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

描述(申请人提供)：重组腺相关病毒(RAAV)是一种用于心肺基因治疗的有吸引力的病毒载体。虽然使用rAAV治疗囊性纤维化(CF)肺部疾病的临床试验到目前为止还没有成功，但人们对使用这种病毒治疗囊性纤维化和其他NHLBI焦点疾病仍有相当大的兴趣。部分原因是由于对控制病毒转导的机制(即编码转基因的表达)的了解很少，rAAV载体在临床上缺乏成功。这项建议旨在描述依赖受体/辅受体的内吞机制，以控制rAAV载体的高效转导，重点是2型血清型(RAAV2)。虽然已经确定了各种rAAV血清型的受体和辅助受体，但控制rAAV内吞作用和细胞内命运的信号机制在很大程度上尚不清楚。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膜间隔处理的效率。来自该项目的初步数据表明，依赖于1521整合素的rAAV2内吞作用比竞争进入途径导致更有效的感染。然而，rAAV2/整合素对接后调节整合素依赖的内吞作用的信号事件仍不清楚。大量文献将整合素的激活与多种细胞内信号蛋白的募集和激活联系在一起，如非受体酪氨酸激酶、粘着斑激酶和c-Src。已知这些整合素效应器可以结合和调节rac1和磷脂酰肌醇3-激酶(PI3K)，这两种途径都被证明可以调节rAAV2的内吞作用和囊泡运输。我们的长期目标是确定通过rAAV2和其他依赖整合素的AAV血清型来提高病毒转导效率的方法。这项拟议的研究将通过确定rAAV2整合素依赖的内吞作用周围的事件以及导致病毒有效核运输和转导的下游信号通路来帮助实现这一点。此外，阐明rAAV感染是如何激活细胞信号的，可能有助于该领域理解对这种病毒的先天性免疫反应的起源--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.