Karakikes Ioannis, Termglinchan Vittavat, Cepeda Diana A, Lee Jaecheol, Diecke Sebastian, Hendel Ayal, Itzhaki Ilanit, Ameen Mohamed, Shrestha Rajani, Wu Haodi, Ma Ning, Shao Ning-Yi, Seeger Timon, Woo Nicole, Wilson Kitchener D, Matsa Elena, Porteus Matthew H, Sebastiano Vittorio, Wu Joseph C
From the Stanford Cardiovascular Institute (I.K., V.T., J.L., S.D., I.I., M.A., R.S., H.W., N.M., N.-Y.S., T.S., N.W., K.D.W., E.M., J.C.W.), Department of Cardiothoracic Surgery (I.K.), Division of Cardiovascular Medicine, Department of Medicine (V.T., J.C.W.), CA; Institute of Stem Cell Biology and Regenerative Medicine (D.A.C., V.S., J.C.W.), Departments of Pediatrics (A.H., M.H.P.), Pathology (K.D.W.), and Obstetrics and Gynecology (V.S.), Stanford University School of Medicine, CA; Berlin Institute of Health, Germany (S.D.); and Max Delbrueck Center, Berlin, Germany (S.D.).
Circ Res. 2017 May 12;120(10):1561-1571. doi: 10.1161/CIRCRESAHA.116.309948. Epub 2017 Feb 28.
Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.
The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.
By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (, and ) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation () in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by in human cardiac myocyte development.
Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.
使用诸如转录激活样效应核酸酶（TALENs）等可编程核酸酶进行靶向基因工程，是在人类基因组中进行精确、位点特异性基因修饰的宝贵工具。
人类诱导多能干细胞（iPSC）和核酸酶介导的基因组编辑等新技术的出现，为体外研究心血管疾病提供了独特的机会。
通过广泛的文献和数据库检索，我们设计了一组TALEN构建体，以敲除88个与心肌病和先天性心脏病相关的人类基因。TALEN对被设计用于在每个基因的起始密码子附近诱导双链DNA断裂，从而破坏起始密码子或在早期编码区域引入移码突变，确保可靠的基因敲除。我们观察到所有构建体均具有活性，并以高频率破坏靶位点。为了说明TALEN介导的敲除技术的实用性，在人类iPSC中高效且特异地敲除了6个个体基因（、和）。通过在患者特异性iPSC衍生的心肌细胞中选择性靶向致病突变（），我们证明了敲除策略在体外改善了扩张型心肌病表型。此外，我们在体外iPSC-心肌细胞中模拟了霍尔特-奥勒姆综合征，并发现了在人类心肌细胞发育中由调控的新途径。
总体而言，我们的研究说明了iPSC和基因组编辑技术在理解基因生物学功能以及人类心血管疾病中遗传变异的病理意义方面的强大结合。本研究中开发的方法、策略、构建体和iPSC系为心血管研究提供了经过验证的、随时可用的资源。
