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

糖原沉积病II型(GSD II)是由单基因缺陷引起的一种典型的代谢性沉积病。小鼠模型的出现为评估基因替代疗法提供了一个论坛。特别是，我们研究了重组腺相关病毒(RAAV)介导的酸性α-葡萄糖苷酶(GAA)基因传递用于肝脏导向纠正GSD II小鼠模型的可行性。在以前的研究中，我们已经 证实了利用rAAV载体将GAA基因转移到心肌和骨骼肌的高效体内实验。最近，我们展示了通过摄取来自肝组织的治疗性GAA蛋白过度表达所产生的分泌蛋白来矫正远端组织(交叉矫正)。然而，我们也注意到，交叉修正的水平可能是 获得的GAA依赖于对所表达的GAA的体液免疫反应水平。在这项研究中，我们建议扩大我们的研究范围，探讨免疫调节的潜力，以提高rAAV介导的、肝脏导向的GSD II基因治疗的有效性。在我们的初步研究中，我们将评估免疫抑制药物的有效性，以防止rAAV来源或输注的重组GAA蛋白的免疫反应。有趣的是，就像其他人和我们一样 注意到一些rAAV介导的对治疗性转基因的免疫耐受的例子，我们建议通过过继转移研究来表征载体诱导对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反应在很大程度上影响了治疗的成功，但载体本身可能影响免疫的严重程度。 回应。为了解决这一问题，我们建议在体外评估六种不同rAAV血清型载体在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反应的特征和检测其潜在的调节作用外，我们还建议表征有助于激发免疫反应的GAA的固有特性，最终目标是确定新的调节机制，从而 导致一种免疫原性较低、更有效的治疗产品。我们将绘制GAA蛋白的抗原表位图，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蛋白中的结构-功能关系不仅可以帮助我们识别抗原性区域，还可以 为GSD II的作用机制和潜在的分子基础提供洞察力。总之，这些研究将在建立一般的肝脏导向rAAV介导的基因治疗，特别是GSD II的治疗的临床相关策略方面产生重要的新信息。

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.