Olgun Hazal Banu, Tasyurek Hale M, Sanlioglu Ahter Dilsad, Sanlioglu Salih
Human Gene and Cell Therapy Center of Akdeniz University Hospitals, Antalya, Turkey.
Methods Mol Biol. 2019;1879:323-345. doi: 10.1007/7651_2018_150.
Lentiviral vectors are becoming preferred vectors of choice for clinical gene therapy trials due to their safety, efficacy, and the long-term gene expression they provide. Although the efficacy of lentiviral vectors is mainly predetermined by the therapeutic genes they carry, they must be produced at high titers to exert therapeutic benefit for in vivo applications. Thus, there is need for practical, robust, and scalable viral vector production methods applicable to any laboratory setting. Here, we describe a practical lentiviral production technique in roller bottles yielding high-titer third-generation lentiviral vectors useful for in vivo gene transfer applications. CaPO-mediated transient transfection protocol involving the use of a transfer vector and three different packaging plasmids is employed to generate lentivectors in roller bottles. Following clearance of cellular debris via low-speed centrifugation and filtration, virus is concentrated by high-speed ultracentrifugation over sucrose cushion.
慢病毒载体因其安全性、有效性以及所提供的长期基因表达，正成为临床基因治疗试验中首选的载体。尽管慢病毒载体的有效性主要由其携带的治疗性基因预先决定，但为了在体内应用中发挥治疗作用，它们必须以高滴度生产。因此，需要适用于任何实验室环境的实用、稳健且可扩展的病毒载体生产方法。在此，我们描述了一种在滚瓶中生产慢病毒的实用技术，可产生用于体内基因转移应用的高滴度第三代慢病毒载体。采用涉及使用转移载体和三种不同包装质粒的磷酸钙介导的瞬时转染方案，在滚瓶中产生慢病毒载体。通过低速离心和过滤清除细胞碎片后，病毒通过在蔗糖垫层上进行高速超速离心进行浓缩。
