Biology of Adeno-Associated Viral Vectors

腺相关病毒载体的生物学

• 批准号: 6853356
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 18.8万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853356
• 关键词: DNA repair; DNA replication; X ray crystallography; adeno associated virus group; aminoacid; capsid; confocal scanning microscopy; cryoelectron microscopy; gene delivery system; gene therapy; helicase; intermolecular interaction; nucleic acid repetitive sequence; nucleic acid sequence; protein localization; protein purification; protein structure function; sialate; site directed mutagenesis; structural biology; transfection /expression vector; virus genetics; virus protein

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.

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。这项工作表明，AAV 可用于将基因有效地转移到原代细胞中。体内细胞，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，该计划项目中所做的工作还导致了新的和 潜在可扩展的 rAAV 培养方法，以及纯化 rAAV 的方法，以产生不含野生型病毒或其他污染物的高滴度重组病毒。这项工作的大部分重点是 AAV2，这是一种血清型，在其可转导的分化细胞类型方面表现出广泛的宿主范围和广泛的趋向性。最近对其他 AAV 血清型的研究表明，它们也具有广泛但略有不同的特征 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。然而，载体靶向仍处于起步阶段，并且由于衣壳信息相对缺乏，在 AAV 方面尤其受到阻碍 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，本提案重点关注 AAV2 衣壳基因的结构功能研究，以及确定与 AAV2 相比最不相似的两种 AAV 血清型（即 AAV4 和 5）的晶体结构。具体目标是： 1) 我们将使用特定于站点的 诱变来鉴定可能参与维持衣壳在 2 倍和 5 倍对称轴完整性的氨基酸。 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有价值的信息 对 AAV 载体在几乎所有基因治疗研究中的使用产生影响。