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）是一种有吸引力的用于心脏和肺部基因治疗的病毒载体。尽管使用rAAV治疗囊性纤维化（CF）肺部疾病的临床试验迄今尚未成功，但使用这种病毒治疗CF和其他NHLBI病灶性疾病仍有相当大的兴趣。在某种程度上，rAAV载体缺乏临床成功是由于对控制病毒转导的机制（即编码转基因的表达）了解甚少。本提案旨在描述控制rAAV载体高效转导的受体/共受体依赖的内吞机制，重点关注2型血清型（rAAV2）。虽然已经确定了各种rAAV血清型的受体和共受体，但控制rAAV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室处理的效率。该项目的初步数据表明，1521-整合素依赖的rAAV2内吞作用比竞争的进入途径更有效地导致感染。然而，在rAAV2/整合素对接后调节整合素依赖性内吞作用的信号事件仍不清楚。大量文献将整合素激活与许多细胞内信号蛋白的募集和激活联系起来，如非受体酪氨酸激酶、局灶黏附激酶（FAK）和c-Src。已知这些整合素效应物结合并调节Rac1和磷酸肌肽3-激酶（PI3K），两者均被证明调节rAAV2的内吞作用和囊泡运输。我们的长期目标是确定能够提高rAAV2和其他整合素依赖性AAV血清型病毒转导效率的方法。拟议的研究将通过确定围绕整合素依赖的rAAV2内吞作用的事件，以及导致病毒有效核转运和转导的下游信号通路来帮助实现这一点。此外，阐明细胞信号是如何被rAAV感染激活的，可能有助于该领域理解对这种病毒的先天免疫反应的起源——这是开发用于基因治疗的rAAV载体的第二个障碍。

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.