Rep/cap gene amplification and high-yield production of AAV in an A549 cell line expressing rep/cap
Rep/cap gene amplification and high-yield production of AAV in an A549 cell line expressing rep/cap
复制标题
DOI：
10.1006/mthe.2001.0591
复制
发表时间：
2002-05-01
影响因子：
12.4
通讯作者：
Wilson, JM
中科院分区：
文献类型：
--
作者：
Gao, GP;Lu, FM;Wilson, JM
稳定表达rep/cap的细胞系是研究腺相关病毒（AAV）生物学和生产AAV载体的重要工具。已经分离出几种rep/cap细胞系，每种细胞系都基于HeLa细胞。用腺病毒感染这些细胞系以产生AAV载体与rep/cap基因的大量扩增相关。对HeLa细胞中存在人乳头瘤病毒（HPV）序列的担忧可能会限制此类细胞系用于生产临床级载体。在这里，我们描述了一个非HeLa衍生的rep/cap细胞系称为K209，产生的稳定转染A549细胞与质粒构建体含有P5 rep/cap盒从AAV2。用腺病毒感染K209细胞会导致rep/cap基因扩增1000倍，并高产率产生腺相关病毒载体。导致rep/cap基因最大扩增和AAV高水平产生的腺病毒感染复数（MOI）在基于HeLa的细胞系中比K209细胞中所需的感染复数高10倍。我们的数据表明，HPV细胞中存在的乳头状瘤衍生的基因产物对于AAV载体的高产量生产是不需要的。
Cell lines stably expressing rep/cap are important tools for studying adeno-associated virus (AAV) biology and producing AAV vectors. Several rep/cap cell lines have been isolated, each of which is based on HeLa cells. Infection of these cell lines with adenovirus for production of AAV vector is associated with substantial amplification of the rep/cap gene. Concerns over the presence of human papilloma viral (HPV) sequences in HeLa cells may limit use of such lines for production of clinical-grade vectors. Here we describe a non-HeLa-derived rep/cap cell line called K209, generated by stable transfection of A549 cells with a plasmid construct containing the P5 rep/cap cassette from AAV2. Infection of K209 cells with adenovirus leads to a 1000-fold amplification of the rep/cap gene with high-yield production of AAV vectors. The multiplicity of infection (MOI) of adenovirus that led to maximum amplification of the rep/cap gene and high-level production of AAV is 10 times higher in the HeLa-based cell line than that required in K209 cells. Our data suggest that papilloma-derived gene products present in HeLa cells are not required for high-yield production of AAV vectors.