EP-4737578-A2 - GENE THERAPY EMPLOYING GENOME EDITING WITH SINGLE AAV VECTOR

Abstract

An adeno-associated virus (AAV) vector for inserting a desired nucleic acid into a nucleic acid in a cell, wherein the nucleic acid in the cell comprises a region consisting of a first nucleotide sequence and a region consisting of a second nucleotide sequence in order in a direction from a 5' end to a 3' end, wherein the vector comprises a first gRNA target sequence, a region consisting of a first nucleotide sequence, the desired nucleic acid, a region consisting of a second nucleotide sequence, a second gRNA target sequence, a cell-specific promoter, a sequence encoding a Cas9 nuclease, an RNA polymerase III promoter, a sequence encoding a first gRNA recognizing the first gRNA target sequence and a sequence encoding a second gRNA recognizing the second gRNA target sequence, wherein the vector yields a nucleic acid fragment comprising a region consisting of a first nucleotide sequence, the desired nucleic acid and the region consisting of the second nucleotide sequence by the Cas9 nuclease, wherein a first nucleotide sequence in the nucleic acid in the cell and a first nucleotide sequence in the vector are linked by a microhomology-mediated joining and a second nucleotide sequence in the nucleic acid in the cell and a second nucleotide sequence in the vector are linked by a microhomology-mediated joining, thereby inserting the desired nucleic acid between the region consisting of the first nucleotide sequence and the region consisting of the second nucleotide sequence in the nucleic acid in the cell.

Inventors

• NISHIGUCHI, KOJI
• NAKAZAWA, TORU
• FUJITA, KOSUKE

Assignees

• NATIONAL UNIVERSITY CORPORATION TOKAI NATIONAL HIGHER EDUCATION AND RESEARCH SYSTEM

Dates

Publication Date

20260506

Application Date

20191108

Claims (12)

1. A method of manufacturing an adeno-associated virus (AAV) vector for inserting a desired nucleic acid into a nucleic acid in a cell, comprising: respectively arranging a first gRNA target sequence, a first PAM sequence, a first nucleotide sequence, the desired nucleic acid, a second nucleotide sequence, a second PAM sequence and a second gRNA target sequence in order in a direction from a 5' end to a 3' end to cause a microhomology-mediated joining (Microhomology-mediated end-joining, MMEJ) with a genomic nucleic acid in the cell; and arranging the vector to further comprise a promoter specific to the cell, a sequence encoding a Cas9 nuclease, a first RNA polymerase III promoter, a sequence encoding a first gRNA recognizing the first gRNA target sequence, a Scaffold sequence, a second RNA polymerase III promoter, a sequence encoding a second gRNA recognizing the second gRNA target sequence and a Scaffold sequence in order in a direction from a 5' end to a 3' end; wherein the vector is configured to comprise a cleavage site that yields, by the Cas9 nuclease, a nucleic acid fragment comprising the desired nucleic acid, the first nucleotide sequence, and the second nucleotide sequence; and wherein the length of the promoter specific to the cell is 500 bases or less.
2. The method of claim 1, comprising introducing, when there is a sequence which is the same as the first and/or second gRNA target sequence between the first nucleotide sequence and the second nucleotide sequence, a mutation in the first and/or second gRNA target sequence so as to avoid cleavage of the first and/or second gRNA target sequence present between the first nucleotide sequence and the second nucleotide sequence by the Cas9 nuclease.
3. The method of claim 1 or 2, comprising introducing, when there is a sequence which is the same as the first and/or second gRNA target sequence between the first nucleotide sequence and the second nucleotide sequence, a mutation in a first PAM sequence adjacent to the first gRNA target sequence and/or a second PAM sequence adjacent to the second gRNA target sequence so as to avoid cleavage of the first and/or second gRNA target sequence present between the first nucleotide sequence and the second nucleotide sequence by the Cas9 nuclease.
4. The method of any one of claims 1 to 3, wherein the promoter specific to the cell is a promoter selected from rhodopsin kinase promoter, RPE65 promoter, Best1 promoter, mGluR6 promoter, cone arrestin promoter, CRALBP1 promoter, Chx10 promoter, rhodopsin promoter, cone opsin promoter, recoverin promoter, synapsin I promoter, myelin basic protein promoter, neuron-specific enolase promoter, calcium/calmodulin-dependent protein kinase II (CMKII) promoter, tubulin α I promoter, platelet-derived growth factor β chain promoter, glial fibrillary acidic protein (GFAP) promoter, L7 promoter and glutamic acid receptor delta 2 promoter; or a promoter having consecutive 50 to 150 base sequence of the promoter; or a promoter consisting of a base sequence that is 90% or more identical with the 50 to 150 base sequence.
5. The method of any one of claims 1 to 4, wherein the number of bases in the first and/or second nucleotide sequence is 5 to 40.
6. A vector manufactured by the method of any one of claims 1 to 5.
7. An adeno-associated virus (AAV) vector for inserting a desired nucleic acid into a nucleic acid in a cell, comprising: a first gRNA target sequence, a first PAM sequence, a first nucleotide sequence, the desired nucleic acid, a second nucleotide sequence, a second PAM sequence and a second gRNA target sequence arranged in order in a direction from a 5' end to a 3' end, wherein the first nucleotide sequence and the second nucleotide sequence cause a microhomology-mediated joining (Microhomology-mediated end-joining, MMEJ) with a genomic nucleic acid in the cell; and a promoter specific to the cell, a sequence encoding a Cas9 nuclease, a first RNA polymerase III promoter, a sequence encoding a first gRNA recognizing the first gRNA target sequence, a Scaffold sequence, a second RNA polymerase III promoter, a sequence encoding a second gRNA recognizing the second gRNA target sequence and a Scaffold sequence arranged in order in a direction from a 5' end to a 3' end; wherein the vector is configured to comprise a cleavage site that yields, by the Cas9 nuclease, a nucleic acid fragment comprising the desired nucleic acid, the first nucleotide sequence, and the second nucleotide sequence; and wherein the length of the promoter specific to the cell is 500 bases or less.
8. The vector of claim 7, which is for treating a disease in a subject.
9. A therapeutic composition for use in treating a disease, comprising a vector manufactured by the method of any one of claims 1 to 5.
10. A promoter comprising the nucleotide sequence set forth in SEQ ID NO: 4.
11. An adeno-associated virus (AAV) vector comprising the promoter of claim 10.
12. A method for retina-specific transcription of a gene, comprising using the promoter of claim 10 as a promoter for the transcription of the gene or the adeno-associated virus (AAV) vector comprising the promoter of claim 10.

Description

[Technical Field] The present invention relates to an adeno-associated virus (AAV) vector for inserting a desired nucleic acid into a corresponding nucleic acid in a cell, a method of introducing a nucleic acid, a cell including the desired nucleic acid and a therapeutic method for treating an ocular disease. [Background Art] An adeno-associated virus (AAV) has been widely used in gene therapy and multiple successful examples of gene supplementation therapy using an AAV against an inherited retinal degeneration have recently been reported (Non Patent Literature 1). As for the therapeutic concept, it is very reasonable to aim to cure a disease by introducing a gene with a normal function to a target retinal cell using an AAV, aiming to compensate for the causal gene with decreased or lost function. Although this therapeutic platform allows to target various genes, many of the frequent pathogenic genes causing retinal degeneration greatly exceed 4000 bp. This is a big problem since there is a restriction in the size of the gene (about 4000 bp or less) that can be effectively delivered by an AAV vector. For example, an EYS gene, which is a pathogenic gene with an overwhelmingly high frequency in Japanese patients with retinitis pigmentosa, is about 10000 bp. This gene cannot be treated by a conventional AAV gene supplementation therapy. Meanwhile, in the recently developed PITCh (Precise Integration into Target Chromosome) method, a homology arm used to precisely insert a DNA fragment to a particular position on the genome is extremely short compared to a conventional homology arm. Thus, a use of this method greatly reduced the size of the element necessary for genome editing therapy (Patent Literature 1, Non Patent Literature 2). [Citation List] [Patent Literature] [PTL 1] WO 2015/068785 [Non Patent Literature] [NPL 1] IOVS, September 2007, Vol.48, No.9[NPL 2] NATURE PROTOCOL, Vol.11, No.1 published on line 17 December 2015, doi:10.1038/nprot.2015.140 [Summary of Invention] [Technical Problem] While there are reports of genome editing gene therapy using an AAV, none has been successful in accurately replacing a mutated sequence with a normal sequence by genome editing with a single AAV vector because the Cas9 gene required for genome editing is too large. The problem to be solved by the present invention is to provide an AAV vector that can introduce a desired nucleic acid to a genome in a cell with a single vector, a method of introducing a nucleic acid using the AAV vector, a cell manufactured by the introduction method and a method for treating a disease using the AAV vector. [Solution to Problem] While a conventional gene therapy assumes introducing a full-length gene by a vector, the inventors of the present invention found out that it is possible to prepare an AAV vector useful for gene therapy or mutation correction of a nucleic acid of a large gene by adopting a method of replacing only an abnormal sequence of a gene using genome editing (excise the abnormal sequence and insert a normal sequence), which led to the establishment of the present invention. In other words, the present invention encompasses the subjects described in the following items. Item 1. An adeno-associated virus (AAV) vector for inserting a desired nucleic acid into a nucleic acid in a cell, wherein the nucleic acid in the cell comprises a region consisting of a first nucleotide sequence and a region consisting of a second nucleotide sequence in order in a direction from a 5' end to a 3' end,wherein the vector comprises a first gRNA target sequence, a region consisting of a first nucleotide sequence, the desired nucleic acid, a region consisting of a second nucleotide sequence, a second gRNA target sequence, a cell-specific promoter, a sequence encoding a Cas9 nuclease, an RNA polymerase III promoter, a sequence encoding a first gRNA recognizing the first gRNA target sequence and a sequence encoding a second gRNA recognizing the second gRNA target sequence,wherein the vector yields a nucleic acid fragment comprising a region consisting of a first nucleotide sequence, the desired nucleic acid and the region consisting of the second nucleotide sequence by the Cas9 nuclease,wherein a first nucleotide sequence in the nucleic acid in the cell and a first nucleotide sequence in the vector are linked by a microhomology-mediated joining and a second nucleotide sequence in the nucleic acid in the cell and a second nucleotide sequence in the vector are linked by a microhomology-mediated joining, thereby inserting the desired nucleic acid between the region consisting of the first nucleotide sequence and the region consisting of the second nucleotide sequence into the nucleic acid in the cell.Item 2. The vector of item 1, wherein the nucleic acid in the cell has a target nucleic acid sequence replaced with the desired nucleic acid between the region consisting of the first nucleotide sequence and the region consisting of the second nucleotide