FREQUENCY-ANALYSIS OF MULTIDRUG RESISTANCE-1 GENE-TRANSFER INTO HUMANPRIMITIVE HEMATOPOIETIC PROGENITOR CELLS USING THE COBBLESTONE AREA-FORMING CELL ASSAY AND DETECTION OF VECTOR-MEDIATED P-GLYCOPROTEIN EXPRESSION BY RHODAMIME-123

Transfer of the multidrug resistance-1 (MDR1) gene into hematopoietic progenitor cells may reduce myelotoxicity of MDR1-related cytotoxic ag ents and therefore allow dose intensification. Mobilized peripheral bl ood progenitor cells (PBPC) can be obtained in ample quantity and are a suitable target cell population, CD34-selected PBPC samples (n = 6) were transduced with cell-free supernatant (SNT) of a cell line produc ing recombinant retrovirus containing the human MDR1 gene, Limiting-di lution long-term cultures were employed that allow continuous monitori ng of stroma-adherent cobblestone areas (CA) and comparison of their f requency in a 5-log range over time, MDR1 provirus integration in CA-c ontaining wells followed single-hit kinetics, According to Poisson sta tistics, proviral DNA was contained in 22% of unselected cobblestone a rea-forming cells (CAFC) at week 6, which represent primitive hematopo ietic precursors, in comparison, 1.0 +/- 0.44% (mean +/- SEM) of week- 6 CAFC were expressing P-glycoprotein at sufficient levels to convey v incristine resistance, suggesting low expression of the retroviral vec tor or splicing of the vector-drived mRNA in hematopoietic progenitor cells, Next we analyzed lineage-committed progenitors, The proviral DN A was detectable in 20-66% of colony-forming units granulocyte-macroph age (CFU-GM) while corresponding percentages (25-52%) of CD34(+) PBPC were in the S/G(2)M phase of the cell cycle at the end of the transduc tion period, The proportion of vincristine-resistant CFU-GM was simila r to the CAFC data and no significant differences were found between v arious MDR1-SNT transduction schedules whereas MDR1 co-cultivation, wh ich served as a positive control, yielded significantly higher proport ions of resistant colonies (5.3 +/- 1.4%, IL-3, 96 hr, p less than or equal to 0.05), Assessment of rhodamine-123 (Rh-123) efflux in the mye lo-monocytic progeny of MDR1-transduced cells mirrored the colony assa y results in the SNT and co-cultivation groups, Less culture effort wa s required in the Rh-123 assay and functional characterization of the transferred P-glycoprotein was possible using cyclosporin A, Further d evelopment toward an effective MDR1 gene therapy should be facilitated by the CAFC assay, which allows estimation of the retroviral gene tra nsfer frequency into primitive hematopoietic cells, and by the Rh-123 assay, which permits tractable side-by-side assessments of numerous MD R1 transduction protocols or different MDR1-SNT lots.