SMBP Group Meeting: Vicky Xu (Imperial College London)

Thursday May 8, 2025, 2:00 PM → 3:00 PM America/New_York

3rd Floor Classroom (162 5th Avenue )

Speaker: Vicky Xu  (Imperial College London)

Topic: Countdown to Package: Molecular Insights Into The Adeno-Associated-Virus Packaging Machinery

Abstract: Recombinant adeno-associated viral (rAAV) vectors are among the most widely used gene therapy vectors. Currently, the bottleneck in rAAV-vector platform lies in manufacturing where inefficient packaging of DNA cargo into capsids, in combination with difficulty in separation of heterogeneous capsids in the downstream purification leads to empty capsid contamination. This contributes to higher risk profile of AAV-based therapies. Here, we use cryoEM to gain molecular understanding of the DNA packaging mechanism mediated by the AAV Replication protein (Rep). Using the smallest Rep isoform Rep40, we sought to understand the interaction of Rep40 with the cis-element for packaging, the inverted-terminal-repeat (ITR) hairpin DNA, and the AAV capsid. Different preparation and processing methods were explored to combat preferred orientation, sample heterogeneity and symmetry mismatch on capsid structures. By solving a series of high resolution cryoEM structures of Rep40 we show that Rep adapts its oligomeric states depending on its role. Rep40 forms a complex with the ITR in 7-mer and 6-mer forms and binds the AAV capsid as a 5-mer. The ITR-bound complexes gave snap-shots of Rep translocating DNA driven by ATP hydrolysis and insights into its DNA unwinding activity; whilst the capsid-bound complexes show a direct interaction of Rep with the capsid which exposes a cryptic ATP binding site. This novel data elucidates a mechanism where a pool of Reps are able to dynamically form different oligomeric states and orchestrate ITR hairpin recognition, duplex unwinding and subsequent capsid association through homomeric interactions. Taken together, this data sheds light on the mechanism of Parvoviral DNA packaging and highlights potential targets for optimization of the packaging process during rAAV production.