In vivo PET imaging of novel engineered AAVs informs capsid design

新型工程 AAV 的体内 PET 成像为衣壳设计提供信息

• 批准号: 10400047
• 负责人: Katherine W Ferrara
• 金额: $ 68.54万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10400047
• 关键词: Address; Affinity; Alzheimer' s Disease; Animals; Antibodies; Binding; Blood - brain barrier anatomy; Brain; Capsid; Capsid Proteins; Cells; Custom; DNA; Data; Dependovirus; Directed Molecular Evolution; Disease; Dose; Drug Kinetics; Encapsulated; Endothelial Cells; Endothelium; Engineering; Evolution; Flow Cytometry; Future; Gastrointestinal tract structure; Gene Delivery; Gene Expression; Gene Proteins; Gene Transfer; Generations; Genetic Crossing Over; Hepatocyte; Herpesvirus 1; Hour; Human; Image; Imaging Techniques; Inbred BALB C Mice; Injections; Intravenous; Joints; Kidney; Kupffer Cells; Label; Laboratories; Liver; Lung; Macaca mulatta; Mediating; Medicine; Methods; Mouse Strains; Mus; Nature; Neurons; Noise; Optical reporter; Optics; Organ; Peptides; Peripheral; Positron-Emission Tomography; Primates; Proteins; Pyruvate Kinase; Radioactive; Rat Strains; Receptor Cell; Reporter; Reporter Genes; Reporting; Rodent; Series; Serotyping; Signal Transduction; Spleen; Structure; Surface; System; Testing; Therapeutic; Time; Tissues; Tracer; Transduction Gene; Transfection; Transgenes; Translating; Variant; Viral; Virus Receptors; adeno-associated viral vector; base; blood-brain barrier crossing; body system; cell type; design; imaging modality; in vivo; innovation; insight; mouse model; nanoscale; nanotherapeutic; neutralizing antibody; novel; particle; receptor; receptor binding; species difference; targeted treatment; therapy development; transcytosis; translational study; uptake

Delivery of gene and protein therapy to the brain has traditionally been extremely limited. Using a directed evolution approach to viral capsid engineering and selection, the Gradinaru group at Caltech identified specific peptides that, when displayed on the surface of modified capsids, enhanced neuronal transduction compared to the conventionally-used adeno-associated virus AAV9, following intravenous (IV) injection in mice. The brain uptake of these novel AAVs studied here by PET imaging by the Ferrara and Gambhir groups at Stanford reaches an extraordinary temporal-peak spatial-maximum of ~35% ID/cc at 4 h post-injection in mouse models. Our quantitative analysis yields a 50-fold enhancement in the brain receptor affinities as compared with earlier AAVs. Recent selections at Caltech have provided additional capsids that transduce the mouse brain with reduced expression in the liver, spleen, kidneys, and lungs. However, strain and species differences in the blood-brain barrier (BBB) and transport of these capsids (e.g. high in most mouse and rat strains but low in BALB/c mice) raise questions as to the nature of transport in primates (including humans). In order to understand species/strain differences and facilitate future translational development of these therapies, we propose novel combined positron emission tomography (PET) imaging techniques that non-invasively assess the pharmacokinetics of the AAV over the first days after injection and the resulting gene expression over months or potentially years. We plan to address key issues by assessing receptor binding, transcytosis and neutralizing antibody (NAb) effects across species. There are several innovative aspects to our approach. First, the radioactive tag used for tracking the capsid is based on a multichelator, increasing the signal-to-noise ratio and allowing us to assess binding to the brain endothelium (key for effective BBB crossing) over the first minutes and hours after injection. Second, PET analysis of AAVs as nanometer-scale therapeutics allows us to non-invasively estimate accumulation and clearance. Our data suggest receptor-mediated accumulation of the engineered AAVs on the brain endothelium over the first few minutes after injection. Third, for real-time reporting on gene transduction, we include a dual reporter system. The HSV1-sr39tk reporter gene has low background in the peripheral tissues with the reporter probe [18F]FHBG. The pyruvate kinase M2 (PKM2) reporter gene has a low background level in the brain and is imaged with [18F]DASA-23, a tracer that freely crosses the BBB. For preliminary studies in rodents we add optical capsid tags and reporter genes to assess cell-specific uptake. Our resulting specific aims are to: 1) validate and apply imaging to assess receptor binding affinity, transcytosis, clearance and transduction across organ systems and 2) image new variants of AAV9 across species to gain insight into the impact of capsid structure. We hypothesize that changes in capsid structure impact both affinity and endothelial transcytosis and that quantifying the binding affinity and transcytosis of multiple variants will inform future analyses of key structural components.

传统上，基因和蛋白质疗法对大脑的输送极其有限。使用定向的 病毒衣壳工程和选择的进化方法，加州理工大学的Gradinaru小组确定了特定的 当展示在修饰衣壳表面时，增强神经元转导的多肽 在小鼠静脉注射(IV)后，与常规使用的腺相关病毒AAV9进行比较。大脑 斯坦福大学的Ferrara和Gambhir小组通过PET成像研究了这些新型AAVs的摄取情况 小鼠在注射后4h达到一个特殊的时间-峰值空间最大值~35%ID/cc 模特们。我们的定量分析显示，大脑感受器的亲和力比 使用较早的AAV。加州理工学院最近的选择提供了更多的衣壳来转导老鼠 在肝、脾、肾和肺中表达减少的脑。然而，菌株和物种的差异 在血脑屏障(BBB)和这些衣壳的运输(例如，在大多数小鼠和大鼠品系中高，但低 在BALB/c小鼠中)提出了关于灵长类动物(包括人类)运输的性质的问题。为了 了解物种/菌株的差异并促进这些疗法的未来翻译开发，我们 提出一种新的非侵入性评估正电子发射断层成像(PET)的新技术 AAV在注射后头几天的药代动力学及由此产生的基因表达 几个月甚至几年。我们计划通过评估受体结合、跨细胞和 中和抗体(NAB)跨物种效应。我们的方法有几个创新方面。 首先，用于追踪衣壳的放射性标签是基于多配位体的，增加了信噪比 比率，并允许我们评估与脑内皮细胞的结合(有效跨越血脑屏障的关键)在第一 注射后几分钟和几小时。其次，将动静脉动静脉作为纳米级疗法的正电子发射计算机断层扫描分析使我们能够 非侵入性估计累积和清除。我们的数据表明，受体介导的细胞内 在注射后的最初几分钟内，对大脑内皮细胞进行了改造的AAVs。第三，对于实时 关于基因转导的报道，我们包括一个双重报告系统。HSV1-sr39tk报告基因低 背景在外周组织中用报告探针[18F]FHBG。丙酮酸激酶M2(PKM2) 报告基因在大脑中的背景水平很低，并用[18F]DASA-23进行成像，这是一种自由的示踪剂 穿过BBB。在啮齿动物的初步研究中，我们添加了光学衣壳标签和报告基因来评估 细胞特异性摄取。我们最终的具体目标是：1)验证和应用成像来评估受体结合 亲和力、跨细胞、清除和跨器官转导和2)AAV9新变种的图像 以深入了解衣壳结构的影响。我们假设衣壳的变化 结构影响亲和力和内皮细胞穿透作用，定量结合亲和力和 多个变异体的跨细胞作用将为未来对关键结构成分的分析提供信息。