Methods of treating phenylketonuria
Provided herein are methods of treating a subject having a disease or disorder associated with a PAH gene mutation. The methods generally comprise administering to the subject a therapeutically effective does of a recombinant adeno-associated virus (rAAV) that can express a phenylalanine hydroxylase (PAH) polypeptide in a cell and thereby restore PAH gene function in the subject.
1. A method for treating a subject having phenylketonuria (PKU), the method comprising administering to the subject an rAAV at a dose of about 6e13 vg/kg to about 1e14 vg/kg, wherein the rAAV comprises:
(a) an AAV capsid comprising:
a capsid protein comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO:11, wherein the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:11 is A, and wherein the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:11 is R;
a capsid protein comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO:11, wherein the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:11 is A, and wherein the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:11 is R; and/or
a capsid protein comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 1-736 of SEQ ID NO:11, wherein the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:11 is A, and wherein the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:11 is R; and
(b) a transfer genome comprising a silently altered PAH coding sequence operably linked to a transcriptional regulatory element, wherein the silently altered PAH coding sequence comprises a nucleotide sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1, and encodes the amino acid sequence set forth in SEQ ID NO:13.
2. The method of claim 1 , wherein:
the transcriptional regulatory element is capable of mediating transcription in a cell of the liver, the kidney, the brain, the pituitary gland, the adrenal gland, the pancreas, the urinary bladder, the gallbladder, the colon, the small intestine, or the breast;
the transcriptional regulatory element comprises a human hepatic control region 1 (HCR1) comprising the nucleotide sequence set forth in SEQ ID NO:2;
the transcriptional regulatory element comprises a human al-antitrypsin (hAAT) promoter comprising the nucleotide sequence set forth in SEQ ID NO:3;
the transcriptional regulatory element comprises an SV40 intron comprising the nucleotide sequence set forth in SEQ ID NO:4; or
the transcriptional regulatory element comprises the nucleotide sequence set forth in SEQ ID NO:5.
3. The method of claim 1 , wherein the transfer genome further comprises an SV40 polyadenylation sequence 3′ to the PAH coding sequence, wherein the SV40 polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO:6.
4. The method of claim 1 , wherein the transfer genome further comprises a 5′ inverted terminal repeat (5′ ITR) nucleotide sequence 5′ of the genome, and a 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the genome, optionally wherein the 5′ ITR nucleotide sequence has at least 95% sequence identity to SEQ ID NO:8, and the 3′ ITR nucleotide sequence has at least 95% sequence identity to SEQ ID NO:9.
5. The method of claim 1 , wherein the transfer genome comprises the nucleotide sequence set forth in SEQ ID NO:7 or 10.
6. The method of claim 1 , wherein the silently altered PAH coding sequence comprises a nucleotide sequence having at least 99% sequence identity to the sequence set forth in SEQ ID NO: 1.
7. The method of claim 1 , wherein:
the AAV capsid comprises a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO:11, a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO:11, and/or a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO:11; or
the amino acid sequence of the capsid protein consists of the amino acid sequence of amino acids 203-736 of SEQ ID NO:11, the amino acid sequence of the capsid protein consists of the amino acid sequence of amino acids 138-736 of SEQ ID NO:11, and/or the amino acid sequence of the capsid protein consists of the amino acid sequence of amino acids 1-736 of SEQ ID NO:11.
8. The method of claim 1 , wherein the rAAV is administered as a single dose or as multiple doses.
9. The method of claim 1 , wherein the rAAV is administered intravenously.
10. The method of claim 1 , wherein the subject is a pediatric or an adult subject.
11. The method of claim 1 , wherein the subject is administered a first immunosuppressant prior to administration of the rAAV, optionally wherein:
the first immunosuppressant is administered one day prior to the administration of the rAAV; and/or
the first immunosuppressant is administered to the subject for about 20 or about 21 weeks.
12. The method of claim 11 , wherein the first immunosuppressant is administered according to the following sequential dosing regimen:
(1) 60 mg/day in prednisolone equivalents for two weeks;
(2) 40 mg/day in prednisolone equivalents for six weeks;
(3) 30 mg/day in prednisolone equivalents for three weeks;
(4) 20 mg/day in prednisolone equivalents for three weeks;
(5) 10 mg/day in prednisolone equivalents for five weeks; and
(6) 5 mg/day in prednisolone equivalents for one or two weeks.
13. The method of claim 12 , wherein the first immunosuppressant is a glucocorticosteroid, optionally wherein the first immunosuppressant is prednisolone.
14. The method of claim 12 , wherein:
if the subject exhibits a level of a liver transaminase that is at least about 2 times the upper limit of normal (ULN) at any time during the sequential dosing regimen, the sequential dosing regimen will be restarted from step (1); or
if the subject exhibits a level of a liver transaminase that is at least about 2 times ULN after completion of step (6), the subject is further administered the first immunosuppressant according to the following sequential dosing regimen:
(7) 60 mg/day in prednisolone equivalents for two to four weeks;
(8) 40 mg/day in prednisolone equivalents for two weeks;
(9) 30 mg/day in prednisolone equivalents for two weeks;
(10) 20 mg/day in prednisolone equivalents for two weeks;
(11) 10 mg/day in prednisolone equivalents for two weeks; and
(12) 5 mg/day in prednisolone equivalents for two weeks.
15. The method of claim 14 , wherein step (7) is performed until the level of the liver transaminase has declined to about or less than about the subject's baseline level of the liver transaminase, optionally wherein:
the subject's baseline level of the liver transaminase is the level of the liver transaminase in the subject prior to receiving the rAAV;
the subject's baseline level of the liver transaminase is within a normal range; and/or
the subject's baseline level of the liver transaminase is at about an upper limit of normal (ULN) for the liver transaminase.
16. The method of claim 14 , wherein the liver transaminase is alanine aminotransferase (ALT) or aspartate aminotransferase (AST), optionally wherein:
the normal range of ALT is from 0 to about 63 U/L;
the normal range of AST is from 0 to about 57 U/L;
the ULN for ALT is from about 30 to about 63 U/L; or
the ULN for AST is about 34 to about 57 U/L.
17. A method for treating a subject having PKU, the method comprising intravenously administering to the subject an rAAV at a dose of about 6e13 vg/kg, about 8e13 vg/kg, or about 1e14 vg/kg, wherein the rAAV comprises:
(a) an AAV capsid comprising:
a capsid protein comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO:11, wherein the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:11 is A, and wherein the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:11 is R;
a capsid protein comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO:11, wherein the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:11 is A, and wherein the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:11 is R; and/or
a capsid protein comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 1-736 of SEQ ID NO:11, wherein the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO:11 is A, and wherein the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO:11 is R; and
(b) a transfer genome comprising the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:10.