Adeno-associated virus compositions for restoring PAH gene function and methods of use thereof

1. A recombinant adeno-associated virus (rAAV) comprising: (a) an AAV capsid comprising an AAV capsid protein; and (b) an rAAV genome comprising: (1) an editing element for editing a target locus in a phenylalanine hydroxylase (PAH) gene, comprising a PAH coding sequence operably linked to a transcriptional regulatory element, wherein the PAH coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 28; (ii) a 5′ homology arm nucleotide sequence positioned 5′ of the editing element, having homology to a first genomic region 5′ to the target locus; and (iii) a 3′ homology arm nucleotide sequence positioned 3′ of the editing element, having homology to a second genomic region 3′ to the target locus.

2. The rAAV of claim 1 , wherein the transcriptional regulatory element is capable of mediating transcription in a hepatocyte, a renal cell, a brain cell, a pituitary gland cell, an adrenal gland cell, a pancreatic cell, a urinary bladder cell, a gallbladder cell, a colon cell, a small intestine cell, or a breast cell, optionally wherein:

the transcriptional regulatory element is endogenous to the PAH gene; or

the transcriptional regulatory element is exogenous to the PAH gene, optionally wherein:

the transcriptional regulatory element is liver specific, optionally wherein the transcriptional regulatory element comprises one or more elements selected from the group consisting of a human albumin promoter, a human transthyretin (TTR) promoter, a human ApoE/C-I hepatic control region (HCR) 1 or 2, a human ApoH promoter, a human SERPINA1 (hAAT) promoter, and a hepatic specific regulatory module thereof;

the transcriptional regulatory element comprises a nucleotide sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 25, 26, 27, and 69;

the transcriptional regulatory element comprises a nucleotide sequence at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 27;

the transcriptional regulatory element comprises the nucleotide sequence set forth in SEQ ID NO: 27; or

the nucleotide sequence of the transcriptional regulatory element consists of the nucleotide sequence set forth in SEQ ID NO: 27.

3. The rAAV of claim 1 , wherein the editing element further comprises an intron element positioned 5′ to the PAH coding sequence and 3′ to the transcriptional regulatory element, optionally wherein the intron element is an exogenous intron element, optionally an SV40 intron element, optionally wherein:

the SV40 intron element comprises a nucleotide sequence at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 29;

the SV40 intron element comprises the nucleotide sequence set forth in SEQ ID NO: 29; or the nucleotide sequence of the SV40 intron element consists of the nucleotide sequence set forth in SEQ ID NO: 29.

4. The rAAV of claim 1 , wherein the editing element further comprises a polyadenylation sequence 3′ to the PAH coding sequence, optionally wherein the polyadenylation sequence is an exogenous polyadenylation sequence, optionally an SV40 polyadenylation sequence, optionally wherein

the SV40 polyadenylation sequence comprises a nucleotide sequence at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 31;

the SV40 polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 31; or

the nucleotide sequence of the SV40 polyadenylation sequence consists of the nucleotide sequence set forth in SEQ ID NO: 31.

5. The rAAV of claim 1 , wherein:

the nucleotide sequence 5′ to the target locus is in an intron of a PAH gene, optionally in intron 1 of a PAH gene; and/or

the nucleotide sequence 3′ to the target locus is in an intron of a PAH gene, optionally in intron 1 of a PAH gene.

6. The rAAV of claim 1 , wherein the PAH gene is a human PAH gene, optionally wherein the human PAH gene is wild-type or a variant PAH gene.

7. The rAAV of claim 1 , wherein the editing element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 25, 26, 27, 29, 31, 50, 51, 52, 69, and 70.

8. The rAAV of claim 1 , wherein:

the 5′ homology arm nucleotide sequence is at least 90% identical to the first genomic region;

the 3′ homology arm nucleotide sequence is at least 90% identical to the second genomic region;

the first genomic region is located in a first editing window, and the second genomic region is located in a second editing window, optionally wherein:

the first editing window consists of the region of the human genome corresponding to the nucleotide sequence set forth in SEQ ID NO: 37;

the second editing window consists of the region of the human genome corresponding to the nucleotide sequence set forth in SEQ ID NO: 38;

the first genomic region consists of the region of the human genome corresponding to the nucleotide sequence set forth in SEQ ID NO: 37; and/or

the second genomic region consists of the region of the human genome corresponding to the nucleotide sequence set forth in SEQ ID NO: 38.

9. The rAAV of claim 1 , wherein:

each of the 5′ and the 3′ homology arm nucleotide sequences independently has a length of about 100 to about 2000 nucleotides;

the 5′ homology arm comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 23;

the nucleotide sequence of the 5′ homology arm consists of the nucleotide sequence set forth in SEQ ID NO: 23;

the 3′ homology arm comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 24; and/or

the nucleotide sequence of the 3′ homology arm consists of the nucleotide sequence set forth in SEQ ID NO: 24.

10. The rAAV of claim 1 , wherein the rAAV genome comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 43.

11. The rAAV of claim 1 , wherein the rAAV genome further comprises a 5′ inverted terminal repeat (5′ ITR) nucleotide sequence 5′ of the 5′ homology arm nucleotide sequence, and a 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the 3′ homology arm nucleotide sequence, optionally wherein:

the 5′ ITR nucleotide sequence is at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 14, and/or the 3′ ITR nucleotide sequence is at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 18; and/or

the rAAV genome comprises the nucleotide sequence set forth in SEQ ID NO: 73, 74, 75, or 76.

12. The rAAV of claim 1 , wherein the rAAV genome comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 45.

13. The rAAV of claim 1 , wherein:

the AAV capsid protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, optionally wherein:

(i) the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Gor Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G;

(ii) (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; and/or

(iii) the capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17;

the AAV capsid protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, optionally wherein:

(i) the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G;

(ii) (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; and/or

(iii) the capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17; and/or

the AAV capsid protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, optionally wherein:

(i) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q;

the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G;

(ii) (a) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; (b) the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y; (c) the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; (d) the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; (e) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (f) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (g) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (h) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (i) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; and/or

(iii) the capsid protein comprises the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

14. A pharmaceutical composition comprising an rAAV of claim 1 .

15. The rAAV of claim 1 , wherein the editing element comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 52.

16. The rAAV of claim 1 , comprising:

(a) an AAV capsid comprising: a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16; a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16; and/or a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 16; and

(b) an rAAV genome comprising a nucleotide sequence that is at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 43.

17. The rAAV of claim 1 , comprising:

(a) an AAV capsid comprising: a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16; a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16; and/or a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 16; and

(b) an rAAV genome comprising a nucleotide sequence that is at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 45.

18. A polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 43, 45, 51, or 52.