EX-VIVO EXPANSION AND SELECTION OF RETROVIRALLY TRANSDUCED BONE-MARROW - AN EFFICIENT METHODOLOGY FOR GENE-TRANSFER TO MURINE LYMPHO-HEMATOPOIETIC STEM-CELLS

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5-FU treatment were incubated under IL-3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU-GM and CFU-S-12 progenitor pools was observed (130- and 15-fold, respectively), with no apparent impairment in long-term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU-GM susceptibility to the infection by supernatants containing a Moloney-derived ecotropic retroviral vector carrying the neo(r) gene. The designed protocol thus involved the infection of freshly harvested 5-FU-treated bone marrow, followed by expansion under IL-3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU-GM and 50 CFU-S-12 per 10(5) bone marrow cells, free from non-genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neo(r) gene and expressing resistance to G418 5 months after the transplantation indicates that long-term lympho-haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self-renewal and differentiation properties. This gene-transfer methodology may improve the development of gene therapy protocols where the purging of non-transduced precursors would guarantee a lasting and uniform expression of exogenous genes.