In mammalian cells, gene transfer is accomplished traditionally by DNA-mediated transfection, a general procedure for introduction of foreign DNA either singly or in combination with a dominant selectable marker. Taking advantage of this, we are studying the transfer of cDNA for human glucocerebrosidase to mouse cells via bovine papilloma virus (BPV) vector and the synthesis, in culture, of large amounts of the enzyme in biologically active form. Using this recombinant product, we are addressing the molecular and cell biological issues related to biosynthesis, compartmentalization, processing and function of lysosomal glucocerebrosidase. These studies complement work on perfecting enzyme replacement techniques. In addition, a major goal of our laboratory is to develop novel therapeutic strategies for inborn errors of metabolism. Of the approaches that have been designed in recent years for this purpose, the most promising one appears to be to infect pluripotent stem cells with chimeric retroviruses carrying a correct copy of the defective gene. Using retroviral shuttle vectors of the pZip neo family in conjunction with the specialized host cell lines, Viz., psi2 and psiAM, that produce helper-free recombinant retroviruses, we have generated high-titer stocks of transmissible chimeric retroviruses carrying a cDNA copy of the human glucocerebrosidase gene. The mouse cell lines as well as the human fibroblasts infected with these replication-defective virus stocks revealed appropriately integrated copies of the proviral DNA and produced antigenically active human glucocere- brosidase protein. We are currently focussing on the manipulation of the insert DNA adduct in the retroviral constructs to obtain enzymatically active protein. The results obtained in this project clearly demonstrate the feasibility of retroviral vector system for human gene transfer and somatic cell gene therapy.
