Impact of the assembly-activating protein (AAP) on molecular evolution of synthetic Adeno-associated virus (AAV) capsids.

Over the last decade, others and we have started to dissect the role of the assembly-activating protein AAP for the formation of Adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, our group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and found AAP to be essential for capsid protein stabilization and generation of functional vector particles. Here, we additionally addressed the lingering question whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame. To this end, we conducted a battery of complementary experiments in which we (i) tested the ability of chimeric AAP from AAVDJ, a hybrid of serotypes 2, 8 and 9, to rescue AAP knock-outs in the three parental serotypes; (ii) measured the functionality of 60 chimeric AAPs extracted from five shuffled, unselected capsid libraries; (iii) assessed whether production of different shuffled libraries, ten wild-type serotypes or 25 individual chimeric capsids can be enhanced by over-expression of AAP cocktails; and (iv) studied the activity of 12 chimeric AAPs isolated from a shuffled library that was iteratively selected <i>in vivo</i> in mouse livers. Collectively, our data demonstrate a remarkable tolerance of AAP for recombination via DNA family shuffling, evidenced by our findings that (i) all chimeric AAPs studied here retained at least partial activity, even in cases where the cognate hybrid capsid may be non-functional, and that (ii) ectopic AAP over-expression did not enhance production of shuffled AAV chimeras or libraries, implying that the inherently encoded hybrid AAP variants are sufficiently active. Together, our work provides compelling evidence that AAP is not rate-limiting during AAV capsid shuffling and thereby relieves a major concern in the field of AAV vector evolution.