Gene therapy for hemophilia A (HA) remains an unrealized therapeutic goal due to outstanding questions regarding safety and long-term efficacy after adeno-associated viral (AAV) vector delivery of the F8 gene to hepatocytes. To address questions of safety, we treated privately owned dogs with a liver directed AAV serotype 8 vector encapsidating a canine F8 gene. These dogs were followed long-term for efficacy of the gene therapy and adverse events related to the vector. One dog (PC9) developed an aggressive lymphoma 3.5 years post-treatment. We rigorously assayed the lymphoma and found no evidence to support a role for the AAV vector in oncogenesis. To address the issue of lost efficacy, we sought to find a high activity F8 transgene that could be used in a gene therapy setting to improve patient outcomes. We hypothesized that selective pressures keep factor VIII (FVIII) activity low. Using criteria established from studies of factor IX-Padua, a factor IX transgene with increased specific activity, we identified 52 amino acids we hypothesized would lead to increased FVIII activity when substituted. A screen of these amino acids found twelve positions that increased specific activity more than two-fold when substituted, and one amino acid location (659) that lead to a six-to-seven-fold increase in specific activity. Three amino acid substitutions (cystine, methionine, and valine) at position 659 were determined to have the greatest increase on FVIII specific activity in myriad clotting assays. Tail clip assays of HA mice after FVIII-K659 variant protein infusion or liver directed AAV gene therapy showed increased hemostatic function of the FVIII-K659 variants in vivo. These studies affirm the safety profile of AAV vectors previously reported, show that FVIII is not optimized for procoagulant activity, and provide proof of concept data for a novel, high activity FVIII transgene for next-generation HA gene therapy.