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

Mutations in the acid β-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 β-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 β-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a β-glucuronidase retroviral vector can be isolated by cell sorting on the basis of β-glucuronidase activity and cultured for further use.In vitro analysis revealed that sorted cells have elevated levels of β-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing β-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.