Adenoviral vector-mediated ß-glucuronidase cDNA transfer to treat MPS VII RPE in vitro

Purpose. To develop an effective therapy for treating glycosaminoglycan (GAG) storage in mucopolysaccharidosis VII (MPS VII) retinal pigment epithelium (RPE) in vitro using adenoviral vector mediated human s-glucuronidase cDNA (Ad-GUSB) transfer. Methods. Ad-GUSB was used to infect RPE at confluency. The transduction condition was optimized varying time of infection and number of infectious particles. The s-glucuronidase (GUSB) activity was measured in transduced cells and media using a fluorogenic substrate. The GAG profiles were examined by metabolically labeling RPE with 35 Na 2 SO 4. Results. Transduced RPE, irrespective of species or disease status, expressed a high level of s-glucuronidase. The expressed enzyme restored normal levels of GAGs in the RPE cells of homozygous affected MPS VII dogs by metabolizing stored GAGs. The over-expressed enzyme (>10000 nmoles/hr/mg) failed to restore normal level of GAGs. A high level of GUSB expression was maintained in vitro at least nine weeks. Conclusions....