HUMAN GENE THERAPY 8:2079-2086 (November 20, 1997) Mary Ann Liebert, Inc. Efficient Gene Transfer in Primitive CD34+/CD38i° Human Bone Marrow Cells Reselected after Long-Term Exposure to GALV-Pseudotyped Retroviral Vector HANNO GLIMM, HANS-PETER KIEM,' BORIS DAROVSKY,^ RAINER STORE,' JtJRGEN WOLF,^ VOLKER DIEHL,2 ROLAND MERTELSMANN, and CHRISTOF VON KALLE ABSTRACT Successful retroviral gene transfer into human hematopoietic stem cells was demonstrated in preliminary clin- ical trials at low efficiency. We have shown previously that gene transfer into committed hematopoietic prog- enitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retroviral vector. Here, we have conducted a systematic study of human hematopoietic progenitor cells after extended transduction with a GALV-pseudotyped retroviral vector. CD34+/CD38''' Cells were transduced for 5 days and reselected according to phenotype after culture and analyzed for cell cycle status, long-term culture-initiating cell (LTC-IC) activity, and gene transfer. Reselection of rare, very primi- tive progenitor cells was successful. Equal to fresh CD34+/CD38'° cells, >90% of reselected CD34+/CD38''' cells were in Gq/Gi. CD34+/CD38"' reselection enriched for LTC-IC (10-fold), as compared to freshly isolated CD34+/CD38'° cells with excellent specificity (82.7% of total LTC-IC were recovered in the reselected CD34+/CD38'" population) and recovery (62% of initial LTC-IC number in CD34+/CD38'" cells were recov- ered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418-resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34+/CD38'° cells. To our knowledge, this is thefirstsystematic analysis of phenotype, function, and cell cycle demonstrating retroviral gene transfer into rare, very primitive human hematopoietic progen- itor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system. OVERVIEW SUMMARY strates the feasibility of efficiently transducing, isolating, and characterizing very primitive hematopoietic progenitor W e have performed a systematic study of retroviral gene cells and should have interesting implications for transfer in primitive human hematopoietic progenitor cells hematopoietic gene therapy. by analyzing the phenotype, function, cell cycle status, and retrovirus transduction after culture. Following extended exposure of CD34+/CD38'" marrow cells to gibbon ape INTRODUCTION leukemia virus (GALV)-pseudotyped retroviral vector, cells were efficiently reisolated that had characteristics of very A fter the successful proof of principle in preliminary primitive progenitor cells, including a CD34+/CD38'° phe- /^.clinical trials, the low efficiency of retroviral gene trans- notype, a very high content of long-term culture-initiating fer obtained in hematopoietic stem cells requires new strategies cells and a low mitotic activity. In this population, func- for efficient isolation, transduction, and analysis of primitive tional and molecular analysis indicate a transduction effi- hematopoietic progenitor cells (Brenner et al, 1993; Deisseroth ciency of more than 40%. The presented strategy demon- et al, 1994; Bordignon et al, 1995; Dunbar et al, 1995; Kohn Department of Intemal Medicine I, Albert-Ludwigs-University, 79106 Freiburg, Gemiany. 'Department of Intemal Medicine I, University of Cologne, 50935 Cologne, Germany 2Fred Hutchinson Cancer Research Center and the University of Washmgton School of Medicme, Seattle, WA 98104. 2079