AAV capsid assembly, viral entry, and viral tropism

AAV 衣壳组装、病毒进入和病毒向性

• 批准号: 7115888
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 31.9万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7115888
• 关键词: adeno associated virus group; confocal scanning microscopy; cryoelectron microscopy; gene expression; genetic promoter element; genetic transcription; laboratory mouse; molecular cloning; mutant; receptor binding; recombinant virus; tissue /cell culture; transfection /expression vector; virus assembly; virus genetics; virus infection mechanism; virus protein; virus receptors

Work from a number of laboratories, including those involved in this program project, has demonstrated that Adeno-associated virus (AAV) holds significant promise for the correction of human disease. AAV2, the most commonly used serotype to data, shows a broad host range and broad tropism. Although a broad host range is useful, it is clearly time to begin exploring ways of developing AAV vectors that have a more restricted or specific tropism, or vectors that have special properties. In particular, it would be extremely helpful if methods could be found to target AAV vectors to specific tissues. Vector targeting is still in its infancy with all vectors and is particularly hampered in the case of AAV by the relative lack of information about capsid assembly, particle entry and intracellular trafficking. To address these problems, this proposal focused in part (during the previous funding period) on developing a comprehensive series of mutations in the AAV2 capsid genes. This provided a resource that can be used to study these problems. More importantly, it will provide valuable information about how and where to insert foreign ligands into the AAV capsid for the purpose of changing the viral tropism. The specific aims are: Specific aim 1: To characterize mutants that are defective in binding the viral cell surface receptors or in intracellular trafficking. In particular, surface residues of the capsid gene involved in heparin binding will be identified, as well as residues involved in endosome escape or nuclear entry. Both molecular biology and confocal and cryoelectron microscopy techniques will be used. Specific aim 2: To characterize mutants defective in assembly of AAV capsids. In particular, attempts will be made to identify the intermediates of capsid assembly and regions of the capsid that are involved in viral assembly. Specific aim 3: Finally,, attempts will be made to change the tropism of the AAV2 capsid by inserting foreign ligands to the capsid coding regions identified as non-essential in the genetic screens. This will include testing of appropriate foreign ligands in vivo to see if the biodistribution of AAV can be significantly changed. It is anticipated that these studies will produce valuable information that will impact on the use of AAV vectors for virtually all gene therapy studies.

许多实验室（包括参与该计划项目的实验室）的工作表明，腺相关病毒（AAV）对于纠正人类疾病具有重大前景。 AAV2 是数据中最常用的血清型，显示出广泛的宿主范围和广泛的趋向性。尽管广泛的宿主范围是有用的，但现在显然是时候开始探索开发具有更受限制或特定向性的 AAV 载体或具有特殊性质的载体的方法了。特别是，如果能够找到将 AAV 载体靶向特定组织的方法，那将非常有帮助。所有载体的载体靶向仍处于起步阶段，并且在 AAV 的情况下尤其受到阻碍，因为相对缺乏有关衣壳组装、颗粒进入和细胞内运输的信息。为了解决这些问题，该提案的部分重点（在上一个资助期间）开发 AAV2 衣壳基因的一系列全面突变。这提供了可用于研究这些问题的资源。更重要的是，它将提供有关如何以及在何处将外源配体插入 AAV 衣壳以改变病毒向性的有价值的信息。具体目标是： 具体目标 1：表征在结合病毒细胞表面受体或细胞内运输方面存在缺陷的突变体。特别是，将鉴定参与肝素结合的衣壳基因的表面残基，以及参与内体逃逸或核进入的残基。将使用分子生物学、共聚焦和冷冻电子显微镜技术。 具体目标 2：表征 AAV 衣壳组装缺陷的突变体。特别是，将尝试鉴定衣壳组装的中间体和参与病毒组装的衣壳区域。 具体目标 3：最后，将尝试通过将外源配体插入遗传筛选中确定为非必需的衣壳编码区来改变 AAV2 衣壳的向性。这将包括在体内测试适当的外源配体，看看 AAV 的生物分布是否可以显着改变。 预计这些研究将产生有价值的信息，影响 AAV 载体在几乎所有基因治疗研究中的使用。