Compositions and methods for treating amyotrophic lateral sclerosis

• US 10,174,328 B2
• Filed: 10/03/2014
• Issued: 01/08/2019
• Est. Priority Date: 10/04/2013
• Status: Active Grant

• Current Assignee: Translate Bio MA Inc. (Sanofi )
• Original Assignee: Translate Bio MA Inc. (Sanofi )
• Inventors: Krieg, Arthur M., Barsoum, James
• Primary Examiner(s): Vivlemore, Tracy
• Application Number:
  US15/026,774
• Publication Number:
  US 20160222391A1
• Time in Patent Office: 1,558 Days
• Field of Search: None
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• 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 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/343 having patterns, e.g. ==--==--==--
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3531 Hydrogen
• CPC Class Codes: C12N 2320/31 Combination therapy
• CPC Class Codes: C12N 2320/33 Alteration of splicing
• CPC Class Codes: C12N 2510/00 Genetically modified cells

Abstract

Methods for modulating expression of SMN1 and/or SMN2 in cells obtained from subjects having ALS or in subjects having ALS using single stranded oligonucleotides are provided. Methods for treating ALS using single stranded oligonucleotides are also provided.

Claims

1. A method of increasing expression of SMN in a cell of a subject having ALS, the method comprising delivering to the cell a first single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, wherein the first single stranded oligonucleotide is up to 21 nucleotides in length, wherein the cell comprises a genetic alteration associated with the ALS in SOD1, ALS2, SETX, FUS/TLS, VAPB, ANG, TDP-43, FIG4, OPTN, ATXN2, VCP, UBQLN2, SIGMAR1, CHMP2B, PFN1 or C9orf72.

2. The method of claim 1, wherein the cell is a motor neuron.

3. The method of claim 1, wherein the first single stranded oligonucleotides comprises a sequence that is 5′

X—

Y—

Z, wherein X is any nucleotide and wherein X is anchored at the 5′

end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 14 nucleotides in length.

4. The method of claim 1, wherein the method further comprises administering a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2.

5. The method of claim 4, wherein the splice control sequence resides in an exon of SMN2.

6. The method of claim 5, wherein the exon is exon 7 or exon 8.

7. The method of claim 4, wherein the splice control sequence traverses an intron-exon junction of SMN2.

8. The method of claim 7, wherein the intron-exon junction is the intron 6/exon 7 junction or the intron 7/exon 8 junction.

9. The method of claim 4, wherein the splice control sequence resides in an intron of SMN2.

10. The method of claim 9, wherein the intron is intron 6 or intron 7.

11. The method of claim 4, wherein the first single stranded oligonucleotide and the second single stranded oligonucleotide are administered simultaneously.

12. The method of claim 4, wherein the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.

13. The method of claim 12, wherein the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond.

14. The method of claim 4, wherein at least one nucleotide of the first or second single stranded oligonucleotide is a nucleotide analogue.

15. The method of claim 4, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.

16. The method of claim 4, wherein the second single stranded oligonucleotide is 8 to 30 nucleotides in length.

17. The method of claim 16, wherein the cell comprises an SMN1 gene that does not have a mutation associated with Spinal Muscular Atrophy (SMA).

18. A method increasing levels of SMN in a subject having ALS, the method comprising administering a first single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene to the subject, wherein the first single stranded oligonucleotide is up to 21 nucleotides in length, and wherein the subject has a mutation in a gene selected from SOD1, FUS/TLS, or TDP-43.

20. The method of claim 18, wherein the single stranded oligonucleotide is administered intrathecally.

21. The method of claim 18, wherein the first single stranded oligonucleotides comprises a sequence that is 5′

X—

Y—

Z, wherein X is any nucleotide and wherein X is anchored at the 5′

end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 14 nucleotides in length.

22. The method of claim 18, wherein at least one nucleotide of the first single stranded oligonucleotide is a nucleotide analogue.

23. The method of claim 18, wherein the method further comprises administering a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2.

24. The method of claim 23, wherein the splice control sequence resides in an exon of SMN2.

25. The method of claim 24, wherein the exon is exon 7 or exon 8.

26. The method of claim 23, wherein the splice control sequence traverses an intron-exon junction of SMN2.

27. The method of claim 26, wherein the intron-exon junction is the intron 6/exon 7 junction or the intron 7/exon 8 junction.

28. The method of claim 23, wherein the splice control sequence resides in an intron of SMN2.

29. The method of claim 28, wherein the intron is intron 6 or intron 7.

30. The method of claim 23, wherein the first single stranded oligonucleotide and the second single stranded oligonucleotide are administered simultaneously.

31. The method of claim 23, wherein the first single stranded oligonucleotide is covalently linked to the second single stranded oligonucleotide through a linker.

32. The method of claim 31, wherein the linker comprises an oligonucleotide, a peptide, a low pH-labile bond, or a disulfide bond.

33. The method of claim 23, wherein at least one nucleotide of the second single stranded oligonucleotide is a nucleotide analogue.

34. The method of claim 23, wherein the first single stranded oligonucleotide or second single stranded oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.

35. The method of claim 33, wherein the second single stranded oligonucleotide is 8 to 30 nucleotides in length.

19. A method of treating ALS in a subject, the method comprising administering a first single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene to the subject, wherein the first single stranded oligonucleotide is up to 21 nucleotides in length, and wherein the subject has a mutation in a gene selected from SOD1, FUS/TLS, or TDP-43.

36. A method for promoting Gem formation in cells having a spliceosome defect, the method comprising:

delivering to the cells a single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, wherein the first single stranded oligonucleotide is up to 21 nucleotides in length; and

evaluating spliceosome integrity in the cells prior to and/or following delivery of the single stranded oligonucleotide to the cells.

37. The method of claim 36, wherein the cell is a motor neuron of a patient having a motor neuron disease.

38. The method of claim 37, wherein the motor neuron disease is ALS or SMA.

39. The method of claim 36, wherein evaluating spliceosome intergrity comprises determining the localization of FUS, TDP-43, SMN, Gemin3, Gemin4 or another Gem marker in the nuclei of the cells.

40. The method of claim 36, wherein the cells comprise an SMN1 gene that does not have a mutation associated with Spinal Muscular Atrophy (SMA).

41. A method of increasing expression of SMN in a cell of a subject having ALS, the method comprising delivering to the cell a first single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, wherein the first single stranded oligonucleotide is up to 21 nucleotides in length, wherein the cell comprises an SMN1 gene that does not have a mutation associated with Spinal Muscular Atrophy (SMA).

42. The method of claim 41, wherein the cell comprises a genetic alteration associated with the ALS in SOD1, ALS2, SETX, FUS/TLS, VAPB, ANG, TDP-43, FIG. 4, OPTN, ATXN2, VCP, UBQLN2, SIGMAR1, CHMP2B, PFN1, or C9orf72.

43. The method of claim 41, wherein the first single stranded oligonucleotides comprises a sequence that is 5′

X—

Y—

Z, wherein X is any nucleotide and wherein X is anchored at the 5′

end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 14 nucleotides in length.

44. The method of claim 41, wherein the method further comprises administering a second single stranded oligonucleotide complementary with a splice control sequence of a precursor mRNA of SMN2.

45. A method for promoting Gem formation in cells having a spliceosome defect, the method comprising delivering to the cells a single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of an SMN gene, wherein the first single stranded oligonucleotide is up to 21 nucleotides in length, wherein the cells comprise an SMN1 gene that does not have a mutation associated with Spinal Muscular Atrophy (SMA).