Enhancing Gene Therapy by Designing Chimeric AAV Virions

描述(申请人提供)：腺相关病毒(AAV)是一种非致病性的细小病毒，是一种有吸引力的基因治疗载体。AAV的血清型在细胞趋向性和长期转基因表达方面表现出多样性，但由于感染效率极低，其医学应用受到限制。AAV是一种低效病毒，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，因为成功的感染依赖于AAV逃脱内体，正确地通过细胞质运输，并将其基因组送到细胞核。目前的研究集中在确定病毒衣壳的功能结构域，使其能够有效地感染特定类型的细胞，以推进理想的病毒粒子的基因治疗设计。三种衣壳蛋白之一的病毒蛋白1(VP1)含有一个独特的部分(VP1up)，该部分具有可能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VP1up的功能尚不完全清楚，但已知相关细小病毒中类似的结构域比AAV中的结构域更强大。因此，提出了以下目标：1)阐明VP1up如何调控AAV的内体逃逸、亚细胞转运和转基因转运；2)通过合理的设计或随机结构域改组，确定VP1up突变是否可以增强AAV的转导；3)在体内建立当替代VP1up为AAV时，组织趋向性是否改变或脑内感染效率是否增加。简而言之，这些目标将通过分子工程结合改变的VP1蛋白的嵌合AAV病毒粒子来实现，然后使用各种分析方法测试这些病毒粒子的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，病毒感染在体外将通过时程免疫荧光在细胞系中成像，在体内将通过跟踪生物发光病毒粒子在小鼠脑中成像。AAV目前正作为一种基因输送载体在几个临床试验中进行测试，最大限度地将其输送到靶组织以进行特定和有效的治疗正变得越来越重要。该项目将有助于更清楚地确定有效的AAV介导的基因在大脑中传递所需的病毒成分的作用。通过操纵这些成分，很快就有可能设计出专门用于向受许多不同遗传疾病和疾病影响的细胞输送治疗药物的病毒粒子。

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.