RNA INTERFERENCE FOR THE TREATMENT OF GAIN-OF-FUNCTION DISORDERS

• US 20110172291A1
• Filed: 12/13/2010
• Published: 07/14/2011
• Est. Priority Date: 09/12/2003
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: ARONIN, Neil, ZAMORE, Phillip D.
• Granted Patent: US 8,680,063 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• 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: C07H 21/02 with ribosyl 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/34 Allele or polymorphism specific uses
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02

Abstract

The present invention relates to the discovery of an effective treatment for a variety of gain-of-function diseases, in particular, Huntington'"'"'s disease (HD). The present invention utilizes RNA Interference technology (RNAi) against polymorphic regions in the genes encoding various gain-of-function mutant proteins resulting in an effective treatment for the gain-of-function disease.

Claims

1. A method of treating a subject having or at risk for a disease characterized or caused by a gain-of-function mutant protein, comprising:

administering to said subject an effective amount of a RNAi agent targeting an allelic polymorphism within a gene encoding said mutant protein, such that sequence-specific interference of said gene occurs;

thereby treating said disease in said subject.

2. The method of claim 1, wherein said gene comprises an expanded trinucleotide repeat region.

3. The method of claim 1, wherein said mutant protein comprises an expanded polyglutamine domain.

4. The method of claim 1, wherein the disease is selected from the group consisting of Huntington'"'"'s disease, spino-cerebellar ataxia type 1, spino-cerebellar ataxia type 2, spino-cerebellar ataxia type 3, spino-cerebellar ataxia type 6, spino-cerebellar ataxia type 7, spino-cerebellar ataxia type 8, spino-cerebellar ataxia type 12, fragile X syndrome, fragile XE MR, Friedreich ataxia, myotonic dystrophy, spinal bulbar muscular disease and dentatoiubral-pallidoluysian atrophy.

5. The method of claim 4, wherein the disease is Huntington'"'"'s disease.

6. The method of claim 5, wherein the RNAi agent targets an allelic polymorphism within the gene encoding a huntingtin protein.

7. The method of claim 5, wherein the RNAi agent targets a polymorphism selected from the group consisting of P1-P5.

8. The method of claim 5, wherein the RNAi agent targets a polymorphism selected from the group consisting of P6-P43.

9. The method of claim 1, wherein the RNAi agent comprises a first strand comprising about 16-25 nucleotides homologous to a region of the gene comprising the polymorphism and a second strand comprising about 16-25 nucleotides complementary to the first strand.

10. The method of claim 1, wherein the effective amount is an amount effective to inhibit the expression or activity of the mutant protein.

11. An RNAi agent comprising a first strand comprising about 16-25 nucleotides homologous to a region of a gene encoding a gain-of-function mutant protein, said region comprising an allelic polymorphism, and a second strand comprising about 16-25 nucleotides complementary to the first strand, wherein the RNAi agent direct target-specific cleavage of a mRNA transcribed from the gene encoding the mutant protein.

12. The RNAi agent of claim 11, which targets a polymorphism within the gene encoding a Huntington protein.

13. The RNAi agent of claim 12, wherein said polymorphism is selected from the group comprising P1-P5.

14. The RNAi agent of claim 12, wherein said polymorphism is selected from the group comprising P6-P43.

15. The RNAi agent of claim 11, wherein the first strand comprises a nucleotide sequence identical to the sequence of the polymorphism.

16. The RNAi agent of claim 11, further comprising a loop portion comprising 4-11 nucleotides that connects the two strands.

17. An isolated nucleic acid molecule encoding the RNAi agent of claim 11.

18. A vector comprising the nucleic acid molecule of claim 17.

19. The vector of claim 18, which is a viral vector, retroviral vector, expression cassette, or plasmid.

20. The vector of claim 18, further comprising an RNA Polymerase III or RNA Polymerase II promoter.

21. The vector of claim 18, wherein the RNA Polymerase III promoter is the U6 or H1 promoter.

23. A host cell comprising the vector of claim 18.

27. A composition comprising the RNAi agent of claim 11, and a pharmaceutically acceptable carrier.

22. A host cell comprising the RNAi agent of claim.

24. The host cell of claim 22, which is a mammalian host cell.

25. The host cell of claim 24, which is a non-human mammalian cell.

26. The host cell of claim 24, which is a human cell.

28. A method for treating a disease or disorder in a subject caused by a gain-of function mutant protein, comprising identifying an allelic polymorphism within a gene encoding said mutant protein and administering to said subject an RNAi agent targeting said polymorphism such that the mutant protein is decreased, thereby treating the subject.

29. A method of silencing a target mRNA encoding a mutant huntingtin (htt) protein in a cell, comprising contacting the cell with effective amount of a RNA silencing agent targeting a heterozygous single nucleotide polymorphism (SNP) within the target mRNA, such that RNA silencing of said mRNA occurs, wherein the SNP has an allelic frequency of at least 35% in a sample population.

30. The method of claim 29, wherein the SNP is present at genomic site RS362331.

31. The method of claim 29, wherein the target mRNA comprises the sequence set forth as SEQ ID NO:

36.

32. The method of claim 29, wherein the target mRNA comprises the sequence set forth as SEQ ID NO:

37.

33. The method of claim 29, wherein the RNA silencing agent is capable of inducing discriminatory RNA silencing.

34. The method of claim 29, wherein the antisense strand of said RNA silencing agent is complementary to the SNP and wherein said RNA silencing agent is capable of substantially silencing the mutant huntingtin protein without substantially silencing the corresponding wild-type huntingtin protein.

35. The method of claim 29, wherein the sample population is of Western European origin.

36. A method of silencing a target mRNA encoding a mutant huntingtin (htt) protein in a cell, comprising contacting the cell with an effective amount of a siRNA targeting a heterozygous single nucleotide polymorphism (SNP) within the target mRNA, such that RNA silencing of said mRNA occurs, wherein the siRNA is selected from the group consisting of:

a. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

34 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

35 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification;

b. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

38 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

39 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification,

c. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

40 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

41 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification, and

d. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

44 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

45 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification.

37. The method of claim 36, wherein the siRNA comprises a lipophilic moiety.

38. The method of claim 37, wherein the lipophilic moiety is a cholesterol moiety.

39. An RNA silencing agent comprising an antisense strand comprising about 16-25 nucleotides homologous to a region of an mRNA encoding a mutant huntingtin (htt) protein, said region comprising a heterozygous single nucleotide polymorphism (SNP) having an allelic frequency of at least 35% in a sample population, wherein the RNA silencing agent is capable of directing RNA silencing of said mRNA.

40. The RNA silencing agent of claim 39, wherein the SNP is present at genomic site RS362331.

41. The RNA silencing agent of claim 39, wherein the target mRNA comprises the sequence set forth as SEQ ID NO:

36.

42. The RNA silencing agent of claim 39, wherein the target mRNA comprises the sequence set forth as SEQ ID NO:

37.

43. The RNA silencing agent of claim 39, which is capable of inducing discriminatory RNA silencing.

44. The RNA silencing agent of claim 39, which is capable of substantially silencing the mutant huntingtin protein without substantially silencing the corresponding wild-type huntingtin protein.

45. The RNA silencing agent of claim 39, wherein the RNA silencing agent is an siRNA.

46. The siRNA of claim 45, comprising a sense strand having a nucleotide sequence identical to the sequence of the SNP.

47. The siRNA of claim 39, wherein the sense strand of the siRNA is identical to the polymorphism at a nucleotide position that is 10 nucleotides from the 5′

end of the antisense strand.

48. The siRNA of claim 39, wherein the sense strand of the siRNA is identical to the polymorphism at a nucleotide position that is 16 nucleotides from the 5′

end of the antisense strand.

49. An siRNA selected from the group consisting of:

a. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

34 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

35 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification;

b. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

38 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

39 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification,

c. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

40 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

41 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification, and

d. an siRNA comprising (i) a sense strand comprising the sequence set forth as SEQ ID NO;

44 or a variant thereof; and

(ii) an antisense strand comprising the sequence set forth as SEQ ID NO;

45 or a variant thereof, said variant comprising at least one nucleotide analog or backbone modification.

50. The siRNA of claim 49, wherein the siRNA comprises a lipophilic moiety.

51. The siRNA of claim 50, wherein the lipophilic moiety is a cholesterol moiety.