GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice. Synopsis Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns. Development of an in vitro phagocytosis assay, exploiting fluorescent virions and advanced imaging techniques, suitable to assess the susceptibility to phagocytosis of different LV versions in different experimental settings. Efficient tropism switch of LV from hepatocytes to liver endothelial cells in vitro and in vivo and shielding from phagocytosis. Reconstitution of FVIII activity in the normal range by a single intravenous administration of GP64-LV equipped with the endogenous endothelial-specific FVIII promoter. Proof-of-concept of stable long-term gene therapy for hemophilia A in both adult and newborn mice. Lentiviral vectors bearing the baculovirus GP64 surface protein and high content of the phagocytosis inhibitor CD47 efficiently transduce liver endothelial cells in vivo and allow stable long-term correction of mice with hemophilia A, both when treated as adults and as newborns.