The isolation of cDNA containing the full sequence encoding human glucocerebrosidase has permitted the use of this enzyme in model studies to correct inherited enzyme deficiences using recombinant methodologies, specifically gene transfer. Particularly suited for gene therapy are those disorders (such as Gaucher's disease) where the storage of undegraded substrate is confined to cells having an accessible precursor population. In these cases, the transfer of normal genes to stem cells in bone morrow would be both rational and desirable. Using information derived from protein studies, the products of gene transfer in mutant cells can be compared to that of normal cells, and the likelihood for success of a particular construct and gene integration rationally predicted. Although we have been successful in utilizing retroviral vectors to transfer and express glucocerebrosidase in host mouse and human cell lines, prerequisites for human gene therapy experiments include sustained expression of the transferred gene during subsequent cell generations and the absence of recombination events detrimental to the host. These aspects are being defined. To better characterize the gene transfer and expression mechanisms Type 2 Gaucher cell lines were utilized as recipients of the retroviral constructs. In this model, monoclonal antibody 8E4 does not recognize the Type 2 variant glucocerebrosidase. Thus, these cells provide a host cell line lacking the normal enzyme epitope recognized by 8E4, and they allow the monitoring of the degree of restoration of both enzyme activity and protein epitopes resulting from gene transfer. Following the demonstration of restored glucocerebrosidase levels to these as well as Type 1 and Type 3 cell lines in culture, the transfer of the glucocerebrosidase gene to bone marrow stem cells will be evaluated using mice and non-human primates. The goal of this research is the application of these recombinant DNA therapeutic strategies to Gaucher's disease and other genetic disorders.
