新型CRISPR/dCas9介导的智能化基因线路精准治疗膀胱癌的实验研究

Bladder cancer is the most common malignant tumor in urinary system, and its occurrence and development is a complex process involving multiple genes. The traditional targeted therapy could not distinguish bladder cancer cells and normal cells based on a single gene. The artificial genetic circuits, which integrate different biological signals as their basic characteristics, have gradually become a new hot spot in the field of cancer research because of their advantages in precision therapy. We have developed a CRISPR-dCas9 “signal conductor” regulated by intracellular signals, and then achieved the construction of genetic circuits that dynamically sensed and integrated a variety of proteins for the first time. However, the system has some potential problems: high-frequency off-target effects, poor transcriptional regulation and narrow endogenous signals-responsive range. We speculate that overcoming the above problems will help to build up intelligent gene regulatory circuits for cancer. This project includes: 1. We will re-design and modify sgRNA and dCas9 and improve the specificity and effectiveness of “signal conductor” ; 2. We will use the in vitro screening technology to identify aptamers that recognize different protein targets of bladder cancer and extend the response range of endogenous signals; 3. We will use optimized "signal conductors" as the fundamental elements to construct genetic circuits that diagnose and treat bladder cancer. The implementation of this project will provide theoretical and scientific bases for constructing genetic circuits treating bladder cancer.

膀胱癌是泌尿系统最常见的恶性肿瘤，其发生、发展是一个多基因参与的复杂过程。针对单一基因的传统靶向治疗，无法有效区分膀胱癌细胞和正常细胞。以整合不同生物信号为基本特征的人工基因线路，因其特有的精准治疗优势，逐渐成为肿瘤研究新热点。我们前期开发了受胞内信号调控的CRISPR-dCas9“信号传导器”，据此搭建的基因线路首次实现对肿瘤多种蛋白信号的动态感应与整合。但该系统存在脱靶率高、转录调控力低、内源信号应答范围窄等潜在问题。我们推测：克服以上不足，有助于搭建智能化肿瘤基因调控线路。本项目拟：1.工程化设计改造sgRNA和dCas9，提高“信号传导器”的特异性和有效性；2. 利用体外筛选技术获得膀胱癌不同蛋白靶标的核酸适体, 扩展对内源信号的应答范围；3.以优化的“信号传导器” 为基本元件，构建膀胱癌基因诊疗线路。该项目的实施将为基因线路精准干预膀胱癌提供理论基础和科学依据。

膀胱癌是泌尿系统最常见的恶性肿瘤，其发生、发展是一个多基因参与的复杂过程。针对单一基因的传统靶向治疗，无法有效区分膀胱癌细胞和正常细胞。以整合不同生物信号为基本特征的人工基因线路，因其特有的精准治疗优势，逐渐成为肿瘤研究新热点。我们前期开发了受胞内信号调控的CRISPR-dCas9“信号传导器”，据此搭建的基因线路首次实现对肿瘤多种蛋白信号的动态感应与整合。但该系统存在脱靶率高、转录调控力低、内源信号应答范围窄等潜在问题。我们推测：克服以上不足，有助于搭建智能化肿瘤基因调控线路。本项目主要完成：1.工程化设计改造sgRNA和dCas9，提高“信号传导器”的特异性和有效性；2. 利用体外筛选技术获得膀胱癌不同蛋白靶标的核酸适体, 扩展对内源信号的应答范围；3.以优化的“信号传导器” 为基本元件，构建膀胱癌基因诊疗线路。该项目的实施将为基因线路精准干预膀胱癌提供理论基础和科学依据。