TREATMENT OF DYSTROPHIN FAMILY RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO DMD FAMILY

• US 20150094356A1
• Filed: 12/09/2014
• Published: 04/02/2015
• Est. Priority Date: 05/08/2009
• Status: Active Grant

• Current Assignee: CURNA, Inc. (Opko Health, Inc.)
• Original Assignee: CURNA, Inc. (Opko Health, Inc.)
• Inventors: KHORKOVA SHERMAN, OLGA, COLLARD, JOSEPH
• Granted Patent: US 9,533,004 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• 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 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/16 Otologicals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 2310/113 targeting other non-coding nucleic acids, e.g. antagomirs
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Dystrophin family, in particular, by targeting natural antisense polynucleotides of Dystrophin family. The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of DMD family.

Claims

1. (canceled)

13. The method of claim 1, wherein the at least one oligonucleotide comprises at least one oligonucleotide sequences set forth as SEQ ID NOS:

8 to 22.

2. (canceled)

3. (canceled)

4. A method of upregulating a function of and/or the expression of a Dystrophin family polynucleotide in patient cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide of 10 to 30 nucleotides in length that targets and specifically hybridizes with a region of a natural antisense oligonucleotide of the Dystrophin family polynucleotide;

thereby upregulating a function of and/or the expression of the Dystrophin family polynucleotide in patient cells or tissues in vivo or in vitro.

5. The method of claim 4, wherein a function of and/or the expression of the Dystrophin family is increased in vivo or in vitro with respect to a control.

6. The method of claim 4, wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a Dystrophin family polynucleotide selected from SEQ ID NOS:

3-6.

7. The method of claim 4, wherein the at least one antisense oligonucleotide targets a natural antisense transcript antisense to coding and/or non-coding nucleic acid sequences of a Dystrophin family polynucleotide.

8. The method of claim 4, wherein the at least one antisense oligonucleotide targets a natural antisense transcript having overlapping and/or non-overlapping sequences of a Dystrophin family polynucleotide.

9. The method of claim 4, wherein the at least one antisense oligonucleotide comprises one or more modifications selected from:

at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified nucleotide, and combinations thereof.

10. The method of claim 9, wherein the one or more modifications comprise at least one modified sugar moiety selected from:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

11. The method of claim 9, wherein the one or more modifications comprise at least one modified internucleoside linkage selected from:

a phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

12. The method of claim 9, wherein the one or more modifications comprise at least one modified nucleotide selected from:

a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative, and combinations thereof.

14. A method of upregulating a function of and/or the expression of a Dystrophin family gene in mammalian cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one short interfering RNA (siRNA) oligonucleotide 19 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for a natural antisense polynucleotide of a Dystrophin family polynucleotide, and, upregulating a function of and/or the expression of Dystrophin family in mammalian cells or tissues in vivo or in vitro.

15. (canceled)

16. (canceled)

17. A synthetic, modified oligonucleotide of 10 to 30 nucleotides in length comprising at least one modification wherein the at least one modification is selected from:

at least one modified sugar moiety;

at least one modified internucleotide linkage;

at least one modified nucleotide, and combinations thereof;

wherein said oligonucleotide is an antisense compound which hybridizes to a natural antisense polynucleotide of a Dystrophin family gene and upregulating the function and/or expression of a Dystrophin family gene in vivo or in vitro as compared to a normal control.

18. The oligonucleotide of claim 17, wherein the at least one modification comprises an internucleotide linkage selected from the group consisting of:

phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

19. The oligonucleotide of claim 17, wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

20. The oligonucleotide of claim 17, wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages.

21. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified nucleotide, said modified nucleotide selected from:

a peptide nucleic acid, a locked nucleic acid (LNA), analogue, derivative, and a combination thereof.

22. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from:

phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof.

23. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from:

peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination, thereof.

24. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified sugar moiety selected from:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

25. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

26. The oligonucleotide of claim 17, wherein the oligonucleotide is of at least about 5 to 30 nucleotides in length and hybridizes to an antisense and/or sense strand of a Dystrophin family polynucleotide wherein said oligonucleotide has at least about 20% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequences of the Dystrophin family polynucleotide.

27. The oligonucleotide of claim 17, wherein the oligonucleotide has at least about 80% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequence of the Dystrophin family polynucleotide.

28. The oligonucleotide of claim 17, wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Dystrophin family polynucleotide in vivo or in vitro, as compared to a normal control.

29. The oligonucleotide of claim 17, wherein the oligonucleotide comprises the sequences set forth as SEQ ID NOS:

8 to 22.

30. A pharmaceutical composition comprising one or more oligonucleotides according to claim 17 and a pharmaceutically acceptable excipient.

31. The composition of claim 30, wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ ID NOS:

8 to 22.

32. The composition of claim 30, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS:

8 to 22.

33. The composition of claim 32, wherein the oligonucleotides set forth as SEQ ID NOS:

8 to 22 comprise one or more modifications or substitutions.

34. The composition of claim 33, wherein the one or more modifications are selected from:

phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof.

35. A method of preventing or treating a disease associated with at least one Dystrophin family polynucleotide and/or at least one encoded product thereof, comprising:

administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Dystrophin family polynucleotide and modulates expression of said at least one Dystrophin family polynucleotide;

thereby preventing or treating the disease associated with the at least one Dystrophin family polynucleotide and/or at least one encoded product thereof.

36. The method of claim 35, wherein a disease associated with the at least one Dystrophin family polynucleotide is selected from:

a muscle disease or disorder (e.g.. Muscular dystrophy including Duchenne'"'"'s muscular dystrophy, Becker'"'"'s muscular dystrophy, Spinal bulbar muscular atrophy, dystrophinopathy, sarcoglycanopathy, limb girdle muscular dystrophy, congenital muscular dystrophy, congenital myopathy, distal myopathy, Symptomatic form of muscular dystrophy of Duchenne and Becker in female carriers myotonic syndrome etc.;

a muscle-wasting disease), a neurological disease or disorder (including a neuromuscular disease or disorder e.g., dystonia, myoclonus-dystonia syndrome, etc.) a disease or disorder associated with altered level of dystrophin or dystrophin DAPC-complex, Left ventricular noncompaction, cancer, a cardiovascular disease or disorder, cardiomyopathy (e.g., sporadic dilated cardiomyopathy, X-linked Dilated Cardiomyopathy (XLDC) etc.), atherosclerosis a cytoskeletal disorder, congenital stationary night blindness and loss of bearing.

37. A method of identifying and selecting at least one oligonucleotide for in vivo administration comprising:

selecting a target polynucleotide associated with a disease state;

identifying at least one oligonucleotide comprising at least five consecutive nucleotides which are complementary to the selected target polynucleotide or to a polynucleotide that is antisense to the selected target polynucleotide;

measuring the thermal melting point of a hybrid of an antisense oligonucleotide and the target polynucleotide or the polynucleotide that is antisense to the selected target polynucleotide under stringent hybridization conditions; and

selecting at least one oligonucleotide for in vivo administration based on the information obtained.