Compositions and methods for intravitreal delivery of polynucleotides to retinal cones
Methods and compositions are provided for intravitreally delivering a polynucleotide to cone photoreceptors. Aspects of the methods include injecting a recombinant adeno-associated virus comprising a polynucleotide of interest into the vitreous of the eye. These methods and compositions find particular use in treating ocular disorders associated with cone dysfunction and/or death.
1. A method for delivering a polynucleotide of interest to a cone photoreceptor in a subject, the method comprising:
delivering into the vitreous of the eye an effective amount of recombinant adeno-associated virus (rAAV) variant comprising the polynucleotide of interest, wherein:
a) the rAAV variant comprises a variant AAV capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO:11) inserted into the GH loop of the VP1 capsid protein; and
b) the polynucleotide of interest comprises a regulatory cassette operably linked to a polynucleotide sequence encoding a therapeutic protein,
wherein the regulatory cassette comprises the sequence:
(SEQ ID NO: 27)
CCTACAGCAGCCAGGGTGAGATTATGAGGCTGAGCTGAGAATATCAAGACT
GTACCGAGTAGGGGGCCTTGGCAAGTGTGGAGAGCCCGGCAGCTGGGGCAG
AGGGCGGAGTACGGTGTGCGTTTACGGACCTCTTCAAACGAGGTAGGAAGG
TCAGAAGTCAAAAAGGGAACAAATGATGTTTAACCACACAAAAATGAAAAT
CCAATGGTTGGATATCCATTCCAAATACACAAAGGCAACGGATAAGTGATC
CGGGCCAGGCACAGAAGGCCATGCACCCGTAGGATTGCACTCAGAGCTCCC
AAATGCATAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAG
GGCATGGGTGTTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAG
TTTGGAGACGGATGGTGGTGACTGGACTATACACTTACACACGGTAGCGAT
GGTACACTTTGTATTATGTATATTTTACCACGATCTTTTTAAAGTGTCAAA
GGCAAATGGCCAAATGGTTCCTTGTCCTATAGCTGTAGCAGCCATCGGCTG
TTAGTGACAAAGCCCCTGAGTCAAGATGACAGCAGCCCCCATAACTCCTAA
TCGGCTCTCCCGCGTGGAGTCATTTAGGAGTAGTCGCATTAGAGACAAGTC
CAACATCTAATCTTCCACCCTGGCCAGGGCCCCAGCTGGCAGCGAGGGTGG
GAGACTCCGGGCAGAGCAGAGGGCGCTGACATTGGGGCCCGGCCTGGCTTG
GGTCCCTCTGGCCTTTCCCCAGGGGCCCTCTTTCCTTGGGGCTTTCTTGGG
CCGCCACTGCTCCCGCTCCTCTCCCCCCATCCCACCCCCTCACCCCCTCGT
TCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTTCTGCAA
GAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTGCCC
CCACAGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAAT
GGGACTTGATCTTCTGTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAG
GTGCAGAACCTACCGCCTTTCCAGGCCTCCTCCCACCTCTGCCACCTCCAC
TCTCCTTCCTGGGATGTGGGGGCTGGCACACGTGTGGCCCAGGGCATTGGT
GGGATTGCACTGAGCTGGGTCATTAGCGTAATCCTGGACAAGGGCAGACAG
GGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCCCC
AGACACCCCACTCCTCCTCTGCTGGACCCCCACTTCATAGGGCACTTCGTG
TTCTCAAAGGGCTTCCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCC
TCAGAGTTGCTTATCTCCCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGG
AGAGGTCGCCCTGTTCAAGGCCACCCAGCCAGCTCATGGCGGTAATGGGAC
AAGGCTGGCCAGCCATCCCACCCTCAGAAGGGACCCGGTGGGGCAGGTGAT
CTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTCCAGCAAATCCCTCTG
AGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGA
GGAGGTCTAAGTCCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGG
GAGCTTTTAAGGTGAAGAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGC
ACCGTGACCCTCAGGTGACGCACCAGGGCCGGCTGCCGTCGGGGACAGGGC
TTTCCATAGCCCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACC
AATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGG
CACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGCCCAG
AGAGGAGACAGGCCGCCACC.
2. The method according to claim 1 , wherein the rAAV variant comprises a VP1 protein having a sequence identity of at least 80% to the polypeptide of SEQ ID NO:19.
3. The method according to claim 2 , wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO:19.
4. The method according to claim 3 , wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO:19.
5. The method according to claim 4 , wherein the VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO:19.
6. The method according to claim 1 , wherein the subject is a primate.
7. A method for expressing a gene product in a cone photoreceptor in a subject, the method comprising:
delivering into the vitreous of the eye an effective amount of recombinant adeno-associated virus (rAAV) variant comprising a polynucleotide that encodes the gene product, wherein:
a) the rAAV variant comprises a variant AAV capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO:11) inserted into the GH loop of the VP1 capsid protein; and
b) the polynucleotide comprises a regulatory cassette operably linked to a polynucleotide sequence encoding the gene product,
wherein the regulatory cassette comprises the sequence:
(SEQ ID NO: 27)
CCTACAGCAGCCAGGGTGAGATTATGAGGCTGAGCTGAGAATATCAAGACT
GTACCGAGTAGGGGGCCTTGGCAAGTGTGGAGAGCCCGGCAGCTGGGGCAG
AGGGCGGAGTACGGTGTGCGTTTACGGACCTCTTCAAACGAGGTAGGAAGG
TCAGAAGTCAAAAAGGGAACAAATGATGTTTAACCACACAAAAATGAAAAT
CCAATGGTTGGATATCCATTCCAAATACACAAAGGCAACGGATAAGTGATC
CGGGCCAGGCACAGAAGGCCATGCACCCGTAGGATTGCACTCAGAGCTCCC
AAATGCATAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAG
GGCATGGGTGTTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAG
TTTGGAGACGGATGGTGGTGACTGGACTATACACTTACACACGGTAGCGAT
GGTACACTTTGTATTATGTATATTTTACCACGATCTTTTTAAAGTGTCAAA
GGCAAATGGCCAAATGGTTCCTTGTCCTATAGCTGTAGCAGCCATCGGCTG
TTAGTGACAAAGCCCCTGAGTCAAGATGACAGCAGCCCCCATAACTCCTAA
TCGGCTCTCCCGCGTGGAGTCATTTAGGAGTAGTCGCATTAGAGACAAGTC
CAACATCTAATCTTCCACCCTGGCCAGGGCCCCAGCTGGCAGCGAGGGTGG
GAGACTCCGGGCAGAGCAGAGGGCGCTGACATTGGGGCCCGGCCTGGCTTG
GGTCCCTCTGGCCTTTCCCCAGGGGCCCTCTTTCCTTGGGGCTTTCTTGGG
CCGCCACTGCTCCCGCTCCTCTCCCCCCATCCCACCCCCTCACCCCCTCGT
TCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTTCTGCAA
GAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTGCCC
CCACAGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAAT
GGGACTTGATCTTCTGTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAG
GTGCAGAACCTACCGCCTTTCCAGGCCTCCTCCCACCTCTGCCACCTCCAC
TCTCCTTCCTGGGATGTGGGGGCTGGCACACGTGTGGCCCAGGGCATTGGT
GGGATTGCACTGAGCTGGGTCATTAGCGTAATCCTGGACAAGGGCAGACAG
GGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCCCC
AGACACCCCACTCCTCCTCTGCTGGACCCCCACTTCATAGGGCACTTCGTG
TTCTCAAAGGGCTTCCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCC
TCAGAGTTGCTTATCTCCCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGG
AGAGGTCGCCCTGTTCAAGGCCACCCAGCCAGCTCATGGCGGTAATGGGAC
AAGGCTGGCCAGCCATCCCACCCTCAGAAGGGACCCGGTGGGGCAGGTGAT
CTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTCCAGCAAATCCCTCTG
AGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGA
GGAGGTCTAAGTCCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGG
GAGCTTTTAAGGTGAAGAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGC
ACCGTGACCCTCAGGTGACGCACCAGGGCCGGCTGCCGTCGGGGACAGGGC
TTTCCATAGCCCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACC
AATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGG
CACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGCCCAG
AGAGGAGACAGGCCGCCACC.
8. The method according to claim 7 , wherein the rAAV variant comprises a VP1 protein having a sequence identity of at least 80% to the polypeptide of SEQ ID NO:19.
9. The method according to claim 8 , wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO:19.
10. The method according to claim 9 , wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO:19.
11. The method according to claim 10 , wherein VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO:19.
12. The method according to claim 7 , wherein the method further comprises detecting the expression of the polynucleotide in the cone photoreceptor.
13. The method according to claim 7 , wherein the subject is a primate.
14. A method for treating or preventing a cone-associated retinal disorder in a subject having or at risk for developing a cone-associated retinal disorder, the method comprising:
administering intravitreally a recombinant adeno-associated virus (rAAV) variant comprising a therapeutic polynucleotide in an amount effective to treat or prevent the cone-associated retinal disorder, wherein:
a) the rAAV variant comprises a variant AAV capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO:11) inserted into the GH loop of the VP1 capsid protein; and
b) the therapeutic polynucleotide comprises a regulatory cassette operably linked to a polynucleotide sequence encoding a therapeutic protein,
wherein the regulatory cassette comprises the sequence:
(SEQ ID NO: 27)
CCTACAGCAGCCAGGGTGAGATTATGAGGCTGAGCTGAGAATATCAAGACT
GTACCGAGTAGGGGGCCTTGGCAAGTGTGGAGAGCCCGGCAGCTGGGGCAG
AGGGCGGAGTACGGTGTGCGTTTACGGACCTCTTCAAACGAGGTAGGAAGG
TCAGAAGTCAAAAAGGGAACAAATGATGTTTAACCACACAAAAATGAAAAT
CCAATGGTTGGATATCCATTCCAAATACACAAAGGCAACGGATAAGTGATC
CGGGCCAGGCACAGAAGGCCATGCACCCGTAGGATTGCACTCAGAGCTCCC
AAATGCATAGGAATAGAAGGGTGGGTGCAGGAGGCTGAGGGGTGGGGAAAG
GGCATGGGTGTTTCATGAGGACAGAGCTTCCGTTTCATGCAATGAAAAGAG
TTTGGAGACGGATGGTGGTGACTGGACTATACACTTACACACGGTAGCGAT
GGTACACTTTGTATTATGTATATTTTACCACGATCTTTTTAAAGTGTCAAA
GGCAAATGGCCAAATGGTTCCTTGTCCTATAGCTGTAGCAGCCATCGGCTG
TTAGTGACAAAGCCCCTGAGTCAAGATGACAGCAGCCCCCATAACTCCTAA
TCGGCTCTCCCGCGTGGAGTCATTTAGGAGTAGTCGCATTAGAGACAAGTC
CAACATCTAATCTTCCACCCTGGCCAGGGCCCCAGCTGGCAGCGAGGGTGG
GAGACTCCGGGCAGAGCAGAGGGCGCTGACATTGGGGCCCGGCCTGGCTTG
GGTCCCTCTGGCCTTTCCCCAGGGGCCCTCTTTCCTTGGGGCTTTCTTGGG
CCGCCACTGCTCCCGCTCCTCTCCCCCCATCCCACCCCCTCACCCCCTCGT
TCTTCATATCCTTCTCTAGTGCTCCCTCCACTTTCATCCACCCTTCTGCAA
GAGTGTGGGACCACAAATGAGTTTTCACCTGGCCTGGGGACACACGTGCCC
CCACAGGTGCTGAGTGACTTTCTAGGACAGTAATCTGCTTTAGGCTAAAAT
GGGACTTGATCTTCTGTTAGCCCTAATCATCAATTAGCAGAGCCGGTGAAG
GTGCAGAACCTACCGCCTTTCCAGGCCTCCTCCCACCTCTGCCACCTCCAC
TCTCCTTCCTGGGATGTGGGGGCTGGCACACGTGTGGCCCAGGGCATTGGT
GGGATTGCACTGAGCTGGGTCATTAGCGTAATCCTGGACAAGGGCAGACAG
GGCGAGCGGAGGGCCAGCTCCGGGGCTCAGGCAAGGCTGGGGGCTTCCCCC
AGACACCCCACTCCTCCTCTGCTGGACCCCCACTTCATAGGGCACTTCGTG
TTCTCAAAGGGCTTCCAAATAGCATGGTGGCCTTGGATGCCCAGGGAAGCC
TCAGAGTTGCTTATCTCCCTCTAGACAGAAGGGGAATCTCGGTCAAGAGGG
AGAGGTCGCCCTGTTCAAGGCCACCCAGCCAGCTCATGGCGGTAATGGGAC
AAGGCTGGCCAGCCATCCCACCCTCAGAAGGGACCCGGTGGGGCAGGTGAT
CTCAGAGGAGGCTCACTTCTGGGTCTCACATTCTTCCAGCAAATCCCTCTG
AGCCGCCCCCGGGGGCTCGCCTCAGGAGCAAGGAAGCAAGGGGTGGGAGGA
GGAGGTCTAAGTCCCAGGCCCAATTAAGAGATCAGATGGTGTAGGATTTGG
GAGCTTTTAAGGTGAAGAGGCCCGGGCTGATCCCACTGGCCGGTATAAAGC
ACCGTGACCCTCAGGTGACGCACCAGGGCCGGCTGCCGTCGGGGACAGGGC
TTTCCATAGCCCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACC
AATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGG
CACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGCCCAG
AGAGGAGACAGGCCGCCACC.
15. The method according to claim 14 , wherein the VP1 protein having a sequence identity of at least 80% to the polypeptide of SEQ ID NO:19.
16. The method according to claim 15 , wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO:19.
17. The method according to claim 16 , wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO:19.
18. The method according to claim 17 , wherein the VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO:19.
19. The method according to claim 14 , wherein the retinal disorder is a cone-associated disorder.
20. The method according to claim 19 , wherein the cone-associated disorder is selected from the group consisting of rod-cone dystrophy; cone-rod dystrophy; progressive cone dystrophy; retinitis pigmentosa (RP); Stargardt Disease; macular telangiectasia, Leber hereditary optic neuropathy, Best's disease; adult vitelliform macular dystrophy; X-linked retinoschisis; a color vision disorder; age-related macular degeneration; wet age-related macular degeneration; geographic atrophy; diabetic retinopathy; a retinal vein occlusion; retinal ischemia; Familial Exudative Vitreoretinopathy (FEVR); COATs disease; and Sorsby's fundus dystrophy.
21. The method according to claim 14 , wherein the subject is a primate.
22. The method according to claim 14 , wherein the retinal disorder is a color vision disorder.
23. The method according to claim 22 , wherein the color vision disorder is blue cone monochromacy.
24. The method according to claim 22 , wherein the color vision disorder is color vision deficiency.