RETROVIRUS-MEDIATED GENE-TRANSFER INTO RHESUS-MONKEY HEMATOPOIETIC STEM-CELLS - THE EFFECT OF VIRAL TITERS ON TRANSDUCTION EFFICIENCY

We have generated a cell line, designated POAM-P1, shedding amphotropi c recombinant retroviruses carrying the human adenosine deaminase (hAD A) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cel ls and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduct ion efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultiva tion of rhesus monkey bone marrow with POAM-P1 cells, successful trans duction could be demonstrated in approximately 10% of myeloid progenit or colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PB MC) and granulocytes in vivo until > 1 year after autologous transplan tation. In addition, the presence of functional hADA enzyme was detect ed in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for >2 years a fter transplantation. Despite the higher retrovirus titer of POAM-P1 c ells as compared to POC-1 cells, no difference was observed in gene tr ansfer efficiency into CFU-C and long-term repopulating stem cells. Th is shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic ste m cells.