Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-β also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture.

中文翻译：

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HEK293T 中的慢病毒载体生产滴度不受诱导细胞内先天免疫的限制。


大多数基因治疗慢病毒载体 (LV) 生产平台采用表达致癌 SV40 大 T 抗原 (TAg) 的 HEK293T 细胞，该抗原被认为可促进质粒介导的基因表达。对其他病毒癌基因的研究表明，TAg 还可能抑制细胞内自主先天免疫系统，该系统在胞质传感器检测到病毒成分后触发防御性抗病毒反应。在这里，我们表明，在 HEK293T 细胞中 HIV-1 衍生的 LV 转染后，特别是通过转基因，可以产生先天反应，但值得注意的是，这对 LV 滴度没有影响。此外，DNA传感通路成分的过度表达导致炎症细胞因子和干扰素（IFN）刺激基因的表达，但没有导致可检测到的IFN或CXCL10，并且对LV滴度没有影响。外源性 IFN-β 也不影响原代 T 细胞中 LV 的产生或转导效率。此外，TAg 的操作不会影响先天抗病毒反应，但 TAg 的稳定表达可促进 HEK293 细胞中载体的产生。我们的研究结果证明了 HEK293T 细胞先天免疫能力的测量，但最重要的是，表明炎症信号的激活与这些细胞中细胞因子的分泌无关。这为 HEK293T 细胞在 LV 制造中的独特适用性提供了新的机制见解。