Mechanisms of Integrin-Linked Transduction by Recombinant Adeno-Associated Virus

描述（由申请人提供）：重组腺相关病毒（rAAV）是一种用于心脏和肺基因治疗的有吸引力的病毒载体。尽管用rAAV进行的囊性纤维化（CF）肺病的临床试验迄今尚未成功，但对使用该病毒治疗CF和其他NHLBI焦点疾病仍有相当大的兴趣。部分地，rAAV载体缺乏临床成功是由于对控制病毒转导的机制（即，编码的转基因的表达）。该提案旨在描述控制rAAV载体高效转导的受体/共受体依赖性内吞机制，重点是2型血清型（rAAV 2）。尽管已经鉴定了各种rAAV血清型的受体和共受体，但是控制rAAV的内吞作用和细胞内命运的信号传导机制在很大程度上是未知的。我们的实验室已经确定rAAV摄取的机制显著影响病毒通过内体隔室的处理效率。该项目的初步数据表明，1521-整合素依赖的rAAV 2的内吞作用导致比竞争进入途径更有效的感染。然而，在rAAV 2/整联蛋白对接后调节整联蛋白依赖性内吞作用的信号传导事件仍不清楚。大量的文献已经将整联蛋白活化与许多细胞内信号传导蛋白的募集和活化联系起来，例如非受体酪氨酸激酶、粘着斑激酶（FAK）和c-Src。已知这些整联蛋白效应物结合并调节Rac 1和磷酸肌醇3-激酶（PI 3 K），这两者均已显示调节rAAV 2内吞作用和囊泡运输。我们的长期目标是确定将增强rAAV 2和其他整合素依赖性AAV血清型的病毒转导效率的方法。拟议的研究将通过确定rAAV 2的整合素依赖性内吞作用周围的事件以及导致病毒有效核转运和转导的下游信号传导途径来帮助实现这一点。此外，澄清细胞信号是如何被激活的rAAV感染可能有助于了解这种病毒的先天性免疫反应的起源，第二个障碍rAAV载体的基因治疗应用的发展领域。 公共卫生相关性：重组腺相关病毒（rAAV）是目前用于治疗许多疾病（包括囊性纤维化和血友病B）的临床试验中涉及的基因校正的主要载体。拟议的研究将调查调节rAAV内吞作用的分子事件，以确定可用于提高基因递送效率的靶点。这些研究对于下一代AAV载体的合理设计和临床上成功的基因转移至关重要。

DESCRIPTION (provided by applicant): Recombinant Adeno-associated virus (rAAV) is an attractive viral vector for gene therapy to the heart and lung. Although clinical trials for cystic fibrosis (CF) lung disease with rAAV have thus far not been successful, considerable interest remains in using this virus to treat CF and other NHLBI focus diseases. In part, the lack of clinical success with rAAV vectors has been due to a minimal understanding of mechanisms that control viral transduction (i.e., expression of an encoded transgene). This proposal seeks to delineate receptor/co-receptor-dependent endocytic mechanisms that control highly efficient transduction by rAAV vectors, with a focus on the type-2 serotype (rAAV2). Although receptors and co-receptors for various rAAV serotypes have been identified, the signaling mechanisms that control endocytosis and the intracellular fate of rAAV are largely unknown. Our laboratory has determined that the mechanism of rAAV uptake significantly influences how efficiently the virus is processed through the endosomal compartment. Preliminary data from this project suggest that 1521-integrin-dependent endocytosis of rAAV2 leads to more efficient infection than do competing pathways of entry. However, the signaling events that regulate integrin-dependent endocytosis following rAAV2/integrin docking remain unclear. A large body of literature has linked integrin activation to the recruitment and activation of numerous intracellular signaling proteins, such as the non-receptor tyrosine kinases, focal adhesion kinase (FAK) and c-Src. These integrin effectors are known to bind and regulate Rac1 and phosphoinositide 3-kinase (PI3K), both of which have been shown to regulate rAAV2 endocytosis and vesicular trafficking. Our long-term goal is to identify approaches that will enhance the efficiency of viral transduction by rAAV2 and other integrin-dependent AAV serotypes. The proposed research will aid in this by identifying the events surrounding integrin-dependent endocytosis of rAAV2, as well as the downstream signaling pathways that lead to efficient nuclear transport and transduction of the virus. In addition, clarifying how cell signaling is activated by rAAV infection may aid the field in understanding the genesis of innate immune responses to this virus-a second barrier to the development of rAAV vectors for gene therapy applications. PUBLIC HEALTH RELEVANCE: Recombinant Adeno-associated virus (rAAV) is a leading vector for gene correction currently involved in clinical trials for the treatment of many disorders, including Cystic Fibrosis, and Hemophilia B. The proposed study will investigate the molecular events that regulate endocytosis of rAAV in an effort to identify targets that can be used to enhance the efficiency of gene delivery. These studies are critical to rational design of the next generation of AAV vectors and clinically successful gene transfer.