Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral membrane-associated accessory protein (MAAP)

Adeno-associated virus (AAV) expresses a membrane-associated accessory protein (MAAP), a small nonstructural protein, that facilitates AAV secretion out of the plasma membrane through an association with extracellular vesicles during AAV egress. Here, we investigated the host proteins that interact with AAV2 MAAP (MAAP2) using APEX2-mediated proximity labeling. We identified two SNARE proteins, Syntaxin 7 (STX7) and synaptosome-associated protein 23 (SNAP23), a vesicle (v-)SNARE and a target (t-)SNARE, respectively, that mediate intracellular trafficking of membrane vesicles aand exhibited associations with MAAP2 in HEK293 cells. We found that MAAP2 indirectly interacted with STX7 or SNAP23, and that the knockout of STX7 or SNAP23 not only enhanced rAAV secretion into the media but also increased total vector yield during rAAV vector production in HEK293 cells. Thus, our study revealed a practical approach for producing higher yields of rAAV vectors from the media, easing downstream processes in rAAV manufacturing.