AAV capsids and their cellular interactions

AAV 衣壳及其细胞相互作用

• 批准号: 10061606
• 负责人: ROBERT MCKENNA
• 金额: $ 41.08万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-17 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10061606
• 关键词: Address; Animal Model; Antibodies; Antibody Binding Sites; Binding; Binding Sites; Biological; Biological Assay; Birds; Brain; Capsid; Cattle; Cell Surface Receptors; Cells; Chiroptera; Clinical; Complex; Cryoelectron Microscopy; Data; Defect; Dependovirus; Disease; Engineering; Epitope Mapping; Epitopes; Evaluation; Exposure to; Eye; Funding; Gene Delivery; Gene Expression; General Population; Genetic Diseases; Glycogen storage disease type II; Gorilla gorilla; Human; Immune; Immune response; Immunity; Immunoglobulin G; In Vitro; Infection; Intravenous Immunoglobulins; Liver; Lung; Mammalian Cell; Maps; Mediating; Modeling; Modification; Molecular; Monoclonal Antibodies; Mus; Organ; Outcome; Parvoviridae; Parvovirus; Patients; Pattern; Phase; Polysaccharides; Population; Primates; Property; Proteins; Resolution; Seroprevalences; Serotyping; Serum; Serum Proteins; Single Stranded DNA Virus; Sinus; Site; Skeletal Muscle; Snakes; Structure; Surface; System; Testing; Therapeutic; Tissues; Treatment Efficacy; Viral; Viral Packaging; Viral Vector; Virus; adeno-associated viral vector; antibody engineering; base; clinical efficacy; clinically relevant; cohort; comparative; gene delivery system; gene therapy; glycosylation; human tissue; image reconstruction; improved; in vivo; member; murine monoclonal antibody; neutralizing antibody; nonhuman primate; novel; preclinical trial; protein expression; receptor; response; success; therapeutic gene; tissue tropism; transduction efficiency; transgene expression; vector; viral gene delivery

The Adeno-associated viruses (AAVs) are ssDNA packaging viruses belonging to the Dependoparvovirus genus of the Parvoviridae. Gene delivery systems based on the AAVs recently entered an exciting phase with the FDA approval of Luxturna, an AAV serotype 2 (AAV2)-based gene therapy for treating a monogenetic defect in the eye. However, the success of Luxturna was ushered by the fact that the eye is an immune privileged organ and direct administration avoids pre-existing host immunity. This remains a significant challenge to the therapeutic efficacy of the AAV gene delivery system. More recently, members of the Bocaparvovirus genus of the Parvoviridae have also been developed as viral gene delivery vectors, also for treating monogenetic diseases. However, high level of seroprevalence of host antibodies against AAVs and bocaviruses (BoVs), at ≥70%, represents a major challenge to full therapeutic realization of both systems. The primary focus of this project has been to characterize the antigenic structures of primate AAVs, using mouse monoclonal antibodies (Mabs), as they relate to capsid determinants of receptor attachment, tissue tropism, and transduction efficiency (gene expression). We pioneered the use of this information for molecular engineering of AAV vectors able to escape antibody recognition and currently under evaluation as potential clinical vectors. However, there is need to confirm that the “polyclonal” information obtained by mapping several mouse Mabs for each AAV serotype studied recapitulates the polyclonal human response. In this renewal application, we will characterize the ability of human and non-human primate (NHP) sera to neutralize or bind and not neutralize vector transduction. This will guide the engineering of antibody escape and/or transduction efficacy and thus therapeutic utility. We expand our viral models to include non-primate AAVs and BoV vectors in an effort to expand the pool of parvoviral vectors available for use. Our three specific aims will ask four new questions: (1) “Do primate antibodies share epitopes with the previously described murine Mabs?” (2) “Do the binding sites of neutralizing and non- neutralizing binding antibodies overlap”? (3) “Do non-primate AAVs naturally escape pre-existing neutralizing primate antibodies and capable of transducing human cells?” And (4) “Can we engineer the antigenic sites on BoV vectors to evade neutralization by antibodies while retaining or improving the parental transduction efficiency?”. We will use cryo-electron microscopy and image reconstruction to determine high-resolution structures of non-primate AAVs and BoV capsids, alone and in complex with glycan receptors, to ≤3 Å resolution and the structures of AAV/BoV capsid – human/NHP antibodies to between 3 to 4 Å resolution. This is routine in our group. We will use the information obtained to engineer vectors that retain their cell binding properties but evade recognition by human/NHP. We will evaluate these vectors in vitro and in vivo in the presence of IgG and IVIG, respectively. We will create new clinical biologics, as was done in the past funding round, to expand the parvovirus viral vector repertoire, thus the number of treatable diseases targets, and cohort of treatable patients.

腺相关病毒（AAVS）是属于依赖类病毒属的ssDNA包装病毒 palvoviridae。基于AAVS的基因输送系统最近与FDA进入了令人兴奋的阶段 批准Luxturna，一种基于AAV血清型2（AAV2）的基因疗法，用于治疗单基因缺陷 眼睛。但是，卢克斯特纳（Luxturna）的成功是因为眼睛是免疫特权器官和 直接管理避免了先前存在的宿主免疫力。这仍然是治疗性的重大挑战 AAV基因输送系统的功效。最近，BOCAPARVOVIRUS属的成员 phvoviridae也已作为病毒基因递送载体开发，也用于治疗单基因疾病。 但是，宿主抗体对AAV和Bocaviruses（BOV）的高含量，≥70％， 对于两种系统的完全治疗实现来说，这是一个主要挑战。该项目的主要重点是 我们要使用小鼠单克隆抗体（mAb）将灵长类动物AAV的抗原结构表征为 它们与衣壳确定受体的附着，组织的乳房和翻译效率（基因）有关 表达）。我们率先将这些信息用于AAV矢量的分子工程可以逃脱 抗体识别，目前被评估为潜在的临床向量。但是，需要 确认通过为每种AAV血清型绘制几个鼠标mAb获得的“多克隆”信息 螺头概括了多克隆人的反应。在此续订应用程序中，我们将表征能力 人类和非人类灵长类动物（NHP）血清的中和，而不是中和矢量翻译。这 将指导抗体逃逸和/或翻译效率的工程，从而导致治疗效用。我们扩展 我们的病毒模型包括非顶峰AAV和BOV矢量，以扩大细小病毒库 向量可供使用。我们的三个具体目标将提出四个新问题：（1）“进行隐私抗体分享 先前描述的鼠mab的表位？ （2）“做中和的结合位点 中和结合抗体重叠”？（3） 灵长类动物抗体并能够转导人细胞？”和（4）“我们可以在 在保留或改善父母转移的同时，通过抗体逃避神经化的BOV载体 效率吗？”。我们将使用低温显微镜和图像重建来确定高分辨率 单独和与聚糖受体复杂的非顶峰AAV和BOV衣壳的结构≤3Å分辨率 以及AAV/BOV衣壳的结构 - 分辨率为3至4Å之间的人/NHP抗体。这是例行程序 在我们的小组中。我们将使用获得的信息，以保留其细胞结合属性但 人类/NHP的逃避认可。我们将在IgG存在的情况下在体外和体内评估这些载体 IVIG分别。我们将像过去的资金一样，创建新的临床生物制剂，以扩展 细节病毒病毒载体库，因此可治疗疾病的靶标数量和可治疗患者队列。