TRICYCLO-DNA ANTISENSE OLIGONUCLEOTIDES, COMPOSITIONS, AND METHODS FOR THE TREATMENT OF DISEASE
Provided are tricyclo-DNA (tc-DNA) AON and methods employing tc-DNA AON for modifying splicing events that occur during pre-mRNA processing. Tricyclo-DNA (tc-DNA) AON are described that may be used to facilitate exon skipping or to mask intronic silencer sequences and/or terminal stem-loop sequences during pre-mRNA processing and to target RNase-mediated destruction of processed mRNA. Tc-DNA AON described herein may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping a mutated exon 23 or exon 51 within a dystrophin gene to restore functionality of a dystrophin protein; in methods for the treatment of Spinal Muscular Atrophy by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein; and in methods for the treatment of Steinert's Myotonic Dystrophy by targeting the destruction of a mutated DM1 mRNA comprising 3′-terminal CUG repeats.
1 . A tricyclo-DNA antisense oligonucleotide (tc-DNA AON) for facilitating the skipping of an exon during processing of a dystrophin pre-mRNA, said tc-DNA AON containing between 10 and 18 tricyclo nucleotides, wherein 8-16 or 6-14 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA intronic splice donor site, wherein 2-8 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA exonic region, and wherein said intronic splice donor site is contiguous with and 5′ to said exonic region.
2 .- 4 . (canceled)
5 . The tc-DNA AON of claim 1 wherein the exon that is skipped during processing of said dystrophin pre-mRNA is exon 23 or exon 51.
6 . The tc-DNA AON of claim 5 wherein, when the skipped exon is exon 23, said tc-DNA AON comprises the nucleotide sequence 5′-AACCTCGGCTTACCT-3′ (SEQ ID NO: 1) or is M23D (+02−13) (SEQ ID NO: 1), and, when the skipped exon is exon 51, said tc-DNA AON comprises a nucleotide sequence selected from the group consisting of 5′-AGAAATGCC ATCTTC-3′ (SEQ ID NO: 2), 5′-AAATGCCATCTTCCT-3′ (SEQ ID NO: 3), and 5′-TGCCATCTTCCTTGA-3′ (SEQ ID NO: 4) or is H51 (+68+82) (SEQ ID NO: 2) or is H51 (+70+84) (SEQ ID NO: 3) is H51 (+73+87) (SEQ ID NO: 4).
7 . A tricyclo-DNA antisense oligonucleotide (tc-DNA AON) for facilitating the inclusion of an atypical exon during processing of an SMN2 pre-mRNA, said tc-DNA AON containing between 10 and 18 tricyclo nucleotides, wherein said tc-DNA AON is complementary to an SMN2 pre-mRNA intronic silencer sequence (ISS).
8 . (canceled)
9 . The tc-DNA AON of claim 7 wherein said atypical exon in said SMN2 pre-mRNA is exon 7 and said intronic silencer sequence is ISS-N1.
10 . The tc-DNA AON of claim 9 wherein said tc-DNA AON comprises the nucleotide sequence 5′-CUUUCAUAAUGCUGG-S′ (SEQ ID NO: 5) or is SMN2i7 (10; 25) (SEQ ID NO: 5).
11 . A tricyclo-DNA antisense oligonucleotide (tc-DNA AON) for facilitating the inclusion of an atypical exon during processing of an SMN2 pre-mRNA, said tc-DNA AON consisting of 10-18 tricyclo nucleotides, wherein said tc-DNA AON is complementary to an SMN2 pre-mRNA terminal stem-loop (TSL).
12 . (canceled)
13 . The tc-DNA AON of claim 11 wherein said atypical exon in said SMN2 pre-mRNA is exon 7 and said terminal stem-loop is TSL2.
14 . The tc-DNA AON of claim 13 wherein said tc-DNA AON comprises the nucleotide sequence 5′-UUAAUUUAAGGAA-3′ (SEQ ID NO: 6) or is SMN2e7 (39; 51) (SEQ ID NO: 6).
15 . A composition for facilitating the skipping of an exon during processing of a dystrophin pre-mRNA, for facilitating the inclusion of an atypical exon during processing of an SMN2 pre-mRNA, or for facilitating the inclusion of an atypical exon during processing of an SMN2 pre-mRNA, said composition comprising:
(a)
when the composition is for facilitating the skipping of an exon during processing of a dystrophin pre-mRNA, a tricyclo-DNA antisense oligonucleotide (tc-DNA AON) containing between 10 and 18 tricyclo nucleotides, wherein 8-16 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA intronic splice donor site, wherein 2-8 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA exonic region, and wherein said exonic region is contiguous with and 3′ to said intronic splice donor site; or
when the composition is for facilitating the inclusion of an atypical exon during processing of an SMN2 pre-mRNA, a tricyclo-DNA antisense oligonucleotide (tc-DNA AON) containing between 10 and 18 tricyclo nucleotides, wherein said tc-DNA AON is complementary to an SMN2 pre-mRNA intronic silencer sequence (ISS); or
when the composition is for facilitating the inclusion of an atypical exon during processing of an SMN2 pre-mRNA, a tricyclo-DNA antisense oligonucleotide (tc-DNA AON) containing between 10 and 18 tricyclo nucleotides, wherein said tc-DNA AON is complementary to an SMN2 pre-mRNA terminal stem-loop (TSL); and
(b) a cell delivery agent.
16 .- 17 . (canceled)
18 . A method for eliminating a mutated exon from a dystrophin mRNA, said method comprising the step of contacting a cell that expresses a dystrophin pre-mRNA with a tricyclo-DNA antisense oligonucleotide (tc-DNA AON), said tc-DNA AON containing between 10 and 18 tricyclo nucleotides, wherein 8-16 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA intronic splice donor site, wherein 2-8 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA exonic region, and wherein said exonic region is contiguous with and 3′ to said intronic splice donor site.
19 . (canceled)
20 . The method of claim 18 wherein the exon that is skipped during processing of said dystrophin pre-mRNA is exon 23 or 51.
21 . The method of claim 20 wherein
when the skipped exon is 23, said tc-DNA AON comprises the nucleotide sequence 5′-AACCTCGGCTTACCT-3′ (SEQ ID NO: 1) or is M23D (+02−13) (SEQ ID NO: 1), and
when the skipped exon is exon 51, said tc-DNA AON comprises a nucleotide sequence selected from the group consisting of 5′-AGAAATGCCATCTTC-3′ (SEQ ID NO: 2), 5′-AAATGCCATCTTCCT-3′ (SEQ ID NO: 3), and 5′-TGCCATCTTCCTTGA-3′ (SEQ ID NO: 4) or is H51 (+68+82) (SEQ ID NO: 2) or is H51 (+70+84) (SEQ ID NO: 3) or is H51 (+73+87) (SEQ ID NO: 4).
22 .- 23 . (canceled)
24 . A method for including an atypical exon within an SMN2 mRNA, said method comprising the step of contacting a cell that is expressing an SMN2 pre-mRNA with a tc-DNA AON that contains between 11 and 18 nucleotides wherein said tc-DNA AON is complementary to an SMN2 pre-mRNA intronic silencer sequence (ISS) or is complementary to an SMN2 pre-mRNA terminal stem-loop (TSL).
25 . (canceled)
26 . The method of claim 24 wherein said atypical exon in said SMN2 pre-mRNA is exon 7 and said intronic silencer sequence is ISS-N1 or said terminal stem-loop is TSL2.
27 . (canceled)
28 . The method of claim 26 wherein said tc-DNA AON is complementary to an ISS, said tc-DNA AON comprises the nucleotide sequence 5′-CUUUCAUAAUGCUGG-S′ (SEQ ID NO: 5) or is SMN2i7 (10; 25) (SEQ ID NO: 5) and when said tc-DNA AON is complementary to a TSL, said tc-DNA AON comprises the nucleotide sequence 5′-UUAAUUUAAGGAA-3′ (SEQ ID NO: 6) or is SMN2e7 (39; 51) (SEQ ID NO: 6).
29 .- 33 . (canceled)
34 . A method for the treatment of Duchene Muscular Dystrophy (DMD) in a patient, said method comprising the step of administering to said patient a tricyclo-DNA (tc-DNA) antisense oligonucleotide (AON);
wherein said tc-DNA AON comprises a nucleotide sequence that is complementary to a dystrophin pre-mRNA intron-exon junction;
wherein said intron-exon junction comprises an intronic splice donor site that is 5′ to an exon;
wherein said exon comprises a nonsense or a frameshift mutation as compared to an exon having a wild-type nucleotide sequence;
wherein said tc-DNA AON facilitates the skipping of said exon during the processing of said dystrophin pre-mRNA to a mature mRNA.
35 . The method of claim 34 wherein:
said tc-DNA AON has a length selected from the group consisting of: (i) between 11 and 18 nucleotides or (ii) 15 nucleotides; or- said tricyclo-DNA oligonucleotide comprises between 6 and 10 nucleotides that are complementary to a splice donor site within an intron; or
said tricyclo-DNA oligonucleotide comprises between 6 and 10 nucleotides that are complementary to 5′ nucleotides within said exon;
or said mutation in said exon is a non-sense mutation or a frame-shift mutation.
36 .- 38 . (canceled)
39 . A method for the treatment of Duchenne Muscular Dystrophy in a patient, said method comprising the step of administering to said patient a tricyclo-DNA oligonucleotide;
wherein said tricyclo-DNA oligonucleotide comprises a sequence of nucleotides that is complementary to an intron-exon junction within a dystrophin pre-mRNA,
wherein said intron-exon junction comprises a splice donor site within intron 51 and 5′ nucleotides within adjacent exon 51 of said dystrophin pre-mRNA,
wherein said dystrophin pre-mRNA comprises a mutation in said exon 51, and
wherein said tricyclo-DNA oligonucleotide is capable of mediating the skipping of said exon 51.
40 . The method of claim 39 wherein;
said intron-exon junction comprises the sequence of SEQ ID NO: 1 ; or
said mutation in said exon 51 is a non-sense mutation or a frame-shift mutation; or
said tricyclo-DNA oligonucleotide comprises the sequence of SEQ ID NO: 2.
41 .- 49 . (canceled)
50 . A method for destroying a DM1 mRNA comprising one or more 3′ CUG amplifications in a cell, said method comprising the step of contacting said cell with a tc-DNA AON comprising 12-21 tricyclo nucleotides wherein said tc-DNA AON is complementary to a mutated DM1 mRNA comprising one or more 3′ CUG amplification(s) and wherein said tc-DNA AON is capable of facilitating the RNAse H-mediated destruction of said DM1 mRNA.
51 . A method for the treatment of Steinert's Myotonic Dystrophy in a patient, said method comprising the step of administering to said patient a tc-DNA AON comprising 12-21 tricyclo nucleotides wherein said tc-DNA AON is complementary to a mutated DM1 mRNA comprising one or more 3′ CUG amplification(s) and wherein said tc-DNA AON is capable of facilitating the RNAse H-mediated destruction of said DM1 mRNA.
52 . A method for correcting abnormal gene expression in a cell of the central nervous system of a subject, the method comprising administering to the subject a tc-DNA antisense oligonucleotide, wherein said tc-DNA antisense oligonucleotide is complementary to a portion of an RNA encoded by said gene, and wherein said tc-DNA antisense oligonucleotide is administered peripherally to the subject in an amount sufficient to correct said abnormal expression.
53 . A method for treating a genetic disease caused by abnormal gene expression in the central nervous system of a subject, the method comprising administering to the subject a tc-DNA antisense oligonucleotide, wherein said tc-DNA antisense oligonucleotide is complementary to a portion of an RNA encoded by said gene, and wherein said tc-DNA antisense oligonucleotide is administered peripherally to the subject in an amount effective to correct said abnormal expression.
54 . A pharmaceutical composition comprising a tc-DNA antisense oligonucleotide wherein said tc-DNA antisense oligonucleotide is complementary to a portion of an RNA encoded by a human gene, and wherein said composition further comprises a pharmaceutically acceptable excipient.
55 . (canceled)
56 . A method according to claim 52 wherein said abnormal gene expression leads to a neuromuscular or musculoskeletal disorder.
57 . A method according to claim 53 wherein said disease is a neuromuscular or musculoskeletal disorder.
58 .- 63 . (canceled)
64 . The method according to claim 52 , wherein said abnormal gene expression results from
an in-frame mutation of an exon,
a mutation disrupting the translational reading frame of the gene, and the tc-DNA facilitates skipping of an exon so as to restore the reading frame;
a deleterious mutation that can be compensated by the inclusion of an atypical exon in the mRNA coded by said gene, and the tc-DNA is complementary to an ISS or TSL present in a pre-mRNA coded by said gene and facilitates inclusion of an atypical exon, or
a mutation resulting in the presence of deleterious 3′ CUG amplification(s) in a mRNA coded by said gene.
65 . The method according to claim 52 , wherein said tc-DNA antisense oligonucleotide is as defined in claim 1 .
66 .- 69 . (canceled)
70 . The method according to any one of claims 53 , wherein said abnormal gene expression results from
an in-frame mutation of an exon,
a mutation disrupting the translational reading frame of the gene, and the tc-DNA facilitates skipping of an exon so as to restore the reading frame;
a deleterious mutation that can be compensated by the inclusion of an atypical exon in the mRNA coded by said gene, and the tc-DNA is complementary to an ISS or TSL present in a pre-mRNA coded by said gene and facilitates inclusion of an atypical exon, or
a mutation resulting in the presence of deleterious 3′ CUG amplification(s) in a mRNA coded by said gene.
71 . The method according to any one of claims 54 , wherein said abnormal gene expression results from
an in-frame mutation of an exon,
a mutation disrupting the translational reading frame of the gene, and the tc-DNA facilitates skipping of an exon so as to restore the reading frame;
a deleterious mutation that can be compensated by the inclusion of an atypical exon in the mRNA coded by said gene, and the tc-DNA is complementary to an ISS or TSL present in a pre-mRNA coded by said gene and facilitates inclusion of an atypical exon, or
a mutation resulting in the presence of deleterious 3′ CUG amplification(s) in a mRNA coded by said gene.