Methods of Inducing Exon Skipping

• US 20170268003A1
• Filed: 04/05/2017
• Published: 09/21/2017
• Est. Priority Date: 03/21/2003
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden
• Original Assignee: Academisch Ziekenhuis Leiden
• Inventors: Van DEUTEKOM, Judith C. T.
• Application Number:
  US15/479,639
• Publication Number:
  US 20170268003A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0016 wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• 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 15/85 for animal cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/30 : Special therapeutic applications
• CPC Class Codes: C12N 2320/33 : Alteration of splicing
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: G01N 33/6887 : from muscle, cartilage or connective tissue

Abstract

Methods for inducing skipping of exons, including exon 51 of the dystrophin gene. Oligonucleotides are used for inducing exon skipping and for treating Duchenne Muscular Dystrophy. Disclosed structures include: (1) h51AON1 (SEQ ID NO: 27; UCAA GGAA GAUG GCAU UUCU), which is 20 bases long, (2) h51AON2 (SEQ ID NO: 28; CCUC UGUG AUUU UAUA ACUU GAU), which is 23 bases long, and (3) the combination of h51AON2 and h45AON5 linked by 10 uracils (i.e., SEQ ID NO: 28 (CCUC UGUG AUUU UAUA ACUU GAU) linked to SEQ ID NO: 16 (GCCC AAUG CCAU CCUG G) by UUUU UUUU UU), which combination is 50 bases long.

Claims

1. A method for treating Duchenne Muscular Dystrophy, said method comprising:

administering to a DMD patient a composition including;

means for inducing skipping of exon 51 of a human dystrophin pre-mRNA; and

a backbone covalently associated with said means for inducing.

2. A method according to claim 1, wherein the backbone comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′

-O-methyl phosphorothioate.

3. A method according to claim 1, wherein the backbone is selected from the group consisting of a morpholino phosphorodiamidate backbone and a 2′

-O-methyl phosphorothioate backbone.

4. A method according to claim 3, wherein said means for inducing skipping includes means for binding to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA.

5. A method according to claim 3, wherein said means for inducing skipping includes means for binding without mismatches to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA.

6. A method according to claim 2, wherein the composition includes an antisense oligonucleotide that is twenty bases in length.

7. A method according to claim 2, wherein the composition includes an antisense oligonucleotide that is twenty-three bases in length.

8. A method according to claim 2, wherein the composition includes an antisense oligonucleotide that is fifty bases in length.

9. A method for treating Duchenne Muscular Dystrophy, said method comprising:

administering to a DMD patient an oligonucleotide including;

means for inducing skipping of exon 51 of a human dystrophin pre-mRNA; and

means for providing the oligonucleotide with resistance to RNaseH.

10. A method according to claim 9, wherein the means for providing the oligonucleotide with resistance to RNaseH comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′

-O-methyl phosphorothioate.

11. A method according to claim 9, wherein the means for providing the oligonucleotide with resistance to RNaseH comprises a backbone selected from the group consisting of a morpholino phosphorodiamidate backbone and a 2′

-O-methyl phosphorothioate backbone.

12. A method according to claim 11, wherein said means for inducing skipping includes means for binding to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA.

13. A method according to claim 11, wherein said means for inducing skipping includes means for binding without mismatches to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA.

14. A method according to claim 10, wherein the oligonucleotide is twenty bases in length.

15. A method according to claim 10, wherein the oligonucleotide is twenty-three bases in length.

16. A method according to claim 10, wherein the oligonucleotide is fifty bases in length.

17. A method for inducing the skipping of exon 51 of the human dystrophin pre-mRNA, said method comprising:

providing a composition including;

means for binding without mismatches to an exon-internal sequence of exon 51 of the human dystrophin pre-mRNA, wherein the exon-internal sequence comprises the sequence of AGUUC CUUCU ACCGU AAAGA; and

a backbone covalently associated with said means for binding.

18. A method according to claim 17, wherein the backbone comprises a chemical modification selected from the group consisting morpholino phosphorodiamidate and 2′

-O-methyl phosphorothioate.

19. A method according to claim 18, wherein said composition is operative in a human muscle cell.

20. A method according to claim 17, wherein the backbone is selected from the group consisting of a morpholino phosphorodiamidate backbone and a 2′

-O-methyl phosphorothioate backbone.

21. A method according to claim 20, wherein said composition is operative in a human muscle cell.

22. A method for inducing the skipping of exon 51 of the human dystrophin pre-mRNA, said method comprising:

providing a composition including an oligonucleotide of between 20 to 50 nucleotides comprising a sequence containing h51AON1 (UCAAG GAAGA UGGCA UUUCU) (SEQ ID NO;

     27) or the DNA thereof, wherein said oligonucleotide binds without mismatches to the complementary sequence of exon 51 of the human dystrophin pre-mRNA.

23. A method according to claim 22, wherein said oligonucleotide further comprises a chemical modification selected from the group consisting of morpholino phosphorodiamidate and 2′

-O-methyl phosphorothioate.

24. A method according to claim 22, wherein said oligonucleotide is selected from the group consisting of a morpholino phosphorodiamidate and a 2′

-O-methyl phosphorothioate.

25. A method according to claim 22, wherein said composition is operative in a human muscle cell.