School of Biochemistry, University of Bristol, 1 Tankard's Close, Bristol BS8 1TD, UK.
GW4 Cryo-EM Facility, University of Bristol, Life Sciences Building, Bristol BS8 1TQ, UK.
Viruses. 2024 Sep 17;16(9):1475. doi: 10.3390/v16091475.
Baculoviral vectors (BVs) derived from multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability.
杆状病毒载体（BVs）源自多种核多角体病毒（AcMNPV），是在哺乳动物细胞中进行多基因传递的一种有吸引力的工具，这对于 CRISPR 技术尤其相关。哺乳动物细胞中的大多数应用都依赖于假型化为水疱性口炎病毒 G 蛋白（VSV-G）的 BVs，以促进有效的内体释放。VSV-G 的表达通常受强激活的 polH 启动子控制。在这项研究中，我们证明 polH 驱动的 VSV-G 表达导致 BVs 的稳定性降低，形态受损，并且在高转导倍数（MOT）下在靶哺乳动物细胞中产生 VSV-G 诱导的毒性。为了克服这些缺点，我们探索了五个替代的病毒启动子，旨在优化假型 BVs 上展示的 VSV-G 水平。我们报告说，Orf-13 和 Orf-81 启动子将 VSV-G 表达降低到 polH 的 5%以下，从而挽救了 BV 的形态和稳定性。在一组人类细胞系中，我们阐明了具有降低的 VSV-G 的 BVs 支持高效的基因传递和 CRISPR 介导的基因编辑，其水平可与先前使用 polH VSV-G 假型 BVs（polH VSV-G BV）获得的水平相媲美。这些结果表明，高效转导哺乳动物细胞并不需要 VSV-G 的过度表达。相比之下，降低的 VSV-G 表达赋予了类似的转导动力学，同时大大提高了 BV 的完整性、结构和稳定性。
