In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.

Yong Wu,Zhuo Li,Xuyun Hu,Xiaolin Wang,Jialun Pang,Zhiqing Hu,Desheng Liang,Bo Liu,Mai Feng,Siyuan Lin-Peng,Lingqian Wu,Fangping Chen

doi:10.1038/srep18865

Abstract

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23–26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.

Highlights

Hemophilia A (HA) is an X-linked recessive congenital bleeding disorder with an occurrence of 1 in 5,000 male births, and affects nearly 80%–85% of patients with hemophilia[1]
We report here an in situ genetic correction of the Inv[22] mutation in HA patient-specific induced pluripotent stem cells by using the transcription activator-like effector nucleases (TALENs), resulting in a rescue of
Dermal fibroblast is the most common initial cell type used for induced pluripotent stem cells (iPSCs) reprogramming, invasive sampling should be avoided for hemophiliac patients

Summary

Introduction

Hemophilia A (HA) is an X-linked recessive congenital bleeding disorder with an occurrence of 1 in 5,000 male births, and affects nearly 80%–85% of patients with hemophilia[1]. With a lower incidence of 1 in 30,000 male births, hemophilia B (HB) has been favored in gene therapy research and a long-term expression of therapeutic levels of FIX has been achieved by adeno-associated viral (AAV)-based gene therapy[4] Such a promising approach is not applicable for HA at the present stage, mainly owing to the size of the FVIII coding sequence that at 7 kb far exceeds the normal packaging capacity of AAV vectors. We report here an in situ genetic correction of the Inv[22] mutation in HA patient-specific induced pluripotent stem cells (iPSCs) by using the transcription activator-like effector nucleases (TALENs), resulting in a rescue of www.nature.com/scientificreports/ Both F8 transcription and FVIII secretion in the endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs

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