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 - 29 . (canceled)
30 . 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 AAV2 VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 11) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein; and
b) the therapeutic polynucleotide comprises a regulatory cassette operably linked to a polynucleotide encoding a therapeutic protein, wherein the regulatory cassette comprises a human L/M opsin Locus Control Region (“LCR”) enhancer and a truncated M-opsin promoter consisting of about 140 nucleotides upstream of the transcription start site.
31 . The method according to claim 30 , wherein the variant AAV2 VP1 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 13) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein.
32 . The method according to claim 30 , wherein the rAAV variant comprises a VP1 protein having a sequence identity of at least 80% to the polypeptide of SEQ ID NO: 19.
33 . The method according to claim 32 , wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO: 19.
34 . The method according to claim 33 , wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO: 19.
35 . The method according to claim 34 , wherein the VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO: 19.
36 . The method according to claim 30 , wherein the cone photoreceptor is a foveal cone.
37 . The method according to claim 30 , wherein the subject is a primate.
38 . 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 AAV2 VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 11) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein; and
b) the therapeutic polynucleotide comprises a regulatory cassette operably linked to a polynucleotide encoding a therapeutic protein, wherein the regulatory cassette comprises a human L/M opsin Locus Control Region (“LCR”) enhancer and a truncated M-opsin promoter consisting of about 140 nucleotides upstream of the transcription start site.
39 . The method according to claim 38 , wherein the variant AAV2 VP1 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 13) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein.
40 . The method according to claim 38 , wherein the rAAV variant comprises a VP1 protein having a sequence identity of at least 80% to the polypeptide of SEQ ID NO: 19.
41 . The method according to claim 40 , wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO: 19.
42 . The method according to claim 41 , wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO: 19.
43 . The method according to claim 42 , wherein VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO: 19.
44 . The method according to claim 38 , wherein the method further comprises detecting the expression of the polynucleotide in the cone photoreceptor.
45 . The method according to claim 38 , wherein the cone photoreceptor is a foveal cone.
46 . The method according to claim 38 , wherein the subject is a primate.
47 . 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 AAV2 VP1 capsid protein comprising the amino acid sequence LGETTRP (SEQ ID NO: 11) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein; and
b) the therapeutic polynucleotide comprises a regulatory cassette operably linked to a polynucleotide encoding a therapeutic protein, wherein the regulatory cassette comprises a human L/M opsin Locus Control Region (“LCR”) enhancer and a truncated M-opsin promoter consisting of about 140 nucleotides upstream of the transcription start site.
48 . The method according to claim 47 , wherein the variant AAV2 VP1 capsid protein comprises the amino acid sequence LALGETTRPA (SEQ ID NO: 13) inserted into the GH loop between amino acids 587 and 588 of the parental AAV2 VP1 capsid protein.
49 . The method according to claim 47 , wherein the VP1 protein has a sequence identity of at least 80% to the polypeptide of SEQ ID NO: 19.
50 . The method according to claim 49 , wherein the VP1 protein has a sequence identity of at least 95% to the polypeptide of SEQ ID NO: 19.
51 . The method according to claim 50 , wherein the VP1 protein has a sequence identity of at least 99% to the polypeptide of SEQ ID NO: 19.
52 . The method according to claim 51 , wherein the VP1 protein has a sequence identity of 100% to the polypeptide of SEQ ID NO: 19.
53 . The method according to claim 47 , wherein the retinal disorder is a cone-associated disorder.
54 . The method according to claim 53 , 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.
55 . The method according to claim 47 , wherein the method further comprises identifying the subject as having a cone-associated disorder.
56 . The method according to claim 47 , wherein the method further comprises detecting an improvement in vision following the administering step.
57 . The method according to claim 47 , wherein the subject is a primate.
58 . The method according to claim 47 , wherein the retinal disorder is a color vision disorder.
59 . The method according to claim 58 , wherein the color vision disorder is blue cone monochromacy.
60 . The method according to claim 58 , wherein the color vision disorder is color vision deficiency.