Vaccines based on mRNA have emerged as potent systems to elicit CD8⁺ T cell responses against various cancers and viral infectious diseases. The efficient intracellular delivery of mRNA molecules encoding antigens into the cytosol of antigen-presenting cells (APCs) is still challenging, requiring cell attachment, active uptake, and subsequent endosomal escape. Here, we report a facile approach for the formulation of peptide-functionalized mRNA polyplexes using copper-free click chemistry to promote presentation of mRNA antigen by dendritic cells (DCs). After screening different membrane active peptides, GALA modified mRNA polyplexes (PPx-GALA) with a size around 350 nm and with a slightly negative surface charge (−7 mV), exhibited the highest EGFP-mRNA transfection in RAW 246.7 macrophages (∼36%) and D1 dendritic cells (∼50%) as compared to polyplexes decorated with melittin or LEDE peptides. Interestingly, we found that PPx-GALA enters DCs through sialic acid mediated endo/phagocytosis, which was not influenced by DC maturation. The PPx-GALA formulation exhibited 18-fold higher cellular uptake compared to a lipofectamine mRNA formulation without inducing cytotoxicity. Live cell imaging showed that PPx-GALA that were taken up by endocytosis induced calcein release from endosomes into the cytosol. DCs treated with PPx-GALA containing mRNA encoding for OVA displayed enhanced T cell responses and DC maturation. Collectively, these data provide a strong rationale for further study of this PPx-GALA formulation in vivo as a promising mRNA vaccine platform.

中文翻译：

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具有后缀合的GALA肽的mRNA多聚体有效地靶向，转染和激活抗原呈递细胞

基于mRNA的疫苗已成为诱导CD8 ^+的强大系统针对各种癌症和病毒感染性疾病的T细胞反应。有效地将编码抗原的mRNA分子在细胞内递送到抗原呈递细胞（APC）的胞质溶胶中仍然具有挑战性，需要细胞附着，主动摄取以及随后的内体逃逸。在这里，我们报告了一种简便的方法，可使用无铜点击化学来促进树突状细胞（DC）提呈mRNA抗原，从而制备肽官能化的mRNA多聚体。在筛选了不同的膜活性肽后，GALA修饰的mRNA多聚体（PPx-GALA）的大小约为350 nm，表面电荷略微为负（-7 mV），在RAW 246.7巨噬细胞中表现出最高的EGFP-mRNA转染率（约36％） ）和D1树突状细胞（约50％），与用蜂毒肽或LEDE肽修饰的多链体相比。有趣的是，我们发现PPx-GALA通过唾液酸介导的内吞/吞噬作用进入DC，不受DC成熟的影响。与lipofectamine mRNA制剂相比，PPx-GALA制剂表现出高18倍的细胞摄取，而不会引起细胞毒性。活细胞成像显示，被胞吞作用吸收的PPx-GALA诱导了钙黄绿素从内体释放到胞质溶胶中。用含有编码OVA的mRNA的PPx-GALA处理的DC表现出增强的T细胞反应和DC成熟。总体而言，这些数据为进一步研究此PPx-GALA配方提供了强有力的理由 与lipofectamine mRNA制剂相比，PPx-GALA制剂表现出高18倍的细胞摄取，而不会引起细胞毒性。活细胞成像显示，被胞吞作用吸收的PPx-GALA诱导了钙黄绿素从内体释放到胞质溶胶中。用含有编码OVA的mRNA的PPx-GALA处理的DC表现出增强的T细胞反应和DC成熟。总体而言，这些数据为进一步研究此PPx-GALA配方提供了强有力的理由 与lipofectamine mRNA制剂相比，PPx-GALA制剂表现出高18倍的细胞摄取，而不会引起细胞毒性。活细胞成像显示，被胞吞作用吸收的PPx-GALA诱导了钙黄绿素从内体释放到胞质溶胶中。用含有编码OVA的mRNA的PPx-GALA处理的DC表现出增强的T细胞反应和DC成熟。总体而言，这些数据为进一步研究此PPx-GALA配方提供了有力的依据体内作为有前途的mRNA疫苗平台。