基于CRISPR-Cas9技术靶向敲除DNMT1甲基化转移酶基因治疗卵巢癌的实验研究

CRISPR-Cas9 targeted genome editing technology is capable of editing the specific genomic nucleotide sequence and has the potential to treat diseases associated genetic abnormalities at the molecular level. The epigenetic DNMT1 gene is a potential target for the treatment of ovarian cancer, and is closely related to the tumorigenesis, progression and drug resistance of ovarian cancer. This project intends to use DNMT1 as the therapeutic target and ovarian cancer-targeted liposomes as a carrier of CRISPR-Cas9 gene, to establish a therapeutic program based CRISPR-Cas9 technology. The activity of gene editing, anti-tumor effect and mechanism, as well as side effects, will be studied in the in-vitro experiments and the treatment experiments of intra-abdominal tumor models. The project has a good basis, and the preliminary study has found that CRISPR-Cas9 system could edit DNMT1 gene specifically in ovarian cancer cells and then reduce the level of genomic methylation in vitro; the liposome/Cas9 compound displayed a good antitumor effect on Paclitaxel-sensitive and resistant ovarian cancer models in vivo. The project will further study the efficiency of gene editing, antitumor mechanism and potential off-target effect of the ovarian cancer-targeted liposomes/Cas9 compound. This study is expected to provide an experimental basis of epigenetic gene regulation and gene therapy for ovarian cancer based on CRISPR-Cas9 technique. This study is also expected to offer new ideas for the precise treatment of ovarian cancer. In addition, the applicant signed as the first, co-first or corresponding author has published 4 papers on journals with impact factors greater than 5.

新型基因组编辑技术CRISPR-Cas9具有治疗基因异常疾病的潜力。DNA甲基化转移酶DNMT1因异常高表达导致抑癌基因失活，诱导卵巢癌的发生和耐药，是卵巢癌治疗的新靶点。本项目拟以DNMT1基因为Cas9的编辑靶点、以卵巢癌靶向脂质体为这一Cas9基因编辑工具的导入系统，建立基于CRISPR-Cas9技术的基因治疗新方案。拟通过体外细胞实验、腹腔内肿瘤模型治疗实验对该方案的基因敲除效率、抗肿瘤作用与机理，以及毒副作用等进行研究。该项目有很好的前期工作基础，已在体外发现Cas9能靶向敲出DNMT1基因，进而降低基因组甲基化水平；对于敏感和耐药两种卵巢癌动物模型，本研究的基因治疗新方案均具有较好的抗肿瘤作用，本项目将进一步对该方案的体内基因敲除效率、抗肿瘤机制以及潜在的脱靶作用等进行研究。此外，申请人以第一、共同第一或通讯作者身份已发表4篇影响因子大于5分的SCI论文。

新型基因组编辑技术CRISPR-Cas9具有治疗基因异常疾病的潜力。DNA甲基化转移酶DNMT1因异常高表达导致抑癌基因失活，诱导卵巢癌的发生和耐药，是卵巢癌治疗的新靶点。本课题拟以DNMT1基因为Cas9的编辑靶点、以卵巢癌靶向脂质体（叶酸脂质体）为这一Cas9基因编辑工具的导入系统，建立基于CRISPR-Cas9技术的基因治疗新方案。通过体外理化性质评价及细胞实验、腹腔内肿瘤模型治疗实验对该方案的基因敲除效率、抗肿瘤作用与机理等进行研究。.叶酸脂质体（F-LP）与靶向DNMT1基因的Cas9基因编辑工具（gDNMT1）在质量比12:1时能形成粒径约150nm、多分散系数约0.14、电位约35mV的F-LP/gDNMT1复合物。该复合物在电镜下观察具有膜结构，呈棒状或球形。在细胞水平的研究发现，F-LP/gDNMT1能够编辑DNMT1基因，通过引入插入或缺失突变从而敲出DNMT1基因，下调DNMT1在细胞核的表达，进而引起5甲基胞嘧啶下调。F-LP/gDNMT1通过腹腔给药在体内能够有效抑制紫杉醇敏感和耐药卵巢癌模型肿瘤的生长，抑瘤率分别达到84%和76%。和阳性对照紫杉醇相比，F-LP/gDNMT1具有更好的安全性。该复合物主要分布在腹腔且维持至少24h，高的药物浓度有利于抗肿瘤作用的保持，而较少扩散至其他脏器避免了毒性的产生。机制研究表明通过在肿瘤组织表达Cas9蛋白，靶向敲出DNMT1基因，导致DNMT1表达下调，诱导下游HP1α、p15INK4b表达下降，从而产生抗肿瘤作用。.本研究表明脂质体能够作为CRISPR-Cas9的递送载体，实现动物体内Cas9的递送。利用CRISPR-Cas9技术靶向敲除DNMT1基因是卵巢癌治疗的一个潜在手段，而非病毒基因递送系统也有望成为CRISPR-Cas9技术实现精准治疗的载体之一。.本项目实施期间发表标注基金的论文14篇，SCI论文11篇，SCI论文第一标注8篇，最高单篇影响因子12.831；SCI论文第二、第三标注分别为2篇、1篇；中文论文3篇，第一、第二标注分别2篇、1篇。申请中国发明专利1项。