transcribed mRNAs hold the promises of many medical applications in disease prevention and treatment, such as replacement or supplement of missing or inadequately expressed endogenous proteins and as preventive vaccines against infectious diseases, therapeutic vaccines, or other protein-based biopharmaceutics for cancer therapy. A safe and efficient delivery system for mRNA is crucial to the success of mRNA therapeutic applications. In this study, we report that InstantFECT, a liposome-based transfection reagent, can pack pseudouridine-incorporated mRNA into nanocomplexes that are highly efficient in mediating transfection in multiple organs after local delivery. High levels of expression of EGFP and luciferase reporters after intratumoral and intramuscular injections were observed, which lasted for up to 96 hrs. Immunogenicity of antigens encoded by mRNA delivered with nanocomplex was investigated by subcutaneous delivery of modified mRNAs encoding adenosine synthase A (AdsA) and a model tumor-associated antigen ovalbumin (OVA). Strong T cell responses were provoked by both mRNAs delivered. Therapeutic and protective treatment with the OVA mRNA-liposome nanocomplex significantly inhibited B16-OVA tumor progression and increased mouse survival. There was no sign of obvious toxicity related to the treatment both in tissue culture and in mice. An intravenous injection of the same dosage of the modified mRNA-lipid nanocomplex showed minimal transfection in major organs, indicating an excellent safety feature as the gene transfer occurred only at the injection sites, whereas intravenous (i.v.) injection with the same amount of mRNA complexed with a commercial transfection reagent Trans-IT showed luciferase expression in the spleen. In summary, InstantFECT cationic liposomes provide a safe and efficient locoregional delivery of mRNA and could be a useful tool for basic research and for the development of mRNA-based therapies.

中文翻译：

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直接局部注射修饰的 mRNA-脂质纳米复合物诱导强烈的免疫反应


转录的 mRNA 在疾病预防和治疗方面具有许多医学应用的前景，例如替代或补充缺失或表达不充分的内源蛋白质，以及作为传染病的预防性疫苗、治疗性疫苗或其他用于癌症治疗的基于蛋白质的生物药剂。安全高效的 mRNA 递送系统对于 mRNA 治疗应用的成功至关重要。在这项研究中，我们报告了 InstantFECT，一种基于脂质体的转染试剂，可以将掺入假尿苷的 mRNA 包装到纳米复合物中，该纳米复合物在局部递送后能够高效地介导多个器官的转染。瘤内和肌内注射后观察到 EGFP 和荧光素酶报告基因的高水平表达，持续长达 96 小时。通过皮下递送编码腺苷合酶 A (AdsA) 和模型肿瘤相关抗原卵清蛋白 (OVA) 的修饰 mRNA，研究了用纳米复合物递送的 mRNA 编码的抗原的免疫原性。传递的两种 mRNA 均引发了强烈的 T 细胞反应。使用 OVA mRNA-脂质体纳米复合物进行治疗和保护性治疗可显着抑制 B16-OVA 肿瘤进展并提高小鼠存活率。在组织培养和小鼠中均未发现与治疗相关的明显毒性迹象。静脉内注射相同剂量的修饰的 mRNA-脂质纳米复合物在主要器官中显示出最小的转染，这表明基因转移仅发生在注射部位，因此具有出色的安全性，而静脉注射等量的 mRNA 复合物则显示出极好的安全性。使用商业转染试剂 Trans-IT 显示脾脏中有荧光素酶表达。 总之，InstantFECT 阳离子脂质体提供了安全有效的 mRNA 局部递送，可能成为基础研究和基于 mRNA 的疗法开发的有用工具。