Codon optimized nucleic acid encoding a retinitis pigmentosa gtpase regulator (rpgr)

Abstract

This invention relates generally to a codon optimized nucleic acid encoding a retinitis pigmentosa GTPase regulator (RPGR) protein. The nucleic acid has enhanced stability during plasmid production relative to a wildtype cDNA encoding the RPGR protein. The invention also relates to expression cassettes, vectors, and host cells comprising the codon optimized nucleic acid. Methods for preparing a recombinant adeno-associated (rAAV) expression vector comprising the codon optimized nucleic acid sequence are also provided. The nucleic acids, expression cassettes, vectors, and host cells provided may be useful in the large scale production of rAAV expression vectors for gene therapy applications.

1 . A polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1 encoding a human retinitis pigmentosa GTPase regulator (RPGR) protein. 
     
     
         2 . An expression cassette comprising the polynucleotide of  claim 1  and an expression control sequence operably linked and heterologous to the nucleic acid sequence. 
     
     
         3 . A vector comprising the polynucleotide of  claim 1 . 
     
     
         4 . The vector of  claim 3 , wherein the vector is a recombinant adeno-associated (rAAV) expression vector. 
     
     
         5 . A recombinant herpes simplex virus (rHSV) comprising the polynucleotide of  claim 1 . 
     
     
         6 . A host cell comprising the polynucleotide of  claim 1 . 
     
     
         7 . The host cell of  claim 6 , wherein the host cell is a mammalian cell. 
     
     
         8 . The host cell of  claim 6 , wherein the host cell is a HeLa cell, a BHK21 cell or a Vero cell. 
     
     
         9 . The host cell of  claim 6 , wherein the host cell is a V27 cell. 
     
     
         10 . The expression cassette of  claim 2 , wherein the expression control sequence is a human interphotoreceptor retinoid-binding protein (IRBP) promoter. 
     
     
         11 . The expression cassette of  claim 10 , wherein the human IRBP promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and directs preferential expression in rods and cones. 
     
     
         12 . The expression cassette of  claim 10 , wherein the human IRBP promoter comprises the nucleic acid sequence of SEQ ID NO: 8. 
     
     
         13 . The polynucleotide of  claim 1 , wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 7. 
     
     
         14 . A method of producing the rAAV expression vector of  claim 4 , comprising
 (a) infecting a host cell with a recombinant herpes simplex virus (rHSV) comprising the nucleic acid sequence of SEQ ID NO: 1;   (b) incubating the host cell; and   (c) following incubation, collecting rAAV from the host cell of step (b).   
     
     
         15 . The method of  claim 14 , wherein the host cell is a HeLa cell, a BHK21 cell or a Vero cell. 
     
     
         16 . The method of  claim 14 , wherein the rHSV further comprises a human IRBP promoter operably linked to the nucleic acid sequence of SEQ ID NO: 1. 
     
     
         17 . The method of  claim 16 , wherein the human IRBP promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 8 and directs preferential expression in rods and cones. 
     
     
         18 . The method of  claim 16 , wherein the human IRBP promoter comprises the nucleic acid sequence of SEQ ID NO: 8. 
     
     
         19 . The method of  claim 14 , wherein the rHSV comprises the nucleic acid sequence of SEQ ID NO: 7.