Extensive length and homology dependent chimerism in pool-packaged AAV libraries

Adeno-associated viruses (AAVs) have emerged as the foremost gene therapy delivery vehicles due to their versatility, durability and safety profile. Here we demonstrate extensive chimerism among complex AAV libraries that are packaged as a pool. The observed chimerism is length- and homology-dependent but capsid-independent, in some cases affecting >60% of packaged AAV genomes. These results have important implications for the design and deployment of functional AAV libraries in research and clinical settings. Overall design: Barcoded plasmid dictionary generation and barcode swap assessment in long-read data.