Adenovirus-mediated Gene Transfer to Liver Grafts

Background. Adenoviral gene therapy in liver transplantation has many potential applications, but current vector delivery methods to grafts lack efficiency and require high titers. In this study, we attempted to improve gene delivery efficacy using three different delivery methods to liver grafts with adenoviral vector encoding the LacZ marker gene (AdLacZ). Methods. AdLacZ was delivered to cold preserved rat liver grafts by: (1) continuous perfusion via the portal vein (portal perfusion), (2) continuous perfusion via both the portal vein and hepatic artery (dual perfusion), and (3) trapping viral perfusate in the liver vasculature by clamping outflow (clamp technique). Results. Using lxl09 plaque-forming units of AdLacZ (multiplicity of infection of 0.4), transduction rate in g-hr preserved liver grafts, determined by 5-bromo-4-chromo-g-indolyl-f1-n-galactopyranoside staining and f1-galactosidase assay 48 hr after transplantation, was best with clamp technique (21.5±2.7% 5-bromo-4-chromo-3-indolyl-f1-n-galactopyranosidepositive cells and 81.1±3.6 Ulg f1-galactosidase), followed by dual perfusion (18.5±1.8%, 66.6±19.4 U/g) and portal perfusion (8.8±2.5%, 19.7±15.4 U/g). Further studies using clamp technique demonstrated a nearmaximal gene transfer rate of 30% at multiplicity of infection of 0.4 with prolonged cold ischemia to 18 hr. Transgene expression was stable for 2 weeks and slowly declined to 7.8±12.1 % at day 28. Lack of inflammatory response was confirmed by histopathological examination and liver enzymes. Transduction was selectively induced in hepatocytes with nearly no extrahepatic transgene expression in the lung and spleen. Conclusions. The clamp technique provides a highly efficient viral gene delivery method to cold preserved liver grafts. This method offers maximal infectivity of adenoviral vector with minimal technical manipulation.