Noguchi Kazuhiro, Ikawa Yasuhiro, Takenaka Mika, Sakai Yuta, Fujiki Toshihiro, Kuroda Rie, Chappell Maxwell, Ghiaccio Valentina, Rivella Stefano, Wada Taizo
Department of Pediatrics, School of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan.
Department of Pediatrics, School of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan.
J Virol Methods. 2023 Apr;314:114689. doi: 10.1016/j.jviromet.2023.114689. Epub 2023 Feb 3.
The development of hematopoietic stem cell (HSCs) gene therapy for DNA repair disorders, such as Fanconi anemia and Bloom syndrome, is challenging because of the induction of HSCs apoptosis by cytokine stimulation. Although the Baboon envelope pseudotyped lentiviral vector (BaEV-Rless-LV) has been reported as a non-stimulatory gene transfer tool, the virus titer of BaEV-Rless-LV is too low for use in clinical applications. Transfected 293 T cells with helper plasmids, including the BaEV-Rless plasmid, showed morphological changes, such as syncytium formation and detachment. To establish a novel protocol for producing a high titer of BaEV-Rless-LV, we optimized three aspects of a basic virus production protocol by focusing on modifying culture conditions and the use of reagents: the virus titer increased 3-fold when the amount of BaEV-Rless plasmid was increased 1.2-fold; the highest titer was obtained when the viral supernatant was harvested at 48-h post-transfection, despite complete syncytium formation and detachment of the 293 T cells; and the use of poly-L-lysine-coated culture plates to enhance the adhesion and proliferation of 293 T cells and prevent detachment doubled the titer. Collectively, our novel protocol resulted in a 10-fold titer increase compared to the basic protocol and may be useful in clinical applications for treating DNA repair disorders.
由于细胞因子刺激会诱导造血干细胞（HSC）凋亡，因此开发用于治疗DNA修复障碍（如范可尼贫血和布卢姆综合征）的造血干细胞基因疗法具有挑战性。尽管据报道狒狒包膜假型慢病毒载体（BaEV-Rless-LV）是一种无刺激的基因转移工具，但BaEV-Rless-LV的病毒滴度过低，无法用于临床应用。用包括BaEV-Rless质粒在内的辅助质粒转染293 T细胞后，细胞出现形态变化，如形成多核巨细胞和细胞脱落。为了建立一种生产高滴度BaEV-Rless-LV的新方案，我们通过关注培养条件的改变和试剂的使用，对基本病毒生产方案的三个方面进行了优化：当BaEV-Rless质粒的用量增加1.2倍时，病毒滴度提高了3倍；尽管293 T细胞完全形成多核巨细胞并发生脱落，但在转染后48小时收获病毒上清液时获得了最高滴度；使用聚-L-赖氨酸包被的培养板来增强293 T细胞的黏附性和增殖能力并防止细胞脱落，使滴度提高了一倍。总体而言，我们的新方案使滴度比基本方案提高了10倍，可能对治疗DNA修复障碍的临床应用有用。
