Codon optimized rpgrorf15 genes and uses thereof

Abstract

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 . 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 90% identical to the nucleotide sequence set forth as SEQ ID NO:1. 
     
     
         2 . The nucleic acid according to  claim 1 , wherein the nucleotide sequence has a codon adaptation index of at least 0.89. 
     
     
         3 . An expression cassette comprising a nucleic acid according to  claim 1 , wherein the nucleotide sequence at least 90% identical to SEQ ID NO:1 is operably linked to an expression control sequence. 
     
     
         4 . The expression cassette of  claim 3 , 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. 
     
     
         5 . The expression cassette of  claim 4 , wherein the expression control sequence is a human G protein-coupled receptor rhodopsin kinase 1 (hGRK) promoter. 
     
     
         6 . The expression cassette of  claim 5 , 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 90% identical to the nucleotide sequence set forth as SEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) an AAV2 terminal repeat. 
     
     
         7 . The expression cassette of  claim 6 , 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. 
     
     
         8 . The expression cassette of  claim 7 , comprising or consisting of the nucleotide sequence of SEQ ID NO:5 or a sequence at least at least 90% identical thereto. 
     
     
         9 . A recombinant adeno-associated virus (rAAV) vector comprising a heterologous nucleic acid comprising the expression cassette according to  claim 3 . 
     
     
         10 . The rAAV vector of  claim 9 , wherein the rAAV vector comprises an AAV capsid of serotype 2, 5 or 8 or a variant thereof. 
     
     
         11 . The rAAV vector of  claim 10 , wherein the rAAV vector comprises a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO:9. 
     
     
         12 . The rAAV vector of  claim 11 , 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 90% identical to the nucleotide sequence set forth as SEQ ID NO:1 and (d) an AAV2 terminal repeat. 
     
     
         13 . The rAAV vector of  claim 12 , 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. 
     
     
         14 . The rAAV vector of  claim 13 , 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. 
     
     
         15 . 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 9  and a pharmaceutically acceptable excipient, whereby the XLRP is treated in the human subject. 
     
     
         16 . 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 12  and a pharmaceutically acceptable excipient, whereby the XLRP is treated in the human subject. 
     
     
         17 . The method according to  claim 16 , 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. 
     
     
         18 . The method according to  claim 17 , 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. 
     
     
         19 . The method according to  claim 18 , wherein the pharmaceutical composition is administered to the subject by periocular, intravitreal, suprachoroidal or subretinal injection. 
     
     
         20 . The method according to  claim 19 , 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. 
     
     
         21 . The method according to  claim 20 , 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. 
     
     
         22 . The method according to according to  claim 21 , 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. 
     
     
         23 . A pharmaceutical composition comprising an rAAV according to  claim 9  and at least one pharmaceutically acceptable excipient. 
     
     
         24 . A pharmaceutical composition comprising an rAAV according to  claim 15  and at least one pharmaceutically acceptable excipient. 
     
     
         25 . The pharmaceutical composition according to  claim 24 , 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.