Codon optimized GLA genes and uses thereof
The present disclosure provides codon optimized nucleotide sequences encoding human 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 treating Fabry disease, the method comprising administering to a human subject with Fabry disease a recombinant adeno-associated virus (rAAV) vector comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion with a length of 5 to 20 amino acids covalently inserted in the GI-loop of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NKTTNKD (SEQ ID NO:7) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding human α-galactosidase A (AGA) protein, wherein said nucleic acid is codon optimized for expression in human cells, wherein said nucleic acid comprises from 5′ to 3′: (a) an AAV2 terminal repeat, (b) a CAO promoter, (c) the nucleotide sequence set forth in SEQ ID NO: 1, (d) a polyadenylation sequence and (e) an AAV2 terminal repeat.
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 (SEQ ID NO:7).
3. The method according to claim 2 , wherein the insertion peptide is LANKTTNKDA (SEQ ID NO:8).
4. The method according to claim 1 , wherein the variant capsid protein comprises a V7081 amino acid substitution to VP1 of AAV2 (SEQ ID NO: 9) 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 3 , wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2 (SEQ ID NO:9), or the corresponding position in the capsid protein of another AAV serotype.
7. The method according to claim 3 , wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO:9) or between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV2 (SEQ ID NO:9) or the corresponding position in the capsid protein of another AAV serotype.
8. The method of claim 1 , wherein said rAAV vector is administered to said subject by intravenous and/or intramuscular injection.
9. The method of claim 1 , wherein the method comprises a single intravenous administration of at least 1×10 8 of the rAAV vector.
10. The method of claim 1 , wherein the method comprises a single intravenous administration of 1×10 12 vg/kg to 1×10 14 vg/kg of the rAAV vector.