ET3i protein challenge in AAV-ET3–treated animals suggests a therapeutic window for AAV-FVIII treatment. (A) Animals treated with either AAV-E06.TTR-ET3 or AAV-HCB-ET3 that did not develop inhibitors by 37 to 42 weeks were challenged with 5 weekly infusions of 1 μg purified ET3i protein. Animals were ranked based on prechallenge FVIII activity, and 1 animal per rank group served as a no challenge control. Naïve hemophilia A animals were immunized alongside the gene therapy–treated animals. Plasma was collected 3 days after every injection and at weeks 6 and 7 after challenge initiation. An additional 1ug ET3i was administered 3 days before sacrifice. (B) Steady-state FVIII activity before ET3i challenge vs. the IgG titer at time of sacrifice in ET3i-challenged animals. The following animals were resistant to challenge: 1433L, 1437LL, 1443N, 1443L, 1455LL, and 1463N. (C) Target AAV-FVIII dose window based on initial FVIII exposure kinetics and steady-state levels relative to thresholds for therapeutic efficacy and immunogenicity to mitigate the risks associated with FVIII levels significantly outside of the normal range. The “minimum effective dose” indicates the minimum dose at which durable and therapeutic steady-state FVIII levels are expected. The “immunogenicity threshold” is determined from the pharmacokinetic model described herein.