RAPID AND EFFICIENT SELECTION OF HUMAN HEMATOPOIETIC-CELLS EXPRESSINGMURINE HEAT-STABLE ANTIGEN AS AN INDICATOR OF RETROVIRAL-MEDIATED GENE-TRANSFER

Recombinant retroviruses offer many advantages for the genetic modific ation of human hematepoietic cells, although their use in clinical pro tocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapid ly characterized and isolated free of residual nontransduced but biolo gically equivalent cells. To address this need, we constructed a murin e stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lin eage marker-negative (lin(-)) normal human marrow or mobilized periphe ral blood cells preactivated by exposure to interleukin-3 (IL-3), IL-6 , and Steel factor in vitro for 48 hours were then infected by coculti vation with these MSCV-HSA producer cells for a further 48 hours in th e presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 2 7%) of those that were still CD34(+) to have acquired the ability to e xpress HSA, The extent of gene transfer to erythroid and granulopoieti c progenitors (burst-forming unit-erythroid and colony-forming unit-gr anulocyte-macrophage), as assessed by the ability of these cells to fo rm colonies of mature progeny in the presence of normally toxic concen trations of G418, averaged 11% and 12%, respectively, in 6 experiments . These values could be increased to 100% and 77%, respectively, by pr ior isolation of the CD34(+)HSA(+) cell fraction and were correspondin gly decreased to an average of 2% and 5%, respectively, in the CD34(+) HSA(-) cells. In addition, the extent of gene transfer to long-term cu lture-initiating cells (LTC-IC) was assessed by G418 resistance. The a verage gene transfer to LTC-IC-derived colony-forming cells in the uns orted population was less than or equal to 7% in 4 experiments. FACS s election of the initially CD34(+)HSA(+) cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34(+)HSA(- ) cells. Transfer of HSA gene expression to a phenotypically defined m ore primitive subpopulation of CD34(+) cells, ie, those expressing lit tle or no CD38, could also be shown by FACS analysis of infected popul ations 24 hours after infection. These findings underscore the potenti al use of retroviral vectors encoding HSA for the specific identificat ion and nontoxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In add ition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry. (C) 1996 by The A merican Society of Hematology.