TRICYCLO-DNA ANTISENSE OLIGONUCLEOTIDES, COMPOSITIONS, AND METHODS FOR THE TREATMENT OF DISEASE

• US 20120149756A1
• Filed: 04/09/2010
• Published: 06/14/2012
• Est. Priority Date: 04/10/2009
• Status: Abandoned Application

• Current Assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm) (Allianz Global Investors GmbH), Centre National De La Recherche Scientifique, Institut de Myologie, Sorbonne Universite
• Original Assignee: Association Institute De Myologie, Centre National De La Recherche Scientifique, Universitã¤T Bern, University Pierre et Marie Curie
• Inventors: Garcia, Luis, Schümperli, Daniel, Leumann, Christian, Furling, Denis, Voit, Thomas
• Application Number:
  US13/263,949
• Publication Number:
  US 20120149756A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/711 Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/712 Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2320/33 Alteration of splicing

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.

Claims

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.

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).

65. The method according to claim 52, wherein said tc-DNA antisense oligonucleotide is as defined in claim 1.

2-4. -4. (canceled)

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).

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).

8. (canceled)

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).

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).

12. (canceled)

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. -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.

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).

19. (canceled)

22-23. -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).

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.

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).

25. (canceled)

27. (canceled)

29-33. -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. -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. -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.

56. A method according to claim 52 wherein said abnormal gene expression leads to a neuromuscular or musculoskeletal disorder.

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.

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.

57. A method according to claim 53 wherein said disease is a neuromuscular or musculoskeletal disorder.

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.

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.

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.

55. (canceled)

58-63. -63. (canceled)

66-69. -69. (canceled)