Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectorsстатья из журнала
Аннотация: Standard protocols for the generation of adenoassociated virus type 2 (AAV-2)-based vectors for human gene therapy applications require cotransfection of cells with a recombinant AAV (rAAV) vector plasmid and a packaging plasmid that provides the AAV rep and cap genes. The transfected cells must also be overinfected with a helper virus, e.g., adenovirus (Ad), which delivers multiple helper functions necessary for rAAV production. Therefore, rAAV stocks produced using these protocols are contaminated with helper adenovirus. The generation of a novel packaging/helper plasmid, pDG, containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids, is described here. Cotransfection of cells with pDG and an AAV vector plasmid was sufficient for production of infectious rAAV, resulting in helper virus-free rAAV stocks. The rAAV titers obtained using pDG as packaging plasmid were up to 10-fold higher than those achieved using conventional protocols for rAAV production. Replacement of the AAV-2 p5 promoter by an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; however, expression of the VP proteins was significantly increased compared with VP levels from standard packaging plasmids. Immunofluorescence analyses showed that the strong accumulation of VP proteins in pDG-transfected cells resulted in enhanced AAV capsid assembly, which is limiting for efficient rAAV production. Furthermore, using a monoclonal antibody highly specific for AAV-2 capsids (A20), an rAAV affinity purification procedure protocol was established. The application of the tools described here led to a significant improvement in recombinant AAV vector production and purification. Two novel tools allowing a simplified and more efficient generation and purification of adenoassociated virus type 2 (AAV-2)-derived vectors are described here. First, the generation of an AAV/adenovirus hybrid plasmid, pDG, which contains all packaging and helper functions required for production of recombinant AAV (rAAV), is reported. Cotransfection of cells with pDG and an AAV vector plasmid led to production of several hundred infectious rAAV particles per transfected cell. Using pDG as a helper plasmid, generation of rAAV was no longer dependent on overinfection of the cells with adenovirus, resulting in helper virus-free rAAV stocks. Second, an rAAV affinity purification procedure based on the monoclonal antibody A20, which specifically recognizes assembled AAV-2 capsids, is described. Detailed analyses of rAAV production using the new packaging and purification tools in comparison with standard protocols are presented.
Год издания: 1998
Издательство: Mary Ann Liebert, Inc.
Источник: Human Gene Therapy
Ключевые слова: Virus-based gene therapy research, Viral Infectious Diseases and Gene Expression in Insects, CAR-T cell therapy research
Другие ссылки: Human Gene Therapy (HTML)
PubMed (HTML)
PubMed (HTML)
Открытый доступ: closed
Том: 9
Выпуск: 18
Страницы: 2745–2760