Flow Cytometry Assay to Monitor Effectiveness of Gene Therapy Correction in Peripheral Blood Mononuclear Cells of Gaucher Disease Type I Patients

Abstract The most common lysosomal storage disorder is Gaucher disease type I (GD-I) which is caused by a genetic defect in the gene encoding the enzyme beta-glucocerebrosidase (GCase). In GD-I, deficient GCase activity results in accumulation of glucocerebrosidases within macrophages of the reticuloendothelial system. We hypothesize that putative precursors for macrophages, monocytes, in peripheral blood of GD-I patients, represent an excellent target population to monitor the effectiveness of novel curative treatments such as lentiviral vector driven gene correction targeting hematopoietic stem and progenitor cells in clinical setting. Here, we present an improved flow cytometry assay that takes advantage of selective uptake of pentafluorobenzoylamino fluorescein di-β-D-glucopyranoside by the CD14+ monocytes resulting in fluorescence when it is hydrolyzed by active GCase, in the presence of formaldehyde (FA), commonly used for intracellular staining and to inactivate pathogens. Using this substrate uptake/FA fixation method our results show a mean of 33.7% ± 3.6% (X±SD) GCase+ cells in GD-1 patients (n=2) compared to 83.7% ± 9.6% (X±SD) in normal controls (n=5). The mean fluorescence intensity (MFI) of the enzymatic activity showed no overlap between untreated GD-1 patients (3480 ± 49) and controls (7187 ± 595.7). Method specificity was demonstrated by complete inhibition of enzyme activity and fluorescence using 400 mM conduritol-B-epoxide, a GCase specific inhibitor. Next, mixtures of normal donor peripheral blood mononuclear cells (PBMC) and GD-1 PBMC were created in vitro to assess the lower limit of detection. As low as 1% normal donor cells could be reliably distinguished within these mixtures. GCase+ monocytes in the mixtures could be identified and quantified based on a higher MFI compared to GD-1 patient cells. Compared to the conventional 4-MU biochemical method, this approach requires only a small blood sample and minimal processing, is rapid and straightforward, and more importantly allows quantification of GCase enzyme activity at individual cells instead of in a bulk cell lysate. These findings suggest this novel flow cytometry method is sensitive and specific to quantify small increases in GCase activity at single cell level, and thus has potential to monitor in vivo the effectiveness of gene therapy for Gaucher disease Type 1 in patients in real time. Additional sample analysis from GD-1 patients and normal donors are in progress to confirm consistency of the method. Disclosures No relevant conflicts of interest to declare.