368. Simultaneous Delivery of Dual AAV9 Vectors Induces Immune Tolerance and Correction of Pompe Disease Pathology
Authors: Phillip A. Doerfler, Adrian G. Todd, Nathalie Clément, Darin J. Falk, Roland W. Herzog, Barry J. Byrne
Journal: Molecular Therapy (2015)

Enzyme replacement therapy (ERT) for Pompe disease (acid α-glucosidase deficiency; GAA) provokes the development of high titer antibodies against the enzyme and contributes to life threatening infusion associated reactions. AAV-mediated gene therapy stands as a more attractive therapeutic option yet AAV-mediated gene transfer can elicit anti-capsid and anti-transgene responses that modulate its effectiveness. To circumvent deleterious immune responses we sought to develop a dual vector system that targets gene expression to specific tissues simultaneously ameliorating the Pompe disease phenotype and facilitate antigen specific tolerance. Two AAV9 vectors containing a liver-specific promoter (LSP) or a cardiac/neuromuscular-specific promoter (desmin; DES) driving human codon-optimized GAA (coGAA) were combined at a ratio of 1 LSP: 9 DES (1:9) or 9 LSP: 1 DES (9:1) and systemically injected at 5×10e13 vg/kg into 4-6 week old Gaa-/- mice. After 8 weeks, physiologic, immunologic, histologic and biochemical assessments were performed as well as the adoptive transfer of Tregs into naïve mice testing for tolerance. GAA activity was significantly increased in AAV treated mice in the heart, diaphragm, liver, quadriceps, sciatic nerve and spinal cord compared to knockouts (p<0.05). The isometric torque generated by the tibialis anterior in all treated groups was significantly greater compared to KO (p<0.05). ECGs revealed the 1:9, but not the 9:1, treated cohort had a normalization of the PR interval similar to age matched wild-type mice (p<0.05). Antibodies against GAA in the 9:1 group were significantly lower than the 1:9 group but titers declined in both groups by weeks 2-6; possibly indicating tolerance. To test for antigen-specific tolerance, Tregs from mice that received AAV9 or vehicle were transferred into naïve recipients. One group of mice did not receive Tregs as a control for non-specific suppression. Challenged with ERT at the clinical dose of 20 mg/kg 18 hours, 2 weeks and 4 weeks post-transfer, anti-GAA IgG1 titers were 2.5-fold lower in mice receiving Tregs from AAV-treated donors compared to mice that received Tregs from vehicle donors or no Tregs (p<0.01). Prior to the third injection all mice had a baseline temperature of 36-37°C. Within 5 minutes, and up to 2 hours later, the body temperature of those mice that received Tregs from vehicle treated mice, or no Tregs, dropped to <35°C whereas those mice that received Tregs from AAV treated mice maintained baseline core temperature (p<0.001). This drop in temperature was also accompanied with outward signs of anaphylaxis: piloerection and hyperventilation. Resolving the immunological complications of gene therapy in conjunction with physiologic correction of systemic disease would typically involve the use of immunosuppressives. Here we described a modality involving two combined AAV9 vectors that, in the 1:9 cohort, resulted in the most complete correction of pathology and tolerance induction. These results lay the framework for the use of multiple vectors to address these separate but equally important aspects of gene therapy for Pompe disease.