Strategies for Sustained Effect of AAV-mediated Correction of Pompe Disease

糖原储存病II型（GSD II）是一种由单基因缺陷引起的典型代谢性储存病。小鼠模型的可用性为评估基因替代疗法提供了一个平台。特别地，我们研究了重组腺相关病毒（rAAV）介导的酸- α葡萄糖苷酶（GAA）基因传递对小鼠GSD II模型进行肝脏定向校正的可行性。在之前的研究中，我们有

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 (cross-correction) 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 immunosuppressive drugs to prevent immune response to 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 adoptive transfer studies with the aim of optimizing rAAV-based therapy. While our previous studies had indicated that immune response to the transgene product substantially influenced the success of therapy, it is possible that the vector itself may have influenced the severity of immune response. To address this, we propose to assess the potential immunogenicity of six different rAAV serotype vectors in primary human dendritic cells in vitro. In addition to analyzing the character of and examination the potential of modulation of the immune response resulting from therapy, we also propose to characterize the inherent properties of GAA that contribute to elicitation of immune response with the eventual goal of identifying novel modulations that would lead to a less immunogenic, more effective therapeutic product. We will map the antigenic 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.