Okada T, Mizukami H, Urabe M, Nomoto T, Matsushita T, Hanazono Y, Kume A, Tobita K, Ozawa K
Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-kawachi, Kawachi, Tochigi 329-0498, Japan.
Biochem Biophys Res Commun. 2001 Oct 19;288(1):62-8. doi: 10.1006/bbrc.2001.5730.
One of the limitations of recombinant adeno-associated virus (rAAV) vector systems for gene therapy applications has been the difficulty in producing the vector in sufficient quantity for adequate evaluation. Since the AAV Rep proteins are cytotoxic, it is not easy to establish stable cell lines that express them constitutively. We describe 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 for rAAV vector production through adenovirus-mediated introduction of a Cre recombinase gene. Without the introduction of the Cre recombinase gene, the cell line was shown to produce neither Rep nor Cap. rAAV vector was produced (1 x 10(9) genome copies/3.5-cm dish) 4 days after the transduction with Cre-expression adenovirus vector together with transfection of AAV vector plasmid. We further showed that the addition of Cap-expression adenovirus vector caused a 10-fold increase in the yield of rAAV vector. This system is also capable of producing rAAV as a transfection-free system by using a small amount of rAAV instead of vector plasmid.
重组腺相关病毒（rAAV）载体系统用于基因治疗时存在的一个局限性是难以大量生产载体以进行充分评估。由于AAV Rep蛋白具有细胞毒性，因此难以建立持续表达它们的稳定细胞系。我们描述了一种新型的源自293细胞的预包装细胞系，该细胞系通过loxP侧翼的CMV启动子持续表达反义rep/cap。通过腺病毒介导的Cre重组酶基因导入，该细胞系被转化为用于生产rAAV载体的包装细胞系，用于表达Rep/Cap。在未导入Cre重组酶基因的情况下，该细胞系既不产生Rep也不产生Cap。在用Cre表达腺病毒载体转导并转染AAV载体质粒4天后，产生了rAAV载体（1×10⁹基因组拷贝/3.5厘米培养皿）。我们进一步表明，添加Cap表达腺病毒载体可使rAAV载体产量提高10倍。该系统还能够通过使用少量rAAV而非载体质粒，作为无转染系统来生产rAAV。
