Genome-wide assays to characterize rAAV integration into human genomic DNA in vivo

Abstract Adeno-associated viral (AAV) vectors are used to treat genetic diseases, expressing therapeutic genes from both extrachromosomal episomes and payloads that integrate into the host genome. Assays were developed to evaluate HR-mediated on-target integration and the potential occurrence of off-target integration. While many studies have addressed elements of these processes, proper characterization requires long-read sequencing to ensure that integrated viral DNA is examined and not the more prevalent episomes. We used Oxford Nanopore to characterize integrated DNA and scan the whole genome for off-target integrations. These assays were applied to cell-based and in vivo models to study vectors that correct phenylketonuria (PKU), caused by loss of phenylalanine hydroxylase ( PAH ). Administration of the human-specific vector in a humanized-liver mouse xenograft model resulted in stable, nuclease-free integration into PAH . Because detection of rare integration events in a much larger pool of episomal DNA is subject to artifacts, careful assay validation was required. A long-read, genome-wide assay capable of detecting on- and off-target vector integrations showed no evidence of off-target integration. Artifactual false positive events were below the limit of blank. These data support rAAV as an investigational therapeutic for genetic diseases and reinforce the need for characterization of integration assays to avoid artifacts.