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

Abstract

Provided herein are adeno-associated virus (AAV) compositions that can restore phenylalanine hydroxylase (PAH) gene function in cell. Also provided are methods of use of the AAV compositions, and packaging systems for making the AAV compositions.

1 . A method for correcting a mutation in a phenylalanine hydroxylase (PAH) gene in a cell, the method comprising transducing the cell with a replication-defective adeno-associated virus (AAV) comprising:
 (a) an AAV capsid; and   (b) a correction genome comprising: (i) an editing element for editing a target locus in the PAH gene; (ii) a 5′ homology arm nucleotide sequence 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 3′ of the editing element having homology to a second genomic region 3′ to the target locus, wherein the cell is transduced without co-transducing or co-administering an exogenous nuclease or a nucleotide sequence that encodes an exogenous nuclease.   
     
     
         2 . The method of  claim 1 , wherein the cell is a hepatocyte, a renal cell, or a cell in the brain, pituitary gland, adrenal gland, pancreas, urinary bladder, gallbladder, colon, small intestine, or breast, optionally wherein the cell is in a mammalian subject and the AAV is administered to the subject in an amount effective to transduce the cell in the subject. 
     
     
         3 . (canceled) 
     
     
         4 . A method for treating a subject having a disease or disorder associated with a PAH gene mutation, the method comprising administering to the subject an effective amount of a replication-defective AAV comprising:
 (a) an AAV capsid; and   (b) a correction genome comprising: (i) an editing element for editing a target locus in the PAH gene; (ii) a 5′ homology arm nucleotide sequence 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 3′ of the editing element having homology to a second genomic region 3′ to the target locus, wherein an exogenous nuclease or a nucleotide sequence that encodes an exogenous nuclease is not co-administered to the subject, optionally wherein the subject or disorder is phenylketonuria, and optionally wherein the subject is a human subject.   
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the editing element comprises at least a portion of a PAH coding sequence, or the editing element comprises a PAH coding sequence, optionally wherein:
 the PAH coding sequence encodes an amino acid sequence set forth in SEQ ID NO: 23;   the PAH coding sequence comprises the sequence set forth in SEQ ID NO: 24;   the PAH coding sequence is silently altered; and/or   the PAH coding sequence comprises the sequence set forth in SEQ ID NO: 25, 116, 131, 132, 138, 139, or 143.   
     
     
         8 - 12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein the editing element comprises a PAH intron-inserted coding sequence, optionally wherein the PAH intron-inserted coding sequence comprises a nonnative intron inserted in a PAH coding sequence, optionally wherein:
 the nonnative intron is selected from the group consisting of a first intron of a hemoglobin beta gene and a minute virus in mice (MVM) intron;   the nonnative intron consists of a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 28-30, and 120-130;   the nonnative intron consists of a nucleotide sequence set forth in any one of SEQ ID NOs: 28-30, and 120-130;   the PAH intron-inserted coding sequence encodes an amino acid sequence set forth in SEQ ID NO: 23; and/or   the PAH intron-inserted coding sequence comprises from 5′ to 3′: a first portion of a PAH coding sequence, the intron, and a second portion of a PAH coding sequence, wherein the first portion and the second portion, when spliced together, form a complete PAH coding sequence, optionally wherein:
 the PAH coding sequence comprises the sequence set forth in SEQ ID NO: 24; 
 the PAH coding sequence is silently altered; 
 the PAH coding sequence comprises the sequence set forth in SEQ ID NO: 25, 116, 131, 132, 138, 139, or 143; 
 the first portion of the PAH coding sequence comprises the amino acid sequence set forth in SEQ ID NO: 64 or 65, and/or the second portion of the PAH coding sequence comprises the amino acid sequence set forth in SEQ ID NO: 66 or 67; and/or 
 the first portion of the PAH coding sequence consist of the amino acid sequence set forth in SEQ ID NO: 64 or 65, and the second portion of the PAH coding sequence consists of the amino acid sequence set forth in SEQ ID NO: 66 or 67. 
   
     
     
         14 - 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the editing element comprises from 5′ to 3′: a ribosomal skipping element, and the PAH coding sequence or the PAH intron-inserted coding sequence, optionally wherein:
 the editing element further comprises a polyadenylation sequence 3′ to the PAH coding sequence or the PAH intron-inserted coding sequence, optionally wherein the polyadenylation sequence is an exogenous polyadenylation sequence, optionally wherein the exogenous polyadenylation sequence is an SV40 polyadenylation sequence, optionally wherein the SV40 polyadenylation sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31-34; 
 the nucleotide 5′ to the target locus is in an exon of the PAH gene; 
 the nucleotide 5′ to the target locus is in exon 1 of the PAH gene; 
 the editing element further comprises a splice acceptor 5′ to the ribosomal skipping element, optionally wherein the nucleotide 5′ to the target locus is in an intron of the PAH gene, optionally intron 1 of the PAH gene; and/or the editing element comprises the nucleotide sequence set forth in SEQ ID NO: 35. 
 
     
     
         25 - 33 . (canceled) 
     
     
         34 . The method of  claim 1 , optionally wherein:
 the 5′ homology arm nucleotide sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the first genomic region;   the 3′ homology arm nucleotide sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% 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 nucleotide sequence set forth in SEQ ID NO: 36 or 45; 
 the second editing window consists of the nucleotide sequence set forth in SEQ ID NO: 36 or 45; and/or 
 the first editing window consists of the nucleotide sequence set forth in SEQ ID NO: 36, and the second editing window consists of the nucleotide sequence set forth in SEQ ID NO: 45; 
   the first genomic region consists of the nucleotide sequence set forth in SEQ ID NO: 36;   the second genomic region consists of the nucleotide sequence set forth in SEQ ID NO: 45; and/or   each of the 5′ and 3′ homology arm nucleotide sequences independently has a length of about 100 to about 2000 nucleotides.   
     
     
         35 - 42 . (canceled) 
     
     
         43 . The method of  claim 1 , wherein the 5′ homology arm comprises: C corresponding to nucleotide −2 of the PAH gene, G corresponding to nucleotide 4 of the PAH gene, G corresponding to nucleotide 6 of the PAH gene, G corresponding to nucleotide 7 of the PAH gene, G corresponding to nucleotide 9 of the PAH gene, A corresponding to nucleotide −467 of the PAH gene, A corresponding to nucleotide −465 of the PAH gene, A corresponding to nucleotide −181 of the PAH gene, G corresponding to nucleotide −214 of the PAH gene, C corresponding to nucleotide −212 of the PAH gene, A corresponding to nucleotide −211 of the PAH gene, G corresponding to nucleotide 194 of the PAH gene, C corresponding to nucleotide −433 of the PAH gene, C corresponding to nucleotide −432 of the PAH gene, ACGCTGTTCTTCGCC (SEQ ID NO: 68) corresponding to nucleotides −394 to −388 of the PAH gene, A corresponding to nucleotide −341 of the PAH gene, A corresponding to nucleotide −339 of the PAH gene, A corresponding to nucleotide −225 of the PAH gene, A corresponding to nucleotide −211 of the PAH gene, and/or A corresponding to nucleotide −203 of the PAH gene. 
     
     
         44 . The method of  claim 1 , wherein the 5′ homology arm comprises:
 (a) C corresponding to nucleotide −2 of the PAH gene, G corresponding to nucleotide 4 of the PAH gene, G corresponding to nucleotide 6 of the PAH gene, G corresponding to nucleotide 7 of the PAH gene, and G corresponding to nucleotide 9 of the PAH gene; 
 (b) A corresponding to nucleotide −467 of the PAH gene, and A corresponding to nucleotide −465 of the PAH gene; 
 (c) A corresponding to nucleotide −181 of the PAH gene; 
 (d) G corresponding to nucleotide −214 of the PAH gene, C corresponding to nucleotide −212 of the PAH gene, and A corresponding to nucleotide −211 of the PAH gene; 
 (e) G corresponding to nucleotide 194 of the PAH gene; 
 (f) C corresponding to nucleotide −433 of the PAH gene, and C corresponding to nucleotide −432 of the PAH gene; 
 (g) ACGCTGTTCTTCGCC (SEQ ID NO: 68) corresponding to nucleotides −394 to −388 of the PAH gene; and/or 
 (h) A corresponding to nucleotide −341 of the PAH gene, A corresponding to nucleotide −339 of the PAH gene, A corresponding to nucleotide −225 of the PAH gene, A corresponding to nucleotide −211 of the PAH gene, and A corresponding to nucleotide −203 of the PAH gene. 
 
     
     
         45 . The method of  claim 44 , wherein the 5′ homology arm comprises the modifications of (c) and (d), (f) and (g), and/or (b) and (h). 
     
     
         46 . The method of  claim 1 , wherein:
 the 5′ homology arm consists of a nucleotide sequence set forth in any one of SEQ ID NOs: 36-44, 111, 115, and 142;   the 3′ homology arm consists of the nucleotide sequence set forth in SEQ ID NO: 45, 112, 117, 144;   the correction genome comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 46-54, 113, 118, 134, 136, and 145;   the correction genome comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 55-63, 114, 119, 135, 137, and 146;   the correction genome consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 55-63, 114, 119, 135, 137, and 146; and/or   the correction 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 has at least 95% sequence identity to SEQ ID NO: 18, and the 3′ ITR nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 19; 
 the 5′ ITR nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 20, and the 3′ ITR nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 21; or 
 the 5′ ITR nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 26, and the 3′ ITR nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 27. 
   
     
     
         47 - 54 . (canceled) 
     
     
         55 . The method of  claim 1 , wherein the AAV capsid comprises an AAV Clade F capsid protein. 
     
     
         56 . The method of  claim 55 , wherein the AAV Clade F capsid protein comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO: 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17, optionally wherein: the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 2 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 2 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 2 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 2 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 2 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 2 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 2 is G,
 optionally wherein:   (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 2 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 2 is G;   (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M;   (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is R;   (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 2 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R; or   (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is C,   optionally wherein the capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17.   
     
     
         57 - 59 . (canceled) 
     
     
         60 . The method of  claim 55 , wherein the AAV Clade F capsid protein comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17, optionally wherein: the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 2 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 2 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 2 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 2 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 2 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 2 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 2 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 2 is G,
 optionally wherein:   (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 2 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 2 is G;   (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M;   (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is R;   (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 2 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R; or   (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is C,   optionally wherein the capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17.   
     
     
         61 - 63 . (canceled) 
     
     
         64 . The method of  claim 55 , wherein the AAV Clade F capsid protein comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of amino acids 1-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, optionally wherein: the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 2 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 2 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 2 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 2 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 2 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 2 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 2 is Q, the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 2 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 2 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 2 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 2 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 2 is G
 optionally wherein:   (a) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 2 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 2 is Q;   (b) the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 2 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 2 is Y;   (c) the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 2 is K;   (d) the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 2 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 2 is S;   (e) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 2 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 2 is G;   (f) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M;   (g) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is R;   (h) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 2 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R; or   (i) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is optionally wherein the capsid protein comprises the amino acid sequence of amino acids C,   1-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.   
     
     
         65 - 67 . (canceled) 
     
     
         68 . The method of  claim 1 , wherein the integration efficiency of the editing element into the target locus is at least 1% when the AAV is administered to a mouse implanted with human hepatocytes in the absence of an exogenous nuclease under standard AAV administration conditions; or the allelic frequency of integration of the editing element into the target locus is at least 0.5% when the AAV is administered to a mouse implanted with human hepatocytes in the absence of an exogenous nuclease under standard AAV administration conditions. 
     
     
         69 . (canceled) 
     
     
         70 . A replication-defective adeno-associated virus (AAV) comprising:
 (a) an AAV capsid; and   (b) a correction genome comprising: (i) an editing element for editing a target locus in the PAH gene; (ii) a 5′ homology arm nucleotide sequence 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 3′ of the editing element having homology to a second genomic region 3′ to the target locus.   
     
     
         71 - 133 . (canceled) 
     
     
         134 . A pharmaceutical composition comprising an AAV of  claim 70 . 
     
     
         135 . A packaging system for recombinant preparation of a replication-defective AAV, wherein the packaging system comprises:
 (a) a Rep nucleotide sequence encoding one or more AAV Rep proteins;   (b) a Cap nucleotide sequence encoding one or more AAV Clade F capsid proteins; and   (c) a correction genome as set forth in  claim 1 , wherein the packaging system is operative in a cell for enclosing the correction genome in the capsid to form the AAV.   
     
     
         136 - 153 . (canceled) 
     
     
         154 . A method for recombinant preparation of a replication-defective AAV, the method comprising introducing the packaging system of  claim 135  into a cell under conditions operative for enclosing the correction genome in the capsid to form the AAV.