Arya Smriti, Lin Qiubin, Zhou Nan, Gao Xiang, Huang Jian-Dong
School of Biomedical Sciences, University of Hong Kong, Hong Kong SAR.
Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, P.R. China.
Mol Ther Nucleic Acids. 2020 Mar 6;19:1098-1109. doi: 10.1016/j.omtn.2019.12.044. Epub 2020 Jan 16.
In vitro 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 in vivo 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 Staphylococcus aureus 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 in vivo locoregional delivery of mRNA and could be a useful tool for basic research and for the development of mRNA-based therapies.
体外转录的mRNA在疾病预防和治疗的许多医学应用中具有广阔前景，例如替代或补充缺失或表达不足的内源性蛋白质，以及作为针对传染病的预防性疫苗、治疗性疫苗或用于癌症治疗的其他基于蛋白质的生物制剂。安全有效的mRNA递送系统对于mRNA治疗应用的成功至关重要。在本研究中，我们报告了InstantFECT，一种基于脂质体的转染试剂，能够将掺入假尿苷的mRNA包装成纳米复合物，这些纳米复合物在局部递送后能高效介导体内多个器官的转染。在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，持续时间长达96小时。通过皮下递送编码金黄色葡萄球菌腺苷合酶A（AdsA）和模型肿瘤相关抗原卵清蛋白（OVA）的修饰mRNA，研究了纳米复合物递送的mRNA所编码抗原的免疫原性。两种递送的mRNA均引发了强烈的T细胞反应。OVA mRNA-脂质体纳米复合物的治疗性和保护性治疗显著抑制了B16-OVA肿瘤进展并提高了小鼠存活率。在组织培养和小鼠中均未出现与治疗相关的明显毒性迹象。静脉注射相同剂量的修饰mRNA-脂质纳米复合物在主要器官中显示出最小的转染，表明具有出色的安全性，因为基因转移仅发生在注射部位，而静脉注射相同量的与商业转染试剂Trans-IT复合的mRNA在脾脏中显示出荧光素酶表达。总之，InstantFECT阳离子脂质体为mRNA提供了一种安全有效的体内局部递送方式，可能是基础研究和基于mRNA疗法开发的有用工具。
