Genome-replicating HC-AdV: A novel high-capacity adenoviral vector class featuring enhanced in situ payload expression

High-capacity adenoviral (HC-AdV) vectors offer large transgene capacities and long-term expression of therapeutics but require high doses due to limited transgene expression. In contrast, replication-competent AdV (RC-AdV) vectors enhance <i>in situ</i> transgene expression by genome replication and increased transcription from amplified genomes. Yet, RC-AdVs are constrained by minimal payload capacity, progeny formation, and toxic protein expression leading to rapid host cell death. To address these limitations, we developed a novel, genome-replicating HC-AdV vector. Therefore, we investigated AdV genome replication independently of progeny particle formation and developed cell-based <i>trans</i>-replication assays, enabling us to probe the requirement for individual AdV proteins in AdV genome replication. We identified seven AdV proteins from the early transcriptional units that promote potent replication of HC-AdV genomes. We then created a genome-replicating HC-AdV vector by encoding an engineered minimal replication system that functionally reconstitutes AdV genome replication. Host cell transduction with our genome-replicating HC-AdV promoted <i>cis</i>-replication of the delivered HC-AdV genome and up to 20-fold increased reporter fluorescence. Our novel vector retained a large transgene capacity (22 kb) and, unlike RC-AdVs, did not induce a cytopathic effect or host cell killing. Together, these data describe a novel delivery platform potentially allowing more efficacious vaccination and vector-mediated therapies.