Development of the gene therapy technologies using adeno-associated virus (AAV)

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We studied the gene transfer methods using adeno-associated virus (AAV).1. Development of the method for AAV vector production: We developed novel packaging cell lines for AAV vector production by regulating the expression of cytotoxic AAV proteins through Cre/loxP system. First, we applied combined Cre/loxP system using variant loxP and wild-type loxP to the simultaneous regulation of Rep and Cap expressions. Second, we developed a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap through the introduction of a Cre recombinase gene.2. Establishment and application of highly sensitive detection method for AAV vector-mediated transgenes : Long PCR (sometimes combined with nested PCR) was conducted with appropriate primer sets located on the D region of ITR. We also collected many samples from the experimental animals which received intramuscular injections of AAV vectors.3. Development of the method for targeted vector integration (TVI) into a defined locus on chromosome 19 using AAV-derived components (ITR and Rep gene) : 293 and K562 cells were transfected with the neoγ gene using the TVI method. We amplified junctional regions between cellular and transgene sequences by Alu-PCR. As a result, no cellular sequences regarded as a common recognition motif of the Rep proteins was found. We also developed mutant Rep-expression vectors with reduced cytotoxicity.