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 lar ge proportion of the mouse haemopoietic system was developed, based on the in vitro expansion of retrovirally infected bone marrow and selec tion of the transduced cells. Bone marrow cells harvested 4 d after 5- FU treatment were incubated under IL-3/SCF stimulation and their growt h dynamic, susceptibility to retroviral infection and reconstitution c apacity evaluated throughout the incubation period. On the third day o f culture a maximum expansion in the CFU-GM and CFU-S-12 progenitor po ols was observed (130- and 15-fold, respectively), with no apparent im pairment in long-term repopulating precursors. This expansion was, how ever, accompanied by a net decrease in the CFU-GM susceptibility to th e infection by supernatants containing a Moloney-derived ecotropic ret roviral vector carrying the neo(r) gene. The designed protocol thus in volved the infection of freshly harvested 5-FU-treated bone marrow, fo llowed by expansion under IL-3/SCF stimulation and selection for resis tance to G418. This procedure allowed us to harvest up to 780 CFU-GM a nd 50 CFU-S-12 per 10(5) bone marrow cells, free from non-genetically marked progenitors. Most of the animals reconstituted with the transdu ced 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 aft er the transplantation indicates that long-term lympho-haemopoietic re populating cells were efficiently transduced and selected in vitro und er conditions that preserve their self-renewal and differentiation pro perties. 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.