1. Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A.
- Author
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Zhao J, Zhou M, Wang Z, Wu L, Hu Z, and Liang D
- Subjects
- Base Sequence, DNA, Ribosomal genetics, Factor VIII chemistry, Factor VIII metabolism, Gene Targeting, Genetic Loci, Genetic Vectors metabolism, Humans, Integrin alpha2 metabolism, Megakaryocytes metabolism, Protein Domains, Sequence Deletion, Transcription Activator-Like Effector Nucleases metabolism, Ectopic Gene Expression, Factor VIII genetics, Hematopoietic Stem Cells metabolism, Hemophilia A genetics, Induced Pluripotent Stem Cells metabolism, Integrin alpha2 genetics, Mesenchymal Stem Cells metabolism, Promoter Regions, Genetic
- 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/10
6 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.- Published
- 2022
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