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Gaucher disease type 1 results from the accumulation of glucocerebroside in macrophages of the reticuloendothelial system, as a consequence of a deficiency in glucocerebrosidase (GC) activity. Recent improvements in the methodologies for introducing foreign genes into bone marrow stem cells have prompted several groups to test the efficacy of gene transfer therapy as a curative treatment for Gaucher disease. Limitations of this approach include the potential for insufficient engraftment of gene-corrected cells and incomplete transduction of hematopoietic stem cells using retroviral gene transfer. Overcoming these obstacles may be critical in the case of treatment for Gaucher disease type 1, because GC transduced cells have not been shown to have a growth advantage over noncorrected cells. Here, we describe the development and application of a novel, fluorescence-activated cell sorter based assay that directly quantitates GC activity at the single cell level. In a test of this application, fibroblasts from a Gaucher patient were transduced, and high expressing cells sorted based on GC activity. Reanalysis of cultured sorted fibroblasts reveals that these cells maintain high levels of enzymatic activity, compared with the heterogeneous population from which they were sorted. The assay is sufficiently sensitive to distinguish GC activity found in Gaucher patient monocytes from that in normal controls. Furthermore, preliminary results indicate that increased GC activity can be detected in transduced, CD34+ enriched peripheral blood mononuclear cells isolated from a Gaucher patient. This method should be a useful addition to current gene therapy protocols as a means to quantitatively assess gene correction of relevant cell populations and potentially purify transduced cells for transplantation.

Recent clinical data have shown that enzyme replacement therapy with macrophage-targeted glucocerebrosidase (GCR) can be effective in treating type 1 Gaucher disease. Sera from 262 patients, repeatedly infused with GCR, were assessed for the presence of antibodies to this therapeutic protein. Patient serum samples obtained at 3-month intervals were assessed by enzyme-linked immunosorbent assay and those with values greater than two standard deviations above the mean value obtained with a pool of normal human sera were further characterized by radioimmunoprecipitation. At the time of these analyses, the duration of patient treatment varied from 3 months to approximately 3 years. Of the 262 patients analyzed, 34 (12.9%) showed IgG antibodies, as confirmed by radioimmunoprecipitation. All patients who seroconverted did so within 1 year of treatment. The predominant antibody developed was the IgG1 subclass. Fourteen patients in the study experienced periodic symptoms suggestive of immediate hypersensitivity. Nine of these 14 patients had antibody to GCR as determined by radioimmunoprecipitation, whereas 5 patients were antibody negative. There was no evidence of the development of IgE antibodies in these 14 patients. The presence of GCR antibodies did not appear to effect efficacy of therapy in any of the patients treated to date.