Antisense compounds and uses thereof

Abstract

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.