SELECTIVE IMMUNOAFFINITY-BASED ENRICHMENT OF CD34(-AFFINITY NERVE GROWTH-FACTOR RECEPTOR (DELTA-LNGFR) GENE() CELLS TRANSDUCED WITH RETROVIRAL VECTORS CONTAINING AN INTRACYTOPLASMATICALLY TRUNCATED VERSION OF THE HUMAN LOW)

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalig nant diseases, To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells , we have used a Moloney murine leukemia virus (Mo-MuLV)-based retrovi ral vector containing the intracytoplasmatically truncated human low-a ffinity nerve growth factor receptor (Delta LNGFR) cDNA as a marker ge ne, Supernatant transduction of CD34(+) cells (mean purity 97%) in fib ronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) t ransduced cells expressing Delta LNGFR (LNGFR(+) cells), After transdu ction, more than 65% of the transduced cells remained CD34(+), Compare d with control (mock-and nontransduced) CD34(+) cells, transduction di d not decrease the cloning efficiency of CD34(+) cells, Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibo dy resulted in >90% LNGFR(+) cells, Further phenotypic characterizatio n of these highly enriched LNGFR(+) cells indicated that the majority co-expressed the CD34 and CD38 antigens, These results show that trans duced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activat ed cell sorting (FACS) analysis and fast and efficiently enriched by i mmunoadhesion using magnetic beads, The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more p rimitive hematopoietic progenitors.