一种新型具有酸敏感的基于聚原酸酯的DNA疫苗载体系统的研制及其作用机制研究

DNA vaccine presents a unique opportunity for immunotherapy against cancer as well as bacterial and viral infections, without the potential limitations of many conventional vaccines. Biodegradable polymers present a promising avenue for DNA vaccine delivery. However, the efficiency of delivery and immunostimulation by current polymers is inadequate in controlling diseases. We recognize that there is a lack of mechanistic understanding of detailed interactions between polymer carriers and the immune system within an in vivo physiologic context.The current proposal will focus on the following two specific aims: 1. To synthesize new poly(ortho ester) polymers incorporating functions and modalities for promoting DNA uptake, antigen production and presentation, and phenotypic maturation of the most potent antigen presenting cells (APCs) - - the dendritic cells (DCs). Here we will exploit the versatility of the POE chemistry to synthesize block copolymers of POE with cationic polyethylenimine (PEI).We will produce DNA-encapsulated polymer micro- and nanoparticles and characterize them in vitro using cultured DCs. 2. To investigate systematically the in vivo mechanisms of POE microspheres carrying model DNA vaccines, quantify and track the time course of critical molecular and cellular events in the early stages of immune activation. We will develop and use novel multi-color flow cytometry and immunostaining assays to quantify the levels of DNA uptake, antigen expression and presentation, APC phenotype and subsets, and the time course of these events as related to activation of T cells by both CD8 and CD4 epitopes.Combining a versatile and tunable biodegradable polymer system with advanced immunological tools and focusing primarily on in vivo investigation, we plan to address several important questions regarding how the timing, location, magnitude, and cells of immune processes may be optimally manipulated through better engineering of the polymer delivery vehicles that will eventually lead to improved in vivo efficacy of DNA-based immunotherapy.

DNA疫苗是一种在肿瘤、细菌和病毒感染的免疫治疗中相较传统疫苗更有优势的新的治疗策略。本项目针对基因免疫治疗研究中DNA疫苗传输系统体内低效这一关键问题，将体内免疫过程机理应用到新型生物可降解聚合物DNA疫苗传输体系的设计中，把具有高基因转染效率的阳离子聚合物聚乙烯亚胺（PEI）接枝到具有酸敏感性的聚原酸酯（POE）上，合成一种新型的、高效的、具有酸敏感性的生物可降解聚原碳酸酯DNA疫苗载体POE-PEI，考察聚合物/DNA疫苗复合物与体外培养的DC细胞之间的相互作用。同时对体内DNA疫苗的摄取、DNA释放及抗原表达、抗原提呈、DC细胞成熟、T细胞激活等关键免疫行为进行深入的研究。通过本项目的研究，将揭示聚合物DNA疫苗载体在体外与DC细胞及在体内与免疫系统的相互作用机制，对开发高效的具有良好临床应用前景的癌症和感染性疾病治疗的聚合物DNA疫苗载体具有非常重要的指导作用。

疫苗递送策略是一种在肿瘤、细菌和病毒感染的免疫治疗中更有优势的新的治疗策略。本项目设计了多种结构与功能不同的聚合物载体，分别在DNA疫苗递送及蛋白疫苗递送方向开展了多项研究，探索将其用于肿瘤免疫治疗的前景。聚合物载体主要包括：具有酸敏感性的生物可降解聚原酸酯DNA 疫苗载体；星型及线性结构的聚甲基丙烯酸氨基乙酯DNA疫苗载体；酸敏感生物降解型半固体聚合物蛋白抗原载体；甘露糖修饰的壳聚糖蛋白疫苗载体；透明质酸修饰的PLGA蛋白疫苗载体；甘露糖修饰的海藻酸钠蛋白疫苗载体等。体外结果显示，这些载体可以成功将DNA或蛋白递送到目标细胞， 并表现出较低的细胞毒性；体内研究显示，蛋白疫苗载体比DNA疫苗载体能够更好的激活持续稳定的抗原特异性免疫应答，对肿瘤具有免疫治疗作用。在该项目的资助下，发表了18篇SCI论文，均标注了基金代码；申请了1项发明专利。项目有多名研究人员参与，培养了研究生6名，两名新加入的项目组成员晋升了职称。项目负责人在国际和国内学术会议上做了学术讲座近20次；作为会议主席之一，举办了3次“国际生物免疫材料大会”。