Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity

Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical m anifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some af fected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of p-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 flu orescence-activated cell sorter-based assay for the quantitative analysis o f beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be is olated by cell sorting on the basis of beta-glucuronidase activity and cult ured for further use. In vitro analysis revealed that sorted cells have ele vated levels of beta-glucuronidase activity and secrete higher levels of cr oss-correcting enzyme than the population from which they were sorted. Tran sduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sort ing and expanded ex vivo. A relatively high percentage of these cells maint ain stable expression after secondary transplantation, yielding significant ly higher levels of enzymatic activity than that generated in the primary t ransplant.