Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration
Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration
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DOI：
10.1016/j.ymthe.2017.05.015
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发表时间：
2017-09-06
影响因子：
12.4
通讯作者：
Tucker, Budd A.
中科院分区：
文献类型：
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作者：
Burnight, Erin R.;Gupta, Manav;Tucker, Budd A.
患者来源的诱导多能干细胞（iPSC）为自体细胞替代提供了巨大的希望。然而，对于许多遗传性疾病，治疗可能需要在移植前进行基因修复。基因组编辑技术对于这一应用是有用的。本研究的目的是开发CRISPR-Cas9介导的基因组编辑策略，以靶向和纠正患者来源的iPSC中三种最常见类型的致病变体：（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，靶向雄性生殖细胞相关激酶（MAK）外显子9中的纯合Mu插入，并证明了患者细胞中视网膜转录本和蛋白质的恢复。我们产生了CRISPR-Cas9介导的非同源末端连接（NHEJ）方法来切除Leber先天性黑蒙的主要贡献者，即CEP 290中的IVS 26隐蔽剪接突变，并证明了患者iPSC中转录物和蛋白质的校正。最后，我们设计了选择性靶向突变型Pro23 His视紫红质（RHO）等位基因的等位基因特异性CRISPR指南，该等位基因在体外递送给患者iPSC和体内猪视网膜后，产生了移码和过早停止，这将阻止致病变体的转录。这项研究中开发的策略将被证明对纠正导致遗传性视网膜变性的基因中的广泛遗传变异有用。
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 Mu 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.