AAV vectors for treatment of dominant retinitis pigmentosa
Aspects of the disclosure relate to methods and compositions for treating retinitis pigmentosa. In some aspects, the disclosure provides compositions and methods for delivering an interfering nucleic acid (for example an interfering RNA) to a subject in order to reduce expression of one or both alleles of an endogenous rho gene (for example a mutant rho allele associated with retinitis pigmentosa) in the subject. In some embodiments, a replacement rho gene that is resistant to the interfering nucleic acid also is delivered to the subject.
1. A synthetic ribonucleic acid (RNA) molecule comprising:
a) a sense strand of sequence
(SEQ ID NO: 1)
CUGCCUACAUGUUUCUGCU
and
an antisense strand of sequence
(SEQ ID NO: 2)
AGCAGAAACAUGUAGGCAG;
b) a sense strand of sequence
(SEQ ID NO: 3)
CCUACAUGUUUCUGCUGAU
and
an antisense strand of sequence
(SEQ ID NO: 4)
AUCAGCAGAAACAUGUAGG;
c) a sense strand of sequence
(SEQ ID NO: 5)
GCAUGGUCAUCAUCAUGGU
and
an antisense strand of sequence
(SEQ ID NO: 6)
ACCAUGAUGAUGACCAUGC;
or
d) a sense strand of sequence
(SEQ ID NO: 7)
GUGGCAUUCUACAUCUUCA
and
an antisense strand of sequence
(SEQ ID NO: 8)
UGAAGAUGUAGAAUGCCAC.
2. The synthetic RNA molecule of claim 1 , wherein the RNA is a small interfering RNA (siRNA).
3. The synthetic RNA molecule of claim 1 , wherein the RNA is a small hairpin RNA (shRNA).
4. The shRNA of claim 3 , having a loop that comprises RNA of sequence UCAAGAG (SEQ ID NO: 9) or RNA of sequence UGUGCUU (SEQ ID NO: 10).
5. The synthetic RNA molecule of claim 1 , wherein the RNA is an artificial micro RNA (miRNA).
6. The artificial miRNA of claim 5 , wherein the artificial miRNA comprises RNA of sequence UGCUGUUGACAGUGAGCGA(X) n UAGUGAAGCCACAGAUGUA(Y) n CUGCCUACUGCCU CGGA (SEQ ID NO: 19), and wherein:
a) (X) n comprises SEQ ID NO: 1 and (Y) n comprises SEQ ID NO: 2;
b) (X) n comprises SEQ ID NO: 3 and (Y) n comprises SEQ ID NO: 4;
c) (X) n comprises SEQ ID NO: 5 and (Y) n comprises SEQ ID NO: 6; or
d) (X) n comprises SEQ ID NO: 7 and (Y) n comprises SEQ ID NO: 8.
7. The synthetic RNA of claim 1 , further comprising an unpaired overhang sequence at the 5′ and/or 3′ end.
8. The synthetic RNA of claim 7 , wherein the unpaired overhang sequence comprises a sequence of repeating bases.
9. The synthetic RNA of claim 8 , wherein the sequence of repeating bases comprises repeating uracil (U) bases.
10. The synthetic RNA of claim 9 , wherein the unpaired overhang sequence is UU.
11. A composition comprising the synthetic RNA of claim 1 .
12. The composition of claim 11 , further comprising one or more physiologically acceptable carriers and/or one or more physiologically acceptable adjuvants.
13. A vector encoding:
a) the shRNA of claim 3 ; or
b) the artificial miRNA of claim 5 .
14. The vector of claim 13 , wherein the shRNA is selected from any one of SEQ ID NOs: 11-18.
15. The vector of claim 13 , wherein the vector is an expression plasmid.
16. The vector of claim 13 , wherein the vector is a viral vector.
17. The viral vector of claim 16 , wherein the viral vector comprises an adeno-associated viral vector.
18. A method of decreasing RHO expression in a subject, the method comprising administering to the subject the composition of claim 11 .
19. A method of treating retinitis pigmentosa (RP) in a subject, the method comprising administering to the subject both:
a) the composition of claim 11 ; and
b) a recombinant RHO gene that does not contain a sequence targeted by an interfering RNA of the composition of a).
20. The method of claim 19 , wherein the recombinant RHO gene is delivered using an rAAV.
21. The method of claim 20 , wherein the interfering RNA and the recombinant RHO gene are delivered using the same rAAV.
22. The method of claim 21 , wherein the interfering RNA and the recombinant RHO gene are both under expression control of a single promoter sequence.
23. The method of claim 22 , wherein the interfering RNA and the recombinant RHO gene are each under expression control of independent promoter sequences.
24. The method of claim 23 , wherein the interfering RNA is shRNA, and wherein the shRNA is under expression control of an RNA polymerase III promoter.
25. The method of claim 23 , wherein the interfering RNA is an artificial miRNA, and wherein the artificial miRNA is under expression control of an RNA polymerase II promoter.
26. The method of claim 24 , wherein the recombinant RHO gene is under expression control of a constitutive or inducible promoter.
27. The method of claim 19 , wherein the subject is a mammal.
28. The method of claim 27 , wherein the mammal is a rodent or a dog.
29. The method of claim 27 , wherein the mammal is a human.
30. A viral vector encoding the shRNA of claim 3 and a recombinant RHO sequence comprising a nucleotide sequence that comprises SEQ ID NO: 42.
31. The viral vector of claim 17 , wherein the viral vector is an AAV2, AAV2/1, AAV2/5, AAV2/8 or AAV2/9 vector.
32. The viral vector of claim 30 , wherein the vector comprises AAV2/5-sc-HOP-RHO 820 -H1-shRNA 820 .
33. The viral vector of claim 31 , wherein the vector encodes:
a) a sense strand of sequence GUGGCAUUCUACAUCUUCA (SEQ ID NO: 7) and an antisense strand of sequence UGAAGAUGUAGAAUGCCAC (SEQ ID NO: 8);
b) a recombinant RHO sequence comprising a nucleotide sequence that comprises SEQ ID NO: 42;
wherein the vector is an AAV2/5 vector.
34. The method of claim 19 , wherein the retinitis pigmentosa is autosomal dominant retinitis pigmentosa (adRP).