同步递送抗原肽及核酸分子的纳米药物载体诱导抗原特异性免疫耐受的研究

Reconstruction of antigen-specific immune tolerance is an ideal method for the treatment of autoimmune diseases (AIDs), and is a hot and challenging topic in recent years. The pathological activation and abnormal proliferation of self-reactive T cells are the key to AID, but are highly dependent on the dual signals presented by both TCR and co-stimulatory molecules simultaneously. If antigen presenting cells (APCs) are only presented with antigen signals but not provided with stimulation signals, antigen-specific immune tolerance will be induced. The key challenge is how to simultaneously and efficiently inhibit co-stimulatory signals while presenting antigens to APCs. Our previous studies showed that the simultaneous delivery of antigenic peptides and gene editing nucleic acid drugs targeting co-stimulatory signals by nanoparticles can effectively inhibit the activation and proliferation of self-reactive T cells and induce antigen-specific immune tolerance. In this project, a library of cationic lipid-assisted polymeric nanoparticles "CLAN" will be prepared by regulating the surface nanoproperties. Nanoparticles will be screened in order to find out the optimized and efficient ones for co-delivery of antigen peptide and siRNA or gene editing systems targeting co-stimulatory signals to APCs. In addition, the ability of inducing antigen specific immune tolerance and the therapeutic efficacy in type I diabetic mice AID model will be studied. This project will provide the basis for the design of nanomedicine in the treatment of AID.

重建抗原特异性免疫耐受是治疗自身免疫性疾病（AID）的理想方案，是近年来的研究热点和难点。自身反应T细胞的病理性激活与异常增殖是导致AID的关键，但高度依赖于TCR及共刺激分子同时呈递的双信号；如能使抗原提呈细胞（APC）仅呈递抗原信号但不提供共刺激信号，将能诱导抗原特异性免疫耐受。其关键挑战是如何在呈递抗原到APC的同时，同步、高效抑制共刺激信号。我们前期研究表明，用纳米载体同步递送抗原肽和靶向共刺激信号的基因编辑核酸药物，能有效抑制自身反应T细胞的激活与增殖，诱导抗原特异性免疫耐受。本项目将在此基础上，制备阳离子脂质辅助的纳米颗粒“CLAN”库，调控其表面纳米特性，在体内外筛选同步、高效递送抗原肽和靶向共刺激信号的siRNA或基因编辑系统到APC的纳米药物载体，诱导抗原特异性免疫耐受，并在小鼠1型糖尿病等AID模型中证明其疗效，揭示其机制，为发展AID治疗的纳米药物设计提供依据。

重建自身抗原特异性免疫耐受是治疗自身免疫性疾病的理想策略，其关键在于如何诱导抗原提呈细胞（APC）在没有共刺激分子的情况下将自身抗原肽呈递给CD4+ T细胞。基于此，我们发展了共递送体系，将自身抗原肽段和干预共刺激分子的核酸药物共递送至树突状细胞（DCs），将其诱导成耐受性表型，重建机体自身抗原特异性免疫耐受。该体系是基于聚乙二醇-聚乳酸/乙醇酸为主要材料，掺入少量阳离子脂质构建阳离子脂质辅助的纳米粒（CLAN），并同时荷载多种药物：针对小鼠1型糖尿病相关抗原肽2.5mi、CRISPR-Cas9质粒（pCas9）以及靶向CD80、CD86和CD40共刺激分子的三个gRNA。采用多种实验方法，我们在体内外证实了该体系能够高效递送肽段和上述核酸药物进入DCs，有效敲低DCs上CD80、CD86和CD40三个共刺激分子的表达，并提呈2.5mi，从而实现了重建2.5mi耐受性DCs，诱导了抗原肽特异性调节性T细胞的产生，降低了CD8+ T细胞的比例和功能，有效预防和缓解小鼠I型糖尿病进程，为改造APC重建自身抗原特异性免疫耐受提供了策略。此外，我们还发展了多种共递送体系，实现了对不同种类药物的联合递送和机体免疫系统功能的调节，用于其他疾病的治疗。基于上述研究，共计在Nano Today、Acta Biomaterialia、Nano Research、Acta Pharmaceutica Sinica B、ACS Applied Materials & Interfaces等期刊发表论文10篇，授权中国发明专利2项，培养博士研究生2名，硕士研究生5名，完成了研究任务和预期目标。