AAV capsid assembly, viral entry, and viral tropism

包括参与该计划项目的实验室在内的许多实验室的工作表明，腺相关病毒（AAV）对纠正人类疾病具有重要意义。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向性。虽然广泛的宿主范围是有用的，但显然是开始探索开发具有更有限或特异性向性的AAV载体或具有特殊性质的载体的方法的时候了。特别是，如果能够找到将AAV载体靶向特定组织的方法，这将是非常有帮助的。所有载体的载体靶向仍处于起步阶段，并且在AAV的情况下由于相对缺乏关于衣壳组装、颗粒进入和细胞内运输的信息而特别受到阻碍。为了解决这些问题，该提案部分集中于（在先前的资助期间）开发AAV2衣壳基因中的一系列全面突变。这提供了一个可以用来研究这些问题的资源。更重要的是，它将提供关于如何以及在何处将外源配体插入AAV衣壳以改变病毒嗜性的有价值的信息。具体目标是： 具体目标1：表征在结合病毒细胞表面受体或在细胞内运输中有缺陷的突变体。特别地，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。将使用分子生物学和共聚焦和冷冻电子显微镜技术。 具体目的2：表征AAV衣壳组装缺陷的突变体。特别地，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到在遗传筛选中鉴定为非必需的衣壳编码区来改变AAV 2衣壳的向性。这将包括在体内测试适当的外源配体，以观察是否可以显著改变AAV的生物分布。 预计这些研究将产生有价值的信息，这些信息将影响几乎所有基因治疗研究中AAV载体的使用。

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.