Le Doux J M, Landazuri N, Yarmush M L, Morgan J R
Georgia Tech/Emory Department of Biomedical Engineering, Atlanta, GA 30332-0535, USA.
Hum Gene Ther. 2001 Sep 1;12(13):1611-21. doi: 10.1089/10430340152528110.
Previously, we have demonstrated that chondroitin sulfate proteoglycans and glycosaminoglycans inhibit retrovirus transduction. While studying the mechanism of inhibition, we found that the combined addition of equal-weight concentrations (80 microg/ml) of Polybrene and chondroitin sulfate C to retrovirus stocks resulted in the formation of a high-molecular-weight retrovirus-polymer complex that could be pelleted by low-speed centrifugation. The pelleted complex contained more than 80% of the virus particles, but less than 0.3% of the proteins that were originally present in the virus stock. Surprisingly, the virus in the complex remained active and could be used to transduce cells. The titer of the pelleted virus, when resuspended in cell culture medium to the starting volume, was three-fold greater than the original virus stock. The selectivity (CFU/mg protein) of the process with respect to virus activity was more than 1000-fold. When the pelleted virus-polymer complex was resuspended in one-eighth of the original volume and used to transduce NIH 3T3 murine fibroblasts and primary human fibroblasts, gene transfer was increased 10- to 20-fold over the original unconcentrated retrovirus stock. The implications of our findings for the production, processing, and use of retrovirus stocks for human gene therapy protocols are discussed.
此前，我们已证明硫酸软骨素蛋白聚糖和糖胺聚糖可抑制逆转录病毒转导。在研究抑制机制时，我们发现向逆转录病毒储备液中同时添加等重量浓度（80微克/毫升）的聚凝胺和硫酸软骨素C，会形成一种高分子量的逆转录病毒-聚合物复合物，该复合物可通过低速离心沉淀。沉淀的复合物包含超过80%的病毒颗粒，但所含蛋白质不到病毒储备液中原始蛋白质的0.3%。令人惊讶的是，复合物中的病毒仍保持活性，可用于转导细胞。将沉淀的病毒重新悬浮于细胞培养基至起始体积时，其滴度比原始病毒储备液高三倍。该过程对病毒活性的选择性（集落形成单位/毫克蛋白质）超过1000倍。当将沉淀的病毒-聚合物复合物重新悬浮于原始体积的八分之一中，并用于转导NIH 3T3小鼠成纤维细胞和原代人成纤维细胞时，基因转移比原始未浓缩的逆转录病毒储备液增加了10至20倍。我们讨论了这些发现对用于人类基因治疗方案的逆转录病毒储备液的生产、加工和使用的意义。
