Cytokine mobilization of peripheral blood stem cells in patients with Gaucher disease with a view to gene therapy

Abstract

As clinical trials for gene therapy in Gaucher disease (GD) begin, questions regarding the biology of the hematopoietic stem cell in this disease remain unanswered. This study demonstrates the ability to mobilize and collect CD34+ cells in three patients with the disorder. Our RAC/FDA-approved clinical trial utilizes mobilized peripheral blood stem cells (PBSC) as the target cells for gene transfer. In this approach, a white blood cell fraction is collected by apheresis, enriched for CD34+ cells, and transduced with a retroviral vector carrying the glucocerebrosidase (GC) gene. Transduced cells from the patient with activity corrected to at least normal levels will be returned to the patient without myelosuppressive therapy. We report here the effect of cytokines in mobilizing PBSC in three patients with GD. Two (patients 1 and 2) were given granulocyte colony-stimulating factor (G-CSF) at a dose of 5 micrograms/kg/d and one (patient 3) was given 10 micrograms/kg/d for 10 days. Leukaphereses were done daily for 5 days and the products enriched for CD34+ cells using the clinical Ceprate (CellPro) column. The CD34+ cells in all fractions were monitored daily during mobilization and leukaphereses. Subset analysis for the expression of Thy-1, CD38, HLA-DR, and CD33 on the CD34+ cells was performed. An increase in CD34+ cells in the peripheral blood was noted from day 5 onward (up to a six-fold increase). Up to a 625-fold enrichment in CD34+ cells in the apheresis product was noted using the clinical Ceprate column. Totals of 1.2, 3.5, and 2.1 x 10(6) CD34+ cells/kg were collected in the three patients. A diminution in the percent of CD34+/Thy-1+ cells was noted with enrichment. In vitro retroviral transduction of the CD34-enriched cells using centrifugation promoted transduction protocol previously described (Bahnson AB et al., Centrifugal enhancement of retroviral-mediated gene transfer. Journal of Virology Methods 54:131, 1995) and modified for clinical use, demonstrated a mean transduction efficiency of 37% (range 8.3-87.1%) in clonogenic cells and up to 50% in long-term culture-initiating cells (LTC-IC) at week 6. Significantly, we have been able to achieve up to a 50-fold increase in the level of GC above deficient levels in the patients' CD34+ enriched cells when maintained in vitro in culture. The study demonstrates that up to a six-fold increase in CD34+ cells in the PB can be achieved with cytokines in patients with GD. CD34+ cells can be collected in numbers sufficient for conventional transplantation and transduced efficiently in vitro. In gene therapy trials for genetic disorders to date, myelosuppressive therapy is not advocated. The clinical trial will demonstrate whether this number of transduced CD34+ cells will be adequate for competitive engraftment of genetically corrected PBSC.