Int J Hematol. 2020 Feb;111(2):225-233. doi: 10.1007/s12185-019-02765-0. Epub 2019 Oct 29.

CRISPR/Cas9-mediated Gene Correction in Hemophilia B Patient-Derived iPSCs

Satoshi Morishige ¹, Shinichi Mizuno ^1 2, Hidetoshi Ozawa ¹, Takayuki Nakamura ¹, Ahmad Mazahery ³, Kei Nomura ¹, Ritsuko Seki ¹, Fumihiko Mouri ¹, Koichi Osaki ¹, Kenichi Yamamura ³, Takashi Okamura ^1 4, Koji Nagafuji ⁵

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 patient-derived 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.