Enhancing Gene Therapy by Designing Chimeric AAV Virions

描述（由申请人提供）：腺相关病毒（AAV）是一种非致病性细小病毒，是一种有吸引力的基因治疗载体。AAV的血清型表现出细胞嗜性和长期转基因表达的多样性，尽管其医学应用受到限制，因为感染是非常低效的。AAV是一种低效的病毒，部分原因是病毒的细胞表面附着和内吞作用并不总是导致转基因表达，因为成功的感染取决于AAV逃离内体，正确地通过胞质溶胶运输，并将其基因组递送到细胞核。目前的研究重点是确定病毒衣壳的功能结构域，允许有效感染特定的细胞类型，以促进基因治疗的理想病毒粒子的设计。病毒蛋白1（VP 1）是三种衣壳蛋白之一，含有一个独特的部分（VP 1up），它具有一个假定的磷脂酶基序和核定位信号，是AAV感染的亚细胞事件所必需的。虽然VP 1up的功能尚不完全清楚，但已知相关细小病毒中的类似结构域比AAV中的结构域更强大。因此，本研究的目的是：1）阐明VP 1up调控AAV内体逃逸、亚细胞运输和转基因递送的机制：2）确定通过合理设计或随机结构域改组突变VP 1up是否可以增强AAV转导;和3）当替代的VP 1up被替换到AAV中时，如果组织嗜性改变或脑中的感染效率增加，则在体内建立。简而言之，这些目标将通过分子工程改造嵌合AAV病毒体来实现，所述嵌合AAV病毒体掺入改变的VP 1蛋白，然后使用多种测定法测试这些病毒体的DNA包装、细胞附着、磷脂酶活性、内体逃逸、核靶向和转基因表达。此外，病毒感染将在体外细胞系中通过时程免疫荧光成像，并在小鼠脑中通过跟踪生物发光病毒体体内成像。AAV目前正在几项临床试验中作为基因递送载体进行测试，并且对于特异性和有效治疗而言，最大化递送到靶组织中变得越来越重要。该项目将有助于更清楚地定义在大脑中有效的AAV介导的基因递送所必需的病毒组分的作用。通过操纵这些成分，很快就有可能设计出专门用于将治疗药物输送到受许多不同遗传疾病影响的细胞的病毒体。

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV), a non-pathogenic parvovirus, is an attractive vehicle for gene therapy. Serotypes of AAV exhibit both diversity in cellular tropism and long-term transgene expression, although its medical application is limited since infection is highly inefficient. AAV is an inefficient virus partially because cell surface attachment and endocytosis of the virus does not always lead to transgene expression, as successful infection depends on AAV escaping the endosome, trafficking correctly through the cytosol, and delivering its genome to the nucleus. Current research focuses on identifying functional domains of the viral capsid that permit efficient infection of specific cell types in order to advance the design of ideal virions for gene therapy. One of three capsid proteins, viral protein 1 (VP1), contains a unique part (VP1up) that possesses a putative phospholipase motif and nuclear localization signal, and is necessary for subcellular events of AAV infection. Though the function of VP1up is not entirely clear, similar domains in related parvoviruses are known to be more robust than those in AAV. Thus, the following aims are proposed: 1) Elucidate the mechanism of how VP1up modulates the endosomal escape, subcellular trafficking, and transgene delivery of AAV; 2) Determine if AAV transduction can be enhanced when VP1up is mutated by rational design or random domain shuffling; and 3) Establish in vivo if tissue tropism is altered or infection efficiency is increased in the brain when an alternative VP1up is substituted into AAV. In short, these aims will be achieved by molecularly engineering chimeric AAV virions that incorporate altered VP1 proteins and then testing these virions for DNA packaging, cellular attachment, phospholipase activity, endosomal escape, nuclear targeting, and transgene expression using a variety of assays. In addition, virus infection will be imaged in cell lines in vitro by time-course immunofluorescence and in mouse brain in vivo by tracking bioluminescent virions. AAV is currently being tested as a gene delivery vehicle in several clinical trials and it is becoming increasingly important to maximize delivery into target tissues for specific and efficient therapy. This project will help to more clearly define the roles of viral components necessary for efficient AAV-mediated gene delivery in the brain. Through manipulating these components, it will soon be possible to design virions that are specialized for delivering therapeutics to cells affected by many different genetic disorders and diseases.