Structural studies of AAV capsids and their glycan receptor interactions

描述（由申请人提供）：腺相关病毒（AAV）与任何疾病无关，并且其包装非基因组DNA和包装不同细胞/组织群体以进行纠正性基因递送的能力引起了人们对了解其基础生物学的极大兴趣。这包括它们的衣壳结构、细胞向性和用于进入、脱壳、复制、DNA包装、衣壳组装和抗体中和的相互作用。目标是提高它们作为载体的特异性和效力。然而，虽然大多数AAV的表征针对血清型2（AAV 2），但对一些最近鉴定的抗原性不同的人和灵长类动物病毒的研究显示，与AAV 2相比，特定细胞类型的转导特性增强。这种特性是由它们的衣壳序列介导的。因此，在为基因治疗群体提供用于开发的潜在AAV载体的更广泛选择的同时，通过鉴定与其不同的组织向性和转导表型以及其抗原反应性相关的其他AAV的衣壳特征，将极大地帮助特异性组织/器官靶向以改善安全性和功效以及工程化更快的转导起始。本提案的总体目标是对AAV衣壳进行结构-功能分析，以鉴定以下特征：（I）确定差异细胞向性;（II）影响转导效率;（III）用于细胞受体识别。该分析还将提供关于保守的衣壳特征的信息，因此对于病毒衣壳组装相互作用的保真度可能是重要的，以及关于决定AAV进化枝组的不同抗原性的衣壳区域的信息。遗传操纵这些特征可以产生新一代的校正病毒基因递送载体，其在组织嗜性和特异性、转导效率以及逃避现有宿主免疫应答的能力方面具有协同改进。为了实现我们的目标，我们将通过X射线晶体学确定AAV进化枝组的代表性成员的衣壳结构，单独地和与鉴定的碳水化合物受体复合，并使用诱变、细胞结合和转导测定以及结合亲和力的生物物理测量功能性地注释参与受体相互作用的AAV衣壳区域。我们已经开发了一个可扩展的杆状病毒系统，用于表达野生型和突变型病毒衣壳。

DESCRIPTION (provided by applicant): The Adeno-associated viruses (AAVs) are not associated with any diseases and their ability to package non-genomic DNA and to transduce different cell/tissue populations for corrective gene delivery has generated significant interest in understanding their basic biology. This includes their capsid structure, cellular tropism and interactions for entry, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The goal is to improve their specificity and efficacy as vectors. However, while the majority of the characterization of the AAVs has been directed at serotype 2 (AAV2), studies on some of the more recently identified antigenically distinct human and primate viruses show enhanced transduction properties for particular cell types compared to AAV2. This property is mediated by their capsid sequence. Thus while providing the gene therapy community with a more expansive choice of potential AAV vectors for development, specific tissue/organ targeting for improving safety profiles and efficacy as well as engineering a faster onset of transduction would be greatly aided by identifying the capsid features of the other AAVs that correlates with their distinct tissue tropism and transduction phenotypes as well as their antigenic reactivities. The overall objective of this proposal is a structure-function analysis of the AAV capsid to identify features that (I) determine differential cell tropism; (II) affect transduction efficiency, (III) and are utilized for cell receptor recognition. The analysis will also provide information on capsid features that are conserved and thus could be important for the fidelity of viral capsid assembly interactions, and on capsid regions that dictate the distinct antigenicity of the AAV clade groups. Genetically manipulating these features could give rise to a new generation of corrective viral gene delivery vectors with synergistic improvements in tissue tropism and specificity, transduction efficiencies, and the ability to evade existing host immune responses. To achieve our objectives, we will determine the capsid structures for representative members of the AAV clade groups, alone and in complex with identified carbohydrate receptors, by X-ray crystallography, and functionally annotate AAV capsid regions involved in the receptor interactions using mutagenesis, cell binding and transduction assays, and biophysical measurements of binding affinity. We have developed a scaleable baculovirus system for the expression of wild type and mutant virus capsids for these studies.