Biology of Adeno-Associated Viral Vectors

许多实验室，包括参与这一项目的实验室的工作已经证明，腺相关病毒(AAV)对纠正人类疾病具有重要的前景，这项工作表明，AAV可以有效地将基因转移到体内的原代细胞中，并且在大多数情况下，转基因的表达似乎是长期的。此外，在该计划项目中所做的工作还导致了新的和 潜在可扩展的培养rAAV的方法，以及产生不含野生型病毒或其他污染物的高滴度重组病毒的纯化rAAV的方法。大部分工作都集中在AAV2上，这种血清型显示出广泛的宿主范围和广泛的趋向性，涉及它可以转导的分化细胞的类型。最近对其他AAV血清型的研究表明，它们也有广泛的，尽管略有不同 向心性。虽然广泛的宿主范围是有用的，但显然是时候开始探索开发具有更受限或特定取向的AAV载体或具有特殊特性的载体的方法了。特别是，如果能找到将AAV载体靶向特定组织的方法，将是非常有帮助的。然而，载体靶向仍处于初级阶段，在AAV病例中，由于相对缺乏关于衣壳蛋白的信息，尤其受到阻碍。 组装、粒子进入和细胞内运输。为了促进我们正在进行的靶向研究，有必要了解AAV衣壳结构的基本生物学。为了解决这些问题，本建议重点研究AAV2衣壳基因的结构功能，并确定与AAV2最不相似的两个AAV4和AAV5血清型的晶体结构。具体目的是：1)我们将使用位点特异性 突变以确定在2折叠和5折叠对称轴上潜在参与维持衣壳完整性的氨基酸。2)我们将确定核定位所需的衣壳蛋白区域。3)我们将用X射线结晶学确定AAV4和AAV5的原子结构，并用低温电子显微镜绘制唾液酸结合区。预计这些研究将产生有价值的信息，将 对使用AAV载体进行几乎所有的基因治疗研究的影响。

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 diseases, This work has shown that AAV can be used to transfer genes efficiently into primary cells in vivo, and that in most cases, expression of the transgene appears to be long lived. Additionally, the work done in this program project has led to the development of new and potentially scalable methods for growing rAAV, as well as methods for purifying rAAV that produce high titer recombinant virus that is free of wild type virus or other contaminants. Much of this work has focused on AAV2, a serotype that shows a broad host range and a broad tropism with respect to the types of differentiated cells that it can transduce. Recent work with other AAV serotypes suggests that they too have a broad although somewhat different 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. However, vector targeting is still in its infancy and is particularly hampered in the case of AAV by the relative lack of information about capsid assembly, particle entry and intracellular trafficking. To facilitate our ongoing studies of targeting, it will be necessary to understand the basic biology of AAV capsid structure. To address these problems, this proposal focuses on structure function studies of the AAV2 capsid genes and on the determination of the crystal structure of the two most dissimilar AAV serotypes when compared to AAV2, namely AAV4 and 5. The specific aims are: 1) We will use site specific mutagenesis to identify the amino acids that are potentially involved in maintaining capsid integrity at the 2 fold and the 5 fold axes of symmetry. 2) We will identify regions of the capsid proteins required for nuclear localization. 3) We will determine the atomic structure of AAV4 and AAV5 using X-ray crystallography and map the sialic acid binding regions using cryoelectron microscopy. 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.