URABE Masashi Jichi Med.Sch., Faculty of Medicine, Research Associate, 医学部, 助手 (40213516)
MANO Hiroyuki Jichi Med.Sch., Faculty of Medicine, Associate Professor, 医学部, 助教授 (90240704)
|Budget Amount *help
¥7,100,000 (Direct Cost : ¥7,100,000)
Fiscal Year 1997 : ¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 1996 : ¥3,700,000 (Direct Cost : ¥3,700,000)
The desirable gene-delivery system for gene therapy should have several features, including 1) integration of the delivered gene in a predictable or site-specific manner into the target genome, 2) long-term expression of the transgene, and 3) the ability to deliver DNA sequences of sufficiently large size to include all the regulatory elements. The development of a novel gene delivery system, termed targeted vector integration (TVI), is currently in progress to fulfilll the above prerequisites. The system is based on adeno-associated virus (AAV) which preferentially integrates into the human genome at a defined locus, called AAVI1, on chromosome 19 (19q13.3-qter). The AAV-Rep proteins are considered to mediate integration of DNA sequence containing AAV-ITR (inverted terminal repeat) into AAVS1 locus, through the formation of a complex between GAGC repeats within ITR and a similar sequence in AAVS1 locus. There are 4 different Rep proteins (Rep78, Rep68, Rep52, and Rep40). Aiming at determining the Rep (s), which confer the ability of site-specific integration of ITR-linked genes, we constructed various plasmids that express individual Rep proteins. These plasmids were co-transfected into 293 cells with the plasmid containing a LacZ expression cassette flanked by ITRs. A PCR-based dot blot assay, Southern analysis, and FISH analysis were conducted to detect site-specific integration. The results showed that the large Rep (78 ot 68) is necessary for the site-specific integration of ITR-linked genes. In addition, mutant Rep proteins with R107A,K136A,or R138A showed the decreased binding to GAGC repeat and no nicking activity. These mutants also lost the site-specific integration activity. The present study will be valuable to develop the more refined TVI system.