高效抑制肿瘤突变BRAF蛋白的新型siRNA的结构修饰策略研究及siRNA联合小分子药物的抗黑色素瘤应用研究

Malignant melanoma is one of the most deadly forms of solid tumors. Over the past three decades, there has been not much improvement in survival for those patients. Hyperactivated Ras/Raf/MEK/ERK signaling pathway is a key regulator of melanoma, providing essential tumor growth and maintenance functions. The mutational activation of BRAF at V600E supports the most important role of this pathway in human oncogenesis (over 60% of melanoma). Small molecular inhibitors targeted to kinases (such as, BRAFV600E mutant, BRAF, or MEK1/2) have significant potential to halt the progression of malignant tumors by blocking this pathway. Unfortunately, most of them had shown serious toxicity to human body due to their lower specific recognization to the targeting kinases, or promoting the dimmer formation of CRAF to activate the ERK pathway. In this proposal we will develop the RNAi based therapy and combinational therapy for malignant melanoma with specific tumor targeting and efficient inhibition property to the hyperactive ERK pathway. Our previous studies found that inhibition of BRAFV600E kinase with RNAi technology could inhibit growth of the tumor growth. However, the best silencing activity of nature siBraf-mu sequence is around 40% due to the mutant position of target gene (brafT1799A). In order to improve its gene silencing activity and serum stability, the novel combinational chemical modification strategies will be developed. Based on our previous structure-activity relationship studies of siRNA, combination of peptide conjugation at 3' terminal both or at sense strand with D-/L- isonucleotides incorporation or phosphorothioate (PS) backbone modification will be developed to enhance bioactivity and stability of the siBraf-mu sequences as therapeutic agent. Not only to the mutant gene, siBraf-wt showed efficient gene silencing activity also to the wild-type braf, which makes it more toxic to normal cells. The tumor targeted peptide-based delivery system will be conjugated to the siBraf-wt for efficient gene silencing in melanoma only. Integrin αvβ3 and MC-1 receptor are reported highly expressed in most melanoma cells. Their peptide ligands, RGD peptides and α-MSH peptides will be incorporated into siBraf-wt to enhance the cell uptake and the gene-silencing capacity of siBraf-wt in melanoma cells with lower cytotoxicity. The specific and efficient silencing braf T1799A and ERK pathway inhibition on tumor cells will be first tested by PCR and western blotting, following by tumorigenesis and angiogenesis studies in vitro and vivo. Melanoma is a complex disease, multiple signaling pathway involved in tumor progression. Combination of siRNA therapy with chemotherapy or targeted therapy (targeted to PI(3)K pathway, receptor tyrosin kinases) will be developed to find the efficient and non-cytotoxic treatment to this malignat melanoma. The information obtained from this study will be translated into other tumor types with ERK pathway mutation.

突变基因是导致表皮黑色素瘤细胞内ERK通路过度活化、造成肿瘤增殖、转移、高度恶性化的主要原因之一。相应的小分子靶向药物能抑制通道的活性、肿瘤生长，但存在脱靶效应、诱发肿瘤的进展等毒性缺憾。本课题拟使用RNAi技术、联合细胞毒/分子靶向药物，发展高效、低毒的肿瘤个性化治疗的新方案。基于前期工作，有机组合优化各种化学修饰方法，发展生物活性明显提高、稳定性高的靶向突变基因的siBraf-mu序列；通过发展黑色素瘤细胞特异的多肽靶头、高效链接方式，研发多肽-siBraf-wt序列；建立合适的细胞、动物水平siRNA活性、特异性筛选模型，发展靶向突变基因的siRNA新修饰策略，应用与恶性肿瘤治疗。根据黑色素瘤特有的肿瘤生物学，针对其他协助肿瘤发展的信号通路、肿瘤生长、转移机制，发展siRNA药物联合小分子药物的治疗恶性黑色素瘤新方案。本项目的研究成果可以衍生应用于与ERK通路密切相关的其他肿瘤治疗。

本研究目标是研发靶向突变蛋白(BRAFV600E)的siRNA类抗肿瘤治疗药物前体。本课题已经筛选出4条生物稳定性显著提高的异核苷、硫代和双肽缀修饰siBraf-mu序列。通过建立茎环引物PCR方法、溶酶体溶酶体逃逸实验等方法，深入讨论了双肽缀合siBraf-mu的体内外药效活性、细胞内代谢和体内代谢等药理活性，揭示其延迟生物活性的分子机制。设计合成了4条具有integrin受体靶向性、胞内可解离的cRGD多肽缀合siBraf-mu (Bis-cRGD-siBraf-mu)序列。发展新型CLD载体/Bis-cRGD-siBraf-mu复合物，理化性质和生物活性研究表明CLD/(21, 5/3)复合物表现高效特异的抑制A375黑色素瘤细胞增殖的活性，具有良好的应用前景。本课题还研发了治疗恶性化程度高的黑色素瘤细胞的多种siRNA和小分子药物联合治疗的新策略。其中siBraf-mu 联合PI3K/Akt/mTOR1/2通路抑制剂对BrafV600E依赖的黑色素瘤细胞的体现显著的协同增殖抑制影响，并深入探讨了联合给药后协同活性的分子机制。通过对黑色素瘤细胞个体化治疗研究，进一步证明有效的治疗需要分析导致肿瘤发生发展的关键因素，对于多因素导致的肿瘤，多靶点用药就能具有较好的效果。