Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.