RAPID RETROVIRAL INFECTION OF HUMAN HEMATOPOIETIC-CELLS OF DIFFERENT LINEAGES - EFFICIENT TRANSFER IN FRESH T-CELLS

In order to develop a clinically feasible gene marking approach, we ha ve used the recently described PINCO retroviral expression system, com posed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Tw o T, five B, one erythromyeloid and three myeloid cell lines were succ essfully infected with % GFP(+) cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility with the my eloid cells being the least efficiently infected. We also infected nor mal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, a nd obtained an average of 30% GFP(+) cells, all present within the CD3 (+) population. with CD4(+) and CD8(+) cells being equally infected. F inally, the tonsillar purified B population showed lower levels of inf ectivity (6%) whereas high susceptibility was shown by normal human um bilical vein endothelial cells (57%). Highly purified CD34(+) cells we re also susceptible, Varying from 6% to 10% GFP(+) cells. Immature mye loid/erythroid progenitors have been infected which stably expressed t he GFP protein during further differentiation in culture. The GFP(+) T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored, In all cases the difference in percentage of GFP(+) cells did not correlate with the percentage of S/G(2)/M cycling cells as determined at the moment of infection or wi th the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the h igh efficiency of infection with respect to normal T lymphocytes (in t his last case higher than previously reported) and the lack of need fo r in vitro selection make this system favourable for clinical developm ent.