Engineering chimeric gene therapy vectors with enhanced packaging capacity

项目摘要 腺相关病毒是一种非致病性的细小病毒，是目前最重要的基因。 治疗载体。随着基因疗法越来越多地被用于治疗各种适应症，AAVs服务于 在传递治疗性DNA方面的关键作用。然而，重组转基因在AAV中的有效包装 载体仍然是成功的基因治疗的最大挑战。首先，AAV的包装能力 衣壳将转基因大小限制在4.7kb以下，排除了许多转基因的传递和基因编辑 盒式磁带。细小病毒科AAV的近亲含有更大的基因组，包括登革热病毒(DNV)，a 自然感染无脊椎动物的细小病毒亚科。我建议设计嵌合的DNV/AAV载体 支持哺乳动物细胞的强大转导，并相对于传统的 AAV载体。然后，我计划调查DNV/AAV转导，并利用这一知识来执行 在DNV/AAV衣壳上定向进化以赋予哺乳动物细胞强大的转导。第二，免疫 大剂量载体注射后AAVs的识别和清除仍然是基因研究中的一个关键问题 提供治疗，因为高剂量往往是在治疗水平上实现转基因表达所必不可少的。 需要大剂量治疗的一个原因是重组AAV载体缺乏效力 已被证明具有较低的感染性与物理颗粒比率的制剂 AAVs包装它们的内源基因组。此前的研究表明，AAV的这些差异 这种产生可能是由于AAV基因组中未特化的顺式作用包装信号所致。因此，我 目的对AAV基因组中赋予增强型基因组的新基序进行无偏见筛选 包装，并研究额外的顺式作用序列的加入如何影响重组载体 制作。总之，这些研究旨在通过加强AAV介导的基因治疗来解决核心问题 可传递的转基因的大小和重组载体生产的效力。

Project Summary Adeno-associated viruses (AAVs) are non-pathogenic parvoviruses that are currently the foremost gene therapy vectors. As gene therapy is increasingly employed to treat a variety of indications, AAVs serve a critical role in delivering therapeutic DNA. However, effective packaging of recombinant transgenes in AAV vectors remains a paramount challenge to successful gene therapy. First, the packaging capacity of the AAV capsid restricts transgene size to under 4.7 kb, precluding delivery of many transgenes and gene editing cassettes. Relatives of AAV within Parvoviridae contain larger genomes, including Densovirus (DNV), a subfamily of parvoviruses that naturally infect invertebrates. I propose to engineer chimeric DNV/AAV vectors that support robust transduction of mammalian cells and enhanced packaging capacity relative to traditional AAV vectors. I then plan to investigate DNV/AAV transduction, and leverage this knowledge to perform directed evolution on DNV/AAV capsids to confer robust transduction of mammalian cells. Second, immune recognition and clearance of AAVs following high dose vector administration remains a key issue in gene therapy delivery, as high doses are often indispensable to achieve transgene expression at therapeutic levels. One reason for the necessity of high dose treatments is the lacking potency of recombinant AAV vector preparations, which have been demonstrated to have lower infectious-to-physical particle ratios relative to AAVs packaging their endogenous genome. Previous studies have shown that these differences in AAV production may be due to uncharacterized cis-acting packaging signals within the AAV genome. Therefore, I aim to perform an unbiased screen for novel motifs within the AAV genome that confer enhanced genome packaging, and to investigate how incorporation of additional cis-acting sequences affects recombinant vector production. Together, these studies aim to address core issues in AAV mediated gene therapy by augmenting both the size of transgenes that can be delivered and the potency of recombinant vector production.