Adeno-Associated Virus Gene Therapy Vectors: Molecular Interactions on Cell Entry

项目摘要/摘要 腺相关病毒(AAV)是一种主要的基因治疗载体。 疾病和嗜好。FDA最近批准了AAV介导的脊肌治疗 萎缩症(SMA)和RPE相关性视网膜营养不良，而150项临床试验正在进行中。然而，效率低下 转导需要高剂量，具有免疫毒性的风险，这在临床试验中已经很明显。 AAV细胞转导所需的全基因组宿主蛋白筛查改变了人们的理解 AAV条目的。到目前为止尚未确定的AAVR是一个关键的受体，而AAVR的下游作用 GPR108最有可能发生内体逃逸。AAVR冷冻-EM结构将被置于生物背景下。 对GPR108的研究将从精确定位AAV和GPR108相关联的进入步骤和细胞位置开始， 以及AAV与其相互作用的域的识别。对结构的处理方法将是整体的。总括 配置将来自AAV与类天然GPR108复合的冷冻电子断层扫描(ET) 纳米盘。这将与AAV的高分辨率冷冻电子显微镜(EM)相结合 表达了GPR108的胞外区。将研究AAV构象转变的触发因素 这释放了病毒隔离的磷脂酶A2以供内体逃逸，因为AAV向细胞核输送。 AAV的抗体中和被广泛认为是通过干扰糖结合或 进入后的步骤。在发现AAVR和几个中和的结合中有重叠后，将重新评估这一点 单克隆抗体。体内普遍的结合模式将首先通过使用冷冻-EM建立 来自混合人血清的多克隆抗体。旨在逃脱的突变将被用来测试 受体结合和进入是否实际上是中和的主要机制，以及是否逃逸到 预先存在的抗体可以设计成快速中和，而不会损害受体介导的细胞进入。 计算机方法，优化原子结构与冷冻-EM图，在AAV研究期间形成原型，将 被开发和传播。模块将实现RSRef算法，在匹配地图方面是独一无二的 值，可细化实验分辨率的3D原子轮廓的总和。这将与Open相结合 源力场，开启了原子分辨低温电子显微镜在氢测定中的新兴应用 键合网络和质子化状态。对于3？以下的决议，消除不必要的限制 自由将被引入，以限制过度适应，这是EM优化中一个被低估的问题。 该项目的主要目标是从分子上了解AAV的宿主相互作用，提供结构 工程的机械基础需要在载体效率和特异性方面进行改进 开发安全的基因疗法。更广泛的间接影响将来自病毒进入的模型系统 这将受到严格的实际测试，以及提高EM结构精度的计算机方法。

Project Summary / Abstract Adeno-associated virus (AAV) is a leading delivery vector for gene therapies of a wide range of genetic disease and predilections. The FDA has recently approved AAV-mediated treatments for spinal muscular atrophy (SMA) and RPE-associated retinal dystrophy, while 150 clinical trials are ongoing. However, inefficient transduction requires high doses with risk of immunotoxicity that has been evident in clinical trials. A genome-wide screen for host proteins, needed for AAV cell transduction, changed the understanding of AAV entry. Hitherto-uncharacterized AAVR was identified as a key receptor, while the downstream role of GPR108 is most likely in endosomal escape. AAVR cryo-EM structure will be put into biological context. Studies of GPR108 will start by pinpointing the entry step and cell location where AAV and GPR108 associate, and identification of the domains with which AAV interacts. The approach to structure will be holistic. Overall configuration will come from cryo-electron tomography (ET) of AAV complexed with native-like GPR108 nanodiscs. This will be integrated with high resolution cryo-electron microscopy (EM) of AAV complexed with expressed extracellular domains of GPR108. Triggers will be investigated of an AAV conformational transition that releases a virally-sequestered phospholipase A2 for endosomal escape, as AAV traffics to the nucleus. Antibody neutralization of AAV is widely considered mediated by interference with glycan-binding or of a post-entry step. This will be reevaluated after finding overlap in the binding of AAVR and several neutralizing monoclonal antibodies. Prevalent in vivo modes of binding will first be established through cryo-EM using polyclonal antibodies from pooled human serum. Mutations, directed towards escape, will be used to test whether receptor-binding and entry are, in fact, primary mechanisms of neutralization, and whether escape to rapid neutralization by pre-existing antibodies can be designed without harm to receptor-mediated cell entry. Computer methods, optimizing atomic structure vs. cryo-EM maps, prototyped during AAV studies, will be developed and disseminated. Modules will implement the RSRef algorithm, unique in matching to map values, a sum of 3D atomic profiles of refinable experimental resolution. This will be combined with open source force fields, opening an emerging application of atomic resolution cryo-EM in determining hydrogen bonding networks and protonation states. For resolutions below 3 Å, restraints eliminating unnecessary freedom will be introduced to limit the over-fitting that is an under-appreciated problem in EM refinement. The project’s main goal is a molecular understanding of AAV’s host interactions, providing structural and mechanistic foundations for engineering needed improvements in vector efficiency and specificity in development of safe gene therapies. Broader collateral impact will come from a model system for viral entry that will be put to rigorous practical test, and computer methods for improving the precision of EM structures.