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

rAAV 生产中膜相关辅助蛋白 (MAAP) 的机制

• 批准号: 10630242
• 负责人: Jianming Qiu
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630242
• 关键词: Amino Acid Sequence; Attenuated; Binding; Biotinylation; COPII-Coated Vesicles; Capsid; Capsid Proteins; Cell Culture Techniques; Cell Nucleus; Cell membrane; Cell secretion; Cell surface; Cells; Clinical; Clinical Trials; Co-Immunoprecipitations; Complement; Complex; Confocal Microscopy; Consumption; Cytoplasm; DNA biosynthesis; Dependovirus; Directed Molecular Evolution; Dose; Engineering; Family; Gelsolin; Gene Delivery; Gene Proteins; Goals; Guidelines; Helper Viruses; Human; Human body; Infection; Initiator Codon; Interferometry; Intervention; Knock-out; Knowledge; Life Cycle Stages; Lytic Phase; Mediating; Medical Research; Medicine; Membrane; Membrane Fusion; Methods; Modification; Mutagenesis; Nonstructural Protein; Nuclear; Nuclear Envelope; Nuclear Export; Open Reading Frames; Organ; Outcome; Parvoviridae; Parvovirus; Pathway interactions; Pharmaceutical Preparations; Phase; Plasmids; Process; Production; Proteins; Proteomics; Reading Frames; Recombinant adeno-associated virus (rAAV); Reporting; Route; Streptavidin; Surface; System; Time; Tissues; Transfection; Translating; Transmembrane Domain; Transportation; Variant; Vesicle; Viral; Viral Genome; Virion; Virus; Virus Diseases; adeno-associated viral vector; alpha helix; experimental study; extracellular; extracellular vesicles; fitness; gene therapy; improved; in vivo; insight; knockout gene; manufacture; neutralizing antibody; novel; protein function; tissue tropism; trafficking; transduction efficiency; ultra high resolution; vector

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.

项目摘要 重组腺相关病毒（rAAV）载体已成为首选的基因递送载体之一 用于临床基因治疗的试剂。到目前为止，两种基于rAAV的药物Luxturna和Zolgensma已经在临床上得到了应用。 已被美国FDA批准，并且已经进行了超过230项使用rAAV载体的人类基因治疗的临床试验。 已经开展或正在开展的活动，有些已取得积极成果。然而，各种壁垒依然存在 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 高产载体生产系统。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衣壳适合度研究中发现的。MAAP是 从来自AAV衣壳蛋白基因编码区的非规范起始密码子翻译，并且已经被 预计属于细小病毒科的依赖细小病毒A和B属，它们具有很高的相似性 氨基酸序列相似性。MAAP通过预测的α- 螺旋跨膜结构域的C-末端。然而，它也定位于核膜， 在细胞核和细胞质中表达。MAAP敲除AAV的感染表明， 被限制在细胞核内。在rAAV生产中MAAP的敲除已经显示MAAP是关键的病毒因子， 通过囊泡分泌途径介导rAAV载体的细胞外出。由于MAAP不与 AAV衣壳，MAAP如何介导AAV病毒体从细胞核到细胞质和从细胞核到细胞外的运输。 细胞表面膜是未知的。从培养基中纯化的rAAV载体是简化的载体- 制造方法，避免了处理细胞的更复杂和耗时的过程 裂解物。并且，从细胞外囊泡（EV）纯化的rAAV载体作为包膜EV-rAAV具有一定的生物学活性。 基因传递的优势。因此，在培养基中细胞外分泌rAAV的有效手段是 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 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，这些对于野生型AAV和rAAV的流出都是重要的。我们将识别主机 与MAAP相互作用的蛋白质，并将决定MAAP介导的途径（核和 细胞的）AAV的流出。成功完成所提出的实验将鉴定宿主蛋白质， 可以靶向这些途径以改善从细胞培养基中纯化rAAV载体。此外，一个完整的 了解MAAP在AAV生命周期中的功能，将使我们对现有知识有更深入的了解 在细小病毒学上。