Lentiviral vectors are one of the most commonly used viral delivery systems for gene therapy. Vesicular stomatitis virus-G envelope glycoprotein (VSV G)-pseudotyped lentiviral vectors have been widely used in clinical studies for treatment of virus infections and genetic deficient diseases. However, the efficiency of lentiviral vector transduction has been long recognized as a limiting factor in clinical gene therapy application, especially in transducing hematopoietic stem cells. MARCH8 (membrane-associated RING-CH 8), an E3 ubiquitin ligase, has been reported to target and downregulate VSV G. Results in this study show that MARCH8 induces ubiquitination and lysosome degradation of VSV G, and knockout of MARCH8 in virus-producing cells increases lentiviral vector transduction by elevating the level of VSV G protein. We then engineered VSV G mutant that has the lysine residues in the cytoplasmic domain substituted for arginine, and showed that this G mutant resists degradation by MARCH8, and allows the enhancement of transduction efficiency of lentiviral vector particles than the parental VSV G protein. This engineered VSV G mutant thus further advances the lentiviral vector system as a powerful tool in gene therapy.

中文翻译：

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工程抗 MARCH8 水疱性口炎病毒糖蛋白增强慢病毒载体转导

慢病毒载体是基因治疗中最常用的病毒递送系统之一。水疱性口炎病毒-G 包膜糖蛋白 (VSV G)-假型慢病毒载体已广泛用于治疗病毒感染和遗传缺陷疾病的临床研究。然而，慢病毒载体转导的效率长期以来一直被认为是临床基因治疗应用的限制因素，特别是在转导造血干细胞方面。据报道，MARCH8（膜相关 RING-CH 8）是一种 E3 泛素连接酶，可靶向和下调 VSV G。本研究的结果表明，MARCH8 诱导 VSV G 的泛素化和溶酶体降解，并在病毒产生过程中敲除 MARCH8细胞通过提高 VSV G 蛋白水平来增加慢病毒载体转导。然后，我们设计了 VSV G 突变体，其细胞质结构域中的赖氨酸残基取代了精氨酸，并表明该 G 突变体可抵抗 MARCH8 的降解，并且与亲本 VSV G 蛋白相比，慢病毒载体颗粒的转导效率更高。因此，这种工程化的 VSV G 突变体进一步推动了慢病毒载体系统作为基因治疗的强大工具。