Codon optimized gla genes and uses thereof

Abstract

The present disclosure provides codon optimized nucleotide sequences encoding hum n alpha-galactosidase A, vectors, and host cells comprising codon optimized alpha-galactosidase A sequences, and methods of treating disorders such as Fabry disease comprising administering to the subject a codon optimized sequence encoding human alpha-galactosidase A.

1 . A method for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product to a kidney cell, brain cell, and/or lung cell, the method comprising contacting the kidney cell, brain cell and/or lung cell with a recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion with a length of 7 to 20 amino acids covalently inserted in the GH-loop of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NKTTNKD and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product, said nucleotide sequence operably linked to a promoter. 
     
     
         2 . The method according to  claim 1 , wherein the insertion peptide has from 1 to 3 spacer amino acids (Y 1 -Y 3 ) at the amino and/or carboxyl terminus of amino acid sequence NKTTNKD. 
     
     
         3 . The method according to  claim 2 , wherein the insertion peptide is LANKTTNKDA. 
     
     
         4 . The method according to  claim 1 , wherein the variant capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO:2) or the corresponding position in the capsid protein of another AAV serotype and wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:4. 
     
     
         5 . The method according to  claim 4 , wherein the variant capsid protein comprises an amino acid sequence at least 95% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:4. 
     
     
         6 . The method according to  claim 1 , wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype. 
     
     
         7 . The method according to  claim 6 , wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype. 
     
     
         8 . The method according to  claim 1 , wherein the promoter is a constitutive promoter. 
     
     
         9 . The method according to  claim 1 , wherein the promoter is a tissue-specific promoter. 
     
     
         10 . A method for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a gee product to the kidney, brain, spinal cord or lung of a primate, the method comprising administering to the primate a recombinant adeno-associated vims (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion with a length of 7 to 20 amino acids covalently inserted in the GH-loop of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NKTTNKD and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product, said nucleotide sequence operably linked to a promoter, or administering to the subject a pharmaceutical composition comprising the rAAV and a pharmaceutically acceptable excipient. 
     
     
         11 . The method according to  claim 10 , wherein the insertion peptide has from 1 to 3 spacer amino acids (Y 1 -Y 3 ) at the amino and/or carboxyl terminus of amino acid sequence NKTTNKD. 
     
     
         12 . The method according to  claim 11 , wherein the insertion peptide is LANKTTNKDA. 
     
     
         13 . The method according to  claim 10 , wherein the variant capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO:2) or the corresponding position in the capsid protein of another AAV serotype and wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:4. 
     
     
         14 . The method according to  claim 13 , wherein the variant capsid protein comprises an amino acid sequence at least 95% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:4. 
     
     
         15 . The method according to  claim 10 , wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype. 
     
     
         16 . The method according to  claim 15 , wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype. 
     
     
         17 . The method according to  claim 10 , wherein the promoter is a constitutive promoter. 
     
     
         18 . The method according to  claim 10 , wherein the promoter is a tissue-specific promoter. 
     
     
         19 . The method according to  claim 10 , wherein the rAAV or pharmaceutical composition is administered to the primate by intravenous injection. 
     
     
         20 . The method according to  claim 10 , wherein the primate is a human.