Escarpe Paul, Zayek Nathalie, Chin Peggy, Borellini Flavia, Zufferey Romain, Veres Gabor, Kiermer Veronique
Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, California 94080, USA.
Mol Ther. 2003 Aug;8(2):332-41. doi: 10.1016/s1525-0016(03)00167-9.
Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient ex vivo and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be demonstrated that lentiviral vector preparations are safe and not contaminated by replication-competent recombinants related to the parental pathogenic virus. Here we describe a sensitive assay for the detection of replication-competent lentiviruses (RCL) in large-scale preparations of HIV-based lentiviral vectors. This RCL assay for lentiviral vectors is based on the principles used for retroviral vectors, using a highly permissive cell line, C8166-45, for RCL amplification and an appropriate positive control virus to establish the assay sensitivity. The assay is capable of detecting 1 RCL infectious unit in a background of 2.5 x 10(8) transducing units of vector in a single test culture. Statistically representative samples from large-scale lentiviral vector productions were assayed using multiple test cultures for each lot. Overall, a total of 1.4 x 10(10) transducing units of vector from 10 independent 14-liter production lots were screened and no RCL was detected. We propose to implement this assay as a release testing for clinical-grade lentiviral vector preparations intended for gene therapy clinical trials.
慢病毒载体已展现出作为基因治疗载体的巨大潜力，可介导高效的体外和体内基因递送，并在分裂细胞和非分裂细胞中实现长期转基因表达。然而，对于临床研究而言，必须证明慢病毒载体制剂是安全的，且未被与亲代致病病毒相关的具有复制能力的重组体污染。在此，我们描述了一种用于检测基于HIV的慢病毒载体大规模制剂中具有复制能力的慢病毒（RCL）的灵敏检测方法。这种针对慢病毒载体的RCL检测方法基于用于逆转录病毒载体的原理，使用高度敏感的细胞系C8166 - 45进行RCL扩增，并使用适当的阳性对照病毒来确定检测灵敏度。该检测方法能够在单个测试培养物中，在2.5×10⁸个载体转导单位的背景下检测到1个RCL感染单位。对于大规模慢病毒载体生产的具有统计学代表性的样本，对每个批次使用多个测试培养物进行检测。总体而言，对来自10个独立的14升生产批次的总共1.4×10¹⁰个载体转导单位进行了筛选，未检测到RCL。我们建议将此检测方法作为用于基因治疗临床试验的临床级慢病毒载体制剂的放行检测方法。
