Gene Transfer Targeting Hepatocytes Using the PiggyBac Transposon System: An Approach towards Hemophilia A Correction and Long Term Expression of Factor VIII.

Abstract 3575

Poster Board III-512

Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBAC can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBAC transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008-15013, 2006). To date, there are no published reports of in vivo gene transfer to mammalian livers using the piggyBAC transposon system. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and the ability to excise itself precisely without leaving a footprint. We hypothesize that a piggyBAC transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PiggyBAC transposons were engineered to carry a hygromycin resistance gene (Hygro), a luciferase expression cassette (PB luciferase), or a human alpha₁ antitrypsin reporter (hAAT). We evaluated co- transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB hygro vector, we demonstrated that the co- transposase generated higher transposition efficiency than an inactive mutant in hepatocytes. We then showed in vivo persistence following hydrodynamic tail-vein injection using firefly luciferase expression driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Luciferase expression measured via in vivo bioluminescence imaging persisted up to eight months in C57Bl/6 liver (duration of experiment). Following partial hepatectomies at 5 months post injection, expression was observed only in animals receiving PB luciferase transposon and an active transposase while expression in those treated with the inactive mutant dropped to background levels supporting that expression was from integrated transgene. We furthered these experiments by introducing PB hAAT via hydrodynamic tail-vein injection as before at either a low (12.5 micrograms each transposon and transposase) or high (50 micrograms each) dose. Serum hAAT levels were measured at 421ng/ml and 365ng/ml via ELISA at 3 months post-injection, respectively. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data represent one of the first studies to show persistent transgene expression in vivo from piggyBAC transposon gene transfer.