Endothelial cell targeting of factor VIII for hemophilia A

ABSTRACT Hemophilia A is an X-linked bleeding disorder caused by a deficiency in clotting factor VIII (FVIII). Current adeno- associated viral (AAV) vector mediated gene therapy approaches for hemophilia A are gene addition approaches that target the liver using promoter elements that result in expression in hepatocytes. However, liver sinusoidal endothelial cells (LSEC) are the natural site of FVIII synthesis, whereas other coagulation factors are expressed in hepatocytes. While AAV delivery of factor IX to hepatocytes for hemophilia B has resulted in stable FIX levels for >10 years, there have been unexpected observations in hemophilia A studies including a lack of a dose response with variability among patients and a significant decline in FVIII expression in the in first three years after AAV delivery. We hypothesize that 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 hemophilia A, there remain unexplored opportunities to improve hemophilia patient outcomes. The goal of this proposal is to target FVIII expression to LSECs and to define the biological consequences of targeting FVIII expression to LSECs compared to hepatocytes. An LSEC targeted genome editing approach (Specific Aim 1) will be compared to an LSEC targeted AAV gene addition approach (Specific Aim 2) using novel tools that we have developed— an engineered AAV capsid with improved LSEC targeting and a lipid nanoparticle conjugated to PECAM-1 that targets endothelial cells. Targeting expression to LSECs or hepatocytes using an AAV approach compared to a genome editing strategy will provide the opportunity to define of the biological differences of expressing FVIII in LSECs compared to hepatocytes (Specific Aim 2). The study of whether chronic liver disease or acute liver injury may affect FVIII expression and different cell types after gene delivery using a genome editing or gene addition approach will also be investigated (Specific Aim 3). Together, these studies will provide the basis for understanding the biology and efficacy of LSEC targeted FVIII expression to support the development of approaches to target FVIII to its endogenous site to overcome the challenges of hemophilia A gene therapy.