高密度核酸-聚酯胶束亲和杂化递送基因药物研究

Gene therapy that uses nucleic acid as a drug to treat disease is a promising strategy for many biomedical purposes. However, there is still lack of ideal vectors that can safely and effectively delivery the functional nucleic acid drugs into the target cells. Indeed, many unsolved issues, such as biosafety and transfection efficiency, remain in both viral and non-viral delivery systems, preventing the translation of gene therapy from fundamental study to clinic use. Particularly, traditional non-viral vectors usually compress and protect the loaded gene against degradation by encapsulating the nucleic acid inside or introducing cationic group on the carriers to form positively-charged nanocomplexes via electrostatic interaction. In most cases, these delivery systems either show low transfection efficiency or lethal cell toxicity, leading into the failure of clinic trials. To circumvent these challenges, herein we propose to construct a new type of non-viral vector that is composed of biodegradable and amphiphilic DNA-grafted polyester block copolymer. In the new approach, multiple DNA strands will be grafted on one terminus of the polyester chain and the resulted DNA brush block copolymer (DBBC) can further self-assemble into micellar structures in aqueous solution. Owning to the existence of dense layer of DNA on micelles’ corona, nucleic acid drug could be loaded on them by Watson-Crick basepairing. Moreover, the loading efficiency, surface density of the nucleic acids, cellular uptake efficiency, and the stability of the gene carrier will be systematically investigated by changing the design of DBBC-based micelles. Based on the optimized structure elucidated in aforementioned study, stimuli-response and cell targeting mechanisms will be further incorporated into the new gene carrier, which endow them with the capabilities of fast and smart delivery of nucleic acid drug in clinic applications.

基因治疗被认为是继蛋白药之后最具前景的治疗方式，但缺乏安全高效的基因药物载体是目前制约基因治疗从基础研究到临床应用的关键所在。传统的基因载体大多通过简单包覆或静电吸附等方式来实现核酸药物的压缩和保护，存在转染效率低、细胞毒性大等问题。本项目拟在前期研究工作的基础上制备一系列结构和功能不同的双亲性多片段DNA接枝聚酯嵌段共聚物，以此为基础自组装形成表面具有高密度核酸的可生物降解核酸-聚酯纳米胶束作为功能性核酸药物载体。此新型基因载体跳出传统载体材料常用的基因负载机制，通过核酸亲和杂化作用实现对siRNA的高效压缩和保护。同时阐明不同纳米胶束结构作为基因载体对siRNA的负载能力、对细胞的转染效率、以及载体系统生理稳定性之间的关系，进而实现功能性核酸的高效安全输送。通过设计多样化的刺激响应机制和细胞靶向机制，赋予新型基因载体快速智能输送核酸药物的能力，为临床的基因治疗提供新的途径。

基于RNAi的基因治疗被认为是继蛋白药之后最具前景的治疗方式之一。但缺乏安全高效的siRNA递送系统是目前制约基因治疗从基础研究到临床应用的关键所在。传统的siRNA载体大多通过简单包覆或静电吸附等方式来实现核酸药物的压缩和保护，存在转染效率低、细胞毒性大等问题。本项研究中我们跳出传统的基因负载机制，提出了利用核酸亲和杂化来构建交联纳米凝胶，从而实现siRNA的有效负载和保护。通过调控siRNA交联剂与DNA接枝聚合物刷的比例，我们能够有效调控纳米凝胶尺寸，并证明纳米凝胶在RNase H酶存在的情况下能够快速实现功能性siRNA的释放。负载特定功能性siRNA序列后， 我们在细胞和动物水平上证实了所制备的siRNA交联纳米凝胶递送系统能够有效地转染细胞，并在肿瘤部位富集，实现了对靶向基因表达的有效调控，进而抑制肿瘤的生长。此外我们还在纳米凝胶上尝试包裹一层聚多巴胺，一方面可以极大地提高纳米凝胶的生理稳定性，同时也赋予了纳米凝胶的光热活性。通过所负载siRNA调控热休克蛋白的表达，实现了在相对低温条件下优异的光热治疗效果。本项目所开发的新型siRNA输送系统具有良好的生物安全性，为临床基因治疗提供了新的途径。