The presence of an autologous marrow stromal cell layer increases glucocerebrosidase gene transduction of long-term culture initiating cells (LTCICs) from the bone marrow of a patient with Gaucher disease.

Gaucher disease is a lysosomal storage disorder resulting form deficiency of the acid beta-glucosidase, glucocerebrosidase (GC). Allogeneic bone marrow transplantation has been beneficial in the treatment of Gaucher patients. Therefore, this disorder may be an ideal candidate for gene therapy by GC gene transduction of hematopoietic stem cells. We sought to increase the extent of gene transfer into CD34+ cells from the marrow of a Gaucher patient using G1GC, a simple retroviral vector containing a normal human GC cDNA. The ability of autologous stromal support and recombinant cytokines to increase the extent of transduction of colony-forming-cells (CFCs) and long-term culture initiating cells (LTCICs) was assessed. The presence of a stromal layer significantly increased the extent of GC gene transfer into 14-day CFCs, as determined by polymerase chain reaction (PCR) of individual colonies (18.8% with stroma versus 5% without, P < 0.001). Stromal support also increased the extent of transduction of LTCICs (10% with stroma versus 0.83% without, P < 0.001). Non-adherent cells from long-term bone marrow cultures initiated with CD34+ progenitors transduced on autologous stroma had higher levels of GC enzyme activity than cultures initiated with cells transduced without stroma. The percentage of cells which were GC positive by immunohistochemistry was also increased (21.1% with stroma versus 2.7% without, P = 0.0003). The addition of cytokines (IL-3, IL-6 and Steel factor) to the transduction, in the presence of stroma, significantly increased the extent of gene transfer into CFCs but not LTCICs. These studies indicate that the GC gene can be effectively transduced into LTCICs by retroviral vectors in the presence of stroma at levels significant for clinical gene therapy trials in patients with Gaucher disease.