Ruan Jinxue, Hirai Hiroyuki, Yang Dongshan, Ma Linyuan, Hou Xia, Jiang Hong, Wei Hongguang, Rajagopalan Carthic, Mou Hongmei, Wang Guoshun, Zhang Jifeng, Li Kui, Chen Yuqing E, Sun Fei, Xu Jie
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, University of Michigan Medical School, 2800 Plymouth Road, Ann Arbor, MI 48109, USA; Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, College of Animal Sciences & Technology, Huazhong Agricultural University, Wuhan, China.
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, University of Michigan Medical School, 2800 Plymouth Road, Ann Arbor, MI 48109, USA; Ann & Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Mol Ther Nucleic Acids. 2019 Jun 7;16:73-81. doi: 10.1016/j.omtn.2019.02.006. Epub 2019 Feb 16.
Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.
囊性纤维化（CF）是一种由囊性纤维化跨膜传导调节因子（CFTR）基因突变引起的致死性常染色体隐性疾病。核酸酶介导的精确基因编辑（PGE）是一种很有前景的CF治疗方法，为此需要一种无病毒载体、药物筛选和报告基因富集的高效策略（无VDR）。在这里，我们比较了不同的转染方法（脂质体转染与电穿孔）和形式（质粒DNA与核糖核蛋白），将CRISPR/Cas9元件与单链寡脱氧核苷酸（ssODN）一起递送至靶向主要CFTR突变位点的临床相关细胞。我们证明，在不同组合中，CRISPR/Cas9和向导RNA（gRNA）核糖核蛋白（Cas9 RNP）的电穿孔是最有效的。通过使用这种无VDR方法，在野生型诱导多能干细胞（iPSC）系中产生dF508、G542X和G551D突变的效率达到了4.8%至27.2%。当将其应用于携带dF508突变的患者来源的iPSC系时，精确校正率超过20%。正如预期的那样，基因校正导致iPSC来源的近端肺类器官以及患者来源的腺癌细胞系CFPAC-1中CFTR功能的恢复。目前的工作证明了基于基因编辑的疗法对CF等单基因疾病的可行性。
