RE-ENGINEERING AAV GENOME PACKAGING

重新设计 AAV 基因组包装

• 批准号: 9919589
• 负责人: Aravind Asokan
• 金额: $ 48.64万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9919589
• 关键词: Address; Affect; Anti-Inflammatory Agents; Biological; Biological Assay; Biology; Capsid; Capsid Proteins; Cell Culture Techniques; Charge; Clinic; Clinical; Clinical Data; Clinical Research; Clinical Trials; DNA; Data; Defective Viruses; Dependovirus; Detection; Development; Directed Molecular Evolution; Dose; Engineering; Europe; Gene Expression; Gene Transfer; Genome; Goals; Hepatotoxicity; Heterogeneity; Human; Hybrids; Immunologics; In Vitro; Indiana; Knowledge; Length; Liver; Maps; Mass Spectrum Analysis; Mediating; Methods; Methylprednisolone; Molecular Virology; Nuclear; Outcome; Parvovirus; Patients; Pattern; Pharmaceutical Preparations; Preparation; Production; Protein Engineering; Protein Subunits; Recombinant adeno-associated virus (rAAV); Recombinants; Regimen; Reporting; Safety; Sampling; Serum; Steroids; Structure; Surface; System; Techniques; Technology; Testing; Toxic effect; Transaminases; Validation; Viral; Viral Genome; Virion; adeno-associated viral vector; arm; density; design; gene therapy; improved; in vivo; insight; mouse model; mutant; novel; particle; preclinical study; prospective; rapid technique; screening; side effect; success; therapy outcome; trafficking; vector; vector genome

ABSTRACT Recombinant adeno-associated viruses (AAV) are promising vectors for human gene therapy. Despite regulatory approval and a rapidly growing pipeline, clinical data have indicated that vector dose-related hepatotoxicity is a significant concern. Recent preclinical studies suggest that heterogeneity in AAV vector preparations, including the presence of defective interfering particles with partial or truncated genomes contribute to such undesirable side effects. Although somewhat resolvable with anti-inflammatory drug regimens, permanent loss of gene expression has been reported. We postulate that improvements to AAV vector design and composition are likely to improve the safety profile. To achieve the latter goals, a better understanding of particle heterogeneity in AAV vectors packaging different genomes as well as the impact of such on AAV biology is essential. In the current proposal, we will first advance a potentially disruptive technology, charge detection mass spectrometry (CDMS) to help map the mass landscape of vector compositions associated with genome packaging. We will then bridge this basic information with new directions in AAV capsid engineering with the goal of improving compositional homogeneity and reducing the potential for side effects. Each aim is tailored to directly interrogate the proposed correlation between the composition of recombinant vectors with viral infectivity in cell culture as well as gene transfer efficiency and toxicity in mouse models. We expect our efforts to (i) offer significant, new insight into parvoviral capsid-genome interactions, (ii) enable the development and validation of a novel analytical technique for rapid screening and quality testing of clinical AAV samples, and (iii) improve AAV gene transfer efficiency and safety profile through capsid engineering.

抽象的 重组腺相关病毒（AAV）是人类基因治疗的有希望的载体。尽管 监管部门的批准和快速增长的管道，临床数据表明载体剂量相关 肝毒性是一个重要问题。最近的临床前研究表明 AAV 载体的异质性 制剂，包括存在具有部分或截短基因组的有缺陷的干扰颗粒 导致这种不良副作用。虽然用消炎药可以稍微缓解 据报道，基因表达永久丧失。我们假设 AAV 的改进 载体设计和组成可能会提高安全性。为了实现后一个目标，更好 了解包装不同基因组的 AAV 载体中的颗粒异质性以及 这样对AAV生物学至关重要。在当前的提案中，我们将首先提出一个可能具有颠覆性的 技术、电荷检测质谱 (CDMS) 来帮助绘制矢量的质量图 与基因组包装相关的组合物。然后我们将把这些基本信息与新的方向联系起来 在 AAV 衣壳工程中，目标是提高成分均质性并降低潜在的 副作用。每个目标都是为了直接询问所提出的组成之间的相关性而定制的 重组载体在细胞培养中具有病毒感染性以及在小鼠中的基因转移效率和毒性 模型。我们希望我们的努力能够（i）为细小病毒衣壳-基因组相互作用提供重要的新见解，（ii） 能够开发和验证一种新颖的分析技术，用于快速筛选和质量测试 临床 AAV 样本，以及 (iii) 通过衣壳提高 AAV 基因转移效率和安全性 工程。