Intravitreal Dosing for Delivery of Polynucleotides to Retinal Cones

1 . A method of treating a color vision deficiency in a human subject in need thereof, comprising intravitreally administering to the subject a recombinant adeno-associated virus (rAAV) vector at a dosage ranging from about 1×10 10 to about 1×10 12 vector genomes (vg)/eye, wherein the rAAV vector comprises a polynucleotide comprising a human L-opsin protein coding sequence operably linked to an M-opsin promoter sequence, and wherein the rAAV vector comprises an AAV2 capsid variant that transduces foveal cone photoreceptors, wherein the polynucleotide comprises a sequence that is at least 95, 98, 99, or 100% identical to the sequence set forth in SEQ ID NO: 3, and which encodes and expresses a biologically-active human L-opsin protein.

2 . The method of claim 1 , wherein the polynucleotide comprises, in a 5′ to 3′ orientation, a 5′ AAV2 inverted terminal repeat (ITR), a human locus control region (LCR) enhancer sequence, a truncated M-opsin promoter sequence, a 5′ untranslated region (UTR) composed of an M-opsin 5′ UTR with an inserted chimeric intron and a strong Kozak sequence, the human L-opsin protein coding sequence, an M-opsin 3′ UTR, an SV40 polyadenylation sequence, and a 3′ AAV2 ITR.

3 . The method of claim 1 , wherein the AAV2 capsid variant is an AAV2.7m8 capsid or AAV2.5T.LSVI capsid that comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to the sequence set forth in SEQ ID NO: 1 or 2, and which transduces foveal cone photoreceptors.

4 . The method of claim 1 , wherein the polynucleotide consists or consists essentially of a sequence that is at least 95, 98, 99, or 100% identical to the sequence set forth in SEQ ID NO: 3, and which encodes and expresses a biologically-active human L-opsin protein.

5 . The method of claim 1 , comprising intravitreally administering the rAAV vector at a dosage of about 5×10 10 , about 1×10 11 , or about 2×10 11 vg/eye, or optionally about 1×10 10 , about 2×10 10 , about 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , or about 1×10 12 vg/eye.

6 . The method of claim 1 , comprising intravitreally administering the rAAV vector at a dosage ranging from about 1×10 10 to about 2×10 10 vg/eye, about 1×10 10 to about 3×10 10 vg/eye, about 1×10 10 to about 4×10 10 vg/eye, about 1×10 10 to about 5×10 10 vg/eye, about 1×10 10 to about 6×10 10 vg/eye, about 1×10 10 to about 7×10 10 vg/eye, about 1×10 10 to about 8×10 10 vg/eye, about 1×10 10 to about 9×10 10 vg/eye, about 1×10 10 to about 1×10 11 vg/eye, about 1×10 10 to about 2×10 11 vg/eye, about 1×10 10 to about 3×10 11 vg/eye, about 1×10 10 to about 4×10 11 vg/eye, about 1×10 10 to about 5×10 11 vg/eye, about 1×10 10 to about 6×10 11 vg/eye, about 1×10 10 to about 7×10 11 vg/eye, about 1×10 10 to about 8×10 11 vg/eye, about 1×10 10 to about 9×10 11 vg/eye, about 1×10 10 to about 1×10 12 vg/eye, about 2×10 10 to about 3×10 10 vg/eye, about 2×10 10 to about 4×10 10 vg/eye, about 2×10 10 to about 5×10 10 vg/eye, about 2×10 10 to about 6×10 10 vg/eye, about 2×10 10 to about 7×10 10 vg/eye, about 2×10 10 to about 8×10 10 vg/eye, about 2×10 10 to about 9×10 10 vg/eye, about 2×10 10 to about 1×10 11 vg/eye, about 2×10 10 to about 2×10 11 vg/eye, about 2×10 10 to about 3×10 11 vg/eye, about 2×10 10 to about 4×10 11 vg/eye, about 2×10 10 to about 5×10 11 vg/eye, about 2×10 10 to about 6×10 11 vg/eye, about 2×10 10 to about 7×10 11 vg/eye, about 2×10 10 to about 8×10 11 vg/eye, about 2×10 10 to about 9×10 11 vg/eye, about 2×10 10 to about 1×10 12 vg/eye, about 3×10 10 to about 4×10 10 vg/eye, about 3×10 10 to about 5×10 10 vg/eye, about 3×10 10 to about 6×10 10 vg/eye, about 3×10 10 to about 7×10 10 vg/eye, about 3×10 10 to about 8×10 10 vg/eye, about 3×10 10 to about 9×10 10 vg/eye, about 3×10 10 to about 1×10 11 vg/eye, about 3×10 10 to about 2×10 11 vg/eye, about 3×10 10 to about 3×10 11 vg/eye, about 3×10 10 to about 4×10 11 vg/eye, about 3×10 10 to about 5×10 11 vg/eye, about 3×10 10 to about 6×10 11 vg/eye, about 3×10 10 to about 7×10 11 vg/eye, about 3×10 10 to about 8×10 11 vg/eye, about 3×10 10 to about 9×10 11 vg/eye, about 3×10 10 to about 1×10 12 vg/eye, about 4×10 10 to about 5×10 10 vg/eye, about 4×10 10 to about 6×10 10 vg/eye, about 4×10 10 to about 7×10 10 vg/eye, about 4×10 10 to about 8×10 10 vg/eye, about 4×10 10 to about 9×10 10 vg/eye, about 4×10 10 to about 1×10 11 vg/eye, about 4×10 10 to about 2×10 11 vg/eye, about 4×10 10 to about 3×10 11 vg/eye, about 4×10 10 to about 4×10 11 vg/eye, about 4×10 10 to about 5×10 11 vg/eye, about 4×10 10 to about 6×10 11 vg/eye, about 4×10 10 to about 7×10 11 vg/eye, about 4×10 10 to about 8×10 11 vg/eye, about 4×10 10 to about 9×10 11 vg/eye, about 4×10 10 to about 1×10 12 vg/eye, about 5×10 10 to about 6×10 10 vg/eye, about 5×10 10 to about 7×10 10 vg/eye, about 5×10 10 to about 8×10 10 vg/eye, about 5×10 10 to about 9×10 10 vg/eye, about 5×10 10 to about 1×10 11 vg/eye, about 5×10 10 to about 2×10 11 vg/eye, about 5×10 10 to about 3×10 11 vg/eye, about 5×10 10 to about 4×10 11 vg/eye, about 5×10 10 to about 5×10 11 vg/eye, about 5×10 10 to about 6×10 11 vg/eye, about 5×10 10 to about 7×10 11 vg/eye, about 5×10 10 to about 8×10 11 vg/eye, about 5×10 10 to about 9×10 11 vg/eye, about 5×10 10 to about 1×10 12 vg/eye, about 6×10 10 to about 7×10 10 vg/eye, about 6×10 10 to about 8×10 10 vg/eye, about 6×10 10 to about 9×10 10 vg/eye, about 6×10 10 to about 1×10 11 vg/eye, about 6×10 10 to about 2×10 11 vg/eye, about 6×10 10 to about 3×10 11 vg/eye, about 6×10 10 to about 4×10 11 vg/eye, about 6×10 10 to about 5×10 11 vg/eye, about 6×10 10 to about 6×10 11 vg/eye, about 6×10 10 to about 7×10 11 vg/eye, about 6×10 10 to about 8×10 11 vg/eye, about 6×10 10 to about 9×10 11 vg/eye, about 6×10 10 to about 1×10 12 vg/eye, about 7×10 10 to about 8×10 10 vg/eye, about 7×10 10 to about 9×10 10 vg/eye, about 7×10 10 to about 1×10 11 vg/eye, about 7×10 10 to about 2×10 11 vg/eye, about 7×10 10 to about 3×10 11 vg/eye, about 7×10 10 to about 4×10 11 vg/eye, about 7×10 10 to about 5×10 11 vg/eye, about 7×10 10 to about 6×10 11 vg/eye, about 7×10 10 to about 7×10 11 vg/eye, about 7×10 10 to about 8×10 11 vg/eye, about 7×10 10 to about 9×10 11 vg/eye, about 7×10 10 to about 1×10 12 vg/eye, about 8×10 10 to about 9×10 10 vg/eye, about 8×10 10 to about 1×10 11 vg/eye, about 8×10 10 to about 2×10 11 vg/eye, about 8×10 10 to about 3×10 11 vg/eye, about 8×10 10 to about 4×10 11 vg/eye, about 8×10 10 to about 5×10 11 vg/eye, about 8×10 10 to about 6×10 11 vg/eye, about 8×10 10 to about 7×10 11 vg/eye, about 8×10 10 to about 8×10 11 vg/eye, about 8×10 10 to about 9×10 11 vg/eye, about 8×10 10 to about 1×10 12 vg/eye, about 9×10 10 to about 1×10 11 vg/eye, about 9×10 10 to about 2×10 11 vg/eye, about 9×10 10 to about 3×10 11 vg/eye, about 9×10 10 to about 4×10 11 vg/eye, about 9×10 10 to about 5×10 11 vg/eye, about 9×10 10 to about 6×10 11 vg/eye, about 9×10 10 to about 7×10 11 vg/eye, about 9×10 10 to about 8×10 11 vg/eye, about 9×10 10 to about 9×10 11 vg/eye, about 9×10 10 to about 1×10 12 vg/eye, about 1×10 11 to about 2×10 11 vg/eye, about 1×10 11 to about 3×10 11 vg/eye, about 1×10 11 to about 4×10 11 vg/eye, about 1×10 11 to about 5×10 11 vg/eye, about 1×10 11 to about 6×10 11 vg/eye, about 1×10 11 to about 7×10 11 vg/eye, about 1×10 11 to about 8×10 11 vg/eye, about 1×10 11 to about 9×10 11 vg/eye, about 1×10 11 to about 1×10 12 vg/eye, about 2×10 11 to about 3×10 11 vg/eye, about 2×10 11 to about 4×10 11 vg/eye, about 2×10 11 to about 5×10 11 vg/eye, about 2×10 11 to about 6×10 11 vg/eye, about 2×10 11 to about 7×10 11 vg/eye, about 2×10 11 to about 8×10 11 vg/eye, about 2×10 11 to about 9×10 11 vg/eye, about 2×10 11 to about 1×10 12 vg/eye, about 3×10 11 to about 4×10 11 vg/eye, about 3×10 11 to about 5×10 11 vg/eye, about 3×10 11 to about 6×10 11 vg/eye, about 3×10 11 to about 7×10 11 vg/eye, about 3×10 11 to about 8×10 11 vg/eye, about 3×10 11 to about 9×10 11 vg/eye, about 3×10 11 to about 1×10 12 vg/eye, about 4×10 11 to about 5×10 11 vg/eye, about 4×10 11 to about 6×10 11 vg/eye, about 4×10 11 to about 7×10 11 vg/eye, about 4×10 11 to about 8×10 11 vg/eye, about 4×10 11 to about 9×10 11 vg/eye, about 4×10 11 to about 1×10 12 vg/eye, about 5×10 11 to about 6×10 11 vg/eye, about 5×10 11 to about 7×10 11 vg/eye, about 5×10 11 to about 8×10 11 vg/eye, about 5×10 11 to about 9×10 11 vg/eye, about 5×10 11 to about 1×10 12 vg/eye, about 6×10 11 to about 7×10 11 vg/eye, about 6×10 11 to about 8×10 11 vg/eye, about 6×10 11 to about 9×10 11 vg/eye, about 6×10 11 to about 1×10 12 vg/eye, about 7×10 11 to about 8×10 11 vg/eye, about 7×10 11 to about 9×10 11 vg/eye, about 7×10 11 to about 1×10 12 vg/eye, about 8×10 11 to about 9×10 11 vg/eye, about 8×10 11 to about 1×10 12 vg/eye, or about 9×10 11 to about 1×10 12 vg/eye.

7 . The method of claim 6 , comprising intravitreally administering the rAAV vector at a dosage ranging from about 6×10 10 to about 6×10 11 vg/eye.

8 . The method of claim 7 , comprising intravitreally administering the rAAV vector at a dosage of about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 vg/eye, about 2×10 11 vg/eye, about 3×10 11 vg/eye, about 4×10 11 vg/eye, about 5×10 11 vg/eye, or about 6×10 11 vg/eye.

9 . The method of claim 7 , comprising intravitreally administering the rAAV vector at a dosage ranging from about 6×10 10 to about 7×10 10 vg/eye, about 6×10 10 to about 8×10 10 vg/eye, about 6×10 10 to about 9×10 10 vg/eye, about 6×10 10 to about 1×10 11 vg/eye, about 6×10 10 to about 2×10 11 vg/eye, about 6×10 10 to about 3×10 11 vg/eye, about 6×10 10 to about 4×10 11 vg/eye, about 6×10 10 to about 5×10 11 vg/eye, about 6×10 10 to about 6×10 11 vg/eye, about 7×10 10 to about 8×10 10 vg/eye, about 7×10 10 to about 9×10 10 vg/eye, about 7×10 10 to about 1×10 11 vg/eye, about 7×10 10 to about 2×10 11 vg/eye, about 7×10 10 to about 3×10 11 vg/eye, about 7×10 10 to about 4×10 11 vg/eye, about 7×10 10 to about 5×10 11 vg/eye, about 7×10 10 to about 6×10 11 vg/eye, about 8×10 10 to about 9×10 10 vg/eye, about 8×10 10 to about 1×10 11 vg/eye, about 8×10 10 to about 2×10 11 vg/eye, about 8×10 10 to about 3×10 11 vg/eye, about 8×10 10 to about 4×10 11 vg/eye, about 8×10 10 to about 5×10 11 vg/eye, about 8×10 10 to about 6×10 11 vg/eye, about 9×10 10 to about 1×10 11 vg/eye, about 9×10 10 to about 2×10 11 vg/eye, about 9×10 10 to about 3×10 11 vg/eye, about 9×10 10 to about 4×10 11 vg/eye, about 9×10 10 to about 5×10 11 vg/eye, about 9×10 10 to about 6×10 11 vg/eye, about 1×10 11 to about 2×10 11 vg/eye, about 1×10 11 to about 3×10 11 vg/eye, about 1×10 11 to about 4×10 11 vg/eye, about 1×10 11 to about 5×10 11 vg/eye, about 1×10 11 to about 6×10 11 vg/eye, about 2×10 11 to about 3×10 11 vg/eye, about 2×10 11 to about 4×10 11 vg/eye, about 2×10 11 to about 5×10 11 vg/eye, about 2×10 11 to about 6×10 11 vg/eye, about 3×10 11 to about 4×10 11 vg/eye, about 3×10 11 to about 5×10 11 vg/eye, about 3×10 11 to about 6×10 11 vg/eye, about 4×10 11 to about 5×10 11 vg/eye, about 4×10 11 to about 6×10 11 vg/eye, or about 5×10 11 to about 6×10 11 vg/eye.

10 . The method of claim 1 , wherein the color vision deficiency is Blue Cone Monochromacy (BCM) or Red-Green Color Blindness.

11 . The method of claim 1 , wherein the subject prior to treatment has impaired color discrimination, decreased visual acuity, nystagmus, and/or photophobia.

12 . The method of claim 1 , wherein the subject prior to treatment has decreased functional L-opsins and M-opsins, inactive L-cones and M-cones, and decreased foveal vision.

13 . The method of claim 1 , wherein the subject is characterized by one or more genetic mutations selected from deletions in the locus control region (LCR), nonhomologous recombination between the L- and M-opsin genes and at least one missense point mutation inactivating the residual gene, optionally a C203R mutation, and/or one or more inactivating mutations within the LCR that inhibit expression of the L-opsin and M-opsin genes.

14 . The method of claim 1 , wherein the subject is a male, optionally about 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, or 60 years of age; or wherein the subject is a pediatric subject, and the dosage is about 2×10 10 vg/eye or 3×10 10 vg/eye.

15 . The method of claim 1 , wherein intravitreally administering the rAAV vector transduces about or at least about 10% to 85% of the foveal cones in the subject with the polynucleotide encoding and expressing the human L-opsin protein.

16 . The method of claim 15 , wherein intravitreally administering the rAAV vector transduces about or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85% of the foveal cones in the subject with the polynucleotide encoding and expressing the human L-opsin protein.

17 . The method of claim 1 , wherein intravitreally administering the rAAV vector improves color discrimination, increases visual acuity, decreases nystagmus, and/or decreases photophobia in the subject.

18 . The method of claim 1 , comprising intravitreally administering to the subject a single dosage per eye of the rAAV vector.

19 . The method of claim 1 , comprising intravitreally administering to the subject a dosage in both eyes simultaneously or nearly so.

20 . The method of claim 1 , comprising intravitreally administering to the subject a dosage in both eyes sequentially, optionally wherein the interval between administering the dosage to the first eye and administering the dosage to the second eye is about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or about or at least about 2, 3, 4, 5, 6, 7, or 8 weeks.

21 . The method of claim 20 , wherein the dosage administered to the second eye is greater than the dosage administered to the first eye, optionally wherein the second dosage is about or at least about 1.5×, 2×, 2.5×, 3×, or 3.5× the first dosage.

22 .- 39 . (canceled)