Development of the method for chromosomal site-specific integration of transgenes using AAV and its application to hematopoietic cells

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Targeted integration of foreign DNA (TVI:targeted vector integration) is desirable for safe gene therapy. Adeno-associated virus (AAV) has the ability to integrate its genome into a defined locus, AAVS1 (19q13.3-qter). The inverted terminal repeat (ITR) at both ends of the AAV genome and Rep proteins are responsible for this site-specific integration. Therefore, two AAV components, Rep and ITR, are utilized to develop the TVI system. A mutant Rep protein that lacks cytotoxicity while retaining the ability to mediate AAVS1-specific integration is an attractive molecule for this system. We constructed plasmids containing the genes encoding mutant Rep proteins, in which all the charged amino acids in the N-terminal region were mutated to alanine. We found several mutants showed lower cytotoxicity, compared to the wild type Rep protein, without losing the ability of the site-specific integration. We are further screening a more suitable mutant Rep for the AAVS1-directed integration of genes. To analyze detailed mechanism of Rep-mediated integration, we amplified junctional regions between cellular and transgene sequences by using an Alu-PCR technique. The TVI system is valuable especially when dividing cells such as hematopoietic cells are transduced and long-term transgene expression is needed. We have showed that this TVI system could introduce a transgene into AAVS1 in K562 cells.