Because mobilized peripheral blood (MPB) represents an attractive source of cells for gene therapy, we investigated the ability of advanced-generation lentiviral vectors (LV) to transfer the Enhanced Green Fluorescent Protein (EGFP) gene into CD34+ cells isolated from MPB in culture conditions that allowed expansion of transplantable human HSC. So far, a few studies reported transduction of MPB cells with LV pseudotyped with VSV-G. However, the critical issue remains whether primitive, hematopoietic repopulating cells have indeed been transduced.

In vitro and in vivo experiments provided here show that MPB CD34+ cells can be effectively transduced by advanced generation LV: they do not lose their proliferation potential, self renewal and multilineage differentiation ability, moreover transgene expression is maintained. Following 48 hour-exposure to FL+TPO+SCF+IL6 and overnight incubation with vector particles, MPB CD34+ cells were further cultured for up to six weeks. During this period of culture the CD34+ cells population, the CD34+/GFP+ fraction and the CFC output were maintained.

Transduction efficiency of NOD/SCID repopulating cells (SRC) was assessed by serial transplants into NOD/SCID mice. Primary mice transplanted with transduced cells showed high level of engraftment (11,5 % CD45+ cells): within the CD45+ cells 11,4% expressed GFP (n=15). Moreover, mice transplanted with transduced, week-1 expanded cells showed higher levels of human engraftment than those transplanted with transduced, non-expanded cells (range from 9.25 % to 10.5 % for transduced basal cells; range from 14, 3 % to 38,19 % for transduced 1-week expanded cells). Engraftment was multilineage, with GFP+/lineage+ cells. Serial transplants were performed with transduced, week 1-expanded cells. Secondary engraftment levels were 1.6 %; 19.3 % of human cells were GFP+ (n=3). Moreover, levels of human engraftment were higher in mice injected with transduced, 1- week expanded cells (3.5 %) rather than in those injected with transduced, non expanded cells (1.2 %). Engraftment was multilineage with GFP+/lINEAGE+ cells.

These results show that LV efficiently transduced HSC, that maintain proliferation and self-renewal ability.

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