ADENO-ASSOCIATED VIRAL VECTOR BASED GENE THERAPY PRODUCT DEVELOPMENT FOR HEMOPHILIA B

Abstract

Hemophilia B is an X-linked bleeding disorder that prevents the blood from clotting normally. It is caused by mutations in the factor IX (FIX) gene leading to low levels or complete loss of FIX expression/activity. It is a rare disease that affects about 1 in 20,000 male births in the USA and approximately 400,000 individuals across the world. The severity of this disease depends on the level of FIX protein expression in the patients and has been classified as mild (5-40% FIX activity), moderate (1-5% FIX activity) or severe (< 1% FIX activity). Patients with mild disease usually bleed only after serious injury, trauma or surgery; and in such patients, the first bleeding episode usually doesn’t occur until adulthood. Individuals with moderate disease, who account for about 15% of the hemophilia population, have bleeding episodes after minor injuries and often have spontaneous bleeding episodes without an obvious cause. People with severe disease also bleed following an injury and have frequent spontaneous bleeding episodes in joints and muscle. Such patients account for about 60% of the hemophilia B population (National Hemophilia Foundation, USA and Centre for Disease Control and Prevention, USA). Replacement therapy for the missing FIX is the only drug modality currently available for the treatment of haemophilia B. It requires frequent infusion of plasma derived or recombinant FIX protein to the patients. Importantly, infusion of plasma derived FIX might entail the risk of patient infection. This study was carried out to develop adeno associated viral vector based gene therapy product for the treatment of Hemophilia B (AAV-FIX). AAV-FIX vectors were expressed in HEK293 cells by triple plasmid co-transfection. Transfected cells were lysed to release the vector in cell lysate which was clarified and subjected to affinity based purification. The purified vectors were analysed by SDS-PAGE and ELISA for the purity and quantity.