Codon optimized rep1 genes and uses thereof

Abstract

The present disclosure provides codon optimized nucleotide sequences encoding human REP1, vectors, and host cells comprising codon optimized REP1 sequences, and methods of treating retinal disorders such as choroideremia comprising administering to the subject a codon optimized sequence encoding human REP1.

1 . A nucleic acid encoding human Rab escort protein-1 (REP1) protein of SEQ ID NO:2 and codon optimized for expression in humans, the nucleic acid comprising the nucleotide sequence set forth as SEQ ID NO: 1 or comprising a nucleotide sequence at least 80% identical thereto. 
     
     
         2 . The nucleic acid according to  claim 1 , wherein the nucleotide sequence has a codon adaptation index of at least 0.94. 
     
     
         3 . An expression cassette comprising the nucleic acid according to  claim 1 , wherein said nucleotide sequence is operably linked to an expression control sequence. 
     
     
         4 . The expression cassette of  claim 3 , wherein the expression control sequence comprises a constitutive promoter or comprises a promoter that directs cell-specific expression of the nucleic acid in rod and cone cells. 
     
     
         5 . The expression cassette of  claim 4 , wherein the expression control sequence comprises a CAG promoter. 
     
     
         6 . The expression cassette of  claim 4 , comprising from 5′ to 3′: (a) a 5′ AAV2 terminal repeat (b) a CAG promoter (c) a nucleotide sequence at least 80% identical to the nucleotide sequence set forth in SEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) a 3′ 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 CAG 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 . A recombinant adeno-associated virus (rAAV) vector comprising a heterologous nucleic acid comprising the expression cassette according to  claim 3 . 
     
     
         9 . The rAAV vector of  claim 8 , wherein the rAAV vector comprises an AAV capsid of serotype 2, 4, 5 or 8 or a variant thereof. 
     
     
         10 . The rAAV vector of  claim 9 , wherein the rAAV vector comprises a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO:9. 
     
     
         11 . The rAAV vector of  claim 10 , wherein the rAAV vector comprises a nucleic acid comprising from 5′ to 3′: (a) a 5′ AAV2 terminal repeat (b) a CAG promoter (c) a nucleotide sequence at least 80% identical to the nucleotide sequence set forth in SEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) a 3′ AAV2 terminal repeat. 
     
     
         12 . The rAAV vector of  claim 11 , wherein the 5′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ ID NO:6 and/or wherein the CAG 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. 
     
     
         13 . A method for treating choroideremia 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 8  and a pharmaceutically acceptable excipient, whereby the choroideremia is treated in the human subject. 
     
     
         14 . The method according to  claim 13 , wherein the rAAV vector comprises a nucleic acid comprising from 5′ to 3′: (a) a 5′ AAV2 terminal repeat (b) a CAG promoter (c) a nucleotide sequence at least 80% identical to the nucleotide sequence set forth in SEQ ID NO:1 (d) an SV40 polyadenylation sequence and (e) a 3′ AAV2 terminal repeat. 
     
     
         15 . The method according to  claim 14 , wherein the 5′ AAV2 terminal repeat has the nucleotide sequence set forth as SEQ ID NO:6 and/or wherein the CAG 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. 
     
     
         16 . The method according to  claim 13 , wherein the rAAV vector comprises an AAV capsid of serotype 2, 4, 5 or 8 or a variant thereof. 
     
     
         17 . The method according to  claim 16 , wherein the rAAV vector comprises a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO:9. 
     
     
         18 . The method according to  claim 13 , wherein the pharmaceutical composition is administered to the subject by periocular, intravitreal, suprachoroidal or subretinal injection. 
     
     
         19 . The method according to  claim 18 , 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. 
     
     
         20 . A pharmaceutical composition comprising the rAAV vector according to  claim 8  and at least one pharmaceutically acceptable excipient.