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.