可控激活内源基因表达的纳米载药系统构建及治疗单倍剂量不足疾病的研究

单倍剂量不足是指一个等位基因突变引起的基因表达量减半，会导致肿瘤和肥胖等疾病。近期发展的CRISPRa系统可直接激活内源基因表达，通过成倍上调未突变等位基因的表达量弥补单倍剂量不足，但面临如何可控激活和体内靶向递送的挑战。申请人前期利用CRISPRa特异性激活了黑色素瘤的基因表达，并构建纳米载体实现CRISPR工具的体内递送（Nat Commun，2018等）。本项目拟在此基础上，利用特定响应元件构建以四环素、咖啡因或远红光为开关的可控CRISPRa系统；并构建纳米载体系统，进行纳米特性调控和靶向基团修饰，通过尾静脉或局部注射筛选针对结肠癌、脂肪和下丘脑中靶细胞的高效纳米载体，实现可控CRISPRa的体内递送。项目将以APC、LEP和MC4R作为靶基因，研究构建的纳米载药系统激活内源基因的可控性及对三种基因杂合模型鼠结肠癌或肥胖的疗效。项目将为基因表达不足的疾病提供可控的基因激活治疗手段。

Haploinsufficiency, in which a single functioning copy of the allele in diploid organisms can only produce half gene product of the normal level when the other copy of the gene is mutated, will consequently lead to cancers, obesity and many other diseases. Recently developed CRISPR activation (CRISPRa) systems, which enable directly activating the expression of all genes, can compensate for haploinsufficiency by exponentially up-regulating the expression of the unmutated copy of the allele. Nevertheless, the gene activating ability of current CRISPRa systems is uncontrollable, usually leading to overexpression of target genes and other serious side effects. Furthermore, efficiently delivering CRISPRa systems into target cells also remains a big challenge for applying CRISPRa in the treatment of haploinsufficiency-caused diseases. In previous studies, the applicants have utilized CRISPRa to specifically activate the expression of chemokine genes (CXCL9, CXCL10 and CXCL11) in melanoma for promoting the infiltration of T cells. In addition, the applicants have prepared a library of cationic lipid-assisted polymeric nanoparticles (CLANs), and screened the optimal CLAN to deliver CRISPR-based tools into macrophages, neutrophils, dendritic cells and cancer cells for treating inflammatory diseases, type II diabetes, etc. (representative works were published in Nat. Commun. (2018) et. al.). Based on these previous studies, the applicants plan to construct three switchable CRISPRa systems for the controllable gene activation using specific responsive elements of tetracycline, caffeine and far-red light from systems biology. To overcome the in vivo delivery challenges of CRISPRa systems, the applicants plan to design and prepare a library of CLAN-based nanocarriers by modulating the nano-properties of CLAN (i.e. size, surface charge and lipid species) and by modifying CLAN with different targeting ligands (i.e. anti-A33, ATS-9R and transferrin). The applicants will screen the optimal CLAN for targeted delivery of these controllable CRISPRa systems into colon cancer cells, adipocytes and hypothalamic nerve cells via systemically or locally injecting different CLANs into mice. Subsequently, the applicants will exploit APC, LEP and MC4R as target genes to investigate the gene activating efficacy and controllability of the three controllable CRISPRa systems delivered by the screened CLANs. Eventually, the applicants will deliver the three controllable CRISPRa systems into APC+/-, LEP+/- and MC4R+/- mouse models using different screened CLANs to treat colon cancer and obesity caused by the haploinsufficiency of APC, LEP and MC4R gene. This project will provide novel nanomedicines for treating diseases caused by insufficient gene expression via controllable gene activation.