Selective atonal gene
delivery improves balance function in a mouse model of vestibular
disease. Gene Ther. 2011 September; 18(9):884-90, incorporated
herein by reference in its entirety.
[0386] Although the above examples have illustrated particular ways
of carrying out embodiments of the invention, in practice persons
skilled in the art will appreciate alternative ways of carrying out
embodiments of the invention, which are not shown explicitly
herein. It should be understood that the present disclosure is to
be considered as an exemplification of the principles of this
invention and is not intended to limit the invention to the
embodiments illustrated.
[0387] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, equivalents
of the specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140364484A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140364484A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References