Comparison of methods for retroviral mediated transfer of glucocerebrosidase gene to CD34+ hematopoietic progenitor cells.

Gaucher disease is an excellent candidate for gene therapy by transduction of hematopoitic stem cells. In this study, we compared methods which allow an increase in transfer of the glucocerebrosidase gene to human hematopoietic progenitor cells. Several techniques were employed, including the use of cytokines, bone marrow stroma, fibronectin, centrifugal enhancement and in vitro long-term culture. The effect of prestimulation with cytokines interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) on transduction of cord blood CD34+ cells was examined. The results suggest that 16-h prestimulation was sufficient for efficient transduction. We examined the effect of bone marrow stroma and fibronectin, both of which increased transduction efficiency up to 36% and 44%, respectively, as measured by PCR for the integrated GC-cDNA in clonogenic cells (9% without any support). Transduction efficiency of 83% was obtained using 2-h centrifugation. Combining centrifugation and in vitro culture in long-term 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 transduction efficiency of 100% after 7-d in vitro culture. This transduction protocol combining centrifugation and in vitro long-term culture is an attractive method and can be applied to clinical trials.

Gaucher's disease: studies of gene transfer to haematopoietic cells.

Transfer of the gene coding for glucocerebrosidase (GC) via a retroviral vector (MFG-GC) to haematopoietic progenitors results in engraftment and life-long expression of the human protein at high levels in transplanted mice. Studies of human CD34 cells were carried out to evaluate their potential use in a gene therapy approach to Gaucher's disease. High transduction efficiency and correction of the enzyme deficiency was possible in CD34 cells obtained from patients with Gaucher's disease. Based on these results, a clinical trial of gene therapy was designed and initiated. Preliminary results of this study indicate the persistence or engraftment of genetically corrected cells in the transplanted patients.