Efficacy of AAV8.SaCas9-mediated hepatic gene targeting in FIX-KO mice treated as newborns and adults. (A) hFIX protein levels in FIX-KO mouse plasma after neonatal temporal vein injection of AAV8.SaCas9 (5 × 10¹⁰ GCs per pup) and AAV8.sgRNA3.hFIXco-Padua donor (2.5 × 10¹¹ GCs per pup) (n = 6). A subset of treated mice (n = 3) was subjected to two-thirds partial hepatectomy 8 weeks postvector treatment. All treated mice were euthanized 32 weeks postvector treatment. Untargeted FIX-KO pups (n = 8) received AAV8.SaCas9 (2.5 × 10¹¹ GCs per pup) and AAV8.control.hFIXco-Padua donor (2.5 × 10¹² GCs per pup). One untargeted mouse died at week 8 from hemorrhage, 3 were euthanized at week 8 for analyses, and the remaining 4 were euthanized at 32 weeks. (B) hFIX activity in FIX-KO mouse plasma after neonatal vector treatment. (C) hFIX protein levels in adult-treated FIX-KO mouse plasma after tail vein injection of AAV8.SaCas9 (5 × 10¹¹ GCs per mouse) and AAV8.sgRNA3.hFIXco-Padua donor (5 × 10¹² GCs per mouse) (n = 5). All treated mice were subjected to two-thirds partial hepatectomy 8 weeks postvector treatment and were euthanized 32 weeks postvector treatment. Untargeted FIX-KO mice (n = 8) received AAV8.SaCas9 (5 × 10¹¹ GCs per mouse) and AAV8.control.hFIXco-Padua donor (5 × 10¹² GCs per mouse). Three of the untargeted mice were euthanized at week 8, and liver samples were harvested for analyses; the remaining 5 mice were euthanized at 32 weeks. (D) hFIX activity in adult-treated FIX-KO mouse plasma. Data are mean ± SD.