489. Treatment for Hemophilia B Using Self-Complimentary AAV8 Vectors

Hemophilia B is an X-linked hemorrhagic disease affecting about 1/30,000 males. The disease manifests as unpredictable, recurrent, and spontaneous bleeding in soft tissues and/or major joints. This recurrent bleeding disease is due to a defect in the blood coagulation factor IX (FIX). Recent clinical trials have showed promise in the safe use of adeno-associated virus (AAV) for gene replacement into muscle or liver. However, these studies showed FIX concentrations that either did not reach therapeutic levels, or were not sustained. New research into the use of alternative AAV serotypes for liver specific transduction and the use of self-complimentary adeno- associated virus (scAAV) for rapid and prolonged gene replacement may help to reach and maintain therapeutic levels of FIX. Recent studies have shown that double-stranded vectors are 10 to 30-fold more robust than their single-stranded counterparts. We have developed an expression cassette that fits within the size limitations (2.3kb) of a scAAV vector, which contains human and canine FIX genes under the control of the tissue specific transthyretin promoter combined with a liver specific synthetic enhancer. This enhancer/promoter combination was found to significantly increase liver specific expression of a reporter gene when compared in hepatoma and non-hepatic cells in tissue culture. Hydrodynamic tail vein injection of the plasmids containing the smaller expression cassette into C57/BL6 mice showed only a 2X lower expression of circulating FIX when compared to the larger ApoE(HCR)hAAT promoter enhancer, a common sequence used for robust liver-based gene expression. The amount of hFIX detectable in mouse serum was 19|[mu]|g/ml and 49|[mu]|g/ml for the TTR and ApoEhAAT constructs, respectively. Construction of a scAAV vector has been completed, and vector using a pseudotyped AAV-8 capsid is being produced. These scAAV-8 vectors will be administered to murine and canine models systems and compared to the ApoE(HCR)hAAT FIX constructs previously tested in dogs and used in a human clinical trial. We expect the new scAAV-8 vectors to be more robust requiring a substantially lower dose to reach sustained therapeutic levels of clotting factor IX.