Engineering the biology of AAV secretion and production

摘要 重组腺相关病毒（AAV）已出现在基因治疗的最前沿， 用于治疗多种疾病的载体。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，仍存在一些挑战 - 最值得注意的是，需要高剂量的AAV以达到治疗效果。这一缺陷造成了 对制造过程造成显著负担，并且还有剂量依赖性临床毒性的风险。为此， 研究AAV生物学的关键方面是很重要的，这些方面可以深刻地影响生产过程、载体、 产量和质量，这反过来又影响临床结果。目前的建议围绕一个关键 问题：AAV如何离开宿主细胞？当与辅助病毒如腺病毒或腺病毒共感染时， 疱疹病毒，野生型AAV经历从潜伏到裂解生命周期的转变，劫持宿主细胞机器 来裂解细胞然而，众所周知的是，在rAAV载体生产过程中，分泌了相当大的部分 进入培养基上清液（作为游离或细胞外囊泡（EV）相关颗粒），同时仍保留一部分 在生产者细胞中。尽管有这些知识，但迫切需要在AAV中进行工艺优化和扩大规模。 生产已经导致在重组AAV生产中采用上游工艺/收获步骤， 涉及生产细胞的去污剂裂解。该过程步骤产生大量的细胞裂解物， 经受负担沉重的下游加工步骤，这可能导致载体产量受损， 质量.最近的工作揭示了一个新的+1移码的开放阅读框架（ORF）在VP 1区， 介导膜相关辅助蛋白（MAAP）表达的AAV cap基因。在当前 我们强调了我们实验室令人兴奋的新发现，这些发现为MAAP在促进AAV方面赋予了新的功能。 离开宿主细胞我们的总体科学前提是基于强有力的支持性证据，即MAAP 通过劫持宿主细胞分泌途径促进AAV流出。因此，目前的建议侧重于进一步 剖析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进化枝的外出因子的作用，（2）确定MAAP的分子结构， MAAP功能和AAV分泌的潜在机制和（3）工程化新的MAAP和稳定的MAAP 用于增强AAV分泌的生产细胞系。我们的首要目标是研究和设计AAV分泌， 简化工艺开发并改善由AAV载体质量确定的临床安全性概况。

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.