GENE THERAPY EMPLOYING GENOME EDITING WITH SINGLE AAV VECTOR

摘要

An adeno-associated virus (AAV) vector which can be used for the insertion of a desired nucleic acid into a nucleic acid in a cell, wherein, when as observed in the direction from the 5'-terminal toward the 3'-terminal, the nucleic acid in the cell contains a region comprising a first nucleotide sequence and a region comprising a second nucleotide sequence, the vector contains a first gRNA target sequence, a region comprising a first nucleotide sequence, the desired nucleic acid, a region comprising a second nucleotide sequence, a second gRNA target sequence, a cell-specific promoter, a sequence encoding Cas9 nuclease, an RNA polymerase III promoter, a sequence encoding first gRNA capable of recognizing the first gRNA target sequence, and a sequence encoding second gRNA capable of recognizing the second gRNA target sequence, the vector can generate a nucleic acid fragment which contains a region comprising a first nucleotide sequence, the desired nucleic acid, and a region comprising the second nucleotide sequence by the action of the Cas9 nuclease, and the first nucleotide sequence in the nucleic acid in the cell and the first nucleotide sequence in the vector are linked to each other through microhomology-mediated end joining and the second nucleotide sequence in the nucleic acid in the cell and the second nucleotide sequence in the vector are linked to each other through microhomology-mediated end joining so that the desired nucleic acid is inserted in a region between the region comprising the first nucleotide sequence and the region comprising the second nucleotide sequence in the nucleic acid in the cell.