Key Points P-selectin–deficient SCD mice are protected from lung vaso-occlusion. P-selectin–deficient SCD mice will be useful in assessing the benefits of anti–P-selectin therapy in diverse complications of SCD.

Chronic liver disease is one of the leading causes of death in the United States. Coagulopathy is often a sequela of chronic liver disease, however, the role and regulation of coagulation components in chronic liver injury remain poorly understood. Clinical and experimental evidence indicate that misexpression of the procoagulant factor VIII (FVIII) is associated with chronic liver disease. Nevertheless, the molecular mechanism of FVIII-induced chronic liver injury progression remains unknown. This review provides evidence supporting a pathologic role for FVIII in the development of chronic liver disease using both experimental and clinical models.

Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor-VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved intravenous administration of FVIII. Gene therapy with adeno associated vectors (AAV) has shown some efficacy in hemophilia A patients. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock out mice by AAV8-GFP. Surprisingly, compared to control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

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Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which carries the danger of transmitting blood-borne diseases. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategy for treating hemophilia has emerged. In patients with severe hemophilia B, intravenous infusion of an adeno-associated viral (AAV) vector encoding factor IX (FIX) under the control of a liver-directed promoter resulted in expression of FIX for a considerable period of time. In hemophilia-A patients, gene treatment utilizing AAV vectors has demonstrated to be less effective than Hemophilia B due to the size of the F8 coding sequence and the decreased release of FVIII protein. Among other concerns high immunogenicity of FVIII with 25-30% of hemophilia A patients forming inhibitors and overexpression of FVIII in hepatocytes triggering a cellular stress response are significantly challenging. A phase 1 clinical trial is now being conducted to examine the AAV8 induced liver directed gene expression strategy to circumvent these challenges. The Factor VIII null mouse has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver. Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. A few of these studies have shown limited effectiveness in hemophilia animal models. Although the coagulation anomaly seen in hemophilia murine model was completely repaired immediately after liver directed adenovirus-mediated treatment, the effect was transient. Additionally, adeno associated virus (AAV8)-FVIII overexpression has been associated with increased cellular stress. In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in FVIII null mouse using recombinant AAV8 vector. Recombinant AAV8 vector delivered through the systemic circulation successfully transduces to target tissues via passing through the permeable barrier of sinusoidal endothelial cell. The bidirectional passage through sinusoidal endothelial cell is mainly supported by the presence of discontinuous fenestrated endothelium. Remarkably, we found that liver directed gene transfer was significantly delayed in FVIII null mice. Using quantitative liver intravital imaging we found that upon AAV8-GFP administration liver sinusoidal endothelial cells shows increased apoptosis. Moreover, structural analysis of the liver sinusoidal endothelial cells using intravital and electron micrograph imaging showed significant structural functional difference in liver sinusoidal endothelial cells of FVIII KO mouse. Work is currently underway to understand how absence of FVIII can affect the LSECs. In conclusion, detailed molecular characterization of LSEC-mediated liver directed gene transfer in a hemophilia mouse model is critical for understanding the efficacy and stability of gene-based hemophilia treatment. Disclosures Sundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.