SIMULTANEOUS UP-REGULATION OF VIRAL RECEPTOR EXPRESSION AND DNA-SYNTHESIS IS REQUIRED FOR INCREASING EFFICIENCY OF RETROVIRAL HEPATIC GENE-TRANSFER

To understand the relative contribution of viral receptor expression a nd cell proliferation in retroviral gene transfer, we created human he patocyte-derived HuH-7.MCAT-1 cell Lines. These cells constitutively e xpress the murine ecotropic retroviral receptor MCAT-1 without changes in morphology or proliferation states. The MCAT-1 receptor is also a cationic amino acid transporter, and the HuH-7.MCAT-1.7 cells showed i ncreased V-max of uptake and steady-state accumulation of the cationic amino acids L-arginine and L-lysine. In HuH-7.MCAT-1 cells, L-arginin e uptake was significantly upregulated by norepinephrine and dexametha sone, and hepatocyte growth factor also increased L-arginine uptake al ong with cellular DNA synthesis. Gene transfer was also markedly incre ased in HuH-7.MCAT-1.7 cells incubated with an ecotropic LacZ retrovir us, and this further increased with hormones and hepatocyte growth fac tor. To define whether viral receptor up-regulation by itself increase d gene transfer, cell cycling was inhibited by a recombinant adenoviru s expressing the Mad transcription factor (AdMad), which is a dominant -negative c-Myc regulator. This restricted cells in G(0)/G(1), without attenuating MCAT-1 activity, as shown by flow cytometry and L-arginin e uptake analysis, respectively. When asynchronously cycling HuH-7.MCA T-1.7 cells were first infected with the AdMad virus and then exposed to the ecotropic LacZ virus, gene transfer was virtually abolished. Th e data indicate that while upregulation of viral receptors can greatly enhance retrovirally mediated gene transfer, DNA synthesis remains an absolute requirement for hepatic gene therapy with this approach.