AAV8-TBG-GFP

Hemophilia A is an inherited bleeding disorder caused by defective or insufficient activity of coagulation factor VIII (FVIII). Adeno-associated vector (AAV) gene therapy has shown some efficacy in patients with hemophilia A. However, limitations remain due to AAV-induced cellular stress, immunogenicity, and reduced persistence of gene expression. Here, researchers examined the efficacy of liver-directed gene transfer of AAV8-GFP in FVIII knockout mice. Surprisingly, FVIII knockout (F8^TKO) mice showed significantly delayed AAV8-GFP transfer in the liver compared to control mice. The delay in liver-directed gene transfer in F8^TKO mice was found to be associated with the loss of liver sinusoidal endothelial cell (LSEC) fenestrations, which resulted in aberrant expression of several sinusoidal endothelial proteins, increased capillarization, and decreased LSEC permeability.

To examine the efficacy, stability, and safety of AAV-mediated liver-directed gene transfer in F8^TKO mice, AAV8–TBG-GFP was injected intraperitoneally, and GFP expression in the liver was analyzed at 15 and 30 days after injection (Figure 1A-B). Previous studies have shown that AAV8–TBG-GFP can successfully transfer to the liver within 10 to 12 days. Surprisingly, GFP immunofluorescence at 15 days after injection (Figure 1C) showed a significant decrease in GFP+ cells in F8^TKO livers compared with control mice. Only the large hepatic veins contained GFP+ cells in F8^TKO livers (Figure 1C). Whole liver enzyme-linked immunosorbent assay (ELISA) also showed that GFP expression in F8^TKO livers was significantly lower compared with control mice (Figure 1D). Similar to the 15-day time point, fewer GFP+ cells were found in F8^TKO livers at 30 days after injection (Figure 1E). However, at day 30, the overall percentage of GFP+ cells was higher than at day 15 (Figure 1E-F), but still significantly lower compared to control mice. Notably, there was GFP staining in the large hepatic veins of F8^TKO mice, which was absent in control livers at 30 days post-injection (Figure 1E). Finally, the reduction in GFP expression was confirmed using western blot analysis, which showed that GFP protein expression was consistently lower in F8^TKO livers compared to controls at both 15 and 30 days post-injection (Figure 1B). Consistent with IP injections, IV injections, even at lower doses, resulted in a similar delay in GFP transduction in F8^TKO animals at 8 weeks, as visualized by immunofluorescence (Figure 1G), ELISA (Figure 1H), and western blot analysis.

Figure 1. AAV8-driven liver-directed gene transfer is significantly delayed in hemophilia A mice. (Kaminski T W, et al., 2022)