Retrovirus-Mediated Gene Transfer into Human CD34+38lowPrimitive Cells Capable of Reconstituting Long-Term CulturesIn Vitroand Nonobese Diabetic–Severe Combined Immunodeficiency MiceIn Vivo

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Abstract
Factors that may improve retroviral transduction of primitive human hematopoietic cells were studied using MFG-based vectors containing a LacZ gene and produced either by a murine (ψ-Crip) or a human (Tasaf) cell line. Cord blood (CB) or bone marrow (BM) CD34+ cells were stimulated and transduced in the presence of three cytokines (interleukin 3 [IL-3], IL-6, and stem cell factor [SCF; c-Kit Ligand]). In the supernatant infection protocol, hematopoietic progenitor cells as measured by X-Gal staining of colony-forming unit cells (CFU-Cs) were transduced more effectively with Tasaf (20%) than with ψ-Crip (8%). In contrast, there was no difference between these two cell lines in a coculture protocol. However, gene transfer into more primitive CD34+CD38¯ subsets and in LTC-IC-derived colonies was low. The use of a large number of cytokines including FLT3-L and PEG–rhMGDF increased the transduction efficiency into CD34+CD38¯-derived CFU-Cs (35% by PCR) or LTC-ICs (10%). A virus pseudotyped with gibbon ape leukemia virus (GALV) envelope further improved gene transfer to 60 and 48% for LacZ+ CFU-C- and LTC-IC-derived colonies, respectively. These conditions of transduction allowed multilineage engraftment of primitive cord blood cells in NOD-SCID mice. Moreover, 10% (at least) of the human hematopoietic cells recovered from the marrow of these immunodeficient animals were transduced. These data suggest that the efficiency of transduction of human hematopoietic primitive cells can be significantly improved by judicious combinations of recombinant cytokines and high retroviral titers. Significant difficulties have been encountered in efforts to infect early hematopoietic stem cells from large animals and humans. Two main parameters are thought to limit transduction efficiency of stem cells: the ratio of infective virus particles relative to target cells and their low replication rate. We used murine (ψ-Crip) and human (Tasaf) cell lines to improve transduction of human primitive cells. Hematopoietic progenitor cells were transduced more effectively with Tasaf rather than ψ-Crip cells in the supernatant protocol when a stromal support was used. However, gene transfer to primitive cell subsets and LTC-IC-derived colonies was low when stimulation was performed using IL-3, IL-6, and stem cell factor. Efficient transduction of these cells was observed when FLT3-L, PEG-rhMGDF, and GM-CSF were added to these cytokines. Consequently, a large number of cytokines is necessary to transduce LTC-ICs and NOD-SCID mouse reconstituting cells.