Features and cellular implication sites of hepatic AAV-cFVIII integration after long-term follow-up in the hemophilia A dog model. (A) Heat map of IS in 10 MB window sizes, demonstrating integration across the canine genome detected by TES; frequencies displayed on a density scale ranging from 0 IS/10 Mb to >697 IS/10 Mb, with highest frequency IS shown in yellow and red. (B) Representation of CIS seen within 10 Mb window sizes on chromosomes 14 (ABCB1), 28 (KCNIP2), and X (F8 and CLIC2). (C-E) No significant dysregulation in gene expression of genes in proximity to CIS in the liver of AAV-cFVIII treated hemophilia A dogs, compared with untreated hemophilia A or normal dogs; quantification by drop-phase ddPCR and normalized to B2M expression. (F) No dysregulation in MET expression in the liver of AAV-cFVIII treated hemophilia A dogs, compared with untreated hemophilia A or normal dogs; quantification by ddPCR and normalized to B2M expression. (G) Comparison of IS frequencies in proximity (≤100 kb) to cancer genes to simulated data sets (n = 10 000), demonstrated this frequency is within the predicted normal range (0.065-0.084). (H) Evaluation of association (P < .05) of IS with areas of chromatin accessibility (ATAC-seq) and methylation status (bisulfite sequencing). N-dog, normal dog; NTC, no template control.