利用多基因缺失型杆状病毒在昆虫细胞中生产腺相关病毒

Production of adeno-associated virus in insect cells using multiple gene deleted baculovirus

摘要:

腺相关病毒(adeno-associated virus, AAV)是基因治疗领域最常使用的病毒载体之一，产量低、成本高是该产业面临的关键瓶颈问题。本研究旨在基于多基因缺失型杆状病毒，建立双病毒感染昆虫细胞以生产AAV的技术体系。首先，进行AAV生产用多基因缺失型重组杆状病毒的构建和扩增，并检测杆状病毒滴度及其感染细胞的效果；然后，使用双杆状病毒共感染昆虫细胞，并优化感染条件；最后，基于优化条件进行AAV生产，并检测评估产量、质量等相关指标。结果表明，AAV生产用多基因缺失型杆状病毒滴度较野生型无差异，感染后细胞存活率下降明显减缓。使用双病毒路线进行AAV优化生产，Bac4.0-1的基因组滴度为1.63×10¹¹ VG/mL，Bac5.0-2的基因组滴度为1.02×10¹¹ VG/mL，较野生型产量分别提升了240%和110%。电镜下，3组均具有正常的AAV病毒形态，且转导活性相近。本研究建立了基于多基因缺失型杆状病毒感染昆虫细胞的AAV生产体系，显著提高了AAV产量，具有一定的应用价值。

Abstract:

Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key bottlenecks such as low yield and high-cost. The aim of this study was to establish a technology system for production of AAV in the double virus infected insects by using multiple-gene deleted baculovirus. First, a multiple gene deleted baculovirus for AAV production was constructed, and the baculovirus titer and its effect on infected cells was examined. Subsequently, the insect cells were co-infected with the double baculovirus and the infection conditions were optimized. At the final stage, we performed AAV production based on optimized conditions, and evaluated relevant parameters including production titer and quality. The results showed that the titer of AAV produced in the multiple gene deleted baculovirus was not different from that of the wild type, but the rate of cell death was significantly slower upon infection. Using the double virus route for optimized production of AAV, the genome titers were 1.63×10¹¹ VG/mL for Bac4.0-1 and 1.02×10¹¹ VG/mL for Bac5.0-2, which were elevated 240% and 110%, respectively, compared with that of the wild-type. Electron microscopy observations revealed that all three groups exhibited normal AAV viral morphology and they showed similar transduction activity. Taken together, we developed an AAV production system based on the infection of insect cells using multiple-gene deleted baculovirus, which significantly improved the virus yield and showed application potential.