Compositions and methods for intravitreal delivery of polynucleotides to retinal cones

Abstract

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.