肿瘤原位重组CRISPR/dCas9的级联响应给药系统研究

跨越肿瘤细胞屏障与胞内有效递释是在体CRISPR基因编辑的关键。本项目以自制富苯硼酸-季铵聚合物（CRP）定量压缩dCas9和sgRNA呈纳米粒，与β-拉帕醌（LAP）共载于pH敏感脂质体中，并仿生锚定肿瘤穿膜肽tLyP-1，构建具有微环境级联响应、原位重组功能的CRISPR/dCas9给药系统。该系统经穿膜肽渗透入瘤，借助胞内低pH爆释LAP，诱导升高微环境活性氧促进CRP氧化解聚，触发原位dCas9和sgRNA快速释放与复合重组，实现在体工程化细胞表达TRAIL，促进细胞间凋亡信号传递，联合活性氧逆转肿瘤免疫耐受微环境。该系统具有高效自组装、定量给药、靶向渗透、级联响应释药，且在体工程化细胞高效便捷、诱导凋亡与免疫应答等优势。项目重点围绕系统构建、功能评价与药效学展开研究，并探讨“渗透递药—微环境调控—级联释药”过程与基因编辑肿瘤系统治疗的联合作用机制，为抗肿瘤治疗提供新思路和新方法。

Crossing tumor-associated cell barrier and effective intracellular delivery are essential for in vivo CRISPR gene editing. In this project, we synthesize phenylboronic acid-rich quaternized polymer (CRP) to quantitatively compress dCas9 and sgRNA as nanoparticle. The nanoparticle generation is encapsulated into pH-sensitive liposomes together with β-lapachone (LAP), followed by bionic surface-modification with tumor penetrating peptide tLyP-1 to formulate CRISPR/dCas9 drug delivery systems, that are provided with tumor microenvironment cascade response and in situ reassembly functions. The obtained nanosystem could penetrate into deep tumor with the aid of penetrating peptide, followed by burst release of LAP upon intracellular acidic microenvironment for ROS elevation and CRP oxidative depolymerization. Afterwards, dCas9 and sgRNA are triggered in situ rapid release and reassembly to realize in vivo engineered cell expression of TRAIL, promotion of intercellular apoptosis signal transmission, and combination with reactive oxygen species for tumor immune tolerance microenvironment reversion. The nanosystem possesses advantages of efficient self-assembly, quantitative dosage, targeted penetration, cascade-responsive drug release, as well as efficient and convenient in vivo cell engineering, and apoptosis promotion plus immune response. For this project, we focus on formulation construction, functional evaluation and pharmacodynamic estimation, and explore the combined action mechanism of "penetrating delivery-microenvironment regulation-cascade responsive drug release" process and gene-editing mediated tumor systemic therapy to provide new ideas and methods for antitumor therapy.