RETROVIRUS-MEDIATED GENE-TRANSFER INTO HUMAN CD34(-TERM CULTURES IN-VITRO AND NONOBESE DIABETIC-SEVERE COMBINED IMMUNODEFICIENCY MICE IN-VIVO()38(LOW) PRIMITIVE CELLS CAPABLE OF RECONSTITUTING LONG)

Factors that may improve retroviral transduction of primitive human he matopoietic cells were studied using MEG-based vectors containing a La cZ gene and produced either by a murine (psi-Crip) or a human (Tasaf) cell line. Cord blood (CB) or bone marrow (BM) CD34(+) cells were stim ulated and transduced in the presence of three cytokines (interleukin 3 [IL-3], IL-6, and stem cell factor [SCF; c-Kit Ligand]). In the supe rnatant infection protocol, hematopoietic progenitor cells as measured by X-Gal staining of colony-forming unit cells (CFU-Cs) were transduc ed more effectively with Tasaf (20%) than with psi-Crip (8%). In contr ast, there was no difference between these two cell lines in a cocultu re protocol. However, gene transfer into more primitive CD34(+)CD38(-) subsets and in LTC-IC-derived colonies was low. The use of a large nu mber of cytokines including FLT3-L and PEG-rhMGDF increased the transd uction efficiency into CD34(+)CD38(-)-derived CFU-Cs (35% by PCR) or L TC-ICs (10%). A virus pseudotyped with gibbon ape leukemia virus (GALV ) envelope further improved gene transfer to 60 and 48% for LacZ(+) CF U-C- and LTC-IC-derived colonies, respectively. These conditions of tr ansduction allowed multilineage engraftment of primitive cord blood ce lls in NOD-SCID mice, Moreover, 10% (at least) of the human hematopoie tic cells recovered from the marrow of these immunodeficient animals w ere 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.