Engineering chimeric gene therapy vectors with enhanced packaging capacity

项目摘要 腺相关病毒（Adeno-associated viruses，AAV）是一种非致病性的细小病毒，是目前最重要的基因 治疗载体随着越来越多地采用基因疗法来治疗各种适应症，AAV起到了重要作用。 在传递治疗性DNA中的关键作用。然而，重组转基因在AAV中的有效包装是不可能的。 载体仍然是成功基因治疗的最大挑战。一、AAV的包装能力 衣壳将转基因大小限制在4.7 kb以下，排除了许多转基因和基因编辑的递送 磁带。细小病毒科中AAV的亲属包含较大的基因组，包括浓核病毒（DNV），一种 自然感染无脊椎动物的细小病毒亚科。我建议设计嵌合DNV/AAV载体 其支持哺乳动物细胞的稳健转导和相对于传统包装增强的包装能力， AAV载体。然后，我计划研究DNV/AAV转导，并利用这些知识进行 在DNV/AAV衣壳上定向进化以赋予哺乳动物细胞的稳健转导。第二，免疫 高剂量载体给药后AAV的识别和清除仍然是基因治疗中的关键问题。 在治疗递送中，高剂量对于实现治疗水平的转基因表达通常是必不可少的。 需要高剂量治疗的一个原因是重组AAV载体缺乏效力 制剂，已经证明相对于 包装其内源基因组的AAV。先前的研究表明，AAV中的这些差异 这种产生可能是由于AAV基因组内未表征的顺式作用包装信号。所以我 目的是在AAV基因组内进行无偏筛选，以获得增强的基因组 包装，并研究额外的顺式作用序列的掺入如何影响重组载体 生产总之，这些研究的目的是解决核心问题，在AAV介导的基因治疗，通过增强 可以递送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.