Contaminating plasmid sequences and disrupted vector genomes in the liver following adeno-associated virus gene therapy

Abstract Adeno-associated viruses (AAVs) are common vectors in gene therapy but can frequently cause liver complications in patients. The mechanisms underlying AAV-related liver toxicity remain poorly understood, posing challenges for effective prevention and intervention. Here we conducted a case study of a child with spinal muscular atrophy type 1 experiencing substantial hepatitis after receiving onasemnogene abeparvovec, undertaking long- and short-read metagenomic sequencing of liver tissue. We identified manufacturing plasmid sequences with complex structures and recombination. Vector genomes had extensive disruption and concatemerization as well as numerous vector–human fusion junctions. We also identified human betaherpesvirus 6B in the liver. Further work and investigation of more patients is needed to establish whether the presence of manufacturing plasmid sequences or helper viruses contribute to the formation of these complex concatemeric DNA structures in the liver, and whether these are a factor in the development of liver toxicity after AAV gene therapy.