CRISPR/Cas9-Mediated in vivo Genetic Correction in a Mouse Model of Hemophilia A

Sanchuan Luo; Zhongxiang Li; Xin Dai; Rui Zhang; Zhibing Liang; Wenzhou Li; Ming Zeng; Jinfeng Su; Jun Wang; Xia Liang; Yong Wu; Desheng Liang

doi:10.3389/fcell.2021.672564

CRISPR/Cas9-Mediated in vivo Genetic Correction in a Mouse Model of Hemophilia A

Front Cell Dev Biol. 2021 Aug 16:9:672564. doi: 10.3389/fcell.2021.672564. eCollection 2021.

Authors

Sanchuan Luo¹, Zhongxiang Li¹, Xin Dai¹, Rui Zhang², Zhibing Liang¹, Wenzhou Li¹, Ming Zeng¹, Jinfeng Su¹, Jun Wang¹, Xia Liang¹, Yong Wu¹, Desheng Liang³

Affiliations

¹ Medical Research Institute, Shenzhen Baoan Women's and Children's Hospital, Jinan University, Shenzhen, China.
² Prenatal Diagnosis Unit, Shenzhen Baoan Women's and Children's Hospital, Jinan University, Shenzhen, China.
³ Hunan Key Laboratory of Medical Genetic, Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China.

Abstract

Hemophilia A (HA), a common bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII), has long been considered an attractive target for gene therapy studies. However, full-length F8 cDNA cannot be packaged efficiently by adeno-associated virus (AAV) vectors. As the second most prevalent mutation causing severe HA, F8 intron 1 inversion (Inv1) is caused by an intrachromosomal recombination, leaving the majority of F8 (exons 2-26) untranscribed. In theory, the truncated gene could be rescued by integrating a promoter and the coding sequence of exon 1. To test this strategy in vivo, we generated an HA mouse model by deleting the promoter region and exon 1 of F8. Donor DNA and CRISPR/SaCas9 were packaged into AAV vectors and injected into HA mice intravenously. After treatment, F8 expression was restored and activated partial thromboplastin time (aPTT) was shortened. We also compared two liver-specific promoters and two types of integrating donor vectors. When an active promoter was used, all of the treated mice survived the tail-clip challenge. This is the first report of an in vivo gene repair strategy with the potential to treat a recurrent mutation in HA patients.

Keywords: CIRSPR/SaCas9; gene editing; gene therapy; hemophilia A; intron 1 inversion.