Nonviral gene delivery to T cells with Lipofectamine LTX
Nonviral gene delivery to T cells with Lipofectamine LTX
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DOI：
10.1002/bit.27686
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发表时间：
2021-02-02
影响因子：
3.8
通讯作者：
Elmer, Jacob
中科院分区：
文献类型：
--
作者：
Harris, Emily;Zimmerman, Devon;Elmer, Jacob
逆转录病毒基因递送广泛用于血液癌症的T细胞疗法。然而，病毒载体制造昂贵，以半随机模式整合基因，并且它们的转导效率在患者之间变化。在这项研究中，比较了几种非病毒基因递送载体，启动子和其他变量，以优化Jurkat和原代T细胞中的非病毒转基因递送和表达。通过在X-VIVO 15培养基中用Lipofectamine LTX转染细胞，使Jurkat细胞的转染最大化至高效率（63.0% +/-10.9%EGFP（+）细胞）。然而，相同的方法在原代T细胞中产生低得多的转染效率（8.1% +/-0.8%EGFP（+））。随后的共聚焦显微镜显示，大多数的lipoplexes没有进入原代T细胞，这可能是由于相对较低的表达水平的硫酸乙酰肝素蛋白聚糖检测通过信使RNA测序。Pyrin和HIN（PYHIN）DNA传感器（例如，在原代T细胞中也检测到高水平的AIM 2和IFI 16），其可以在结合细胞质DNA后诱导凋亡或抑制转录。因此，原代T细胞的转染似乎在细胞摄取或细胞质中的DNA感测水平上受到限制。这两个因素都应该考虑在未来的病毒和非病毒T细胞基因传递方法的发展。
Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semirandom patterns, and their transduction efficiency varies between patients. In this study, several nonviral gene delivery vehicles, promoters, and additional variables were compared to optimize nonviral transgene delivery and expression in both Jurkat and primary T cells. Transfection of Jurkat cells was maximized to a high efficiency (63.0% +/- 10.9% EGFP(+) cells) by transfecting cells with Lipofectamine LTX in X-VIVO 15 media. However, the same method yielded a much lower transfection efficiency in primary T cells (8.1% +/- 0.8% EGFP(+)). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans detected via messenger RNA-sequencing. Pyrin and HIN (PYHIN) DNA sensors (e.g., AIM2 and IFI16) that can induce apoptosis or repress transcription after binding cytoplasmic DNA were also detected at high levels in primary T cells. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake or DNA sensing in the cytoplasm. Both of these factors should be considered in the development of future viral and nonviral T cell gene delivery methods.