CRISPR/Cas9-mediated gene correction in hemophilia B patient-derived iPSCs
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ORIGINAL ARTICLE
CRISPR/Cas9‑mediated gene correction in hemophilia B patient‑derived iPSCs Satoshi Morishige1 · Shinichi Mizuno1,2 · Hidetoshi Ozawa1 · Takayuki Nakamura1 · Ahmad Mazahery3 · Kei Nomura1 · Ritsuko Seki1 · Fumihiko Mouri1 · Koichi Osaki1 · Kenichi Yamamura3 · Takashi Okamura1,4 · Koji Nagafuji1 Received: 24 July 2019 / Revised: 16 October 2019 / Accepted: 17 October 2019 © Japanese Society of Hematology 2019
Abstract The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is an efficient genome-editing tool that holds potential for gene therapy. Here, we report an application of this system for gene repair in hemophilia B (HB) using induced pluripotent stem cells (iPSCs). We prepared targeting plasmids with homology arms containing corrected sequences to repair an in-frame deletion in exon 2 of the factor IX (F9) gene and transfected patientderived iPSCs with the Cas9 nuclease and a guide RNA expression vector. To validate the expression of corrected F9, we attempted to induce the differentiation of iPSCs toward hepatocyte-like cells (HLCs) in vitro. We successfully repaired a disease-causing mutation in HB in patient-derived iPSCs. The transcription product of corrected F9 was confirmed in HLCs differentiated from gene-corrected iPSCs. Although further research should be undertaken to obtain completely functional hepatocytes with secretion of coagulation factor IX, our study provides a proof-of-principle for HB gene therapy using the CRISPR/Cas9 system. Keywords Clustered regularly interspaced short palindromic repeats · CRISPR-associated proteins · Gene editing · Hemophilia B · Induced pluripotent stem cells
Introduction Hemophilia B (HB) is an inherited, X-linked recessive bleeding disorder, with an incidence of 1 in 30,000 male births. HB occurs due to a deficiency in or absence of the Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12185-019-02765-0) contains supplementary material, which is available to authorized users. * Koji Nagafuji [email protected]‑u.ac.jp 1
Division of Hematology and Oncology, Department of Medicine, Kurume University School of Medicine, 67 Asahi‑machi, Kurume 830‑0011, Japan
2
Center for Advanced Medical Innovation, Kyushu University, 3‑1‑1 Maidashi, Higashi‑Ku, Fukuoka 812‑8582, Japan
3
Institute of Resource Development and Analysis, Kumamoto University, 2‑2‑1 Honjo, Chuo‑ku, Kumamoto 860‑0811, Japan
4
Center for Hematology and Oncology, St. Mary’s Hospital, 422 Tsubuku‑Honmachi, Kurume 830‑8543, Japan
functional coagulation factor IX (FIX), caused by mutations in the factor IX (F9) gene located on chromosome Xq27.1. About 80% of severe HB is caused by missense, nonsense, or frameshift mutations in F9 [1]. HB is well suited for gene therapy and viral vectors such as adeno-associated viral (AAV) vector have been reported by several groups [2–5]. However, there have been some potential limitations in its use for replacing current therapies. In pa
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