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 nucleic acid encoding human α-galactosidase A (AGA) protein, wherein said nucleic acid is codon optimized for expression in human cells, wherein said nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1 or comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, wherein said nucleic acid is expressed in a host cell at a greater level of expression than the wild type GLA nucleotide sequence of SEQ ID NO: 3 in said host cell under the same conditions.
2. The nucleic acid of claim 1 , wherein said nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1.
3. An expression cassette comprising the nucleic acid of claim 1 and an expression control sequence operably linked and heterologous to said nucleic acid sequence.
4. The expression cassette of claim 3 , wherein said expression control sequence is a constitutive promoter.
5. The expression cassette of claim 3 , wherein said expression control sequence is a promoter that directs preferential expression of said nucleic acid in a muscle cell.
6. A vector comprising the expression cassette of claim 3 .
7. The vector of claim 6 , wherein said vector is a recombinant adeno-associated (rAAV) vector.
8. The vector of claim 7 , wherein said rAAV vector comprises an AAV capsid of serotype 1, 2, 6, or 8 or a variant thereof.
9. The vector of claim 8 , wherein said rAAV vector comprises an AAV2 capsid or a variant thereof.
10. The vector of claim 9 , wherein said AAV2 capsid variant comprises the amino acid sequence of SEQ ID NO: 4.
11. The vector of claim 10 , wherein said rAAV vector comprises a nucleic acid comprising from 5′ to 3′: (a) an AAV2 terminal repeat, (b) a CAG promoter, (c) the nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, (d) a polyadenylation sequence and (e) an AAV2 terminal repeat.
12. The vector of claim 11 , wherein said rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 1.
13. The vector of claim 12 , wherein said nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 6.
14. A host cell comprising the vector of claim 12 .
15. The host cell of claim 14 , wherein said host cell is a CHO cell, a HEK293T cell, a HEK293 cell, a HeLa cell, a BHK21 cell or a Vero cell and/or wherein said host cell is grown in a suspension or adherent culture and/or wherein said host cell is a cardiac or skeletal muscle cell.
16. A pharmaceutical composition comprising the vector of claim 6 and at least one pharmaceutically acceptable excipient.
17. A pharmaceutical composition comprising the vector of claim 12 and at least one pharmaceutically acceptable excipient.
18. A pharmaceutical composition comprising the vector of claim 13 and at least one pharmaceutically acceptable excipient.
19. A method for treating Fabry disease, the method comprising administering to a human subject with Fabry disease the pharmaceutical composition of claim 17 .
20. The method of claim 19 , wherein said pharmaceutical composition is administered to said subject by intravenous and/or intramuscular injection.
21. A method for treating Fabry disease, the method comprising administering to a human subject with Fabry disease the pharmaceutical composition of claim 18 .
22. The method of claim 21 , wherein said pharmaceutical composition is administered to said subject by intravenous and/or intramuscular injection.
23. The method of claim 22 , wherein the method comprises a single intravenous administration of 1×10 12 vg/kg to 1×10 14 vg/kg of the rAAV vector.