The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice
Recombinant adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes can integrate into the host genome, but the theoretical risk of tumorigenesis may depend on vector regulatory features. A mouse model was used to investigate long-term kinetics and integration profiles of an AAV serotype 5 (AAV5) vector that mimics key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95 percent) of vector genome reads identified by target enrichment sequencing were derived from episomes, and mean integration frequency was 2.70 (standard deviation, 1.24) integrations per 1000 cells. Longitudinal integration analysis suggested AAV5 vector integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal and only 5.46 percent of integrations had a common integration site order greater than or equal to 5, suggesting random distributions when looking at a 50 kb genomic window. No integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver (P = 1x10-4) and less enriched for genes with low or no liver expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations.