SUSTAINED RETROVIRAL GENE MARKING AND EXPRESSION IN LYMPHOID AND MYELOID CELLS DERIVED FROM TRANSDUCED HEMATOPOIETIC PROGENITOR CELLS

The expression of antiviral genes in human hematopoietic stem or proge nitor cells has been proposed as a strategy for gene therapy of AIDS. To be successful, this strategy requires safe and efficient transfer o f the therapeutic gene into hematopoietic cells and gene expression ha s to be maintained in HIV susceptible cells following differentiation. We have used retroviral vectors to transfer the gene for a transdomin ant inhibitor of HIV replication (RevM10) into CD34(+) stem/progenitor cells isolated from human umbilical cord blood (UCB). Following trans duction, cells were allowed to differentiate either in vitro in clonog enic assays and long-term stromal cell cultures or in human thymus imp lanted in immunodeficient scid/scid mice in vivo (SCID-hu). Following differentiation and expansion, multiple lineages of cells were shown t o carry the transgene. A higher percentage of gene-marked progenitor c ells (10-30% in most cases) were detected in methylcellulose colony as says and in long-term stromal cell cultures (1-5%). In contrast, gene- marked T cells derived from transduced CD34(+) cells in a SCID-hu mode l were detected at an even lower frequency (0.01-1%). RevM10 RNA expre ssion was detected in CD34(+) cells immediately after transduction and was maintained after in vitro differentiation of those cells into CD1 4(+) myeloid cells. In T cells, the RevM10-specific RNA was detectable by RT-PCR and also by semiquantitative RNase protection. These findin gs demonstrate that LTR-driven gene expression is sustained in relevan t cells derived from retrovirus-transduced hematopoietic progenitor ce lls after extensive differentiation in vitro and in vivo and suggest t hat stringent in vivo, rather than in vitro assays, may be a better pr eclinical system to improve gene marking and expression in hematopoiet ic cells.