Mechanism of the Membrane-Associated Accessory Protein (MAAP) in rAAV Production

项目总结 重组腺相关病毒(RAAV)载体已成为首选的基因载体之一 用于临床基因治疗的药物。到目前为止，两种基于rAAV的药物，Luxturna和Zolgensma，已经 获得美国FDA批准，已有230多项使用rAAV载体进行的人类基因治疗临床试验 已经开展或正在进行的一些活动取得了积极成果。然而，各种障碍依然存在。 RAAV在人类应用中用作载体需要解决的问题，包括缺乏简化的 高产媒介生产系统。最近，一种新的AAV非结构蛋白，膜相关蛋白， 辅助蛋白(MAAP)是在一项全面的AAV衣壳健身研究中发现的。MAAP是 从AAV衣壳蛋白基因编码区的非规范起始密码子翻译而来，并已被 预测属于细小病毒科的DependoparvoVirus A和B属，它们有很高的 氨基酸序列的相似性。MAAP通过预测的α-1与细胞表膜结合 C末端的螺旋跨膜结构域。然而，它也定位于核膜，并且是 在胞核和胞浆中均有表达。MAAP基因敲除AAV的感染显示衣壳 被限制在细胞核内。MAAP在rAAV生产中的敲除表明MAAP是一个关键的病毒因子 通过囊泡分泌途径介导rAAV载体的细胞外流。因为MAAP不会与 AAV衣壳，MAAP如何介导AAV病毒粒子从细胞核到细胞质和胞外的运输 细胞表面膜尚不清楚。从培养基中纯化的rAAV载体是一种简化的载体-- 制造方法，避免了更复杂和耗时的电池处理过程 裂解物。从胞外囊泡(EV)中提纯的rAAV载体具有一定的特异性 在基因传递方面的优势。因此，细胞外分泌rAAV是一种有效的方法 将有助于建立简化的高产载体生产系统，并提高EV-rAAV的生产。我们的 总体假设是MAAP促进AAV核输出、细胞质运输和细胞表面 膜出口，这对野生型AAV和rAAV的出口都是重要的。我们将确定主机 与MAAP相互作用的蛋白质，并将决定MAAP介导的路线(包括核和 细胞)AAV的出口。成功完成拟议的实验将识别宿主蛋白质和 可靶向提高细胞培养上清液中rAAV载体纯化的途径。此外，还提供了完整的 了解MAAP在AAV生命周期中的功能将使我们对当前的知识有更深的了解 在细小病毒学方面。

PROJECT SUMMARY Recombinant adeno-associated virus (rAAV) vectors have emerged as one of the preferred gene delivery agents for clinical gene therapy. To date, two rAAV-based drugs, Luxturna and Zolgensma, have been approved by the US FDA, and over 230 clinical trials of human gene therapy using rAAV vectors have been carried out or are ongoing, and some have yielded positive outcomes. However, various barriers still remain that need to be resolved for rAAV to be used as a vector in human applications, including lack of a simplified high yield vector production system. Recently, a novel AAV nonstructural protein, membrane-associated accessory protein (MAAP), was discovered during a comprehensive AAV capsid fitness study. MAAP is translated from a non-canonical start codon from the AAV capsid protein gene-encoding region, and has been predicted to be in the genera of the Dependoparvovirus A&B of the Parvoviridae family, which share a high similarity in amino acid sequence. MAAP is associated with the cell surface membrane through a predicted α- helix transmembrane domain at the C-terminus. However, it is also localized to the nuclear membrane and is expressed in both the nucleus and the cytoplasm. Infection of a MAAP knockout AAV showed that the capsid is restricted in the nucleus. Knockout of MAAP in rAAV production has shown MAAP is a key viral factor that mediates cellular egress of rAAV vectors through vesicle secretion pathways. As MAAP does not interact with the AAV capsid, how MAAP mediates transportation of AAV virions from the nucleus to cytoplasm and out of the cell surface membrane is unknown. rAAV vectors purified from the culture medium is a simplified vector- manufacturing method, avoiding the more complex and time-consuming process of the treatment of the cell lysates. And, rAAV vectors purified from extracellular vesicle (EV) as enveloped EV-rAAV possess certain advantages in gene delivery. Therefore, an efficient means to extracellularly secret rAAV in the culture medium will help establish a simplified high yield vector production system and improve EV-rAAV production. Our overall hypothesis is that MAAP facilitates AAV nuclear export, cytoplasmic trafficking, and cell surface membrane exit, which are all important for egress of both the wild-type AAV and rAAV. We will identify host proteins that interact with MAAP, and will determine the route of the MAAP-mediated (both nuclear and cellular) egress of AAV. Successful fulfillment of the proposed experiments will identify host proteins and pathways that can be targeted to improve rAAV vector purification from cell culture medium. In addition, a full understanding of the MAAP function in the AAV life cycle will provide deeper insight into our current knowledge in parvovirology.