PB0166 - Liver directed gene therapy in hemophilia A mice is dependent on Notch pathway driven endothelial development

Background: Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. The Factor VIII null mouse has been effective in the development of liver directed novel gene therapy techniques. FVIII is predominantly produced in the liver sinusoidal endothelial cells (LSECs). Thus, liver directed adenoviral have been studied by several groups to understand the gene delivery method in hemophilia.

Aims: In this study we evaluated the efficacy of liver driven gene-transfer mechanism in F8TKO mouse using recombinant-AAV8 vector.

Methods: AAV8–TBG-GFP was administered, and GFP expression was analyzed in the liver 15- and 30-days post injection.

Results: Immunofluorescence of GFP at day 15 and day 30 post-injection revealed significantly fewer GFP+ cells in the liver of F8TKO compared with control mice. Moreover, Caspase-3 staining revealed significant enrichment (p=0.05) in the liver of F8TKO mice compared with control mice after AAV8-GFP administration suggestive of increased LSEC apoptosis. To further confirm apoptosis of LSECs, we next used intravital imaging to visualize LSECs in F8TKO mice at baseline and upon AAV8-GFP administration. TXR-dextran and AF488–anti-CD31 antibody were administered to visualize hepatic blood flow and LSECs, respectively. At baseline, both control and F8TKO mice showed normal blood flow and enriched expression of AF488–anti-CD31. However, there were fewer AF488–anti-CD31+endothelial cells in F8TKO than control mice at day 15 post–AAV8-GFP treatment, suggestive of apoptosis of LSECs in F8TKO mice post–AAV8-GFP administration. Scanning electron microscopy revealed dramatic reduction in endothelial fenestration in the liver of F8TKO as compared with control mouse liver. Mechanistically we show significant upregulation of Notch pathway components by RT-PCR IHC and western in the liver of F8TKO mice.

Conclusion(s): In conclusion, we demonstrate molecular characterization of LSEC-mediated liver-directed gene transfer in a null hemophilia A mouse, which is critical for understanding the efficacy of AAV-based hemophilia treatment.