| Co-Investigator(Kenkyū-buntansha) |
SHIMADA Takashi Nippon Medical School, Department of Biochemistry and Molecular Biology, Profess, 教授 (20125074)
ENDO Fumio Kumamoto University School of Medicine, Department of Pediatrics, Professor, 医学部, 講師 (00176801)
YAMAMOTO Tetsuro Kumamoto University Graduate School of Medical Sciences, Division of Molecular P, 医学部, 教授 (60112405)
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| Research Abstract |
To achieve the efficient and persistent gene therapy for ornithine transcarbamylase deficiency (OTCD), we have tried to prepare recombinant adenoassociated virus (rAAV) vector. At first, we prepared two vector plasmids which had a GAG promotor and a hOTCcDNA with a TK neo (5.35kb, OTC2) or not (4.4kb, OTC3) between ITRs. To evaluate these plasmids, we prepared rAAV with each plasmid as previously described (Current protocols in human genetics).
rAAV vector with OTC2 was prepared at lO^4cfu/ml, but no enzymal activity was obtained. Another vector with OTC3 was obtained at 10^5 particles/ml and 2*10^5 particles of this vector expressed 1.265mumo1/mg-protein/hr of OTC activity in 10^6 cells of HepG2 (endogeneous activity ; O.02-O.03mumol/mg-protein/hr).
However, this concentration was insufficient for in vivo gene therapy. To obtain higher titered rAAV, we tried to prepare new packaging cell lines with vector plasmid and helper plasmid and new constraucts of vector plasmid.
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