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Detection and isolation of gene-corrected cells in Gaucher disease via a
fluorescence-activated cell sorter assay for lysosomal glucocerebrosidase
activity
M Lorincz, LA Herzenberg, Z Diwu, JA Barranger and WG Kerr
Department of Genetics, Stanford University Medical School, CA, USA.
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.
Volume 89,
Issue 9,
pp. 3412-3420,
05/01/1997
Copyright © 1997 by The American Society of Hematology
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