Mechanistic Studies of AAP and Capsid Assembly of AAV Vectors

摘要 腺相关病毒(AAV)作为人类基因治疗的载体越来越受到人们的欢迎。 然而，达到治疗效果所需的高媒介剂量的要求对 具有成本效益的临床用AAV载体的制造。AAV载体的基本过程 生产是病毒粒子组装，60个VP衣壳蛋白亚基形成二十面体蛋白的过程 壳牌随后对AAV载体基因组进行包装。不幸的是，这一基本过程仍然知之甚少。 因此迫切需要阐明AAV载体产生的过程和机制。是这样的 了解这一点还有可能发现生产高滴度和高质量AAV的关键 向量。在这方面，2010年有一项范式转变的发现，即AAV帽基因表达 装配激活蛋白(AAP)，一种以前未知的非结构蛋白，促进衣壳 集合。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，AAV衣壳的组装过程在 不同的AAV血清型，即在AAP发现前时代建立的AAV衣壳组装的知识 使用AAV2的研究不能翻译到其他AAV血清型的衣壳组装。我们的初步数据显示 挑战了长期以来关于AAV衣壳在核仁中组装的教条，并揭示了显著的 衣壳组装过程中依赖于血清型的异质性。此外，越来越多的人意识到 关于AAP在促进衣壳组装之外所扮演的其他角色。在此，为了推进我们的 了解AAV载体并提高其在基因治疗中的有效利用，我们寻求彻底 了解AAV衣壳的组装过程和AAP在AAV病毒粒子组装中的多方面作用 载体，使用包括高通量突变、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的质谱学。 此外，我们将探索各种潜在的策略来提高AAV载体的产量和质量 通过操纵各种途径，我们将在项目中确定。因此，这个项目的具体目标是 有：(Aim1)全面了解AAP生物学中病毒粒子组装过程中的AAV载体 不同AAV血清型的差异；(目标2)鉴定参与AAV衣壳组装的宿主细胞蛋白； 以及(目标3)探索通过操纵AAV载体来提高AAV载体的产量和质量的新策略 病毒粒子组装过程。我们的项目不仅将解决关于 AAV载体的病毒粒子组装，也大大加深了我们对AAV-宿主相互作用的理解 将军。该项目还有可能发现改进媒介生产的新策略。

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.