Biology of Adeno-Associated Viral Vectors

来自许多实验室的工作，包括参与该计划项目的实验室，已经证明腺相关病毒（AAV）对纠正人类疾病具有重要的希望。这项工作表明，AAV可以用于将基因有效地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存在的。此外，在该计划项目中所做的工作导致了新的和 用于培养rAAV的潜在可扩展的方法，以及用于纯化rAAV的方法，所述方法产生不含野生型病毒或其它污染物的高滴度重组病毒。这项工作的大部分集中在AAV2上，AAV2是一种血清型，其显示出广泛的宿主范围和相对于其可以抑制的分化细胞类型的广泛向性。最近的工作与其他AAV血清型表明，他们也有广泛的，但有些不同， 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。然而，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蛋白的信息而受到特别的阻碍。 组装、颗粒进入和细胞内运输。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，该提议集中于AAV2衣壳基因的结构功能研究和与AAV2相比时两种最不相似的AAV血清型（即AAV4和5）的晶体结构的测定。具体目标是：1）我们将使用特定的网站 诱变以鉴定潜在参与在2倍和5倍对称轴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的区域。3)我们将使用X射线晶体学确定AAV4和AAV5的原子结构，并使用冷冻电子显微镜绘制唾液酸结合区域。预计这些研究将产生宝贵的信息， 对几乎所有基因治疗研究中使用AAV载体的影响。

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.