酯酶响应型聚合物载体应用于肿瘤基因治疗的研究

Cancer gene therapy is a new effective treatment, but the delivery system is its hurdle to real applications. The applicant has demonstrated that using esterase-responsive charge-reversal polymer as the carrier, the suicide gene could be highly transfected in tumor cells but not in tumor associated fibroblasts in a tumor, realizing tumor cell-selective gene therapy and much higher therapeutic efficacy than several first-line chemotherapy drugs. The drawback of this system, however, is that the polymer was not very stable in water, deteriorating its esterase-selectivity and utility in drug formulation; the in vivo delivery system was only for local intraperitoneal gene delivery. Therefore, the goal of this proposal is to synthesize water-stable, highly selective and sensitive esterase-responsive cationic polymers and fabricate effective tumor targeting gene delivery system for systemic cancer gene therapy. Along this line, a water-stable methylene-phenol ether ester will be used as the esterase-responsive moiety, whose stability and esterase selectivity and responsiveness will be tuned by the acyl structure. A phospholipid-coated polyplex-based gene delivery system will be fabricated and its lipid-membrane will be tailed to be capable of membrane fusion with cells to bypass the lysosome-traps for high gene transfection efficiency. The system will be further pegylated and functionalized with tumor-targeting group CRGDK to obtain high tumor targeting ability after intravenous administration. The gene delivery systems will be evaluated in vitro and in vivo to infer the structure-property-efficacy relationship for further optimization to finally obtain the cancer gene delivery system with potent anticancer efficacy. This work will make great contributions to the clinical translation of cancer gene therapy.

基因治疗是安全有效的肿瘤治疗新手段，其瓶颈是基因输送系统。申请人利用酯酶响应电荷反转型阳离子聚合物为载体，使自杀基因在肿瘤细胞内高表达而在成纤维细胞中不表达，实现了肿瘤内细胞选择性基因治疗，获得了远高于化疗药物的疗效。但是，该聚合物的稳定性和酯酶选择性不够，不能满足制剂应用要求，其输送系统只是以腹腔治疗为目的。因此，本项目的研究目标是设计合成高稳定性、高酯酶响应性阳离子聚合物，并以此制备高肿瘤靶向性和高效基因输送系统，应用于肺癌和结肠癌的系统基因治疗。为此，本项目将以酚基-甲撑醚基酯为酯酶触发基团，通过调节羧酸基团来优化其稳定性和酯酶选择性；构建磷脂包裹膜融合入胞型基因输送系统，使其避开溶酶体陷阱而提高基因转染效率；通过PEG化和引入靶向基团提高其长循环和肿瘤靶向性，利用动物模型进行综合评价，最终获得高抗肿瘤活性的基因输送系统。本研究将为解决肿瘤基因治疗的瓶颈难题、推动其临床应用做出贡献。

非病毒载体应用于肿瘤基因治疗的瓶颈在于高转染效率的载体的设计和体内应用。本项目设计并合成了酯酶响应聚合物载体PQDEA，它能够响应肿瘤细胞和巨噬细胞内的酯酶，发生电荷反转快而速释放DNA，实现高效转染。主要包括三方面的工作：（1）酯酶响应型聚合物载体包载TRAIL质粒应用于晚期宫颈癌模型的基因治疗。利用宫颈癌细胞高表达酯酶的特点，设计合成了酯酶响应型聚合物载体PQDEA,用于包载自杀基因TRAIL质粒。脂质化后的TRAIL/聚合物复合物可用于治疗晚期宫颈癌腹腔扩散肿瘤，能够高效转染宫颈癌细胞使其表达自杀蛋白TRAIL，诱导肿瘤细胞凋亡，抑瘤效果优于临床一线化疗药物紫杉醇、顺铂和盐酸伊立替康。研究成果发表于《Biomacromolecules杂志》 （本人为论文第一作者和共同通讯作者，期刊影响因子为6.092）。（2）肿瘤细胞和肿瘤相关巨噬细胞(TAMs)双转染非病毒载体应用于癌症免疫基因治疗。利用TAMs和肿瘤细胞胞浆高表达酯酶的特点，设计了可同时高效转染肿瘤细胞和TAMs的基因输送系统，用于输送促炎因子白介素12(IL12)质粒。该基因输送体系可主动靶向肿瘤组织，同时高效转染肿瘤细胞和TAMs，使二者都成为IL12的生产“工厂”，瘤内高浓度的IL12将巨噬细胞M1/M2比例提高了4倍以上，显著延缓肿瘤的生长并使小鼠的生存期增加一倍,是领域内首次实现TAMs和肿瘤细胞双重高效转染的非病毒基因递送体系。研究成果发表于《Advanced Materials》杂志（本人为论文第一作者，期刊影响因子为27.398）。（3）在资助期间，首次发现了能够同时激活肿瘤免疫及降低肿瘤细胞PD-L1表达的雷公藤红素纳米乳，实现了不依赖于PD-L1抗体的肿瘤免疫治疗。研究成果发表于《Biomaterials》上（本人为论文第一作者，期刊影响因子为10.317）。