COMP ARISEN OF METHODS FOR RETROVIRAL-MEDIATED TRANSFER OF GLUCOCEREBROSIDASE GENE TO CD34(+) HEMATOPOIETIC PROGENITOR CELLS

Gaucher disease is an excellent candidate for gene therapy by transduc tion of hematopoitic stem cells. In this study, we compared methods wh ich allow an increase in transfer of the glucocerebrosidase gene to hu man hematopoietic progenitor cells. Several techniques were employed, including the use of cytokines, bone marrow stroma, fibronectin, centr ifugal enhancement and in vitro long-term culture. The effect of prest imulation with cytokines interleukin-3 (IL-3), interleukin-6 (IL-6) an d stem cell factor (SCF) on transduction of cord blood CD34(+) cells w as examined. The results suggest that 16-h prestimulation was sufficie nt far efficient transduction. We examined the effect of bone marrow s troma and fibronectin, both of which increased transduction efficiency up to 36% and 44%, respectively, as measured by PCR for the integrate d GC-cDNA in clonogenic cells (9% without any support). Transduction e fficiency of 83% was obtained using 2-h centrifugation. Combining cent rifugation and in vitro culture in longterm bone marrow culture media containing cytokines (IL-3/IL-6/SCF), CD34+ cells from cord blood and peripheral blood of 3 Gaucher patients were transduced weekly for 21 d . The results of 6 separate experiments consistently demonstrated tran sduction efficiency of 100% after 7-d in vitro culture. This transduct ion protocol combining centrifugation and in vitro long-term culture i s an attractive method and can be applied to clinical trials.