粒径可控型多肽基因载体的构建及其转染机理研究

Exploring the method to promote transfection efficiency and targeting ability of non-viral vectors efficiently is a major problem for gene therapy. The previous research of applicant demonstrated that peptide vectors with suitable size could enhance the uptake and transfection efficiency of target genes in targeted cells obviously. And it lays the foundation for revolutionary enhancement of transfection efficiency. Moreover, there has been no systematic report focusing on the transfection mechanism and influence rules of cellular delivery and gene expression of peptide vectors. In this study, to overcome various physiological barriers during gene delivery, a series of novel multifunctional targeting peptides consisting of cell-penetrating peptide, endosomolytic peptide, nuclear localization signal and tumor targeting peptide will be constructed by solid phase synthesis based on the obtained peptide vectors. Modern molecular biology technologies will be applied to analyze the cellular uptake and intracellular delivery of peptide/DNA complexes. The mechanism of the effects on gene expression level caused by cellular delivery of the complexes will be investigated. And the influence on cellular delivery and transfection efficiency brought by the peptide sequence and complex size will be further elucidated. Finally, the transfection mechanism, structure-activity relationship and influence rules of gene delivery carried out by peptide vectors will be revealed. Furthermore, the new mode of sequence design and size regulation of efficient peptide vectors will be built, which may promote the development of basic research and clinical applications of gene therapy.

有效提高非病毒载体的转染效率和靶向能力，是当今基因治疗领域面临的重大难题。申请人研究表明粒径适中的多肽载体能显著增强靶细胞中目的基因的内吞效率和转染水平，为转染效率实现突破性的提高奠定了基础。同时，多肽载体细胞转运和基因表达的相关转染机理与影响规律研究尚未见系统报道。本项目拟针对基因传递过程中需要克服的各种生理屏障，根据前期多肽载体材料的设计基础，通过固相合成构建一系列含有细胞穿膜肽、内含体逃逸肽、核定位信号肽和肿瘤靶向肽的新型多功能靶向多肽。利用现代分子生物学手段，考察多肽/DNA复合物的细胞摄取和胞内传递过程，探寻复合物的细胞转运过程对基因表达水平的影响机制，阐明多肽序列和复合物粒径对细胞转运和转染效率的影响。最终揭示多肽载体基因传递的转染机理、构效关系和影响规律，建立高效多肽基因载体序列设计与粒径调控的新模式，促进基因治疗基础研究和临床应用研究的发展。

构建高效低毒的非病毒载体对基因治疗的成败至关重要。本项目通过设计合成得到功能型组合长肽PKKKRKVH7R8，研究发现PKKKRKVH7R8的毒性小，并能将质粒DNA的转染效率提升181.62倍。对比具有不同粒径和电位的PKKKRKVH7R8/DNA复合物的转染结果表明，较小的粒径和较高的正电性有利于复合物的转染。PKKKRKVH7R8与各活性短肽的转染效率差异说明组合长肽能有效利用各短肽的促转染能力，实现转染效率的增强。细胞内吞研究表明，在4 h内，PKKKRKVH7R8/DNA复合物能够有效实现载体系统的跨膜转运，并广泛分布于细胞质区域。向PKKKRKVH7R8/DNA复合物体系中引入钙离子，将会增大复合物的粒径，降低复合物的电位，导致其稳定性减弱和转染效率下降。为了进一步提高PKKKRKVH7R8的转染效率，选择构建了低毒的PKKKRKVH7R8/海藻酸钠/碳酸钙复合基因载体，通过优化复合物中海藻酸钠和PKKKRKVH7R8的比例，其转染效率最高可达质粒DNA的7976.71倍，在PKKKRKVH7R8的基础上，转染能力得到显著增强。此外，本项目还初步阐明了抗癌活性物质T-2毒素导致癌细胞凋亡与生长抑制的机理，为构建基因/药物共传递的抗癌多肽载体系统，以及肿瘤靶向多肽的筛选与设计提供理论依据。本项目构建得到的高效低毒多肽载体系统为新型非病毒基因载体的设计提供了有益的参考，而T-2毒素抑癌机理的阐明为后续基因治疗的载体与辅助活性物质的创新奠定了基础。