Multiple exon skipping compositions for dmd

Abstract

Provided are antisense molecules capable of binding to a selected target site in the human dystrophin gene to induce exon skipping, and methods of use thereof to treat muscular dystrophy.

It is claimed: 
     
         1 . A composition for use in producing skipping of exon 44 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 1-20, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 44, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         2 . The composition of  claim 1 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 8, 11 and 12. 
     
     
         3 . The composition of  claim 1 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         4 . A composition for use in producing skipping of exon 45 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 21-76 and 612 to 624, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 45, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         5 . The composition of  claim 4 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 27, 29, 34 and 39. 
     
     
         6 . The composition of  claim 5 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 29 and 34. 
     
     
         7 . The composition of  claim 4 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         8 . A composition for use in producing skipping of exon 46 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 77-125, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 46, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         9 . The composition of  claim 8 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 77-105. 
     
     
         10 . The composition of  claim 9 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 82, 84-87, 90, 96, 98, 99 and 101. 
     
     
         11 . The composition of  claim 8 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         12 . A composition for use in producing skipping of exon 47 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 126-169, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 47, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         13 . The composition of  claim 12 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 126-149. 
     
     
         14 . The composition of  claim 13 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 126, 128-130, 132, 144 and 146-149. 
     
     
         15 . The composition of  claim 12 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         16 . A composition for use in producing skipping of exon 48 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 170-224 and 634, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 48, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         17 . The composition of  claim 16 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 170-201 and 634. 
     
     
         18 . The composition of  claim 17 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 176, 178, 181-183, 194 and 198-201. 
     
     
         19 . The composition of  claim 16 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         20 . A composition for use in producing skipping of exon 49 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 225-266, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 49, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         21 . The composition of  claim 20 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 225-248. 
     
     
         22 . The composition of  claim 21 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 227, 229, 234, 236, 237 and 244-248. 
     
     
         23 . The composition of  claim 20 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         24 . A composition for use in producing skipping of exon 50 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 267-308, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 50, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         25 . The composition of  claim 24 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 277, 287, 290 and 291. 
     
     
         26 . The composition of  claim 25 , wherein the compound contains the sequence consisting of SEQ ID NO: 287. 
     
     
         27 . The composition of  claim 24 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         28 . A composition for use in producing skipping of exon 51 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 309-371, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 51, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         29 . The composition of  claim 28 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 324, 326 and 327. 
     
     
         30 . The composition of  claim 29 , wherein the compound contains the sequence consisting of SEQ ID NO: 327. 
     
     
         31 . The composition of  claim 28 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         32 . A composition for use in producing skipping of exon 52 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 372-415, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 52, a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         33 . The composition of  claim 32 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 372-397. 
     
     
         34 . The composition of  claim 33 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 379-382, 384, 390 and 392-395. 
     
     
         35 . The composition of  claim 32 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         36 . A composition for use in producing skipping of exon 53 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 416-475 and 625-633, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 53 a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         37 . The composition of  claim 36 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 428, 429 and 431. 
     
     
         38 . The composition of  claim 37 , wherein the compound contains a sequence consisting of SEQ ID NO: 429. 
     
     
         39 . The composition of  claim 36 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         40 . A composition for use in producing skipping of exon 54 in the processing of human dystrophin pre-processed mRNA, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 476-519, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 54 a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         41 . The composition of  claim 40 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 476-499. 
     
     
         42 . The composition of  claim 41 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 479-482, 484, 489 and 491-493. 
     
     
         43 . The composition of  claim 40 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         44 . A composition for use in producing skipping of exon 55 in the processing of human dystrophin-gene pre-processed mRNA transcript, comprising
 a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS: 520-569 and 635, and capable of forming with the complementary mRNA sequence in the dystrophin-gene exon 55 a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C.   
     
     
         45 . The composition of  claim 44 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 520-546 and 635. 
     
     
         46 . The composition of  claim 45 , wherein the compound contains a sequence selected from the group consisting of SEQ ID NOS: 524-528, 537, 539, 540, 542 and 544. 
     
     
         47 . The composition of  claim 44 , wherein the compound is conjugated to an arginine-rich peptide having a sequence selected from the group consisting of SEQ ID NOS: 570-578. 
     
     
         48 . A method of treating muscular dystrophy in a subject, comprising administering to the subject an effective amount of a substantially uncharged antisense compound containing 20-35 morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, comprising a sequence selected from the group consisting SEQ ID NOS:1 to 569 and 612 to 635, and capable of forming with the complementary mRNA sequence in a dystrophin-gene exon a heteroduplex structure between said compound and mRNA having a Tm of at least 45° C., wherein the exon is selected from the group consisting of exons 44-55. 
     
     
         49 . The method of  claim 48 , wherein the muscular dystrophy is Duchenne's muscular dystrophy (DMD). 
     
     
         50 . The method of  claim 48 , wherein the muscular dystrophy is Becker muscular dystrophy (BMD). 
     
     
         51 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 1-20, and the exon is exon 44. 
     
     
         52 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 21-76 and 612 to 624, and the exon is exon 45. 
     
     
         53 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 77-125, and the exon is exon 46. 
     
     
         54 . The method of  claim 48 , wherein the sequence selected from the group consisting SEQ ID NOS: 126-169, and the exon is exon 47. 
     
     
         55 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 170-224 and 634, and the exon is exon 48. 
     
     
         56 . The method of  claim 48 , wherein the sequence selected from the group consisting SEQ ID NOS: 225-266, and the exon is exon 49. 
     
     
         57 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 267-308, and the exon is exon 50. 
     
     
         58 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 309-371, and the exon is exon 51. 
     
     
         59 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 372-415, and the exon is exon 52. 
     
     
         60 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 416-475 and 625-633, and the exon is exon 53. 
     
     
         61 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 476-519, and the exon is exon 54. 
     
     
         62 . The method of  claim 48 , wherein the sequence is selected from the group consisting SEQ ID NOS: 520-569 and 635, and the exon is exon 55. 
     
     
         63 . The method of  claim 48 , wherein the sequence comprises SEQ ID NO:287. 
     
     
         64 . The method of  claim 48 , wherein the compound is conjugated to an arginine-rich peptide. 
     
     
         65 . The method of  claim 64 , wherein the arginine-rich peptide comprises a sequence selected from the group consisting of SEQ ID NOS: 570-578.