Intravenous administration of adenovirus serotype 5 (Ad5) during gene therapy trials results in liver tropism and hepatotoxicity, limiting Ad5 applications in gene therapy. Previous research has shown that coagulation factor X (FX) acts as a bridge between the Ad5 virion and hepatocytes causing this liver tropism. The GLA domain of FX binds to the hexon on the virion surface, while the serine protease (SP) domain of FX binds to heparin sulfate proteoglycans (HSPG) on the surface of hepatocyte cells. For FX to bind the Ad5 hexon proper γ-carboxylation of the GLA domain is essential as it allows for the correct folding of the GLA domain in the presence of Ca²⁺. However, ablating the FX interaction with Ad5 results in the Ad5 virion being cleared by the immune system.

My project was to design a novel FX variant to retarget the Ad5 virion towards other cell types while maintaining immune protection by covering the Ad5 hexon. Prostate cancer cells were chosen as the target model for retargeting as my co-supervisor had experience working with prostate cancer models. A Flpin-293 cell line was validated to show it could gamma-carboxylate my FX constructs. Surface plasmon resonance (SPR) was used to compare FX truncated and point mutation constructs to identify regions of interest where point mutations could be introduced into FX to reduce Ad5 hexon dissociation. The 51MKKGH55 region of the GLA domain was discovered to be an area where point mutations could be introduced. In addition, loss of the FX SP domain did not significantly impact FX binding to the Ad5 hexon. In conclusion, based on my results and publicly available FX-Ad5 complex and GLA domain structural data, several possible point mutations were recommended for improving FX/Ad5 hexon binding as well as a suitable retargeting domain to target prostate cancer cells.