Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A.

Junya Zhao,Miaojin Zhou,Zujia Wang,Lingqian Wu,Desheng Liang,Zhiqing Hu

doi:10.3390/ijms23020623

Abstract

Hemophilia A (HA) is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). Gene therapy is a hopeful cure for HA; however, FVIII inhibitors formation hinders its clinical application. Given that platelets promote coagulation via locally releasing α-granule, FVIII ectopically expressed in platelets has been attempted, with promising results for HA treatment. The B-domain-deleted F8 (BDDF8), driven by a truncated ITGA2B promoter, was targeted at the ribosomal DNA (rDNA) locus of HA patient-specific induced pluripotent stem cells (HA-iPSCs). The F8-modified, human induced pluripotent stem cells (2bF8-iPSCs) were differentiated into induced hematopoietic progenitor cells (iHPCs), induced megakaryocytes (iMKs), and mesenchymal stem cells (iMSCs), and the FVIII expression was detected. The ITGA2B promoter-driven BDDF8 was site-specifically integrated into the rDNA locus of HA-iPSCs. The 2bF8-iPSCs were efficiently differentiated into 2bF8-iHPCs, 2bF8-iMKs, and 2bF8-iMSCs. FVIII was 10.31 ng/106 cells in lysates of 2bF8-iHPCs, compared to 1.56 ng/106 cells in HA-iHPCs, and FVIII was 3.64 ng/106 cells in 2bF8-iMSCs lysates, while 1.31 ng/106 cells in iMSCs with CMV-driven BDDF8. Our results demonstrated a high expression of FVIII in iHPCs and iMSCs derived from hiPSCs with site-specific integration of ITGA2B promoter-driven BDDF8, indicating potential clinical prospects of this platelet-targeted strategy for HA gene therapy.

Highlights

Hemophilia A (HA) is an X-linked recessive genetic disorder that affects approximately 1 in 5000 newborn boys
Between the 5 long homologous arm (935 bp) and the 3 short homologous arm (591 bp), the 889 bp megakaryocyte-specific promoter of ITGA2B gene driving the B-domaindeleted F8 (BDDF8) and a promoterless neomycin resistance (Neo) cassette flanked by locus of X-overinP1 (LoxP) sites were contained (Figure 1A)
The Neo cassette contained an encephalomyocarditis virus internal ribosomal entry site (EMCV-IRES), which enabled Neo gene expression under the control of the endogenous RNA polymerase I (Pol I) promoter after homologous recombination, so that the gene-targeted human iPSCs (hiPSCs) could be enriched by this promoter trapping

Summary

Introduction

Hemophilia A (HA) is an X-linked recessive genetic disorder that affects approximately 1 in 5000 newborn boys. There is no cure, and the main clinical treatment is replacement therapy with intravenous injections of plasma-derived or recombinant coagulation factor VIII (FVIII) protein. As the half-life of FVIII in the plasma is only 12 h [2], patients require repeated infusions, which makes this treatment very costly and brings a heavy economic and psychological burden to them and their families. The novel EHL-factors and the non-factor therapies are reducing the burden of the life-long treatment without impacting the long-term therapeutic solution of gene therapy in hemophilia [3]. A major complication is the formation of FVIII inhibitor, which affects approximately 25% to 30% of HA patients, decreasing its coagulation efficacy [4,5]

Methods

Results

Conclusion