Evaluation of the Human Factor IX Gene Therapy Vector TAK-748 in Hemophilia: Results from Non-Clinical Studies in Factor IX Knockout Mice and Rhesus Monkeys

Introduction: Adeno-associated virus (AAV)-based factor IX (FIX) gene therapy has the potential to provide clinical benefit in patients with hemophilia B. TAK-748 is a novel next-generation AAV vector for FIX gene therapy. The vector design includes the insertion of 3 hepatocyte-specific cis-regulatory elements to increase the strength of the liver-specific transthyretin promoter driving expression of a human FIX transgene.

Aims: The objectives of this study were to investigate the TAK-748 dose-response relationship for FIX activity, and evaluate its efficacy, in FIX knockout (KO) mice and rhesus monkeys.

Methods: Male FIX KO mice (N=12/group) received single intravenous doses of TAK-748 (7.4×10¹⁰, 1.5×10¹¹, 7.4×10¹¹, or 1.5×10¹² vector genomes [vg]/kg) or buffer. Blood samples were taken on days 7, 14, 28, 42, and 56 for the analysis of plasma FIX activity. At the end of the observation period, the bleeding phenotype was assessed by a tail-tip bleeding assay. The viral transduction efficiency of TAK-748 in liver tissue was analyzed by quantitative real-time polymerase chain reaction. Safety assessments included monitoring for clinical signs, and histopathological analysis of selected organs (liver, spleen, kidney, and heart).

Male rhesus monkeys (N=3/group) were administered single TAK-748 intravenous bolus injections (3.8×10¹¹, 9.5×10¹¹, or 1.9×10¹² vg/kg). Blood samples were collected before dosing and weekly after dosing up to week 18. Plasma FIX activity, human (hu)FIX antigen, and anti-hu-Padua FIX neutralizing antibodies were analyzed.

Results: No clinical signs or deaths were recorded in animals treated with TAK-748, and there were no TAK-748-related histopathological findings in the tissues collected from the mice.

FIX activity levels in plasma from FIX KO mice treated with buffer were below the lower limit of quantification. Administration of TAK-748 resulted in a dose-dependent increase in mean plasma FIX activity, and supraphysiologic mean FIX levels up to 41.0 IU/mL (1.5×10¹² vg/kg). In the tail-tip bleeding assay, blood loss was significantly reduced in the TAK-748 groups at dose levels above 7.4×10¹⁰ vg/kg (P<0.05 vs. buffer control).

Administration of TAK-748 to rhesus monkeys resulted in a dose-dependent increase in mean plasma FIX activity and antigen, and peak levels of huFIX expression were detected 2-4 weeks after treatment. Mean huFIX activity was 0.3, 0.6, and 1.9 IU/mL after treatment with 3.8×10¹¹ vg/kg, 9.5×10¹¹ vg/kg, and 1.9×10¹² vg/kg TAK-748, respectively. A significant reduction in FIX activity and huFIX protein was observed in most of the animals starting about 4 weeks after dosing with TAK-748. In most animals, anti-huFIX Padua neutralizing antibody titers were detected at about week 6 of the study and correlated with the preceding reductions in huFIX expression.

Conclusions: Treatment with TAK-748 resulted in dose-dependent increases in plasma FIX activity and was efficacious in FIX KO mice and rhesus monkeys. There were no treatment-related safety findings.