Glycogen storage disease type II (GSD II) is a prototypic metabolic storage disease resulting from a single gene defect. The availability of a mouse model has provided a forum in which to assess gene replacement therapy. In particular, we have investigated the feasibility of recombinant adeno-associated virus (rAAV)-mediated gene delivery of acid-alpha glucosidase (GAA) for liver-directed correction of a mouse model of GSD II. In previous studies, we have demonstrated high-efficacy in vivo gene transfer of GAA to heart and skeletal muscle using rAAV vectors. Recently, we have shown correction of distal tissues (crosscorrection) via uptake of secreted protein resulting from over-expression of therapeutic GAA protein from hepatic tissue. However, we also noted that the levels of cross-correction that could be achieved were dependent on the level of humoral immune response to the expressed GAA. In this study, we propose to extend our studies and examine the potential of immune modulation to improve the efficacy of rAAV mediated, liver-directed gene therapy for GSD II. In our initial studies, we will evaluate the efficacy of a novel immunosuppressive regimen using rapmycin and IL-10 target CD4+ CD25+T-regulatory cells for immune non-responsiveness rAAV-derived or infused recombinant GAA protein. Interestingly, as others and we have noted some instances of rAAV-mediated immune tolerance to a therapeutic transgene, we propose to characterize the mechanism of vector-induced tolerance to GAA through the hepatic route of gene transfer and induction of T-regulatory cells. In a new set of experiments, we propose to study a clinically relevant strategy for B-cell depletion by use of Rituxan in a mouse model of Pompe. We will map the T-cell epitopes of the GAA protein and correlate those findings to the structure of the protein by determining the crystal structure of GAA. Understanding the structure-function relationship in the GAA protein would not only allow us to identify regions of antigenicity, but also provide insight into the mechanism of action and potentially the molecular basis of GSD II. Together, these studies will yield important new information in establishing clinically relevant strategies for liver-directed rAAV-mediated gene therapy in general, and for the treatment of GSD II, in particular.
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