Gene therapy for retinitis pigmentosa
Provided herein are methods for treating retinitis pigmentosa using an AAV particles encoding miR-708. In one aspect, viral particles are administered to the eye of a human subject; for example, by subretinal injection. Viral particles comprising AAV5 capsids or mutants thereof are contemplated.
1. A rAAV particle comprising a rAAV vector comprising a nucleic acid encoding a miR-708 and a nucleic acid encoding rhodopsin, wherein the nucleic acid encoding rhodopsin comprises a substitution, insertion or deletion of nucleic acid in the miR-708 target sequence wherein the miR-708 target sequence is SEQ ID NO:19.
2. The rAAV particle of claim 1 , wherein the nucleic acid encoding miR-708 is operably linked to a promoter.
3. The rAAV particle of claim 2 , wherein the promoter comprises a RK promoter or an opsin promoter, or a chicken β-actin (CBA) promoter.
4. The rAAV particle of claim 1 , wherein the nucleic acid encoding rhodopsin is operably linked to a promoter.
5. The rAAV particle of claim 4 , wherein the promoter comprises a RK promoter or an opsin promoter.
6. The rAAV particle of claim 5 , wherein i) the nucleic acid encoding miR-708 and the nucleic acid encoding rhodopsin are operably linked to one RK promoter or opsin promoter; or ii) the nucleic acid encoding miR-708 is operably linked to a first RK promoter or a first opsin promoter and the nucleic acid encoding rhodopsin is operably linked to a second RK promoter or a second opsin promoter.
7. The rAAV particle of claim 2 , wherein a sequence derived from a minute virus of mouse (MVM) intron is located 3′ to the promoter.
8. The rAAV particle of claim 2 , wherein the promoter further comprises
i) a CMV enhancer;
ii) a sequence derived from a photoreceptor specific transcription factor;
iii) a sequence derived from a rod photoreceptor specific transcription factor;
iv) a sequence derived from a neural retinal basic zipper factor;
v) a sequence derived from a cone rod homeobox-containing transcription factor sequence;
vi) a CMV enhancer and at least one or more of a sequence derived from a photoreceptor specific transcription factor, a sequence derived from a rod photoreceptor specific transcription factor, a sequence derived from a neural retinal basic zipper factor; or a sequence derived from a cone rod homeobox-containing transcription factor sequence;
vii) a neural retinal basic leucine zipper factor, a CMV enhancer and an Opsin promoter (−500 to +17);
viii) a neural retinal basic leucine zipper factor, a CMV enhancer, an Opsin promoter (−500 to +17), and an MVM intron;
ix) a CMV enhancer comprising SEQ ID NO:29;
x) a neural retinal basic leucine zipper factor sequence comprising SEQ ID NO:30;
xi) a sequence derived from a cone rod homeobox-containing transcription factor sequence comprising SEQ ID NO:28;
xii) a CMV enhancer comprising SEQ ID NO:29 and at least one or more of a sequence derived from a photoreceptor specific transcription factor, a sequence derived from a rod photoreceptor specific transcription factor, a sequence derived from a neural retinal basic zipper factor comprising SEQ ID NO:30; a sequence derived from a cone rod homeobox-containing transcription factor sequence comprising SEQ ID NO:28;
xiii) a neural retinal basic leucine zipper factor comprising SEQ ID NO:30, a CMV enhancer comprising SEQ ID NO:29 and an Opsin promoter (−500 to +17) comprising SEQ ID NO:22; or
xiv) a neural retinal basic leucine zipper factor comprising SEQ ID NO:30, a CMV enhancer comprising SEQ ID NO:29, an Opsin promoter (−500 to +17) comprising SEQ ID NO:22, and an MVM intron comprising SEQ ID NO:23.
9. The rAAV particle of claim 1 , wherein the nucleic acid encoding miR-708 is embedded in an intron.
10. The rAAV particle of claim 1 , wherein the nucleic acid encoding miR-708 comprises an endogenous miR-708 scaffold or a miR-155 scaffold.
11. The rAAV particle of claim 1 , wherein the rhodopsin is human rhodopsin.
12. The rAAV particle of claim 1 , wherein the substitution, insertion or deletion reduces or prevents recognition by miR-708.
13. The rAAV particle of claim 1 , wherein the rhodopsin lacks the 3′ UTR miR-708 target sequence.
14. The rAAV particle of claim 1 , wherein expression of the rhodopsin is refractory to suppression by miR-708.
15. The rAAV particle of claim 1 , wherein the nucleic acid encoding miR-708 comprises the nucleic acid of SEQ ID NO:1, or a nucleic acid having about at least 85% identity to SEQ ID NO:1 and the nucleic acid encoding the rhodopsin comprises nucleic acid of SEQ ID NO:3 or a nucleic acid having about 85% identity to SEQ ID NO:3.
16. The rAAV particle of claim 1 , wherein the rhodopsin comprises the amino acid sequence of SEQ ID NO:2, or amino acid sequence having about at least 85% identity to SEQ ID NO:2.
17. The rAAV particle of claim 1 , wherein the AAV viral particle comprises a recombinant viral genome comprising a polynucleotide of SEQ ID NO:5, or a polynucleotide having about at least 85% identity to SEQ ID NO:5.
18. The rAAV particle of claim 1 , wherein the AAV viral particle comprises a recombinant viral genome comprising a polynucleotide of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, or a polynucleotide having about at least 85% identity to SEQ ID NO:6 SEQ ID NO:7, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.
19. The rAAV particle of claim 1 , wherein the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2_R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1/AAV2 chimeric AAV, bovine AAV, mouse AAV, or rAAV2/HBoV1 serotype capsid.
20. The rAAV particle of claim 1 , wherein the rAAV vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype inverted terminal repeats (ITRs).
21. The rAAV particle of claim 1 , wherein the ITR and the capsid of the rAAV viral particles are derived from different AAV serotypes.
22. The rAAV particle of claim 21 , wherein the rAAV viral particle comprises an AAV-5 capsid or an AAV-5 tyrosine mutant capsid, and wherein the vector comprises AAV2 ITRs.
23. A composition comprising the rAAV particle of claim 1 .
24. A composition comprising a first rAAV particle comprising a rAAV vector comprising a nucleic acid encoding a miR-708 and a second rAAV particle comprising a rAAV vector comprising nucleic acid encoding rhodopsin, wherein the nucleic acid encoding rhodopsin comprises a substitution, insertion or deletion of nucleic acid in the miR-708 target sequence wherein the miR-708 target sequence is SEQ ID NO:19.
25. A kit comprising an effective amount of the rAAV particle according to claim 1 .
26. A kit comprising an effective amount of the composition of claim 24 .
27. An article of manufacture comprising an effective amount of the rAAV particle according to claim 1 .
28. An article of manufacture comprising an effective amount of the composition of claim 24 .