JPWO2019152843A5

JPWO2019152843A5 -

Info

Publication number: JPWO2019152843A5
Application number: JP2020541887A
Authority: JP
Publication date: 2022-02-09

Claims

(a) AAV capsids containing replication-deficient adeno-associated virus (AAV) capsid proteins,
(b) With the modified genome
A replication-deficient adeno-associated virus (AAV) containing the above-mentioned modified genome.
(i) Editing elements for editing target loci within the PAH gene
(ii) A 5'homologous arm nucleotide sequence on the 5'side of the editing element having homology to the first genomic region on the 5'side of the target locus.
Editing having homology to the first genomic region on the 5'side of the target locus, wherein the first genomic region is located within the first editing window having the nucleotide sequence set forth in SEQ ID NO: 36. The 5'homologous arm nucleotide sequence on the 5'side of the element,
(iii) A 3'homologous arm nucleotide sequence on the 3'side of the editing element having homology to the second genomic region on the 3'side of the target locus.
An edit having homology to the second genomic region on the 3'side of the target locus, wherein the second genomic region is located within a second edit window having the nucleotide sequence set forth in SEQ ID NO: 45. 3'homologous arm nucleotide sequence on the 3'side of the element
Including,
Replication-deficient adeno-associated virus (AAV).

The editing element contains at least a portion of the PAH code sequence and, if necessary,
The PAH coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 23.
The PAH coding sequence comprises and / or the sequence set forth in SEQ ID NO: 24.
The PAH coding sequence has a silent variation, and optionally the PAH coding sequence comprises the sequence set forth in SEQ ID NO: 25, 116, 131, 132, 138, 139, or 143.
The editing element contains a PAH intron insertion code sequence, and optionally the PAH intron insertion code sequence contains an unnatural intron inserted into the PAH code sequence, optionally.
The unnatural intron is selected from the group consisting of the first intron of the hemoglobin beta gene and the mouse microvirus (MVM) intron.
The unnatural intron consists of a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 28-30 and 120-130.
The unnatural intron comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 28-30 and 120-130.
The PAH intron insertion coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 23 and / or
The PAH intron insertion coding sequence comprises a first portion of the PAH coding sequence, an intron, and a second portion of the PAH coding sequence from 5'to 3', the first portion and the first portion. When the two parts are spliced together, they form a complete PAH coding sequence and, if necessary,
The PAH coding sequence comprises the sequence set forth in SEQ ID NO: 24.
The PAH coding sequence has a silent variation, and optionally 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 is the amino acid sequence set forth in SEQ ID NO: 66 or 67. Including, and / or
The first portion of the PAH coding sequence comprises the amino acid sequence set forth in SEQ ID NO: 64 or 65, and 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 editing element comprises a ribosome skipping element and the PAH coding sequence or the PAH intron insertion coding sequence from 5'to 3'.
The AAV of claim 1.

The editing element further comprises a polyadenylation sequence on the 3'side of the PAH coding sequence or the PAH intron insertion coding sequence, and if necessary,
The polyadenylation sequence is an extrinsic polyadenylation sequence, and if necessary, the extrinsic polyadenylation sequence is an SV40 polyadenylation sequence, and if necessary, the SV40 polyadenylation sequence is SEQ ID NO: 31-. Containing a nucleotide sequence selected from the group consisting of 34,
The nucleotide on the 5'side of the target locus is in the exon of the PAH gene.
The nucleotide on the 5'side of the target locus is in exon 1 of the PAH gene.
The editing element further comprises a splice acceptor on the 5'side of the ribosome skipping element, if necessary.
The nucleotide on the 5'side of the target locus is in the intron of the PAH gene and / or
The nucleotide on the 5'side of the target locus is in intron 1 of the PAH gene and / or
The editing element comprises the nucleotide sequence set forth in SEQ ID NO: 35.
The AAV according to claim 1 or 2.

The 5'homologous arm nucleotide sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the first genomic region.
The 3'homologous arm nucleotide sequence is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the second genomic region.
The first genomic region comprises the nucleotide sequence set forth in SEQ ID NO: 36.
The second genomic region comprises the nucleotide sequence set forth in SEQ ID NO: 45.
Each of the 5'and 3'homologous arm nucleotide sequences independently has a length of about 100-about 2000 nucleotides.
The 5'homologous arm corresponds to 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, and nucleotide 7 of the PAH gene. G, 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, said. 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, G of the PAH gene. C corresponding to nucleotide-433, C corresponding to nucleotide-432 of the PAH gene, ACGCTGTTCTTCGCC (SEQ ID NO: 68) corresponding to nucleotides -394 to -388 of the PAH gene, and nucleotide -341 of the PAH gene. A, 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 corresponding to nucleotide -203 of the PAH gene. Including A,
The 5'homologous arm
(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 the above. 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 the A corresponding to nucleotide-203 of the PAH gene.
And optionally, the 5'homologous arm comprises modifications of (c) and (d), (f) and (g), and / or (b) and (h).
The 5'homologous arm comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 36-44 and 115.
The 3'homologous arm comprises the nucleotide sequence set forth in SEQ ID NO: 45 or 117, and / or
The modified genome comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 46-54, 118, and 136.
The AAV according to any one of claims 1 to 3.

The modified genome has a 5'reverse end repeat (5'ITR) nucleotide sequence on the 5'side of the 5'homologous arm nucleotide sequence and a 3'reverse end repeat (3' reverse end repeat (3') on the 3'side of the 3'homologous arm nucleotide sequence. Further contains 3'ITR) nucleotide sequence, if necessary,
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. ,
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.
The modified genome comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 55-63, 119, and 137, and / or.
The modified genome comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 55-63, 119, and 137.
The AAV according to any one of claims 1 to 4.

The AAV capsid protein has at least 95% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17. Contains an amino acid sequence with, as needed,
(i) The amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 2 is C, and the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H, and the amino acid of SEQ ID NO: 2. The amino acid in the capsid protein corresponding to 312 is Q and the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 2 is A and in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2. The amino acid of is N, the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 2 is S, and 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 590 of SEQ ID NO: 2 is R, corresponding to amino acid 626 of SEQ ID NO: 2. The amino acid in the capsid protein is G or Y, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is. The amino acid is R, the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 2 is K, and 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.
(ii)
(a) Whether 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, and the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N, and the amino acid of SEQ ID NO: 2 Whether the amino acid in the capsid protein corresponding to 505 is R and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M.
(c) Whether 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 of SEQ ID NO: 2 The amino acid in the capsid protein corresponding to amino acid 706 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, 6, 7, 10, 11, 12, 13, 15, 16, or 17.
The AAV capsid protein is at least 95% of 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. Containing an amino acid sequence with sequence identity, if necessary,
(i) The amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 2 is R, and the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 2 is D, and the amino acid of SEQ ID NO: 2. The amino acid in the capsid protein corresponding to 206 is C and the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H and in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 2. The amino acid in the capsid protein is Q, 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 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, and the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I, corresponding to amino acid 505 of SEQ ID NO: 2. The amino acid in the capsid protein is R, the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 2 is R, and 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, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is R and the sequence. The amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 2 is K, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is C, or amino acid 718 of SEQ ID NO: 2. The amino acid in the corresponding capsid protein is G,
(ii)
(a) Whether 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, and the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N, and the amino acid of SEQ ID NO: 2 Whether the amino acid in the capsid protein corresponding to 505 is R and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M.
(c) Whether 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 of SEQ ID NO: 2 The amino acid in the capsid protein corresponding to amino acid 706 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 is at least the amino acid sequence of amino acids 1 to 736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. Contains an amino acid sequence with 95% sequence identity, if required,
(i) 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 65 of SEQ ID NO: 2 is I, and the amino acid of SEQ ID NO: 2. The amino acid in the capsid protein corresponding to 68 is V and the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 2 is R and in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 2. The amino acid in the capsid protein is L, the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 2 is R, and 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, and the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 2 is H, which corresponds to amino acid 312 of SEQ ID NO: 2. The amino acid in the capsid protein is Q, 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 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, and the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 2 is I and SEQ ID NO: 2. The amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: R is R, and the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 2 is R, and the amino acid corresponding to amino acid 626 of SEQ ID NO: 2 is the capsid. The amino acid in the protein is G or Y, the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 2 is. The amino acid in the capsid protein that is R and corresponds to amino acid 690 of SEQ ID NO: 2 is K, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 2 is C or SEQ ID NO: The amino acid in the capsid protein corresponding to amino acid 718 of 2 is G,
(ii)
(a) Whether 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) Whether 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) Whether 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) Whether 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) Whether 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, and the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 2 is N, and the amino acid of SEQ ID NO: 2 Whether the amino acid in the capsid protein corresponding to 505 is R and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 2 is M.
(g) Whether 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 of SEQ ID NO: 2 The amino acid in the capsid protein corresponding to amino acid 706 is C and / or
(iii) The amino acid sequence of amino acids 1 to 736 of the capsid protein of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17 including,
The AAV according to any one of claims 1 to 5.

When the AAV is administered to mice implanted with human hepatocytes under standard AAV administration conditions in the absence of exogenous nucleases, the efficiency of integration of the editing element into the target locus is at least 1. % And / or the edit to the target locus when the AAV is administered to mice implanted with human hepatocytes under standard AAV administration conditions in the absence of exogenous nucleases. The AAV according to any one of claims 1 to 6, wherein the built-in allele frequency of the element is at least 0.5%.

A pharmaceutical composition comprising the AAV according to any one of claims 1 to 7 .

A method for correcting a mutation in the phenylalanine hydroxylase (PAH) gene in a cell, which comprises the step of transducing the cell with AAV according to any one of claims 1 to 7.
A method in which the cells are transduced with an exogenous nuclease, or a nucleotide sequence encoding an exogenous nuclease, without co-transduction or co-administration.

The cells are hepatocytes, renal cells, or cells in the brain, pituitary gland, adrenal gland, pancreas, bladder, bile sac, colon, small intestine or breast, and / or the cells are cells of a mammalian subject. The method of claim 9, wherein AAV is administered to the subject in an amount effective for transduction into the subject's cells.

The use of AAV according to any one of claims 1 to 7 for the treatment of a subject suffering from a disease or disorder associated with a PAH gene mutation.
An extrinsic nuclease, or a nucleotide sequence encoding an extrinsic nuclease, is not co-administered to said subject, use.

The disease or disorder is phenylketonuria and / or
The use according to claim 11, wherein the subject is a human subject.

A packaging system for recombinant preparation of replication-deficient AAV.
(a) Rep nucleotide sequences encoding one or more AAV Rep proteins,
(b) Cap nucleotide sequence encoding one or more AAV capsid proteins according to claim 6.
(c) With the modified genome according to any one of claims 1 to 6.
A packaging system comprising, and effective for encapsulating the modified genome in the capsid in the cell to form the AAV.

A first vector containing the Rep nucleotide sequence and the Cap nucleotide sequence and a second vector containing the modified genome are included.
The Rep nucleotide sequence encodes the AAV2 Rep protein, and optionally the AAV2 Rep protein is 78/68 or Rep 68/52, and / or the AAV2 Rep protein is AAV2 of SEQ ID NO: 22. It comprises an amino acid sequence having the least sequence identity percent with respect to the Rep amino acid sequence, the minimum sequence identity percent being at least 70% over the length of the amino acid sequence encoding the AAV2 Rep protein, and / or. ,
A third vector is further included, wherein the third vector is a helper virus vector and, if necessary,
The helper virus vector is an independent third vector,
The helper virus vector is integrated with the first vector,
The helper virus vector is integrated with the second vector,
The third vector contains a gene encoding a helper viral protein and / or
Helper viruses are selected from the group consisting of adenovirus, herpesvirus, vaccinia virus, and cytomegalovirus (CMV), as needed.
The helper virus is an adenovirus, and the adenovirus genome contains, or optionally, one or more adenovirus RNA genes selected from the group consisting of E1, E2, E4 and VA.
The helper virus is a herpes simplex virus (HSV), and the HSV genome is, if necessary, among the HSV genes selected from the group consisting of UL5 / 8/52, ICPO, ICP4, ICP22 and UL30 / UL42. Including one or more,
The packaging system according to claim 13.

The first vector and the third vector are contained in the first transfection plasmid.
The second vector and the third vector are contained in the second transfect plasmid.
The nucleotides of the first vector and the third vector are cloned into a recombinant helper virus or
The nucleotides of the second vector and the third vector are cloned into a recombinant helper virus.
The packaging system according to claim 14.

The method according to any one of claims 13 to 15 , which is a method for recombinant preparation of replication-deficient AAV, under conditions effective for encapsulating the modified genome in the capsid to form the AAV. The method comprising the step of introducing the packaging system of the above into cells.