OLIGONUCLEOTIDES FOR TREATING EXPANDED REPEAT DISEASES

• US 20140303238A1
• Filed: 11/30/2012
• Published: 10/09/2014
• Est. Priority Date: 11/30/2011
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Linsley, Peter, Hanson, Gunnar J., Leppert, Brian James
• Granted Patent: US 10,066,228 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• 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: C07F 9/65583 each of the hetero rings containing nitrogen as ring hetero atom
• CPC Class Codes: C07F 9/65616 containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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 2310/323 modified ring structure
• 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 2320/34 Allele or polymorphism specific uses
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention provides for a method for selectively reducing the expression of a mutant mRNA and/or protein having an expanded nucleotide repeat relative to a wild-type mRNA, comprising contacting a cell with an antisense oligonucleotide of sufficient length and complementarity to the expanded nucleotide repeat. More particularly it relates to selectively reducing the expression of mutant Huntington protein associated with Huntington'"'"'s disease. The antisense oligonucleotide comprising either a nucleotide or a repeated three nucleotide sequence as defined in the claims.

Claims

1. An antisense oligonucleotide of 10-40 nucleotides in length having a sequence complementary to an expanded DNA repeat which is associated with a human disease, wherein the antisense comprises a nucleotide having a formula:

2. The antisense oligonucleotide of claim 1, where Nu is selected from the group consisting of adenine, guanine, thymine, uracil, cytosine, and hypoxanthine.

3. The antisense oligonucleotide of claim 2, wherein Nu is thymine or uracil.

4. The antisense oligonucleotide of claim 1, wherein R₁ is selected from the group consisting of

5. The antisense oligonucleotide of claim 1, wherein the nucleotide has the formula:

6. The antisense oligonucleotide of claim 5, wherein Nu is thymine.

7. The antisense oligonucleotide of claim 1, comprising a sequence selected from the group consisting of (CCG)n, (CTG)n, (TTC)n, (NGC)n, (GNC)n, (CAGG)n, (AGAAT)n, and (CGCG₄CG₄)n, wherein N is any nucleotide and n is from 3 to 10.

8. The antisense oligonucleotide of claim 1, wherein the human disease is selected from the group consisting of Huntington'"'"'s disease, amyotrophic lateral sclerosis (ALS) and Myotonic dystrophy type 1 and type 2.

9. The antisense oligonucleotide of claim 8, comprising a sequence (GCT)₇.

10. The antisense oligonucleotide of claim 9, wherein Ry is a G nucleotide.

11. A pharmaceutical composition comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.

12. A method of treating Huntington'"'"'s disease in a subject comprising administering the composition of claim 11.

20. A method of diagnosing a hereditary disorder associated with an expanded DNA repeat by Northern Blotting comprising the steps of:

g. extraction of total RNA from a homogenized tissue sample or cells;

h. isolating the mRNA;

i. separating the mRNA by size using gel electrophoresis;

j. transferring the mRNA to a membrane;

k. immobilizing the mRNA to the membrane; and

l. using a probe to detect an expanded repeat, wherein the probe is an oligonucleotide of claim 1.

13. An antisense oligonucleotide comprising a repeated three nucleotide sequence having the formula:

14. The antisense nucleotide of claim 13, wherein Nu is thymine.

15. The antisense oligonucleotide of claim 13, wherein the three nucleotide sequence is selected from the group consisting of (CCG), (CTG), (TTC), (NGC), and (GNC), wherein N is any nucleotide.

16. The antisense oligonucleotide of claim 13, wherein the repeated three nucleotide sequence is (GCT)₇.

17. The antisense oligonucleotide of claim 13, wherein Ry is a G nucleotide.

18. A pharmaceutical composition comprising the antisense oligonucleotide of claim 13 and a pharmaceutically acceptable carrier.

19. A method of treating Huntington'"'"'s disease in a subject comprising administering the composition of claim 18.