ABSTRACT The development of transformative therapeutics, including the possibility of a cure though gene therapy, is the sole mission of Expression Therapeutics and continues to be a major research and development activity in the overall $10 B USD hemophilia market space. However, progress in the field has been limited by significant hurdles including the size, complexity, instability, immunogenicity and biosynthetic inefficiency of coagulation factor VIII (FVIII). Through the study of existing vertebrate species and, even more recently, predicted ancestral FVIII variants, we have identified differentials in molecular, cellular, immunological and biochemical properties that are predicted to be pharmacologically beneficial. Based on these data, as well as existing knowledge of FVIII biosynthesis and mechanism of action, we have bioengineered a novel FVIII candidate, An-53, for both protein infusion and gene-based therapies. In the current proposal, we seek to compare the performance of An-53 to existing FVIII candidates including the hybrid human/porcine FVIII, ET3, and B domain deleted human FVIII in lentiviral vector and adeno-associated viral vector gene therapy platforms. Additionally, we will perform critical immunogenicity assessments that, together with the gene transfer performance data will facilitate lead candidate selection by Expression Therapeutics.