HEMATOPOIETIC STEM CELL-BASED GENE-THERAPY FOR ACQUIRED-IMMUNODEFICIENCY-SYNDROME - EFFICIENT TRANSDUCTION AND EXPRESSION OF REVM10 IN MYELOID CELLS IN-VIVO AND IN-VITRO

Gene delivery via the hematopoietic stem cell (HSC) offers an attracti ve means to introduce antiviral genes into both T cells and macrophage s for acquired immunodeficiency syndrome (AIDS) gene therapy, An ampho tropic retroviral vector encoding a bicistronic gene coexpressing RevM 10 and the murine CD8 alpha' chain (lyt2) was developed to transduce H SC/progenitor cells. After transduction of CD34(+) cells isolated from human umbilical cord blood, the lyt2 molecule detected by flow cytome try was used to monitor the level of gene transduction and expression and to enrich RevM10 expressing cells by cell sorting without drug sel ection. Using this quantitative method, high levels of gene transducti on and expression (around 20%) were achieved by high-speed centrifugat ion of CD34(+) cells with the retroviral supernatant (spinoculation), After reconstitution of human bone marrow implanted in SCID mice (SCID -hu bone) with the transduced HSC/progenitor cells, a significant numb er of donor-derived CD14(+) bone marrow cells were found to express th e RevM10/lyt2 gene. Finally, replication of a macrophage-tropic human immunodeficiency virus-type 1 (HIV-1) isolate was greatly inhibited in the lyt2(+)/CD14(+) cells differentiated from transduced CD34(+) cell s after the enrichment of lyt2(+) population. Thus, the RevM10 gene di d not appear to inhibit the differentiation of HSC/progneitor cells in to monocytes/ macrophages. The level of retrovirus-mediated RevM10 exp ression in monocytes/macrophages derived from transduced HSCs is suffi cient to suppress HIV-1 replication. (C) 1997 by The American Society of Hematology.