Engineering the biology of AAV secretion and production

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.

Grant Number: 5R01EB034573-04
NIH Institute/Center: NIH
Principal Investigator: Aravind Asokan