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.

来自许多实验室的工作，包括参与该计划项目的实验室，证明与腺相关病毒（AAV）对纠正人类疾病具有巨大的希望。 AAV2是数据最常用的血清型，显示了广泛的宿主范围和广泛的向上主义。尽管广泛的宿主范围很有用，但显然是时候开始探索开发具有更受限制或特定的对流的AAV向量或具有特殊属性的向量的方法了。特别是，如果可以找到将AAV向量靶向特定组织的方法，那将非常有帮助。向量靶向仍处于所有矢量的起步阶段，并且在AAV的情况下，由于相对缺乏有关衣壳组件，粒子进入和细胞内运输的信息，因此尤其受到阻碍。为了解决这些问题，该提案部分集中在（在上一个资金期间），以在AAV2 Capsid基因中开发一系列的突变。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国配体插入AAV Capsid的宝贵信息，以改变病毒性的性质主义。具体目的是： 特定目的1：表征在结合病毒细胞表面受体或细胞内运输中有缺陷的突变体。特别是，将鉴定出与肝素结合的衣壳基因的表面残基，以及与内体逃生或核进入的残基。将使用分子生物学以及共焦和冷冻电子显微镜技术。 特定目的2：表征在AAV capsids组装中有缺陷的突变体。特别是，将尝试识别涉及病毒装配的衣壳组件和衣壳区域的中间体。 特定目标3：最后，将通过将外核配体插入遗传筛选中确定为非必需品的衣壳编码区域来尝试改变AAV2 CAPSID的对流。这将包括在体内对适当的外国配体进行测试，以查看AAV的生物分布是否可以显着更改。 预计这些研究将产生有价值的信息，这些信息将影响使用AAV矢量实际上所有基因治疗研究。