Determining the Minimally Effective Dose of a Clinical Candidate Adeno-Associated Virus Vector in a Mouse Model of Hemophilia A

Hemophilia A, a bleeding disorder, affects 1:5,000 males and is caused by a deficiency of human blood coagulation factor VIII (hFVIII). Studies in mice and macaques identified AAVhu37.E03.TTR.hFVIIIco-SQ.PA75 as a clinical candidate gene therapy vector to treat hemophilia A. In this study, we sought to determine the minimally effective dose (MED) of this vector in a hemophilia A mouse model. Mice received one of four vector doses (3 × 10¹¹-1 × 10¹³ genome copies [GCs]/kg) via intravenous tail vein injection; one cohort received vehicle as a control. Animals were monitored daily after vector/vehicle administration. Blood samples were collected to evaluate hFVIII activity levels and anti-hFVIII antibodies. Animals were sacrificed and necropsied on days 28 and 56; tissues were harvested for histopathological examination and blood was collected for serum chemistry panel analysis. We found no significant differences in liver transaminase levels in mice administered any vector dose compared to those administered vehicle (except for one group administered 3 × 10¹¹ GC/kg). Total bilirubin levels were significantly elevated compared to the vehicle group following two vector doses at day 56 (1 × 10¹² and 1 × 10¹³ GC/kg). We observed no vector-related gross or histological findings. Most microscopic findings were in the vehicle group and considered secondary to blood loss, an expected phenotype of this mouse model. Since we observed no dose-limiting safety markers, we determined that the maximally tolerated dose was greater than or equal to the highest dose tested (1 × 10¹³ GC/kg). Since we detected hFVIII activity in all cohorts administered vector, we conclude that the MED is 3 × 10¹¹ GC/kg-the lowest dose evaluated in this study.