ANTISENSE COMPOUNDS AND USES THEREOF
The present invention provides compounds comprising oligonucleotides complementary to a pyruvate kinase M transcript. Certain such compounds are useful for hybridizing to a pyruvate kinase M transcript, including but not limited to a pyruvate kinase M transcript in a cell. In certain embodiments, such hybridization results in modulation of splicing of the pyruvate kinase M transcript. In certain embodiments, such compounds are used to treat one or more symptoms associated with cancer.
1 . A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region, wherein the complementary region comprises at least 8 contiguous nucleobases and is complementary to an equal-length portion of a target region of a PK-M transcript.
2 . The compound of claim 1 , wherein the target region of the PK-M transcript comprises at least a portion of exon 10 of the PK-M transcript.
3 . The compound of claim 1 or 2 , wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.
4 . The compound of any of claims 1 to 3 , wherein the complementary region of the modified oligonucleotide comprises at least 10 contiguous nucleobases.
5 . The compound of any of claims 1 to 3 , wherein the complementary region of the modified oligonucleotide comprises at least 15 contiguous nucleobases.
6 . The compound of any of claims 1 to 3 , wherein the complementary region of the modified oligonucleotide comprises at least 20 contiguous nucleobases.
7 . The compound of any of claims 1 - 6 , wherein the nucleobase sequence of the oligonucleotide is at least 80% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.
8 . The compound of any of claims 1 - 6 , wherein the nucleobase sequence of the oligonucleotide is at least 90% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.
9 . The compound of any of claims 1 - 6 , wherein the nucleobase sequence of the oligonucleotide is 100% complementary to an equal-length region of the PK-M transcript, as measured over the entire length of the oligonucleotide.
10 . The compound of any of claims 1 - 9 , wherein the target region is within exon 10 of the PK-M transcript.
11 . The compound of any of claims 1 - 10 , wherein the target region is within nucleobase 29153 and nucleobase 29281 of SEQ ID NO.: 1.
12 . The compound of any of claims 1 - 10 , wherein the target region is within nucleobase 29158 and nucleobase 29262 of SEQ ID NO.: 1.
13 . The compound of any of claims 1 - 10 , wherein the target region is within nucleobase 29164 and nucleobase 29188 of SEQ ID NO.: 1.
14 . The compound of any of claims 1 - 10 , wherein the target region is within nucleobase 29261 and nucleobase 29279 of SEQ ID NO.: 1.
15 . The compound of any of claims 1 - 10 , wherein the target region is within nucleobase 29168 and nucleobase 29183 of SEQ ID NO.: 1.
16 . The compound of any of claims 1 - 15 , wherein the nucleobase sequence of the antisense oligonucleotide comprises any one of SEQ ID NOs: 4 to 36.
17 . The compound of any of claims 1 - 16 , wherein the modified oligonucleotide comprises at least one modified nucleoside.
18 . The compound of claim 17 , wherein at least one modified nucleoside comprises a modified sugar moiety.
19 . The compound of claim 18 , wherein at least one modified sugar moiety is a 2′-substituted sugar moiety.
20 . The compound of claim 19 , wherein the 2′-substitutent of at least one 2′-substituted sugar moiety is selected from among: 2′-OMe, 2′-F, and 2′-MOE.
21 . The compound of any of claims 17 - 20 , wherein the 2′-substituent of at least one 2′-substituted sugar moiety is a 2′-MOE.
22 . The compound of any of claims 1 - 18 , wherein at least one modified sugar moiety is a bicyclic sugar moiety.
23 . The compound of claim 22 , wherein at least one bicyclic sugar moiety is LNA or cEt.
24 . The compound of any of claims 18 - 23 , wherein at least one sugar moiety is a sugar surrogate.
25 . The compound of claim 24 , wherein at least one sugar surrogate is a morpholino.
26 . The compound of claim 24 , wherein at least one sugar surrogate is a modified morpholino.
27 . The compound of any of claim 1 - 26 , wherein the modified oligonucleotide comprises at least 5 modified nucleosides, each independently comprising a modified sugar moiety.
28 . The compound of claim 27 , wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.
29 . The compound of claim 27 , wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.
30 . The compound of claim 27 , wherein each nucleoside of the modified oligonucleotide is a modified nucleoside, each independently comprising a modified sugar moiety
31 . The compound of any of claims 1 - 30 , wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.
32 . The compound of any of claims 1 - 31 , wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are different from one another.
33 . The compound of any of claims 1 - 32 , wherein the modified oligonucleotide comprises a modified region of at least 5 contiguous modified nucleosides.
34 . The compound of claim 33 , wherein the modified oligonucleotide comprises a modified region of at least 10 contiguous modified nucleosides.
35 . The compound of claim 33 , wherein the modified oligonucleotide comprises a modified region of at least 15 contiguous modified nucleosides.
36 . The compound of claim 33 , wherein the modified oligonucleotide comprises a modified region of at least 20 contiguous modified nucleosides.
37 . The compound of any of claims 32 - 36 , wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2′-F, 2′-OMe, 2′-MOE, cEt, LNA, morpholino, and modified morpholino.
38 . The compound of any of claims 33 - 37 , wherein the modified nucleosides of the modified region each comprise the same modification as one another.
39 . The compound of claim 38 , wherein the modified nucleosides of the modified region each comprise the same 2′-substituted sugar moiety.
40 . The compound of claim 38 , wherein the 2′-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2′-F, 2′-OMe, and 2′-MOE.
41 . The compound of claim 39 , wherein the 2′-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2′-MOE.
42 . The compound of claim 38 , wherein the modified nucleosides of the region of modified nucleosides each comprise the same bicyclic sugar moiety.
43 . The compound of claim 42 , wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt.
44 . The compound of claim 38 , wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.
45 . The compound of claim 44 , wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a morpholino.
46 . The compound of claim 44 , wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a modified morpholino.
47 . The compound of any of claims 1 - 46 , wherein the modified nucleotide comprises no more than 4 contiguous naturally occurring nucleosides.
48 . The compound of any of claims 1 - 46 , wherein each nucleoside of the modified oligonucleotide is a modified nucleoside.
49 . The compound of claim 48 wherein each modified nucleoside comprises a modified sugar moiety.
50 . The compound of claim 49 , wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.
51 . The compound of claim 50 , wherein the modified nucleosides of the modified oligonucleotide each comprise the same 2′-substituted sugar moiety.
52 . The compound of claim 51 , wherein the 2′-substituted sugar moiety of the modified oligonucleotide is selected from 2′-F, 2′-OMe, and 2′-MOE.
53 . The compound of claim 52 , wherein the 2′-substituted sugar moiety of the modified oligonucleotide is 2′-MOE.
54 . The compound of claim 50 , wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.
55 . The compound of claim 54 , wherein the bicyclic sugar moiety of the modified oligonucleotide is selected from LNA and cEt.
56 . The compound of claim 50 , wherein the modified nucleosides of the modified oligonucleotide each comprises a sugar surrogate.
57 . The compound of claim 56 , wherein the sugar surrogate of the modified oligonucleotide is a morpholino.
58 . The compound of claim 56 , wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.
59 . The compound of any of claims 1 - 58 , wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
60 . The compound of claim 59 , wherein each internucleoside linkage is a modified internucleoside linkage.
61 . The compound of claim 59 or 60 , comprising at least one phosphorothioate internucleoside linkage.
62 . The compound of claim 60 , wherein each internucleoside linkage is a modified internucleoside linkage and wherein each internucleoside linkage comprises the same modification.
63 . The compound of claim 62 , wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.
64 . The compound of any of claims 1 - 63 comprising at least one conjugate.
65 . The compound of any of claims 1 - 64 consisting of the modified oligonucleotide.
66 . The compound of any of claims 1 - 65 , wherein the compound modulates splicing of the PK-M transcript.
67 . The compound of any of claims 1 - 66 , having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 4 to 36.
68 . The compound of any of claims 1 - 66 , having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 4 to 17.
69 . The compound of any of claims 1 - 66 , having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 18 to 28.
70 . The compound of any of claims 1 - 66 , having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 29 to 36.
71 . The compound of any of claims 1 - 66 , having a nucleobase sequence comprising SEQ ID NO. 32.
72 . The compound of any of claims 1 - 66 , having a nucleobase sequence comprising SEQ ID NO. 7.
73 . The compound of any of claims 1 - 66 , having a nucleobase sequence comprising SEQ ID NO. 24.
74 . A pharmaceutical composition comprising a compound according to any of claims 1 - 73 and a pharmaceutically acceptable carrier or diluent.
75 . The pharmaceutical composition of claim 74 , wherein the pharmaceutically acceptable carrier or diluent is sterile saline.
76 . A method of modulating splicing of a PK-M transcript in a cell comprising contacting the cell with a compound according to any of claims 1 - 75 .
77 . The method of claim 76 , wherein the cell is in vitro.
78 . The method of claim 76 , wherein the cell is in an animal.
79 . The method of any of claims 76 - 78 , wherein inclusion of exon 9 is increased.
80 . The method of any of claims 76 - 79 , wherein exclusion of exon 10 is increased.
81 . The method of any of claims 76 - 79 , wherein inclusion of exon 10 is decreased.
82 . The method of any of claims 76 - 81 , wherein PK-M1 mRNA expression is increased.
83 . The method of any of claims 76 - 82 , wherein PK-M2 mRNA expression is decreased.
84 . A method of modulating the expression of PK-M in a cell, comprising contacting the cell with a compound according to any of claims 1 - 75 .
85 . The method of claim 84 , wherein PK-M1 expression is increased.
86 . The method of claim 84 or 85 , wherein PK-M2 expression is decreased.
87 . The method of claim 84 , wherein the cell is in vitro.
88 . The method of claim 84 , wherein the cell is in an animal.
89 . A method of inducing apoptosis in a cell, comprising contacting the cell with a compound according to any of claims 1 - 75 .
90 . The method of claim 89 , wherein the cell is in vitro.
91 . The method of claim 89 , wherein the cell is in an animal.
92 . A method comprising administering the compound according to any of claims 1 - 67 or the pharmaceutical composition of claim 74 or 75 to an animal.
93 . The method of claim 92 , wherein the administration is intracerebroventricular.
94 . The method of claim 92 , wherein the administration is into the central nervous system.
95 . The method of any of claims 92 - 94 , wherein the animal has one or more symptoms associated with cancer.
96 . The method claim 95 , wherein the cancer is glioblastoma.
97 . The method of claim 96 , wherein the administration results in amelioration of at least one symptom of cancer.
98 . The method of any of claims 91 - 97 , wherein the animal is a mouse.
99 . The method of any of claims 91 - 97 , wherein the animal is a human.
100 . A method of preventing or retarding the growth of a cancerous tumor, comprising administering the compound according to any of claims 1 - 73 or the pharmaceutical composition of claim 74 or 75 to an animal in need thereof.
101 . The method of claim 100 , wherein the animal is a mouse.
102 . The method of claim 100 , wherein the animal is a human.
103 . The method of claims 100 to 102 , wherein the cancerous tumor comprises glioblastoma.
104 . Use of the compound according to any of claims 1 - 73 or the pharmaceutical composition of claim 74 or 75 for the preparation of a medicament for use in the treatment of cancer.
105 . Use of the compound according to any of claims 1 - 73 or the pharmaceutical composition of claim 74 or 75 for the preparation of a medicament for use in the amelioration of one or more symptoms cancer.
106 . The use of claim 104 or 105 , wherein the cancer is glioblastoma.