RAAV-based compositions and methods for treating amyotrophic lateral sclerosis
The invention relates to inhibitory nucleic acids and rAAV-based compositions, methods and kits useful for treating Amyotrophic Lateral Sclerosis.
1. A synthetic miRNA that targets a C9orf72 RNA transcript, wherein the synthetic miRNA specifically binds to a nucleic acid sequence of the RNA transcript, wherein the nucleic acid sequence of the RNA transcript bound by the synthetic miRNA is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2, 6, or 7, and wherein the synthetic miRNA further comprises flanking regions of miR-155.
2. The synthetic miRNA of claim 1 , wherein the RNA transcript is a pre-mRNA transcript.
3. The synthetic miRNA of claim 1 , wherein the RNA transcript comprises a G 4 C 2 hexanucleotide repeat.
4. The synthetic miRNA of claim 2 , wherein the pre-mRNA transcript is a C9orf72 V 1 isoform transcript.
5. The synthetic miRNA of claim 2 , wherein the pre-mRNA transcript is a C9orf72 V 3 isoform transcript.
6. The synthetic miRNA of claim 2 , wherein the pre-mRNA transcript is not a C9orf72 V 2 isoform transcript.
7. The synthetic miRNA of claim 1 , wherein the RNA transcript comprises an intron.
8. A recombinant nucleic acid encoding a synthetic miRNA that targets a C9orf72 RNA transcript, wherein the synthetic miRNA specifically binds to a nucleic acid sequence of the RNA transcript, wherein the nucleic acid sequence of the RNA transcript bound by the synthetic miRNA is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2, 6, or 7, and wherein the nucleic acid sequence encoding the synthetic miRNA is flanked by AAV inverted terminal repeats (ITRs).
9. The recombinant nucleic acid of claim 8 , further comprising a promoter operably linked with a region encoding the synthetic miRNA.
10. The recombinant nucleic acid of claim 9 , wherein the promoter is a tissue-specific promoter.
11. The recombinant nucleic acid of claim 9 , wherein the promoter is an RNA polymerase II promoter, optionally wherein the polymerase II promoter is a chicken β-actin (CBA) promoter.
12. The recombinant nucleic acid of claim 9 , wherein the promoter is an RNA polymerase III promoter, optionally wherein the polymerase III promoter is a U6 promoter.
13. A recombinant adeno-associated virus (rAAV) comprising a recombinant nucleic acid encoding a synthetic miRNA that targets a C9orf72 RNA transcript, wherein the synthetic miRNA specifically binds to a nucleic acid sequence of the RNA transcript, wherein the nucleic acid sequence of the RNA transcript bound by the synthetic miRNA is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2, 6, or 7, and further comprising AAV inverted terminal repeats (ITRs).
14. The rAAV of claim 13 , further comprising one or more capsid proteins of one or more AAV serotypes selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, and variants thereof.
15. The rAAV of claim 13 , wherein the synthetic miRNA comprises flanking regions of miR-155.
16. The rAAV of claim 13 , wherein the RNA transcript is a pre-mRNA transcript.
17. The rAAV of claim 13 , wherein the RNA transcript comprises a G 4 C 2 hexanucleotide repeat.
18. The rAAV of claim 13 , wherein the RNA transcript comprises an intron.
19. A composition comprising the rAAV of claim 13 .