Engineering the biology of AAV secretion and production

摘要 重组腺相关病毒(AAV)已成为基因治疗的前沿领域。 治疗多种疾病的媒介。尽管3种不同的AAV基因治疗产品获得批准 对于眼部(Luxturna)、神经肌肉(Zolgensma)和代谢(Glybera)障碍，仍然存在几个挑战 -最值得注意的是，需要大剂量的AAV才能达到治疗效果。这一缺陷强加了 制造过程中的巨大负担，以及剂量依赖性临床毒性的风险。为此， 重要的是研究AAV生物学的关键方面，这些方面可以深刻地影响制造过程，媒介 产量和质量，这反过来又影响临床结果。目前的提案围绕着一个关键 问题-AAV是如何离开宿主细胞的？在与辅助病毒(如腺病毒或 疱疹病毒，野生型AAV经历了从潜伏到裂解的生命周期的转变，劫持了宿主细胞机器 来裂解细胞。然而，众所周知，在rAAV载体生产过程中，有相当一部分被分泌 进入培养上清液(作为游离或胞外囊泡(EV)相关颗粒)，同时仍保留一部分 在生产者细胞内。尽管有这些知识，但AAV迫切需要流程优化和扩大规模 制造已导致在重组AAV生产中采用上游工艺/收获步骤， 包括生产细胞的洗涤剂裂解。这个过程步骤产生大量的细胞裂解物，然后 受到下游处理步骤的沉重负担，这可能导致载体产量和 质量。最近的工作揭示了在VP1区有一个新的+1移码开放阅读框架(ORF) 介导膜相关辅助蛋白(MAAP)表达的AAV帽基因。在当前 提案，我们强调了来自我们实验室的令人兴奋的新发现，这些发现赋予MAAP在促进AAV方面的新功能 从宿主细胞中流出。我们的总体科学前提是基于强有力的证据，即MAAP 通过劫持宿主细胞的分泌途径促进AAV的外泄。因此，目前的提案侧重于进一步 对MAAP介导的AAV胞外分泌机制进行剖析。该提案的具体目标是 (1)剖析MAAP在不同AAV分支中作为出口因子的作用；(2)确定分子水平 MAAP功能和AAV分泌的机制以及(3)工程新型MAAP和稳定的MAAP 增强甲型肝炎病毒分泌的产生细胞系。我们的首要目标是研究和改造AAV分泌物以 简化流程开发并改善由AAV载体质量决定的临床安全性。

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.