新型RBP131脂质体应用于mRNA体内外给药的性能与机理研究

Gene therapy includes three generations of technologies that are based on plasmid DNA, RNA interference, messenger RNA (mRNA), respectively. mRNA-based gene therapy is becoming a new research hotspot in recent years. However, lack of safe and efficient in vivo delivery system, as a bottle-neck problem, hampers its extensive application and drug development. Previously, the applicant developed a novel lipid-based liver-targeting delivery system called RBP131, which is extremely high efficient, safe and is not reported in any publication up to now. In consideration of the novelty of RBP131 and the unmet requirement of mRNA delivery system, the applicant plans to investigate the capability and mechanism of RBP131 in mediating liver-targeted delivery of mRNA in vitro and in vivo in proposed project. First, mRNA will be prepared and stabilized through in vitro transcription. Then we will evaluate the mRNA load ability of RBP131, characterize the physico-chemical properties of RBP131/mRNA nano-system, and explore the efficiency and mechanism of RBP131 in mediating cell transfection in vitro. Furthermore, we will assess the safety/toxicity of this delivery system both in vitro and in vivo, investigate mRNA expression pattern mediated by RBP131 in mouse liver and so as to explore its intrinsic mechanism. Finally, we will apply this system to hypoglycaemia mouse model; examine its curative effect to alleviate the hypoglycaemia symptoms, which may provide a new therapeutic protocol for those diabetes patients suffered long-term hypoglycaemia. Moreover, this project would provide a novel general liver-targeted mRNA delivery tool with high efficiency and perfect safety.

广义的基因治疗包括基于质粒DNA、RNA干扰、信使RNA的三代技术，基于mRNA的基因治疗近年来正成为新的研究热点，但限制其广泛应用与开发的关键技术瓶颈在于缺乏安全、高效的体内给药系统。前期研究中，申请人获得了全球尚无报道的、具有突出肝靶向给药能力的新型RBP131脂质体载体。本项目拟结合RBP131的新颖性与mRNA给药的紧迫性，研究RBP131应用于mRNA体内外输送的效果及其内在机理。项目通过体外转录获得稳定化的mRNA，并制备RBP131/mRNA纳米体系，表征其理化性质，研究其细胞转染的效果与机理，考察其体内外安全性；并在小鼠体内探索RBP131介导mRNA肝靶向投递与基因表达的能力及其内在机制；最后在低血糖小鼠模型上递送胰高血糖素mRNA，研究其改善低血糖症状的治疗效果，以期为长期低血糖的糖尿病人的治疗提供一种新方案，更为广泛的肝靶向mRNA给药与应用提供高效、安全的新工具。

基于mRNA的基因治疗近年来正成为新的研究热点，但mRNA不够稳定、以及存在如何实现安全高效的体内递送的关键技术难题。本项目中，承担团队首先建立了mRNA的体外转录技术平台，构建了三种（红色荧光蛋白RFP、萤火虫荧光素酶Luciferase、促红细胞生长素EPO）的PCR质粒，且就其中的EPO表达序列进行了密码子优化，基于该系列质粒，研究者利用抗倒转帽类似物（anti-reverse-cap analogue, ARCA）进行体外转录得到mRNA，在HepG2、Hela等细胞上研究了RFP、Luciferase、EPO的表达效果，同时在动物水平，通过利用研究者前期获得的优秀核酸递送脂质体RBP131包裹Luciferase或EPO mRNA，建立了脂质体包裹mRNA的制备工艺，阐明了制剂理化性质，并成功的在小鼠体内实现了靶蛋白的表达，其中密码子优化的EPO mRNA表达丰度较未优化的mRNA显著提升；最后，研究者还考察了RBP131负载核酸在小鼠体内的初步安全性，证明了其理想的生物相容性。.整体上，该项目的实施，证实了密码子优化、ARCA体外转录对mRNA稳定性与表达效果的增强作用，阐明了RBP131对mRNA的有效包裹及体内递送效力，为基于mRNA的基因治疗、疫苗研究奠定了良好的理论与技术基础，为贫血病人的治疗提供了潜在的新疗法、新方案。