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Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Deficient in Expression of Immediately Early Genes.
Molecular Therapy - Methods & Clinical Development ( IF 4.6 ) Pub Date : 2020-03-17 , DOI: 10.1016/j.omtm.2020.03.014
Seiji Kuroda 1, 2, 3 , Yoshitaka Miyagawa 1 , Yuriko Sato 1 , Motoko Yamamoto 1 , Kumi Adachi 1 , Hiromi Kinoh 1 , William F Goins 4 , Justus B Cohen 4 , Joseph C Glorioso 4 , Nobuhiko Taniai 2 , Hiroshi Yoshida 3 , Takashi Okada 5
Affiliation  

Non-toxic herpes simplex virus (HSV) vectors can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this issue, we previously developed a novel production cell line, called U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the process of vector manufacturing on this platform, we evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced by culturing conditions. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a greatly improved vector yield and quality and showed elevated transgene expression in cultures of primary dorsal root ganglion neurons. Taken together, our optimized production approach emerges as a relevant protocol for high-yield and high-quality preparation of non-toxic HSV-based gene therapy vectors.

中文翻译:


用于生产缺乏立即早期基因表达的非细胞毒性 JΔNI5 HSV 载体的方案优化。



无毒的单纯疱疹病毒(HSV)载体可以通过所有立即早期(IE)基因的功能性删除来产生,从而提供具有基因治疗潜力的良性载体。然而,多个 IE 基因的缺失引起了生产问题,从而限制了这些载体的临床应用。为了解决这个问题,我们之前通过慢病毒转导具有两个必需 HSV IE 基因 ICP4 和 ICP27 的人骨肉瘤 U2OS 细胞,开发了一种新型生产细胞系,称为 U2OS-ICP4/27。为了优化该平台上的载体制造过程,我们评估了 U2OS-ICP4/27 的多个细胞培养参数,以实现高滴度和高质量生产无毒 HSV 载体,结果表明这些载体的产量和功能可以显着提高受培养条件影响。我们还发现几种化合物可以增强无毒 HSV 载体的复制及其从生产细胞释放到上清液中。值得注意的是,通过我们优化的方案产生的载体显示出极大提高的载体产量和质量,并且在初级背根神经节神经元的培养物中显示出升高的转基因表达。总而言之,我们优化的生产方法成为高产和高质量制备无毒 HSV 基因治疗载体的相关方案。
更新日期:2020-03-17
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