Cerqueira Carla, Thompson Cynthia D, Day Patricia M, Pang Yuk-Ying S, Lowy Douglas R, Schiller John T
Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Mol Ther Methods Clin Dev. 2017 Apr 19;5:165-179. doi: 10.1016/j.omtm.2017.04.005. eCollection 2017 Jun 16.
Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer papillomavirus L1/L2 capsids. We investigated the incorporation of several DNA forms into a variety of different papillomavirus types, including human and animal types. Our results show that papillomavirus capsids can package and transduce linear or circular DNA under defined conditions. Packaging and transduction efficiencies were surprisingly variable across capsid types, DNA forms, and assembly reaction conditions. The pseudoviruses produced by these methods are sensitive to the same entry inhibitors as cell-derived pseudovirions, including neutralizing antibodies and heparin. The papillomavirus vector production systems developed in this study generated as high as 10 infectious units/mg of L1. The pseudoviruses were infectious both in vitro and in vivo and should be compatible with good manufacturing practice (GMP) requirements.
乳头瘤病毒衣壳能够包装各种各样的非病毒DNA质粒，并将包装好的遗传物质递送至细胞，这使其成为靶向基因递送载体的理想候选者。然而，目前方法所产生的乳头瘤病毒载体不太可能适用于临床应用。我们开发了一种化学成分明确、无细胞的、基于乳头瘤病毒的载体生产系统，该系统能够将纯化的质粒DNA（假基因组）整合至高滴度乳头瘤病毒L1/L2衣壳中。我们研究了几种DNA形式在多种不同乳头瘤病毒类型中的整合情况，包括人类和动物类型。我们的结果表明，在特定条件下，乳头瘤病毒衣壳能够包装并转导线性或环状DNA。在衣壳类型、DNA形式和组装反应条件方面，包装和转导效率存在惊人的差异。通过这些方法产生的假病毒对与细胞来源的假病毒相同的进入抑制剂敏感，包括中和抗体和肝素。本研究中开发的乳头瘤病毒载体生产系统产生的病毒滴度高达每毫克L1有10个感染单位。这些假病毒在体外和体内均具有感染性，并且应该符合药品生产质量管理规范（GMP）要求。
