MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD
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.
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.