Olgun Hazal Banu, Tasyurek Hale M, Sanlioglu Ahter D, Sanlioglu Salih
Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Antalya, Turkey.
Methods Mol Biol. 2019;1879:347-365. doi: 10.1007/7651_2018_154.
Lentiviral vectors (LVs) have been increasingly used in clinical gene therapy applications particularly due to their efficient gene transfer ability, lack of interference from preexisting viral immunity, and long-term gene expression they provide. Purity of LVs is essential in in vivo applications, for a high therapeutic benefit with minimum toxicity. Accordingly, laboratory scale production of LVs frequently involves transient cotransfection of 293T cells with packaging and transfer plasmids in the presence of CaPO. After clearance of the cellular debris by low-speed centrifugation and filtration, lentivectors are usually concentrated by high-speed ultracentrifugation in sucrose cushion. Concentrated viral samples are then purified by anion exchange chromatography (AEX) after benzonase treatment to remove the residual cellular DNA. Here, we describe an improved practical method for LV purification using AEX, useful for experimental studies concerning gene and stem cell therapy.
慢病毒载体（LVs）因其高效的基因转移能力、不受既往病毒免疫干扰以及能实现长期基因表达，在临床基因治疗应用中越来越受到青睐。慢病毒载体的纯度对于体内应用至关重要，因为这能在毒性最小的情况下实现高治疗效益。因此，实验室规模生产慢病毒载体通常涉及在磷酸钙存在的情况下，将293T细胞与包装质粒和转移质粒进行瞬时共转染。通过低速离心和过滤清除细胞碎片后，慢病毒载体通常在蔗糖垫层中通过高速超速离心进行浓缩。然后，在经核酸酶处理以去除残留的细胞DNA后，通过阴离子交换色谱法（AEX）对浓缩的病毒样本进行纯化。在此，我们描述了一种使用AEX纯化慢病毒载体的改进实用方法，该方法对基因和干细胞治疗的实验研究很有用。
