RNA sequencing of isolated mouse CD8 T cells treated with IL2, IL7 or IL15. RNA sequencing of isolated mouse CD8 T cells treated with IL2, IL7 or IL15

The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4+ and CD8+ T cells in vitro. By pre- treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo. Overall design: CD8 T cells were isolated from the spleens of C57BL/6 mice and cultured with IL2, IL7 or IL15 for 48 hours. RNA was extracted and sequenced.

脂质纳米颗粒（lipid nanoparticles, LNP）用于封装并递送信使核糖核酸（mRNA）已成为一项重要的治疗学进展。除疫苗领域外，LNP-mRNA体系还有诸多其他应用场景。例如，通过抗CD5抗体修饰脂质纳米颗粒（CD5/tLNP），可实现将mRNA有效递送至T细胞以表达目标蛋白。由于静脉注射CD5/tLNP诱导的蛋白阳性T细胞比例相对较低（4%~20%），本研究旨在探索提升mRNA诱导翻译效率的方法。研究发现，体外实验中使用抗CD3抗体激活T细胞可提升CD5/tLNP转染后的蛋白表达水平，但该效果在体内实验中未显现。细胞因子可改善T细胞状态并激活T细胞，因此本研究以mCherry mRNA作为报告基因，发现无论小鼠还是人源T细胞，在白细胞介素7（IL7）培养条件下，体外实验中CD4+及CD8+ T细胞的递送mRNA蛋白表达水平均显著提升。通过先给小鼠全身性注射IL7，再给予tLNP给药，我们观察到体内T细胞的mCherry蛋白表达量显著升高。对体外经IL7处理的小鼠T细胞进行转录组分析后发现，与蛋白质翻译相关的基因组通路得到增强。进一步实验证实，在IL7（而非IL2或IL15）培养的T细胞中，通过电穿孔递送mCherry mRNA后，蛋白表达水平升高，证明其翻译能力得到提升。上述数据表明，IL7可选择性增强T细胞内的蛋白质翻译过程，该特性可用于提升tLNP递送的mRNA在体内的表达水平。整体实验设计：从C57BL/6小鼠脾脏中分离CD8+ T细胞，分别使用IL2、IL7或IL15培养48小时，随后提取RNA并进行测序。