Adenovirus-mediated gene transfer to liver grafts - An improved method to maximize infectivity

Background Adenoviral gene therapy in liver transplantation has many potent ial applications, but current vector delivery methods to grafts lack effici ency and require high titers. In this study, we attempted to improve gene d elivery efficacy using three different delivery methods to liver grafts wit h adenoviral vector encoding the LacZ marker gene (AdLacZ). Methods. AdLacZ was delivered to cold preserved rat liver grafts by: (1) co ntinuous perfusion via the portal vein (portal perfusion), (2) continuous p erfusion 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 1x10(9) plaque-forming units of AdLacZ (multiplicity of infe ction of 0.4), transduction rate in 3-hr preserved liver grafts, determined by 5-bromo-4-chromo-3-indolyl-beta-D-galactopyranoside staining and beta-g alactosidase assay 48 hr after transplantation, was best with clamp techniq ue (21.5+/-2.7% 5-bromo-4-chromo-3-indolyl-beta-D-galactopyranoside- positi ve cells and 81.1+/-3.6 U/g beta-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 near-maximal gene transfer rate of 30% at multiplicity of infection of 0.4 with prolonge d cold ischemia to 18 hr. Transgene expression was stable for 2 weeks and s lowly 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 transgen e expression in the lung and spleen. Conclusions. The clamp technique provides a highly efficient viral gene del ivery method to cold preserved liver grafts. This method offers maximal inf ectivity of adenoviral vector with minimal technical manipulation.