Here we describe the development of an optimized human Factor IX (hF.IX) gene expression cassette under the control of a human liver-specific transthyretin promoter covalently flanked by AAV inverted terminal repeats (ITRs) with no open ends (optNE-TTR-hF.IX), which mediated ~sixfold higher hF.IX levels than that from a linear TTR-hF.IX DNA construct in human hepatoma cells up to four-weeks post-transfection. Here, F9 is linked to hepatocellular carcinoma.