基于含铂高分子纳米胶束的癌症CRISPR/Cas9基因编辑治疗

CRISPR-Cas9 system, enabling sequence-specific genome editing, is an emerging technology for disease treatment, and the combination of CRISPR-Cas9 genome editing system and chemotherapy would hold originality and tremendous promise for enhanced cancer therapy. However, the safety and efficiency of co-delivery of drug and CRISPR-Cas9 system are limited by the drawbacks of carriers’ material, controlled release fashion and drug resistance of tumor. Hence, in this project, reduction-responsive and visible light-responsive platinum-backboned polycationic micelles are prepared for delivery of CRISPR-Cas9 systems through electrostatic interaction to sequence-specific edit and knock out cisplatin-resistant genome. For the first time, we realize the controllable co-delivery of drug and CRISPR-Cas9 system by using a single polymeric carrier for combination cancer therapy. Under intracellular reductive microenvironment/remote visible light irradiation, the platinum-backboned micelles would simultaneously undergo responsive release of active platinum(II) drug and fast degradation of the polycationic backbones to improve CRISPR-Cas9 system unpacking and endosomal escaping. The synergistic effect of platinum(II) drug and CRISPR-Cas9 system would be fully realized, resulting in enhanced anticancer efficacy and even cisplatin resistance reversal. The cancer treatment effect, synergistic mechanism and toxicity of CRISPR/Cas9 genome editing system based on the different stimuli-responsive platinum-backboned micelles are in-depth studied, providing experimental basis for solving the scientific problems including controllable co-delivery of drug and CRISPR-Cas9 system, efficiently combating cisplatin resistance and delivery safety.

CRISPR/Cas9系统是一项新兴的序列特异性基因编辑技术，将其与化疗联合用于癌症的增强治疗极具原创性和应用前景。然而基因编辑系统与化疗药物纳米共传输的安全性和有效性受到载体材料、可控释放、肿瘤耐药性等问题限制。本项目拟制备还原和可见光刺激响应性主链含铂聚阳离子纳米胶束并静电负载可特异性敲除铂药耐药基因序列的基因编辑系统，创新性地实现高分子纳米可控共传输基因编辑系统和化疗药物用于联合癌症治疗；通过细胞内还原微环境或外部可见光照诱导含铂纳米胶束主链结构迅速崩解，同时实现可控响应的活性铂药释放和基因编辑系统逃逸与卸载，充分发挥它们间协同增效作用，逆转肿瘤铂药耐药性。深入研究基于不同刺激响应性含铂高分子纳米胶束的癌症CRISPR/Cas9基因编辑治疗的效果、协同作用机理以及毒性，为CRISPR/Cas9系统和化疗药物的可控共传输、肿瘤铂药耐药性有效逆转以及安全性等关键科学问题奠定实验研究基础。

CRISPR/Cas9系统是一项新兴的序列特异性基因编辑技术，将其与化疗联合用于癌症的增强治疗极具原创性和应用前景。然而基因编辑系统与化疗药物纳米共传输的安全性和有效性受到载体材料、可控释放、肿瘤耐药性等问题限制。因此，在本项目中，我们制备了可刺激响应链消除的主链含铂聚阳离子纳米胶束并静电负载可特异性敲除EZH2基因序列的CRISPR/Cas9基因编辑系统 (NPCSPt/pEZH2)，用于前列腺癌的联合化疗及基因编辑治疗。实验结果表明，NPCSPt/pEZH2能有效地从内溶酶体中逃逸出来，并在肿瘤细胞内还原微环境下，通过Pt(IV) 还原响应性，促进NPCSPt/pEZH2中的骨架聚合物以链粉碎方式降解，造成NPCSPt/pEZH2的解体，从而触发pEZH2和Pt(II)在细胞内有效释放。与预期的一样，NPCSPt/pEZH2在体内外均能对EZH2基因进行高效编辑，显著抑制EZH2蛋白表达。随后， H3K27me3蛋白的表达量随着EZH2的沉默而减少，导致肿瘤细胞核中染色质结构变成更松散的状态，从而增加了释放出来的Pt(II)与细胞核中DNA接近的机会，使铂和DNA的加和量 (Pt-DNA) 显著增加，导致更多的癌细胞凋亡。细胞和动物实验表明，NPCSPt/pEZH2可以显著抑制前列腺癌细胞的增殖并进一步有效抑制前列腺癌皮下移植瘤的生长。因此，该纳米平台不仅为CRISPR/Cas9系统提供了一种很有前途的非病毒纳米载体，而且扩大了基因编辑-化疗联合治疗癌症的潜力。