Optimizing Non-Viral Gene Delivery to T Cells with Lipofectamine LTX. Optimizing Non-Viral Gene Delivery to T Cells with Lipofectamine LTX

Retroviral gene delivery is widely used in CAR-T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semi-random patterns, and their transduction efficiency is highly variable. In contrast, non-viral delivery methods could offer a potentially less expensive and safer alternative for CAR-T cell gene delivery. In this study, several different cationic vehicles, promoters, and additional culture conditions were compared to optimize non-viral transgene delivery and expression in both Jurkat and primary T cells. In addition, confocal microscopy and next-generation sequencing experiments were also conducted to detect putative methods of transfection resistance in Jurkat and primary T cells. Transfecting Jurkat cells in X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection efficiency in Jurkat cells (63.0±10.9% EGFP+). However, this protocol yielded a much lower transfection efficiency (8.07±0.76% EGFP+) in primary T cells. Confocal microscopy and mRNA-sequencing experiments revealed that a majority of lipoplexes did not enter the primary T cells, perhaps due to relatively low expression levels of heparan sulfate proteoglycans (HSPGs). Primary T cells also expressed high levels of PYHIN DNA sensors (e.g., AIM2, IFI16), which can induce apoptosis when bound to cytoplasmic DNA. Primary T cells are more resistant to transfection with Lipofectamine than Jurkat T cells. Transfection of primary T cells appears to be hindered by decreased expression of HSPGs and high expression of PYHIN DNA sensors. Both of these factors should be considered in the development of future viral and non-viral T cell gene delivery methods. Overall design: 3 cell lines, 6 samples per cell line with (3) untransfected and (3) transfected samples (n=3) (18 samples overall)

逆转录病毒基因递送（Retroviral gene delivery）广泛应用于血液系统恶性肿瘤的嵌合抗原受体T细胞（CAR-T cell）疗法。然而，病毒载体（viral vector）制备成本高昂，基因整合模式呈半随机性，且转导效率（transduction efficiency）波动幅度极大。与之相对，非病毒递送方法（non-viral delivery method）可为CAR-T细胞基因递送提供一种成本更低、安全性更优的潜在替代方案。本研究对比了多种不同的阳离子载体（cationic vehicle）、启动子（promoter）及附加培养条件，以优化Jurkat细胞（Jurkat cell）与原代T细胞（primary T cell）中的非病毒转基因递送与表达效果。此外，本研究还开展了共聚焦显微镜（confocal microscopy）与下一代测序（next-generation sequencing）实验，以探究Jurkat细胞与原代T细胞中潜在的转染抗性（transfection resistance）机制。在X-VIVOTM 15培养基（X-VIVOTM 15 media）中使用Lipofectamine LTX对Jurkat细胞进行转染，可获得较高的转染效率（63.0±10.9% EGFP+）。但该方案在原代T细胞中的转染效率却显著更低（8.07±0.76% EGFP+）。共聚焦显微镜与mRNA测序（mRNA-sequencing）实验结果显示，大部分脂质复合物（lipoplex）无法进入原代T细胞，这可能与硫酸乙酰肝素蛋白聚糖（heparan sulfate proteoglycans, HSPGs）的表达水平相对较低有关。原代T细胞还高表达PYHIN DNA传感器（PYHIN DNA sensor），这类传感器结合细胞质DNA后可诱导细胞凋亡（apoptosis）。相较于Jurkat T细胞，原代T细胞对Lipofectamine介导的转染抗性更强。原代T细胞的转染障碍似乎源于HSPGs表达下调与PYHIN DNA传感器高表达这两个因素，在未来开发病毒与非病毒T细胞基因递送方法时，需同时考虑这两点。实验整体设计：设置3种细胞系，每个细胞系包含6个样本，其中3个为未转染样本、3个为转染样本（每组生物学重复数n=3），总计18个样本。