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

糖原累积病II型（GSD II）是由单基因缺陷引起的原型代谢性储存病。小鼠模型的可用性提供了一个评估基因替代疗法的论坛。特别是，我们已经研究了重组腺相关病毒（rAAV）介导的酸性α-葡萄糖苷酶（GAA）的基因递送的可行性，用于肝脏定向校正GSD II的小鼠模型。在以前的研究中，我们 证明了使用rAAV载体将GAA体内基因转移至心脏和骨骼肌的高效性。最近，我们已经显示了通过从肝组织中过度表达治疗性GAA蛋白而引起的分泌蛋白的摄取对远端组织的校正（交叉校正）。然而，我们也注意到，交叉校正的水平， 实现的免疫应答依赖于对表达的GAA的体液免疫应答的水平。在这项研究中，我们建议扩展我们的研究，并检查免疫调节的潜力，以提高rAAV介导的，肝脏定向基因治疗GSD II的疗效。在我们的初步研究中，我们将评估免疫抑制药物预防对rAAV衍生或输注的重组GAA蛋白的免疫应答的功效。有趣的是，我们和其他人 注意到rAAV介导的对治疗性转基因的免疫耐受的一些实例，我们建议通过过继转移研究来表征载体诱导的对GAA的耐受的机制，目的是优化基于rAAV的治疗。虽然我们以前的研究表明，对转基因产物的免疫应答实质上影响了治疗的成功，但载体本身可能影响了免疫应答的严重程度。 反应为了解决这个问题，我们建议评估六种不同rAAV血清型载体在体外原代人树突状细胞中的潜在免疫原性。除了分析治疗引起的免疫应答的调节的特性和检查其潜力之外，我们还建议表征GAA的固有特性，其有助于引发免疫应答，最终目标是鉴定新的调节， 从而产生免疫原性更低、更有效治疗产品。我们将绘制GAA蛋白的抗原表位，并通过确定GAA的晶体结构将这些发现与蛋白质的结构相关联。了解GAA蛋白的结构与功能关系不仅使我们能够识别抗原性区域，而且还可以 提供深入了解的作用机制和潜在的GSD II的分子基础。总之，这些研究将产生重要的新信息，在建立临床相关的策略，肝脏定向rAAV介导的基因治疗一般，并为GSD II的治疗，特别是。

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.