Structure-Function of AAV - a Viral Gene Therapy Vector

描述(申请人提供)：腺相关病毒(AAV)是体内和体外基因治疗的主要候选载体，用于细胞递送DNA以纠正遗传错误、缺陷或疾病易感性。我们的长期目标是了解病毒-宿主之间的相互作用，这些相互作用调节了AAV进入特定细胞和组织的效率。这个基金会将支持广泛寻求的目标，即以自然不允许的细胞为靶标，并减少非靶标组织中转基因表达的不利影响。目的1着重于细胞进入的第一步，即与主要受体的附着，即几种人AAVs的蛋白多糖硫酸肝素(HS)。它建立在我们的AAV-2、-3和-6的结构以及这些原本同源的病毒的HS结合位点上意想不到的多样性的基础上。结晶学和电子显微镜(EM)将被用来描述AAV中进化的HS相互作用的多样性，将结构与测量的肝素亲和力、细胞附着和细胞进入相关联，通过体外转基因表达来测量。我们的目标是阐明连接的结构亲和力决定因素，这些决定因素调节进入效率和特定细胞类型的不同敏感性。我们的理解将通过工程来检验 具有改变的HS相互作用的载体，并表征它们在代表性细胞系的面板中的附着和进入。目的2阐明人多克隆中和抗体反应中的主要抗原决定簇。我们的AAV-DJ是一种嵌合重组蛋白，被选择用来逃避汇集的中和血清，其结构在EM观察到的可能占主导地位的AAV2单抗表位周围发生了变化。它开辟了一条通往人类多克隆相互作用的直接途径：从随机单点突变的文库中选择中和血清逃逸。将通过对AAV结构上的突变进行聚类来绘制优势抗原点的图谱。目的2将对体内人类基因治疗的抗中和载体的设计产生影响，并将指导单抗复合体的结构研究，使其能够最好地模拟对人类患者重要的相互作用。目的3重点研究参与细胞进入第二步的辅助受体。我们的第一个目标是使越来越多的被各种证据所牵连的完整膜蛋白清单合理化。基因组学方法，包括基因捕获、基因表达克隆和RNA干扰，将被用来对相互作用对病毒进入的功能重要性进行排名。它们还将有助于区分AAV的直接相互作用和间接影响rAAV转基因表达的细胞途径的调节。在通过互补和其他方法确认之后，将表达胞外结构域结构，用于结合亲和力的测量和通过低温EM进行结构表征或通过氢/氢交换质谱仪进行足迹表征。因此，目标3将促进对AAV生物学中一个鲜为人知的方面的基本理解，这一方面很可能在载体的细胞靶向方面也被证明是重要的。

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is a leading candidate vector for in vivo and ex vivo gene therapy, for cellular delivery of DNA to correct genetic errors deficiencies or predispositions to disease. Our long term goal is to understand the virus-host interactions that mediate the efficiency with which AAV enters specific cells and tissues. This foundation will support the widely sought goals of targeting naturally non-permissive cells, and reducing the adverse effects of transgene expression in off-target tissues. Aim 1 focuses on the first step in cell entry, attachment to the primary receptor, which is heparan sulfate (HS) proteoglycan for several human AAVs. It builds on our structures of AAV-2, -3 & -6, and unexpected diversity in the HS binding site of these otherwise homologous viruses. Crystallography and electron microscopy (EM) will be used to characterize the diversity of HS interactions that has evolved in AAV, correlating structure with measured heparin affinity, cell attachment and cell entry, measured by in vitro transgene expression. Our goal is to elucidate the structure-affinity determinants of attachment that modulate the efficiency of entry and the differential susceptibility of specific cell types. Our understanding will be tested by engineering vectors with altered HS interactions, and characterizing their attachment and entry in a panel of representative cell lines. Aim 2 will elucidate the major antigenic determinants in a human polyclonal neutralizing antibody response. Our structure of AAV-DJ, a chimeric recombinant selected to escape pooled neutralizing serum, shows change localized around a likely dominant AAV2 monoclonal epitope visualized by EM. It opens a direct path to human polyclonal interactions: selection for neutralizing serum escape from a library of random single-site mutants. Dominant antigenic sites will be mapped through clustering of mutations on the AAV structure. Aim 2 will have impact in the design of neutralization-resistant vectors for in vivo human gene therapies, and will guide structural studies of MAb complexes to those that best model interactions that are important in human patients. Aim 3 focuses on co-receptors involved in the second step of cell entry. Our first goal is to rationalize a growing list of integral membrane proteins that have been implicated by various lines of evidence. Genomic approaches, including gene trapping, cDNA expression cloning and RNA interference, will be used to rank the functional importance of interactions to viral entry. They will also help distinguish direct AAV interactions from modulation of cellular pathways indirectly affecting rAAV transgene expression. Following confirmation by complementation and other methods, ecto-domain constructs will be expressed for measurement of binding affinities and for structural characterization by cryo-EM or footprinting by hydrogen / deuterium exchange mass spectrometry. Thus, Aim 3 will advance the basic understanding of a poorly understood aspect of AAV biology that will likely also prove important in cellular targeting of vectors.