Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in coagulation factor VIII (FVIII). Therapies aimed at even modest increases in clotting factor levels are associated with substantial improvement of the severe disease phenotype. The current treatment for HA is protein replacement therapy, however, the therapeutic landscape is rapidly changing for this disorder. Gene therapy approaches for adeno-associated viral (AAV) vector delivery of FVIII are in clinical trials. Interestingly, these clinical studies target expression of FVIII to hepatocytes using hepatocyte specific promoter elements which is based on the assumption that FVIII is synthesized in hepatocytes. However, the primary site of FVIII synthesis was identified as the liver sinusoidal endothelial cell (LSEC). While preclinical and clinical studies have demonstrated that hepatocytes can synthesize functional FVIII, it has been challenging to produce high levels of FVIII after hepatocyte targeted expression. In the ongoing AAV clinical studies for HA, there have been several unexpected observations. The first successful AAV-hFVIII trial with the most patients (>13 subjects) and the longest follow-up (>3 years, ongoing) observed a significant decrease in FVIII expression. All of the available clinical data from three trials to date shows a lack of a vector dose response with significant variability among subjects. Importantly, these findings were not observed in the AAV-FIX clinical studies suggesting that there is additional complexity to the delivery and expression of FVIII. These unanticipated findings in the clinical studies may be related to the site of FVIII synthesis. Thus, while significant hurdles have been overcome in gene therapy for HA, unexplored opportunities for improved hemophilia patient outcomes remain. The goal of this proposal is to target FVIII expression to LSECs using AAV vectors to study if there are biological differences in expressing FVIII in LSECs and hepatocytes and to study the efficacy of AAV-FVIII delivery to LSECs. FVIII expression will be targeted to LSECs using novel promoter elements (Specific Aim 1), novel AAV capsids that more specifically target AAV to these cells will be identified (Specific Aim 2) and the biological differences between hepatocyte and LSEC derived FVIII expression after AAV delivery will be investigated (Specific Aim 3). Together, these studies will provide the basis for understanding the biology and efficacy of LSEC targeted AAV-FVIII expression to support the development of this therapeutic approach for hemophilia A.

Public Health Relevance

Gene therapy for hemophilia is a strategy for providing continuous levels of clotting factor in the circulation to prevent bleeding episodes. The current approaches target the expression of the defective protein (factor VIII) to hepatocytes in the liver for gene therapy for hemophilia A but the primary natural site of factor VIII synthesis is in endothelial cells in the liver. The goal of this project is to determine the biological and therapeutic differences between endothelial cell and hepatocyte-derived factor VIII after gene therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Therapeutic Approaches to Genetic Diseases Study Section (TAG)
Program Officer
Kindzelski, Andrei L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Children's Hospital of Philadelphia
United States
Zip Code