Mechanistic Studies of AAP and Capsid Assembly of AAV Vectors

总结 腺相关病毒（AAV）作为人类基因治疗的载体越来越受欢迎。 然而，实现治疗功效所需的高载体剂量的要求对免疫治疗提出了挑战。 用于临床用途的AAV载体的成本有效的制造。AAV载体的基本过程 生产是病毒体装配，60个VP衣壳蛋白亚基形成二十面体蛋白的过程 外壳，然后进行AAV载体基因组包装。不幸的是，人们对这一基本过程仍然知之甚少 迫切需要阐明AAV载体生产的过程和机制。等 了解这一点还可能揭示生产高滴度和高质量AAV的关键 向量。在这方面，2010年有一个范式转变的发现，即AAV cap基因表达 组装激活蛋白（AAP），一种以前未鉴定的非结构蛋白， 组装件.出乎意料的是，我们小组和其他人在后AAP中对AAV衣壳组装的研究 发现时代已经令人信服地证明了AAV衣壳组装过程在 不同的AAV血清型，也就是说，在前AAP发现时代建立的AAV衣壳组装的知识， 使用AAV 2的研究不能转化为其它AAV血清型的衣壳组装。我们的初步数据显示 挑战了长期以来的AAV衣壳在核仁中组装的教条，并揭示了显著的 衣壳组装过程中的依赖于衣壳型的异质性。此外，人们越来越欣赏 AAP除了促进衣壳组装外还发挥着其他作用。在这里，为了推进我们的 了解AAV载体，提高其在基因治疗中的有效利用，我们寻求彻底 了解AAV衣壳组装过程和AAP在AAV病毒体组装中的多方面作用 载体，使用稳健的方法，包括高通量诱变，定向进化，条形码， 基于BioID的邻近标记，以及最先进的自下而上和自上而下质谱法。 此外，我们将探索各种潜在的策略，以提高AAV载体的产量和质量 通过操纵我们将在项目中确定的各种途径。因此，该项目的具体目标 （目的1）为了全面了解AAV载体的病毒体组装过程中的AAP生物学， （目的2）鉴定参与AAV衣壳组装的宿主细胞蛋白; 和（目的3）探索新的策略，以提高产量和质量的AAV载体，通过操纵 病毒粒子组装过程。我们的项目将不仅解决有关的机制， 病毒粒子组装的AAV载体，而且也大大促进了我们的理解AAV-宿主相互作用， 将军该项目也有可能发现新的策略，以提高载体生产。

SUMMARY Adeno-associated viruses (AAV) have become increasingly popular as vectors for human gene therapy. However, the requirement for high vector doses needed to achieve therapeutic efficacy poses a challenge to cost-effective manufacturing of AAV vectors for clinical use. The fundamental process of AAV vector production is virion assembly, the process by which 60 VP capsid protein subunits form an icosahedral protein shell followed by AAV vector genome packaging. Unfortunately, this basic process remains poorly understood and there is an urgent need to elucidate the process and mechanisms underlying AAV vector production. Such an understanding also could potentially uncover the key to production of high titer and high-quality AAV vectors. In this regard, there was a paradigm-shifting discovery in 2010 that the AAV cap gene expresses assembly-activating protein (AAP), a previously unidentified non-structural protein that promotes capsid assembly. Unexpectedly, studies on AAV capsid assembly from our group and others in the post-AAP discovery era have convincingly demonstrated that the AAV capsid assembly process is not conserved among different AAV serotypes, that is, knowledge of AAV capsid assembly built in the pre-AAP discovery era through studies using AAV2 is not translatable to capsid assembly of other AAV serotypes. Our preliminary data has challenged the long-standing dogma of AAV capsid assembly in the nucleolus and revealed significant serotype-dependent heterogeneity in the capsid assembly process. In addition, there is a growing appreciation for additional roles that AAP plays beyond promoting capsid assembly. Here, in order to advance our understanding of AAV vectors and improve their effective utilization in gene therapy, we seek to thoroughly understand the AAV capsid assembly process and the multifaceted roles of AAP in virion assembly of AAV vectors, using robust approaches including high-throughput mutagenesis, directed evolution, barcoding, proximity-based labeling based on BioID, and state-of-the-art bottom-up and top-down mass spectrometry. Furthermore, we will explore various potential strategies to enhance the yield and quality of AAV vectors through manipulation of various pathways we will identify in the project. Thus, the specific aims of this project are: (Aim1) To comprehensively understand AAP biology in the process of virion assembly of AAV vectors that varies across different AAV serotypes; (Aim 2) To identify host cell proteins involved in AAV capsid assembly; and (Aim 3) To explore novel strategies to enhance the yield and quality of AAV vectors by manipulating the process of virion assembly. Our project will not only address fundamental questions about the mechanism of virion assembly of AAV vectors, but also substantially further our understanding of AAV-host interactions in general. The project also has potential to discover novel strategies to improve vector production.