Episomal AAV-cFVIII vector forms detected in the liver after long-term follow-up, with correlation between vector copies and FVIII expression. (A) Circular full-length AAV-cFVIII VGs, detected by drop-phase ddPCR following treatment with PS-DNase and KpnI to enrich circular genomes and quantify full-length monomers. (B) Strong correlation between circular full-length VG copies and terminal FVIII:C levels measured by chromogenic substrate assay. For ELI, where terminal FVIII:C was unavailable, a mean of earlier FVIII:C on-study was used. (C) Strong correlation (r = 0.83; P < .0001) between full-length episomal VG copies and cFVIII mRNA expression, supporting episomal AAV-cFVIII as the primary source of FVIII expression. (D) Southern blotting, following PS-DNase and BamHI, demonstrates full-length AAV-cFVIII VGs in an H-T configuration (band size 4 kb). Sample from JUN contains 2 fragments (∼1600 and 850 bp) suggesting AAV-cFVIII truncation. (E) TES comparing frequencies of V-V, possibly episomal (blue) to vector-canine genome V-G, integrated (orange) sequencing reads demonstrating the majority resulted from V-V (episomal) reads.