多功能性支化聚（β-氨基酯）mRNA基因载体的构建及其在大疱疹表皮松懈症治疗中的应用

Messenger RNA (mRNA) is the template for the synthesis of protein, and thus of great potential for gene therapy. However, lack of safe and efficienct mRNA delivery vector is one of the major bottlenecks that severely limit its translation to clinical applications. Previously, by employing A2 type amine, B3 type triacrylate and C2 type diacrylate, we successfully synthesized a series of highly branched poly(β-amino ester)s via the A2+B3+C2 type michael addition reaction, and evaluated DNA transfection performance of the synthesized HPAE systematically. Results show that in a variety of cell types, HPAE always exhibited excellent gene transfection efficiency and low cytotoxicity. Despite of the great progress, however, the development of HPAE as mRNA delivery vectors has not yet been explored. In this project, we aim to develop new and multifunctional HPAE as mRNA delivery vector. We intend to optimize the monomer combinations, feed ratios along with incorporating functional components, to faciliate HPAE formation of multiple hydrogen bonding with mRNA, strong hydrophobic interaction with cell membrane and degradation by glutathione, so as to navigate the core barriers associated with mRNA delivery including easy of degradation by RNase, low polyplex cellular uptake efficiency and difficulty in mRNA release in cytoplasm, ultimately screen out high performance mRNA vectors, especially for keratinocyte and fibroblast transfection. We will also evaluate the mRNA transfection performance of the optimized HPAE in vivo by using a recessive dystrophic epidermolysis bullosa (RDEB) animal model. Understanding of the correlation of HPAE composition and structure with their mRNA transfection performance will provide new insights into the development of non-viral mRNA delivery vector, also highly desirable mRNA vectors for the treatment of RDEB in clinic.

制约mRNA基因治疗临床应用的核心瓶颈之一是缺乏有效的载体。前期工作中，我们通过A2+B3+C2类型迈克尔加成反应合成了高度支化聚（β-氨基酯）（HPAE），并对其DNA转染性能进行了详细研究。结果表明，在多种细胞中，HPAE均显示出优异的转染性能。但在开发HPAE用于mRNA递送方面，我们尚未进行任何探索和研究。本项目拟在前期工作基础上，开发与mRNA具有多重氢键、与细胞膜有强疏水相互作用并且可被谷胱甘肽还原的多功能HPAE用于mRNA递送，以克服mRNA递送过程中易被核酸酶降解、复合物纳米粒子细胞内吞效率差以及不易释放等核心屏障，优选出特别是针对皮肤角质细胞和成纤维细胞的高效mRNA载体，并以大疱疹表皮松懈症为疾病模型进行评价。本项目拟阐明的多功能HPAE组成和结构与mRNA转染性能之间的关系，对开发非病毒mRNA载体具有重要借鉴意义，并且能够开发出临床亟需的RDEB基因治疗载体。

本项目主要针对基因转染过程中易被核酸酶降解、复合物纳米粒子细胞内吞效率低以及压缩的基因在细胞质中无法及时释放等核心屏障，利用已建立的A2+B3+C2合成平台设计开发新的多功能性HPAE，并对其进行特异性功能化修饰改性，用于基因的递送。在项目开展过程中：1）合成了超过150中具有不同化学组成、支化度及末端基团的HPAE，结果表明具有中等分子量、支化度和疏水组分的HPAE呈现出更高的DNA及mRNA亲和性，形成的复合物纳米粒子具有更高的Zeta电位和更小的粒径，细胞内吞和内涵体逃逸效率更高，可归结于该类HPAE与磷脂膜具有更强的相互作用及更高的分子柔韧性，因而可压缩具有不同尺寸的DNA及mRNA实现高效基因递送，这为开发可同时递送DNA及mRNA的高效非病毒基因载体提供了一种新思路，也初步证明了优选的HPAE可递送功能基因用于大疱疹表皮松懈症基因治疗的潜力；2）通过对引入二硫键及疏水烷基链对HPAE进行特异性功能化修饰，促进复合物纳米粒子的细胞内吞、内涵体逃逸、DNA释放并降低细胞毒性，使得HPAE在ADSCs等较为脆弱且难转染的细胞中呈现出优异的基因转染效率和安全性，为该类细胞的高效转染提供了一种新策略。此外，ADSCs分泌的NGF可有效促进PC12细胞形成神经突，也为帕金森氏综合症等神经退行性疾病的基因治疗提供了一种思路；3）开发了单环状聚（β-氨基酯）（SPAE）并对其DNA递送性能进行了探究，阐明了单环结构对聚（β-氨基酯）（LPAE）的DNA亲和性、复合物纳米粒子尺寸、Zeta电位、基因转染效率及细胞毒性的影响，为开发具有环形拓扑结构的聚（β-氨基酯）基因递送载体提供了重要的借鉴。项目执行过程中取得了多重研究成果，以通讯作者在Advanced Functional Materials、ACS Macro Letters及Chemical Communications等期刊发表论文8篇，申请中国专利6项，授权2项，指导了多名博士研究生及硕士研究生，与国内外同行开展了广泛的学术交流。部分成果已与相关企业初步达成转让协议，为研究成果的向临床转化奠定了基础。

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