METHODS AND COMPOSITIONS FOR TREATMENT OF FRAGILE X SYNDROME

Abstract

Methods for alleviating symptoms in a Fragile X Syndrome (FXS) patient using adeno-associated viral (AAV) 9 viral particles encoding a wild-type human fragile X mental retardation 1 (FMR1) protein (human FMRP). Also provided herein are methods to determine suitable doses of AAV9 viral particles for a FXS patient to alleviate at least one symptom associated with FXS, as well as methods for monitoring treatment efficacy.

Description

Claims

1. A method for treating Fragile X Syndrome (FXS), comprising administering to a human patient having FXS an effective amount of a plurality of adeno-associated viral (AAV) 9 viral particles, wherein the AAV9 viral particles comprise a single-stranded AAV DNA vector, which comprises a nucleotide sequence encoding a wild-type human fragile X mental retardation 1 (FMR1) protein (human FMRP), wherein the nucleotide sequence is in operable linkage to a promoter, and wherein the AAV DNA vector expresses the wild-type human FMR1 in the brain of the human patient after infection by the AAV9 viral particles.

2. The method of claim 1, wherein the AAV DNA vector is a self-complementary AAV vector.

3. The method of claim 1, wherein the AAV DNA vector is a standard AAV vector.

4. The method of claim 1, wherein the promoter is a hybrid of a chicken β-actin promoter and a CMV promoter.

5. The method of claim 1, wherein the promoter is a human phosphoglycerate kinase (hPGK) promoter.

6. The method of claim 1, wherein the AAV DNA vector further comprises one or more regulatory elements regulating expression of human FMRP.

7. The method of claim 6, wherein the one or more regulatory elements comprises a human β-globin intron sequence, one or more polyA signaling sequences, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), or a combination thereof.

8. The method of claim 7, wherein the one or more polyA signaling sequences comprise a human β-globin polyA signaling sequence, an SV40 polyA signaling sequence, or a combination thereof.

9. The method of claim 4, wherein the AAV DNA vector does not contain a WPRE.

10. The method of claim 1, wherein the AAV DNA vector is a standard AAV vector comprising a hybrid of a chicken β-actin promoter and a CMV promoter in operable linkage to the nucleotide sequence encoding the human FMRP, a WPRE and an SV40 polyA signaling sequence downstream to the nucleotide sequence encoding the human FMRP.

11. The method of claim 1, wherein the AAV DNA vector is a standard AAV vector comprising a hybrid of a chicken β-actin promoter and a CMV promoter in operable linkage to the nucleotide sequence encoding the human FMRP, and an SV40 polyA signaling sequence downstream to the nucleotide sequence encoding the human FMRP, and wherein the AAV DNA vector does not contain a WPRE.

12. The method of claim 1, wherein the AAV DNA vector is a standard AAV vector comprising is a human phosphoglycerate kinase (hPGK) promoter in operable linkage to the nucleotide sequence encoding the human FMRP, a human β-globin intron sequence upstream to the nucleotide sequence encoding the human FMRP, and SV40 polyA signaling and human β-globin polyA signaling sequences downstream to the nucleotide sequence encoding the human FMRP, and wherein the AAV DNA vector does not contain a WPRE.

13. The method of claim 1, wherein the AAV DNA vector further comprises one or more microRNA-target sites (MTSs) specific to one or more tissue-selective microRNAs to suppress expression of the wild-type FMRP in non-brain tissues.

14. The method of claim 13, wherein the one or more MTSs comprise MTS of miR-122, MTS of miR-208a, MTS of miR-208b-3p, MTS of miR-499a-3p, or a combination thereof.

15. The method of claim 1, wherein the wild-type human FMRP is human FMRP isoform 1.

16. The method of claim 1, wherein the human FMRP is a fragment of a wild-type human FMRP comprising the N-terminus 1-297 amino acid residues.

17. The method of claim 1, wherein the AAV9 viral particles are administered to the human patient by intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or a combination thereof.

18. The method of claim 1, wherein the AAV9 viral particles are administered to the human patient via at least two administration routes.

19. The method of claim 18, wherein the at least two administration routes are selected from the group consisting of: (a) intracerebroventricular injection and intravenous injection;(b) intrathecal injection and intravenous injection;(c) intra-cisterna magna injection and intravenous injection; and(d) intra-parenchymal injection and intravenous injection.

20. The method of claim 1, wherein prior to the administration, the human patient is subject to electroencephalogram (EEG), behavioral and/or cognitive neurorehabilitation assessment, or a combination thereof for determining phenotypic severity of the disease.

21. The method of claim 20, wherein the method further comprises, prior to the administering step, subjecting the human patient to electroencephalogram (EEG), behavioral and/or cognitive neurorehabilitation assessment, or a combination thereof.

22. The method of claim 21, wherein the method further comprises determining dosage of the AAV9 viral particles and/or delivery routes based on the EEG analysis, the behavioral and/or cognitive assessment, or the combination thereof.

23. The method of claim 1, wherein the human patient has been undergoing or is undergoing a treatment comprising a GABA receptor agonist, a PI3K isoform-selective inhibitor, a MMP9 antagonist, or a combination thereof.

24. The method of claim 1, further comprising administering to the human patient an effective amount of a GABA receptor agonist, a PI3K isoform-selective inhibitor, a MMP9 antagonist, or a combination thereof.

25. The method of claim 1, further comprising subjecting the human patient to EEG after administration of the AAV9 viral particles to monitor treatment efficacy.

26. The method of claim 1, further comprising subjecting the human patient to behavioral and/or cognitive neurorehabilitation.

27. The method of claim 26, wherein the neurorehabilitation is performed after administration of the AAV9 viral particles.

28. The method of claim 1, wherein the human patient is a human child.

29. An adeno-associated viral (AAV) vector, comprising: (i) an AAV backbone, which comprises a 5′ inverted terminal repeats (ITR) and a 3′ ITR;(ii) a nucleotide sequence encoding a wild-type human fragile X mental retardation 1 (FMR1) protein;(iii) a promoter in operable linkage to (ii); and(iv) one or more microRNA-target sites (MTSs) specific to one or more tissue-selective microRNAs to suppress expression of the wild-type FMRP in non-brain tissues.

30. The AAV vector of claim 29, which is a self-complementary AAV vector.

31. The AAV vector of claim 29, wherein the promoter is a hybrid of a chicken β-actin promoter and a CMV promoter.

32. The AAV vector of claim 29, wherein the one or more MTSs comprise MTS of miR-122, MTS of miR-208a, MTS of miR-208b-3p, MTS of miR-499a-3p, or a combination thereof.

33. The AAV vector of claim 29, wherein the wild-type human FMRP is human FMRP isoform 1.

34. A self-complementary adeno-associated viral (AAV) vector, comprising: (i) an AAV backbone, which comprises a 5′ inverted terminal repeats (ITR) and a truncated 3′ ITR, either one of which or both of which are truncated;(ii) a nucleotide sequence encoding a wild-type human fragile X mental retardation 1 (FMR1) protein (human FMRP), wherein the wild-type FMRP is FMRP isoform 1; and(iii) a promoter in operable linkage to (ii).

35. The self-complementary AAV vector of claim 34, further comprising one or more microRNA-target sites (MTSs) specific to one or more tissue-selective microRNAs to suppress expression of the wild-type FMRP in non-brain tissues.

36. The self-complementary AAV vector of claim 34, wherein the promoter is a hybrid of a chicken b-actin promoter and a CMV promoter.

37. The self-complementary AAV vector of claim 34, wherein the one or more MTSs comprise MTS of miR-122, MTS of miR-208a, MTS of miR-208b-3p, MTS of miR-499a-3p, or a combination thereof.

38. A standard adeno-associated viral (AAV) vector, comprising: (i) an AAV backbone, which comprises a 5′ inverted terminal repeats (ITR) and a 3′ ITR;(ii) a nucleotide sequence encoding a wild-type human fragile X mental retardation 1 (FMR1) protein;(iii) a promoter in operable linkage to (ii); and(iv) one or more regulatory elements regulating expression of the FMRP.

39. The AAV vector of claim 38, wherein the promoter is a hybrid of a chicken β-actin promoter and a CMV promoter or a human phosphoglycerate kinase (hPGK) promoter.

40. The AAV vector of claim 38, wherein the one or more regulatory elements comprises a human β-globin intron sequence, one or more polyA signaling sequences, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), or a combination thereof.

41. The AAV vector of claim 40, wherein the one or more polyA signaling sequences comprise a human β-globin polyA signaling sequence, an SV40 polyA signaling sequence, or a combination thereof.

42. The AAV vector of claim 38, wherein the AAV DNA vector does not contain a WPRE.

43. The AAV vector of claim 38, wherein the AAV vector comprises a hybrid of a chicken β-actin promoter and a CMV promoter in operable linkage to the nucleotide sequence encoding the human FMRP, a WPRE and an SV40 polyA signaling sequence downstream to the nucleotide sequence encoding the human FMRP.

44. The AAV vector of claim 38, wherein the AAV vector comprises a hybrid of a chicken β-actin promoter and a CMV promoter in operable linkage to the nucleotide sequence encoding the human FMRP, and an SV40 polyA signaling sequence downstream to the nucleotide sequence encoding the human FMRP, and wherein the AAV DNA vector does not contain a WPRE.

45. The AAV vector of claim 38, wherein the AAV vector comprises a human phosphoglycerate kinase (hPGK) promoter in operable linkage to the nucleotide sequence encoding the human FMRP, a human β-globin intron sequence upstream to the nucleotide sequence encoding the human FMRP, and SV40 polyA signaling and human β-globin polyA signaling sequences downstream to the nucleotide sequence encoding the human FMRP, and wherein the AAV DNA vector does not contain a WPRE.

46. An adeno-associated viral (AAV) 9 viral particle, comprising an AAV9 capsid encapsulating a single-stranded AAV DNA vector, wherein the AAV DNA vector is set forth in claim 29.

47. A pharmaceutical composition, comprising the AAV9 viral particle of claim 46 and a pharmaceutically acceptable carrier.