Recombinant adeno associated virus encoding clarin-1 and uses thereof
Aspects of the disclosure relate to compositions and methods for treating hereditary hearing loss and/or vision loss, for example, due to Usher syndrome, Type 3A. In some embodiments, the disclosure provides a recombinant adeno-associated virus comprising: (i) an AAV-S capsid protein, and (ii) an isolated nucleic acid comprising a transgene (e.g., a transgene for expressing a clarin-1 protein). The present disclosure also provides methods of treating hereditary hearing loss and/or vision loss (e.g., Usher Syndrome, Type 3A) using the same.
1. A recombinant adeno-associated virus (rAAV), wherein the rAAV comprises:
(i) an AAV capsid protein comprising an amino acid sequence at least 95% identical to amino acids 203-743 of SEQ ID NO: 3, wherein the amino acid sequence comprises S, T, T, L, Y, S, and P at the positions corresponding to positions 589, 590, 591, 592, 593, 594, and 595, respectively, of SEQ ID NO: 3; and
(ii) a nucleic acid comprising two adeno-associated virus inverted terminal repeats (ITRs) flanking a transgene, wherein the transgene comprises a promoter operably linked to a nucleotide sequence encoding a human clarin-1 protein.
2. The rAAV of claim 1 , wherein the human clarin-1 protein comprises the amino acid sequence of any one of SEQ ID NO: 5-9.
3. The rAAV of claim 1 , wherein the nucleotide sequence encoding the human clarin-1 protein is at least 80% identical to the nucleotide sequence of SEQ ID NO: 10 or 19.
4. The rAAV of claim 1 , wherein the transgene further comprises a 5′ untranslated region (5′ UTR).
5. The rAAV of claim 4 , wherein the 5′ UTR is a 5′ UTR of a CLRN gene.
6. The rAAV of claim 5 , wherein the 5′ UTR comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 16.
7. The rAAV of claim 1 , wherein the transgene further comprises a 3′ untranslated region (3′ UTR).
8. The rAAV of claim 7 , wherein the 3′ UTR is a 3′ UTR of a CLRN gene.
9. The rAAV of claim 8 , wherein the 3′ UTR comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 17.
10. The rAAV of claim 1 , wherein the promoter is a constitutive promoter, an inducible promoter, or a tissue specific promoter.
11. The rAAV of claim 1 , wherein the transgene further comprises an enhancer, an intron, and/or a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
12. The rAAV of claim 1 , wherein the AAV ITRs are ITRs of one or more serotypes selected from the group consisting of AAV2, AAV3, AAV4, AAV5, and AAV6.
13. The rAAV of claim 1 , wherein the AAV capsid protein has tropism for inner ear cells and/or eye cells.
14. A cell comprising the rAAV of claim 1 .
15. A pharmaceutical composition comprising the rAAV of claim 1 and a pharmaceutically acceptable carrier.
16. A method for treating Usher syndrome Type 3A or a CLRN-associated disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV of claim 1 .
17. A recombinant adeno-associated virus comprising:
(i) an AAV capsid protein comprising an amino acid sequence at least 95% identical to amino acids 203-743 of SEQ ID NO: 3, wherein the amino acid sequence comprises S, T, T, L, Y, S, and P at the positions corresponding to positions 589, 590, 591, 592, 593, 594, and 595, respectively, of SEQ ID NO: 3; and
(ii) a nucleic acid comprising, from 5′ to 3′:
(a) a 5′ ITR;
(b) a Human Cytomegalovirus Major Immediate-Early Enhancer (CMV IE enhancer);
(c) a Chicken beta-actin (CBA) promoter;
(d) a beta-actin exon;
(e) a chimeric intron;
(f) a 5′ UTR;
(g) a Kozak sequence;
(h) a nucleotide sequence encoding a human clarin-1 protein;
(i) a 3′ UTR;
(j) a bovine growth hormone poly A signal; and
(k) a 3′ ITR.