BackgroundHemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities.MethodsTo investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters.ResultsTo examine tissue specificity, the expression of a B-domain deleted humanF8(F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDDand LV-ITGA-F8BDDin the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. InF8knockout mice (F8KO mice,F8nullmice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDDand LV-Gp-F8BDDachieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDDdisplayed a low FVIII inhibitory response in the treatedF8nullmice.ConclusionsThe LV-VEC-F8BDDexhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in theF8nullmice, thus has a great potential for clinical applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-023-00680-z.