Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease

Abstract

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.