小分子肽基因载体的研究

Gene therapy is an innovational approach for devastating inherited or acquired diseases treatment through delivering therapeutic genes to targeted cells and replacing the disorder genes, where conventional therapy, such as radiotherapy or chemotherapy, met strong resistance. Gene therapy holds great potential to cure diseases such as cancer, monogenic disorder, cardiovascular disease, infectious disease, neurological disease and ocular disease etc. However, the negatively charged cell membrane inhibits the entry of naked DNA due to its electronegativity, and the unprotected DNA will be rapidly degraded by nucleases present in plasma, so that the major challenge in gene therapy is the development of powerful gene delivery vectors with low toxicity, high transfection efficiency and controllability. In this study, novel peptide-based gene carriers will be designed and synthesized for overcoming in vitro and in vivo barriers in gene delivery. Several biocompatable and functional peptides (DNA binding peptides, targeting peptides, cell-penetrating peptides, endosomolytic peptides, nuclear localization signal peptides, etc.) will be introduced to form the combination vectors via advanced methods, like solid phase synthesis, peptide self-assembly and plasmid recombination. Moreover, we will design and synthesize several fusion peptides, build peptide library, investigate the transfection capability of peptides. Self-assembly behavior of bio-active membrane-spanning amphiphilic peptide/gene self-assembled complexes and their effects on gene therapy will be studied under different physiological microenvironments. After selection of the optimal peptides, these peptides and therapeutic gene sequences will be utilized to construct novel fusion plasmid vectors. These resulted plasmid vectors could transform and amplify in E. coli, and express therapeutic proteins that efficiently overcome physiological barriers in transfected cells. Furthermore, efficiency and mechanism of gene and protein delivery for tumor therapy using high efficient and low toxic peptide vectors will be investigated by in vitro biological experiments as well as in vivo animal testing. This study will greatly promote basic research and encourage clinical applications of gene therapy.

基因治疗是针对传统药物无法治愈的遗传病和后天疾病的重要医疗手段，构建低毒、高效、可控的非病毒基因载体对于治疗和预防先天免疫不全症、癌症、心血管疾病、神经退行性疾病等具有重要意义。本项目拟针对基因转运过程中需克服的各种生理屏障，采用固相合成、多肽自组装、质粒构建重组的方法，将生物相容性好且具备不同功能的结合肽、靶向肽、穿膜肽、内涵体溶解肽、核定位信号肽等引入基因载体中。设计并人工合成多种融合肽，构建肽库，探索多肽在基因转染上的规律；通过将兼具生物效应的两亲性跨膜肽与基因进行复合自组装，研究不同生理微环境下的自组装行为及其对基因治疗的影响；在筛选出理想多肽的基础上，构建多肽与治疗蛋白的融合质粒载体，通过生物发酵获得能高效穿越生理屏障的治疗蛋白；进一步通过体外生物实验和体内动物实验，探讨多肽载体高效低毒地运载基因和蛋白进行肿瘤治疗的机理和效果，推进基因治疗的基础研究和应用基础研究。

合成了系列功能化多肽基因载体，研究了不同的序列、结构的多肽对基因传递性能的影响，对基因载体的结构和功能进行了优化。合成了具有特定功能的多种功能肽及融合肽，其功能包括肿瘤细胞靶向、细胞器（细胞核、线粒体）靶向、内涵体/溶酶体逃逸、穿膜等，构建了一系列具有肿瘤靶向、协同响应性能的多肽类和多肽杂化类基因传递系统、基因及其它治疗剂的共传递系统，研究了传递系统的传递效率及抑制肿瘤效果，所制备的功能化传递系统能高效地将治疗剂传递到肿瘤细胞，在体外及动物体内均能有效抑制肿瘤生长。此外，设计合成了具有特定治疗功能（线粒体凋亡、抗肿瘤）的治疗肽，直接利用多肽材料本身的生物活性进行疾病治疗，同时对其进行功能化，赋予其肿瘤细胞及细胞器靶向功能。设计构建了多种功能化诊疗一体化纳米系统、功能化多模态治疗纳米系统，以克服肿瘤治疗中的耐药、乏氧等肿瘤微环境不利因素对治疗的影响等问题。本研究为多肽类生物材料在疾病治疗方面的研究提供了理论依据。