Engineering the biology of AAV secretion and production

AAV 分泌和生产的生物学工程

• 批准号: 10656028
• 负责人: Aravind Asokan
• 金额: $ 45.7万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656028
• 关键词: Adenoviruses; Adoption; Biological Assay; Biology; CRISPR-mediated transcriptional activation; Capsid; Cell Culture Techniques; Cell Fraction; Cell Line; Cell physiology; Cell secretion; Cells; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Confocal Microscopy; Culture Media; Cytolysis; DNA Packaging; DNA Sequence; Data; Dependovirus; Detergents; Development; Disease; Dose; Electron Microscopy; Engineering; Ensure; Exclusion; Family; Foundations; Genes; Genome; Goals; Harvest; Helper Viruses; Herpesviridae; Human; Integration Host Factors; Inverted Terminal Repeat; Kinetics; Knowledge; Life Cycle Stages; Ligation; Lytic; Maps; Mass Spectrum Analysis; Mediating; Membrane; Metabolic; Molecular; Molecular Virology; Open Reading Frames; Outcome; Parvoviridae; Pathway interactions; Phylogenetic Analysis; Process; Production; Property; Protein Engineering; Proteins; Recombinant adeno-associated virus (rAAV); Recombinants; Risk; Role; Safety; Secretory Cell; Secretory Component; Serotyping; Signal Transduction; Single Stranded DNA Virus; Structural Models; Structural Protein; Toxic effect; Treatment Efficacy; Viral; Viral Genome; Viral Packaging; Virion; Virus Assembly; Work; adeno-associated viral vector; arm; co-infection; cost; exosome; experience; extracellular; extracellular vesicles; gene therapy; high throughput screening; improved; laboratory experiment; manufacture; manufacturing process; nanoparticle; nanopore; neuromuscular; novel; particle; process improvement; process optimization; protein function; scale up; stable cell line; success; technology platform; vector

ABSTRACT Recombinant adeno-associated viruses (AAV) have emerged at the forefront of gene therapy as promising vectors for treating a wide spectrum of diseases. Despite the approval of 3 different AAV gene therapy products for ocular (Luxturna), neuromuscular (Zolgensma) and metabolic (Glybera) disorders, several challenges remain – most notably, the need for high doses of AAV to achieve therapeutic efficacy. This drawback imposes a significant burden on manufacturing processes and also the risk of dose dependent clinical toxicity. To this end, it is important to study key aspects of AAV biology that can profoundly influence manufacturing processes, vector yield and quality, which in turn impacts clinical outcomes. The current proposal is centered around one key question – how does AAV exit the host cell? Upon co-infection with a helper virus such as Adenovirus or Herpesvirus, wild type AAV undergoes a transition from a latent to lytic life cycle, hijacking the host cell machinery to lyse the cell. However, it is well known that during rAAV vector production, a significant fraction is secreted into media supernatant (as free or extracellular vesicle (EV)-associated particles), while a fraction is still retained within the producer cell. Despite this knowledge, the urgent need for process optimization and scale up in AAV manufacturing has resulted in adoption of upstream process/harvest steps in recombinant AAV production that involve detergent lysis of producer cells. This process step generates large quantities of cell lysate that is then subject to heavily burdened downstream processing steps that can result in compromised vector yield and quality. Recent work has revealed a novel +1 frameshifted open reading frame (ORF) in the VP1 region of the AAV cap gene that mediates expression of the membrane-associated accessory protein (MAAP). In the current proposal, we highlight exciting new findings from our lab that assign a novel function to MAAP in promoting AAV egress from host cells. Our overall scientific premise is based on strong supportive evidence that MAAP promotes AAV egress by hijacking host cell secretory pathways. Thus, the current proposal is focused on further dissecting the mechanism of MAAP-mediated AAV extracellular secretion. Specific goals of the proposal are to (1) dissect the role of MAAP as an egress factor for different AAV clades, (2) determine the molecular mechanisms underlying MAAP function and AAV secretion and (3) engineer novel MAAPs and stable MAAP producer cell lines for enhanced AAV secretion. Our overarching goal is to study and engineer AAV secretion to streamline process development and improve the clinical safety profile as determined by AAV vector quality.

抽象的 正如承诺 用于治疗多种疾病的媒介。尽管批准了3种不同的AAV基因治疗产品 对于眼部（Luxturna），神经肌肉（Zolgensma）和代谢（Glybera）疾病，仍然存在一些挑战 - 最值得注意的是，需要高剂量的AAV来实现治疗效率。这个缺点不可能 大量伯恩在制造过程中以及依赖剂量临床毒性的风险。为此， 研究AAV生物学的关键方面非常重要 产量和质量，这反过来影响临床结果。当前的建议集中在一个密钥围绕 问题 - AAV如何退出宿主单元？与辅助病毒（例如腺病毒或 疱疹病毒，野生型AAV经历了从潜在的裂解生命周期的过渡，劫持了宿主细胞机械 裂解细胞。但是，众所周知，在RAAV矢量生产过程中，分泌很大一部分 进入培养基上清液（作为免费或细胞外囊泡（EV）相关的颗粒），而馏分仍保留 在生产者单元中。尽管有这些知识，但迫切需要在AAV中进行过程优化和扩展 制造业导致采用了重组AAV生产的上游过程/收获步骤 涉及确定生产细胞的裂解。此过程步骤生成大量的细胞裂解物，然后 受到严重燃烧的下游处理步骤，可能导致载体产量受损和 质量。最近的工作揭示了一个新颖的+1框架开放式阅读框（ORF） AAV CAP基因介导与膜相关辅助蛋白（MAAP）的表达。在电流中 提案，我们重点介绍了我们实验室中令人兴奋的新发现，这些发现将新颖的功能分配给促进AAV 从宿主细胞出口。我们的整体科学前提是基于有力的支持证据 通过劫持宿主细胞秘书途径来促进AAV出口。这是当前的提议集中于进一步 解剖MAAP介导的AAV细胞外分泌的机制。该提案的具体目标是 （1）剖析MAAP作为不同AAV进化枝的出口因子的作用，（2）确定分子 MAAP功能和AAV分泌的基础机制以及（3）工程师的新型垫和稳定的MAAP 生产者细胞系用于增强AAV分泌。我们的总体目标是研究和研究AAV分泌 简化过程开发并改善由AAV矢量质量确定的临床安全性。