COMPARATIVE-ANALYSIS OF RETROVIRAL-MEDIATED GENE TRANSDUCTION INTO CD34(-BLOOD HEMATOPOIETIC PROGENITORS IN THE PRESENCE AND ABSENCE OF GROWTH-FACTORS() CORD)

Hematopoietic stem and progenitor cells present in umbilical cord bloo d at the birth of a child are efficiently transduced ex vivo by genes using retroviral vectors in combination with exposure of these cells t o combinations of growth factors, Because retroviral-mediated gene tra nsduction of adult bone marrow and blood hematopoietic stem and progen itor cells is greatly enhanced by growth factors, we evaluated the pos sibility that cord blood progenitors, which have extensive proliferati ve and replating capacity, could be efficiently transduced with a TK n eo gene in a retroviral vector in the absence of growth factors, and a lso determined the influence of exogenously added growth factors on th is transduction. Highly purified CD34(+) (62% pure) cord blood cells i solated by magnetic bead separation were cultured in suspension for 72 hours with viral supernatant in the absence and presence of interleuk in-3 (IL-3), IL-6, and steel factor, Evaluation of progenitor cell-der ived colonies and polymerase chain reaction (PCR)/Southern analysis of the TK neo gene in resultant colony cells demonstrated that some gene transduction was apparent in the absence of growth factors (12.8-14.3 % by PCR), but that this was greatly enhanced (40.0-44.4%) by addition of growth factors, Reverse transcription PCR analysis of the expressi on of IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor genes in this population of cells suggested that the transduction, al though at a lower efficiency, in the absence of added growth factors m ight in part be due to ''constitutive'' and viral supernatant-induced expression of these cytokine genes in the CD34(+)-enriched cell popula tion, The results suggest that like adult bone marrow and blood progen itors, those present in cord blood require exogenous addition of growt h factors for enhanced efficiency of retroviral-mediated gene transduc tion.