Abstract
Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated successfully with enzyme replacement therapy (ERT). Gene therapy has been proposed as an alternative to ERT due to the presumed advantages of continuous, endogenous production of the therapeutic enzyme. GLA production in the liver and its therapeutic efficacy in the Fabry mouse have been demonstrated previously with various viral vector systems. In consideration of the potential advantages of using the salivary glands as endogenous GLA biosynthesis sites, we explored the feasibility of this approach in the Fabry mouse. GLA -/0 or -/- mice received an adenoviral vector (2 × 10(10) or 1 × 10(9) viral particles) expressing GLA to the right submandibular gland via oral cannulation of the submandibular duct. Four days later, animals were sacrificed; saliva, plasma, kidney, liver, and brain were collected and assayed using ELISA, Western blot, and a GLA enzymatic activity assay using both traditional fluorescence methods and isotope dilution mass spectrometry by following the U.S. EPA Method 6800. GLA activity was significantly elevated in the serum and liver of both treatment groups, and improvement in the kidney was marginally significant (P < 0.069) in the high-dose group. Notably, we found that liver and salivary gland produce different glycoforms of the GLA transgene. Only small numbers of adenoviral genomes were observed in the livers of treated animals, but in four of 14 in the high-dose groups, liver levels of adenovirus exceeded 20 copies/μg, indicating that the sequestration in the salivary gland was imperfect at high doses. Taken together, these results indicate that the salivary gland-based gene therapy for Fabry disease is promising, and further studies with advanced viral vector gene delivery systems (e.g., adeno-associated virus) for long-term treatment appear to be warranted.