Development of an AAV DNA-based synthetic vector for the potential gene therapy of hemophilia in children.

Recombinant AAV serotype vectors and their variants have been or are currently being used for gene therapy for hemophilia in several phase I/II/III clinical trials in humans. However, none of these trials have included children with hemophilia since the traditional liver-directed AAV gene therapy will not work in these patients because of the following reasons: (i) Up until age 10-12, the liver is still growing and dividing, and with every cell division, the AAV vector genomes will be diluted out due to their episomal nature; and (ii) Repeated gene delivery will be needed, but repeat dosing, even with an ideal AAV vector is not an option because of pre-existing antibodies to AAV vectors following the first administration. 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. In future studies, encapsidation of the optNE-TTR-hF.IX DNA in liver-targeted synthetic liposomes, may provide a viable approach for the potential gene therapy for hemophilia in children.