GROWTH-FACTORS AND STROMAL SUPPORT GENERATE VERY EFFICIENT RETROVIRALTRANSDUCTION OF PERIPHERAL-BLOOD CD34(+) CELLS FROM GAUCHER PATIENTS

We have achieved high-efficiency gene transfer into nonmobilized perip heral blood (PB) CD34(+) cells from patients with Gaucher's disease us ing a clinically acceptable retroviral supernatant transduction protoc ol. In our studies, bone marrow(BM) and PB CD34(+) cells were transduc ed using a high titer (10(8) particles/mL) retroviral supernatant once a day for 4 consecutive days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), with or without an irradiated allog eneic BM stromal layer, The growth factors alone resulted in 29% +/- 1 0% gene transfer of PB CD34(+) clonogenic cells in contrast with 71% /- 17% gene transfer efficiency using stroma with the growth factors: a 2.5-fold increase, The increase in gene transfer efficiency was less prominent when BM CD34(+) cells were used (40% +/- 16% without and 57 % +/- 8% with stroma, a 1.5-fold increase), The overall transduction e fficiency of both PB and BM CD34(+) cells was lower when the cells wer e transduced over a stromal cell layer without added growth factors. T he combination of IL-3, IL-6, and SCF with stroma transduced 75% of pr imitive long-term culture initiating cells (PB LTC-ICs) in comparison with 34% of LTC-ICs when IL-3, IL-6, and SCF were used without stromal support. Using this clinically acceptable supernatant/cytokines/strom a transduction protocol, correction of the glucocerebrosidase (GC) def iciency in the progeny cells of PB LTC-ICs from Gaucher's-disease pati ents has been accomplished, Efficient transduction of the PB CD34(+) c ells using this transduction protocol may allow repeated delivery of ' 'GC-corrected'' hematopoietic stem and progenitor cells to Gaucher's-d isease patients.