Stephen A. Wynn Institute for Vision Research and Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52241, USA.
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 01451, USA.
Mol Ther. 2017 Sep 6;25(9):1999-2013. doi: 10.1016/j.ymthe.2017.05.015. Epub 2017 Jun 12.
Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.
患者来源的诱导多能干细胞 (iPSC) 在自体细胞替代方面具有巨大的应用前景。然而，对于许多遗传性疾病，在移植前可能需要进行遗传修复。基因组编辑技术在这一应用中非常有用。本研究旨在开发 CRISPR-Cas9 介导的基因组编辑策略，以靶向和纠正患者来源的 iPSC 中三种最常见的致病变异类型：(1) 外显子，(2) 深内含子，和 (3) 显性功能获得。我们开发了一种同源定向修复策略，靶向男性生殖细胞相关激酶 (MAK) 外显子 9 中的同源 Alu 插入，并证明了患者细胞中视网膜转录本和蛋白的恢复。我们生成了一种 CRISPR-Cas9 介导的非同源末端连接 (NHEJ) 方法来切除 Leber 先天性黑矇的主要致病因素，CEP290 的内含子 26 隐匿剪接突变，并证明了患者 iPSC 中转录本和蛋白的纠正。最后，我们设计了等位基因特异性的 CRISPR 向导，该向导可以选择性地靶向突变的 Pro23His 视蛋白 (RHO) 等位基因，该等位基因在体外递送至两个患者 iPSC 以及体内猪视网膜后，会产生移码并提前终止，从而阻止致病变异的转录。本研究中开发的策略将有助于纠正引起遗传性视网膜变性的基因中广泛的遗传变异。
