PROJECT SUMMARY Hemophilia A (HemA) results from a deficiency of factor VIII (FVIII) gene (abbreviated as F8 in constructs). Traditional treatment of HemA patients is repeated infusions of FVIII, which is costly, inconvenient, short-term, and incompletely effective. In addition, approximately 25% of treated patients develop anti-FVIII immune responses. Development of inhibitory antibodies (inhibitors) significantly increases morbidity and lowers life quality for these patients. Treatment for hemophilia inhibitor patients includes use of bypassing agents or immune tolerance induction (ITI) with high doses of FVIII or new therapies with bioengineered factor Xa, a humanized bispecific antibody (emicizumab; ACE910), and others. However, frequent infusions of costly reagent are required, and potential long-term side effects still need to be evaluated. Compared to drug or protein therapy, gene therapy can achieve a prolonged therapeutic effect with only one or several treatments over the lifetime. AAV-mediated gene therapy showed very promising results in clinical trials however is not accessible to a significant portion of patients including pediatric patients and patients with anti-AAV or anti-FVIII antibodies. Recently ex vivo HSC gene therapy targeting FVIII expression in megakaryocytes (Megs) corrected the bleeding diathesis even in the presence of inhibitors. This is because FVIII is stored in α-granules and only released at the injury sites during platelet activation, therefore protected from circulating inhibitors. However, several limitations exist for ex vivo gene therapy. Our approach to direct long-term expression of FVIII in platelets is intraosseous (IO) infusion of lentiviral vectors (LVs) (IO-LV gene therapy) carrying a FVIII transgene driven by a Meg-specific promoter GpIbα (G-F8-LVs). In this in vivo gene therapy protocol, hematopoietic stem cells (HSCs) were efficiently transduced by LVs in situ, resulting in FVIII expression in Megs and storage in platelet α-granules. A single IO infusion of G-F8-LV leads to phenotype correction in HemA mice. In vivo delivery of LVs can avoid many difficulties and potential toxicities encountered by ex vivo gene therapy including low engraftment potential and pre-conditioning of the subject which induces thrombocytopenia, a particularly undesirable complication for hemophilia patients. In vivo gene therapy can bypass this significant hemostatic risk. Previously we have demonstrated very promising results to correct hemophilia phenotype following IO-LV gene therapy in HemA mice both with and without pre-existing inhibitors. In order to develop a clinically feasible protocol for human applications, we propose to enhance the efficacy and safety of this strategy in HemA mice. Furthermore, we will examine the efficacy and safety in human cells using novel humanized NSGW41 and HemA/NSG/VWFRH/RH transgenic mice. Lastly, we have initiated the study of IO-LV gene therapy in HemA dogs. We will further improve...