Clark K R, Voulgaropoulou F, Johnson P R
Department of Pediatrics, College of Medicine, Children's Hospital Research Foundation, Children's Hospital, Columbus, OH 43205, USA.
Gene Ther. 1996 Dec;3(12):1124-32.
Adeno-associated virus (AAV) vectors are being developed for in vivo and ex vivo gene transfer to human cells. At present, widespread usage of AAV vectors is limited primarily by difficulties in generating recombinant virions on a scale sufficient for in-depth preclinical and clinical trials. However, recent work in several laboratories suggests that this technical obstacle should be overcome in the near future. As a result, it can be anticipated that the interest in AAV vectors will expand, Thus, it becomes important to develop assay systems that will permit accurate quantification of the infectivity of AAV vectors derived from a variety of sources. We have developed an assay using a cell line that expresses AAV helper functions (rep and cap) upon induction by adenovirus infection. This assay system is based on the replication of input rAAV genomes rather than transgene expression (transduction). Thus, infectivity titrations in this system yield an estimation of rAAV infectious particles irrespective of the promoter or transgene present in the vector genome. Moreover, this assay method is more sensitive than conventional methods being used in other laboratories.
腺相关病毒（AAV）载体正被开发用于体内和体外向人类细胞的基因转移。目前，AAV载体的广泛应用主要受到难以大规模生产重组病毒颗粒的限制，而这一规模足以用于深入的临床前和临床试验。然而，多个实验室最近的研究表明，这一技术障碍在不久的将来应该能够被克服。因此，可以预期对AAV载体的兴趣将会增加。所以，开发能够准确量化源自多种来源的AAV载体感染性的检测系统变得很重要。我们开发了一种检测方法，使用一种细胞系，该细胞系在腺病毒感染诱导后表达AAV辅助功能（rep和cap）。这个检测系统基于输入的重组腺相关病毒（rAAV）基因组的复制，而不是转基因表达（转导）。因此，该系统中的感染性滴定可以估算rAAV感染性颗粒的数量，而与载体基因组中存在的启动子或转基因无关。此外，这种检测方法比其他实验室使用的传统方法更灵敏。
