Duan Deming, Ji Yongpiao, Zhou Mengjie, Gao Jimin
School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China.
Sheng Wu Gong Cheng Xue Bao. 2019 Jul 25;35(7):1307-1316. doi: 10.13345/j.cjb.190127.
Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.
基因治疗是一个快速发展的领域。最广泛使用的外源基因转移技术是慢病毒介导的基因治疗。就安全性而言，慢病毒载体已从第一代发展到第三代。制备高滴度的慢病毒仍然困难。在本研究中，使用Fibra-Cel片载体作为HEK293T细胞载体基质，并将几个无菌细胞培养旋转器组合在滚瓶机上进行培养，以扩大贴壁细胞的规模。通过逐一筛选第三代慢病毒包装过程中优化慢病毒滴度的因素，使病毒滴度最大化。Fibra-Cel片载体成功用作HEK293T细胞粘附的基质，以大规模培养贴壁细胞。筛选了通过滚瓶大规模制备第三代慢病毒的最佳条件，并中试规模生产了三批慢病毒。从质粒转染到病毒收获，慢病毒的生产时间从120小时缩短至54小时；在成本方面，使用滚瓶机代替生物反应器，成本更低且整个过程无需重复灭菌。成功生产的安全、有效和低成本操作将为慢病毒的大规模制备提供技术基础，从而为其临床应用奠定坚实基础。
