11. Transposition from a Gene-Deleted Adenoviral Vector Results in Phenotypic Correction in a Canine Model for Hemophilia B

Abstract

Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences offer high transduction efficiencies but were hampered by transient phenotypic correction at a non-toxic dose. For persistent hemostatic correction in hemophilia B dogs, our current study utilized a novel gene-deleted adenoviral vector system which combines adenoviral vectors for high transduction efficiencies and for the first time in a larger animal the Sleeping Beauty (SB) transposase for somatic integration and stable transgene expression. We generated the HD adenoviral vector FTC/TcFIX/attB/(FRT)2 in which a transposon with a canine factor IX (cFIX) expression cassette is flanked by FRT sites. In the presence of Flp recombinase and the recently developed hyperactive transposase HSB5 (Yant et al., unpublished), the transgene undergoes Flp mediated excision followed by HSB5 mediated somatic integration.To analyze transgene persistence in vivo C57Bl/6 mice were co-transfused with 2x10e9 transducing units (TU) of the vector FTC/TcFIX/attB/(FRT)2 and 7×10e8 TU of a second HD vector which encodes HSB5 and Flp. Control mice received an inactive version of SB, respectively. Rapid cell cycling of mouse hepatocytes was induced by performing a two-thirds partial hepatectomy and by injecting CCl(4). Seventy-five days post-injection we detected serum levels of cFIX of up to 2000 ng/ml in the active SB group and no cFIX in the control mice. This indicated that integration of the transgene from the episomal adenoviral vector genome into the host genome occurred.To test for persistence of hemostatic correction of the bleeding diathesis in hemophila B dogs we have co-injected 5.4x10e11 TU of the HD adenoviral vector FTC/TcFIX/attB/(FRT)2 and 2.6x10e11 TU of the HSB5 and Flp encoding vector. This equals a total dose of 1.6×10e13 viral particles (vps) and 1x10e12 vps/kg body weight. We measured plasma cFIX levels of up to 3900ng/ml (normal level = 5000ng/ml) and observed complete phenotypic correction of the factor IX deficiency. The whole blot clotting time (WBCT) was reduced from 60min to 18.5min on day 35, the length of the study to date. This was in sharp contrast to our previous studies in hemophilia B dogs in which we used a non-integrating HD vector. In that study we observed a 3-fold increase of the WBCT 35 days post-injection which indicates that in our current study transposition stabilized phenotypic correction. In contrast to previous studies using early generation adenoviral vectors and keeping in mind that for the first time the in vivo performance of the SB transposase in a larger animal was evaluated, we observed no vector-related elevation of liver enzymes and no fall in platelet counts. Taken together, this study demonstrates that this adeno-transposon hybrid vector system will be important for treating genetic diseases.