Single Cell Analysis and Selection of Living Retrovirus Vector-corrected Mucopolysaccharidosis VII Cells Using a Fluorescence-activated Cell Sorting-based Assay for Mammalian β-Glucuronidase Enzymatic Activity

Matthew C Lorincz,Michael K Parente,Mario Roederer,Garry P Nolan,Zhenjun Diwu,David I.K Martin,Leonard A Herzenberg,John H Wolfe

doi:10.1074/jbc.274.2.657

Abstract

Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

Highlights

The lysosomal storage disorders classified as mucopolysaccharidoses (MPS)1 result from deficiencies in a number of en
Somatic gene therapy using retrovirus vector-mediated transduction of hematopoietic stem cells followed by autologous transplantation of irradiated recipients showed that as little as 2% of normal enzyme levels could completely reverse the pathology in some major organs (10)
To determine whether FDGlcu is hydrolyzed by mammalian ␤-glucuronidase in viable cells, ␤-glucuronidase-negative 3521 fibroblasts or 3521 fibroblasts transduced with the ␤-glucuronidase double copy retrovirus vector DCH␤H, which express high levels of ␤-glucuronidase (13), were incubated in the presence of FDGlcu and analyzed by fluorescence-activated cell sorting (FACS)

Summary

EXPERIMENTAL PROCEDURES

Cells and Tissue Culture—Murine MPS VII skin fibroblasts (3521), fibroblasts isolated from a heterozygous mouse (3522) (13), and primary cultures of BL/6 fibroblasts were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum, 50 nM 2-mercaptoethanol, 100 units/ml penicillin, 0.05 ␮M streptomycin, and 2.0 mM glutamine. 3521 fibroblasts were transduced with the retroviral expression vector DCH␤H (DCH␤H-3521) as described previously (13). After 3.5 days in culture, supernatant and cells were harvested and assayed as described below. Adherent 3521 fibroblasts were washed in phosphate-buffered saline and harvested by incubation in the presence of 0.2– 0.5 ml of 0.25% trypsinEDTA for 5 min at 37 °C. The cells were harvested by trypsinization and washed once with phosphate-buffered saline, and 5 ϫ 106 cells/ml were resuspended in Dulbecco’s modified Eagle’s medium containing 10% FBS, 5 mM Hepes, 100 ␮g penicillin/streptomycin/fungizone, 1 ␮l (10 ␮g/ml) fibroblast growth factor-2 (Promega), 0.25 ␮l (0.8 mg/ml) epidermal growth factor (Boehringer Mannheim), and 0.5 ml (3 mg/ml) collagen in a dish with evenly separated fibers. After incubation at 37 °C for 30 min in the presence of 5% CO2, 2 volumes of medium were added, and the incubation was continued for 2 days, by which time the neo-organ contracted into a semisolid gel. After 4 weeks, individual neoorgan were removed, single cell suspensions were made by mincing and dispase digestion, and the explant cultures were evaluated for enzyme expression

RESULTS

DISCUSSION

Sorted secondary