AAV capsids and their cellular interactions

AAV 衣壳及其细胞相互作用

基本信息

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
项目类别：
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

项目摘要

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 (AAV) 是属于依赖细小病毒属的 ssDNA 包装病毒基于 AAV 的细小病毒科基因传递系统的研究最近进入了 FDA 的激动人心的阶段。 Luxturna 获批，这是一种基于 AAV 血清型 2 (AAV2) 的基因疗法，用于治疗单基因缺陷然而，Luxturna 的成功是由于眼睛是一个免疫特权器官，并且是一个事实。直接给药避免了预先存在的宿主免疫，这仍然是治疗的一个重大挑战。 AAV 基因传递系统的功效最近，博卡细小病毒属的成员。细小病毒科也已被开发为病毒基因传递载体，也用于治疗单基因疾病。然而，针对 AAV 和博卡病毒 (BoV) 的宿主抗体血清阳性率很高，≥70%，代表了这两个系统的全面治疗实现的重大挑战。使用小鼠单克隆抗体 (Mab) 来表征灵长类 AAV 的抗原结构，如它们与受体附着、组织向性和转导效率的衣壳决定因素有关（基因我们率先利用这些信息对能够逃脱的 AAV 载体进行分子工程。抗体识别并目前正在评估其作为潜在的临床载体。确认通过对每种 AAV 血清型的多个小鼠单克隆抗体进行图谱获得的“多克隆”信息研究概括了多克隆人类反应在这个更新应用中，我们将描述这种能力。人类和非人类灵长类动物 (NHP) 血清中和或结合但不中和载体转导。将指导抗体逃逸和/或转导功效的工程设计，从而扩大治疗效用。我们的病毒模型包括非灵长类 AAV 和 BoV 载体，以扩大细小病毒库我们的三个具体目标将提出四个新问题：（1）“灵长类动物抗体是否共享？与先前描述的鼠单克隆抗体的表位？”(2)“中和性和非-的结合位点吗？ (3)“非灵长类 AAV 会自然地逃避预先存在的中和作用吗？灵长类抗体并能够转导人类细胞？”以及（4）“我们可以设计其抗原位点吗？ BoV 载体可逃避抗体的中和作用，同时保留或改善亲本转导效率？”。我们将使用冷冻电子显微镜和图像重建来确定高分辨率。非灵长类 AAV 和 BoV 衣壳的结构，单独或与聚糖受体复合，分辨率≤3 Å 以及 AAV/BoV 衣壳 – 人/NHP 抗体的结构，分辨率为 3 至 4 Å 这是常规操作。在我们的小组中，我们将利用获得的信息来设计保留其细胞结合特性的载体。我们将在 IgG 存在的情况下在体外和体内评估这些载体。我们将分别创建新的临床生物制剂，就像上一轮融资所做的那样，以扩大规模。细小病毒病毒载体库，即可治疗疾病目标的数量以及可治疗患者的队列。