CODON OPTIMIZED RPGRORF15 GENES AND USES THEREOF
The present disclosure provides codon optimized RPGRorf15 sequences, vectors, and host cells comprising codon optimized RPGRorf15 sequences, and methods of treating retinal disorders such as XLRP comprising administering to the subject a codon optimized RPGRorf15 sequence.
1 - 41 . (canceled)
42 . A nucleic acid encoding human retinitis pigmentosa GTPase regulator (RPGR) protein of SEQ ID NO:2 and codon optimized for expression in humans, the nucleic acid comprising a nucleotide sequence at least 91% identical to the nucleotide sequence set forth as SEQ ID NO:1.
43 . The nucleic acid according to claim 42 , wherein the nucleotide sequence has a codon adaptation index of at least 0.89.
44 . An expression cassette comprising a nucleic acid according to claim 42 , wherein the nucleotide sequence at least 91% identical to SEQ ID NO:1 is operably linked to an expression control sequence.
45 . The expression cassette of claim 44 , wherein the expression control sequence is a constitutive promoter or is a promoter that directs preferential expression of the nucleic acid in rod and cone cells.
46 . The expression cassette of claim 45 , wherein the expression control sequence is a human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter.
47 . The expression cassette of claim 46 , comprising from 5′ to 3′: (a) an AAV2 terminal repeat (b) an hGRK promoter of SEQ ID NO:4 (c) codon optimized RPGRorf15 gene comprising a nucleotide sequence at least 91% identical to the nucleotide sequence set forth as SEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) an AAV2 terminal repeat.
48 . The expression cassette of claim 47 , wherein the 5′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ ID NO:6 and/or wherein the hGRK promoter has the nucleotide sequence set forth as SEQ ID NO:4 and/or wherein the SV40 polyadenylation sequence has the nucleotide sequence set forth as SEQ ID NO:8 and/or wherein the 3′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ ID NO: 7.
49 . The expression cassette of claim 48 , comprising or consisting of the nucleotide sequence of SEQ ID NO:5 or a sequence at least at least 91% identical thereto.
50 . A recombinant adeno-associated virus (rAAV) vector comprising a heterologous nucleic acid comprising the expression cassette according to claim 44 .
51 . The rAAV vector of claim 50 , wherein the rAAV vector comprises an AAV capsid of serotype 2, 5 or 8 or a variant thereof.
52 . The rAAV vector of claim 51 , wherein the rAAV vector comprises a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO:9.
53 . The rAAV vector of claim 52 , wherein the rAAV vector comprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat (b) an hGRK promoter (c) codon optimized RPGRorf15 gene comprising a nucleotide sequence at least 91% identical to the nucleotide sequence set forth as SEQ ID NO:1 and (d) an AAV2 terminal repeat.
54 . The rAAV vector of claim 53 , wherein the 5′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ ID NO:6 and/or wherein the hGRK promoter has the nucleotide sequence set forth as SEQ ID NO′4 and/or wherein the SV40 polyadenylation sequence has the nucleotide sequence set forth as SEQ ID NO:8 and/or wherein the 3′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ ID NO:7.
55 . The rAAV vector of claim 54 , wherein the rAAV vector comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO:5 or a sequence at least 90% identical thereto.
56 . A method for treating XLRP in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of pharmaceutical composition comprising rAAV vector according to claim 50 and a pharmaceutically acceptable excipient, whereby the XLRP is treated in the human subject.
57 . A method for treating XLRP in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of pharmaceutical composition comprising an rAAV vector according to claim 53 and a pharmaceutically acceptable excipient, whereby the XLRP is treated in the human subject.
58 . The method according to claim 57 , wherein the 5′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ NO:6 and/or wherein the hGRK promoter has the nucleotide sequence set forth as SEQ ID NO:4 and/or wherein the SV40 polyadenylation sequence has the nucleotide sequence set forth as SEQ ID NO:8 and/or wherein the 3′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ ID NO:7.
59 . The method according to claim 58 , wherein the rAAV vector comprises (i) a capsid comprising a capsid protein comprising or consisting of the sequence of SEQ ID NO:9 and (ii) a nucleic acid comprising a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO:5.
60 . The method according to claim 59 , wherein the pharmaceutical composition is administered to the subject by periocular, intravitreal, suprachoroidal or subretinal injection.
61 . The method according to claim 60 , wherein the vector is administered to the subject at a dosage from about 10 10 vector genomes (vg)/eye to about 10 13 vg/eye.
62 . The method accordion to claim 61 , wherein the vector is administered to the subject at a dosage from about 1×10 11 vg/eye to about 5×10 12 vg/eye.
63 . The method according to according to claim 62 , wherein the vector is administered to the subject at a dosage from about 3×10 11 vg/eye or at a dosage of about 1×10 12 vg/eye.
64 . A pharmaceutical composition comprising an rAAV according to claim 50 and at least on pharmaceutically acceptable excipient.
65 . A pharmaceutical composition comprising an rAAV according to claim 53 and at least one pharmaceutically acceptable excipient.
66 . The pharmaceutical composition according to claim 65 , wherein the pharmaceutical composition comprises between 10 9 vg and 10 14 vg of the rAAV or comprises between 10 10 vg and 10 13 vg of the rAAV, or comprises about 3×10 11 vg or about 1×10 12 vg of the rAAV.