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.