Miki Toshio, Vazquez Ludivina, Yanuaria Lisa, Lopez Omar, Garcia Irving M, Ohashi Kazuo, Rodriguez Natalie S
Department of Surgery, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 509A, Los Angeles, CA 90033-9141, USA.
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Stem Cells Int. 2019 Apr 1;2019:6978303. doi: 10.1155/2019/6978303. eCollection 2019.
Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler's disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neo) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neo sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neo removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.
1型黏多糖贮积症（MPS-1），也称为胡勒氏病，是一种先天性代谢紊乱疾病，由α-L-艾杜糖醛酸酶（IDUA）基因突变引起，该突变导致溶酶体酶功能丧失，无法降解糖胺聚糖。在此，我们利用诱导多能干细胞（iPSC）和CRISPR/Cas9技术，以MPS-1模型小鼠细胞为例，展示了离体基因编辑疗法的概念验证。患病的iPSC由基因敲除小鼠胚胎成纤维细胞产生，这些细胞在Idua基因的外显子VI中携带一个破坏新霉素抗性基因盒（Neo）。使用双向导RNA去除Neo序列，并使用不同长度的供体模板重建外显子VI序列。采用基于定量PCR的筛选方法鉴定Neo的去除情况。通过桑格测序进一步确认了无任何插入缺失突变的序列恢复情况。诱导成纤维细胞分化后，基因校正的iPSC来源的成纤维细胞表现出与野生型iPSC来源的成纤维细胞相当的Idua功能。Idua缺陷细胞能够重编程为iPSC，并且通过CRISPR/CAS9介导的基因校正维持了多能性。这些结果支持了使用iPSC和CRISPR/Cas9技术对MPS-1患者进行离体基因编辑治疗的概念。
