In vivo ET3-specific activity after AAV-ET3 treatment in different anti-ET3 immune response scenarios. (A) The mean ET3 antigen levels 5 days after AAV-ET3 delivery are significantly higher in animals that subsequently formed inhibitors compared with animals that did not. Error bars indicate standard deviation (2-tailed t test, P = .0008). (B) This graph plots ET3 antigen vs. FVIII activity 5 days after AAV-ET3 delivery, with a combined linear regression slope of 8559 IU/mg. Animals that subsequently formed inhibitors are indicated by red dots, and there was no significant difference in day 5 linear regression slopes between animals that did and did not form inhibitors. (C) This graph plots ET3 antigen vs. FVIII activity through week 28 for all animals that did not form inhibitors after AAV-ET3 delivery. The linear regression slope is 9376 IU/mg. (D-E) These graphs profile the median in vivo ET3 specific activity and IgG titer over time before ET3i challenge (D) or during the ET3i challenge course (E). These data are stratified by the type of anti-ET3 immune response observed. The IQR error bars were removed for visual clarity but are shown in supplemental Figures 4 and 5. (F) There is no significant difference in in vivo ET3-specific activity at time of death for animals that demonstrated a multiphasic inhibitor response after AAV-ET3 delivery (blue) and AAV-ET3–treated animals that were resistant to ET3i challenge (purple) compared with the AAV-ET3–treated + no ET3i challenge control animals (green) (Kruskal-Wallis H test, P = .5874).