Adeno-associated virus compositions for PAH gene transfer and methods of use thereof
Provided herein are adeno-associated virus (AAV) compositions that can express a phenylalanine hydroxylase (PAH) polypeptide in a cell, thereby restoring the PAH gene function. Also provided are methods of use of the AAV compositions, and packaging systems for making the AAV compositions.
1. An adeno-associated virus (AAV) genome comprising, from 5′ to 3′: (a) an AAV2 5′ inverted terminal repeat (ITR) nucleotide sequence; (b) a transcriptional regulatory element comprising, from 5′ to 3′, a human hepatic control region | (HCR1) and a human a1-antitrypsin (hAAT) promoter; (c) a silently altered phenylalanine hydroxylase (PAH) coding sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 71, wherein the silently altered PAH coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 23; and (d) an AAV2 3′ ITR nucleotide sequence.
2. The AAV genome of claim 1 , wherein the transcriptional regulatory element further comprises an intron.
3. The AAV genome of claim 2 , wherein the intron is an SV40 intron or a minute virus of mouse (MVM) intron.
4. The AAV genome of claim 1 , wherein the transcriptional regulatory element comprises a nucleotide sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-33, 35, and 37-39.
5. The AAV genome of claim 1 , wherein the transcriptional regulatory element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-33, 35, and 37-39.
6. The AAV genome of claim 1 , wherein the transcriptional regulatory element comprises, from 5′ to 3′, the nucleotide sequences of SEQ ID NOs: 29, 30, and 31.
7. The AAV genome of claim 1 , wherein the transcriptional regulatory element comprises, from 5′ to 3′, the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 31.
8. The AAV genome of claim 1 , wherein the AAV genome further comprises a polyadenylation sequence 3′ to the PAH coding sequence.
9. The AAV genome of claim 8 , wherein the polyadenylation sequence is an SV40 polyadenylation sequence.
10. The AAV genome of claim 9 , wherein the SV40 polyadenylation sequence comprises a nucleotide sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, and 45.
11. The AAV genome of claim 10 , wherein the SV40 polyadenylation sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, and 45.
12. The AAV genome of claim 1 , wherein the 5′ ITR nucleotide sequence has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 26, and the 3′ ITR nucleotide sequence has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 27.
13. The AAV genome of claim 1 , wherein the 5′ ITR nucleotide sequence has the nucleotide sequence of SEQ ID NO: 26, and the 3′ ITR nucleotide sequence has the nucleotide sequence of SEQ ID NO: 27.
14. The AAV genome of claim 13 , wherein the AAV genome comprises a nucleotide sequence having at least 95% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 75, 77, 79, 81, 83, 87, and 90.
15. A replication-defective AAV comprising: an AAV capsid comprising a capsid protein; and an AAV genome of claim 1 .
16. The AAV of claim 15 , wherein the AAV capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16.
17. The AAV of claim 15 , wherein the AAV capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16.
18. The AAV of claim 15 , wherein the AAV capsid protein comprises the amino acid sequence of amino acids 1-736 of SEQ ID NO: 16.
19. A pharmaceutical composition comprising the AAV of claim 15 .
20. An adeno-associated virus (AAV) genome comprising a nucleotide sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47, 49, 86, and 89.