RE-ENGINEERING AAV GENOME PACKAGING

摘要 重组腺相关病毒（AAV）是一种很有前途的人类基因治疗载体。尽管 监管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，包括存在部分或截短基因组的缺陷性干扰颗粒 会导致这种不良的副作用。虽然用消炎药可以缓解 在一些治疗方案中，已经报道了基因表达的永久丧失。我们假设AAV的改进 载体设计和组成可能会改善安全性。为了实现后一个目标， 理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生物学是必不可少的。在目前的提案中，我们将首先提出一个潜在的破坏性建议， 技术，电荷检测质谱（CDMS），以帮助绘制矢量的质量景观 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，目的是改善组成的均一性并降低 副作用.每一个目标都是量身定制的，以直接询问 在细胞培养中具有病毒感染性以及在小鼠中具有基因转移效率和毒性的重组载体 模型我们希望我们的努力（i）提供重要的，新的见解细小病毒衣壳-基因组相互作用，（ii） 能够开发和验证一种新的分析技术，用于快速筛选和质量检测， 临床AAV样品，和（iii）通过衣壳蛋白提高AAV基因转移效率和安全性 工程.

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.