145. A Comparison of scAAV8-TT-034 Mediated Transduction and shRNA Expression in Human Liver Biopsy Samples versus a Chimeric Mouse Model with Humanized Liver

Background: TT-034 is a DNA-directed RNA interference (ddRNAi) agent designed for the treatment of chronic HCV infection and is currently being tested in a phase I/IIa clinical study. TT-034 is comprised of a vector that expresses three independent short hairpin RNAs (shRNAs) simultaneously targeting three well-conserved regions of the HCV genome. The recombinant genome is packaged in a self-complementary aden-associated virus serotype 8 (AAV8) capsid with tropism for hepatic tissues and delivered as a single dose intravenous (IV) infusion.Methods: Chimeric mouse models in which human hepatocytes replace the majority of mouse hepatocytes are used to study human hepatic function. In order to assess validity of dose/transduction relationships in this murine model to those observed in a human clinical study, chimeric mice were infected with identical doses of TT-034 used in a phase I/IIa study using the same clinical lot of TT-034. In the clinical study, eight subjects have received a single IV infusion of TT-034 at 4.00E10, 1.25E11, 4.00E11 or 1.25E12 vg/kg. At 21 days post dosing, a liver biopsy was collected to assess TT-034 DNA levels and shRNA expression by qPCR. PXB chimeric mice (Phoenix Bio) repopulated with a minimum of 80% hepatocytes were dosed identically with the TT-034 drug product (5 groups, n=4). After 21-28 days, the livers of two mice in each group were removed and hand curated to purify human hepatic tissues (u003e93% purity). The liver tissues of the other two mice were dissociated and human hepatocytes were enriched to u003e99% using mouse hepatocyte-capturing Dynabeads. TT-034 DNA and shRNA expression were assessed by qPCR.Results: In the human study, modest levels of TT-034 DNA copies were detected in the 3 subjects dosed at 1.25E11 vg/kg, yielding 0.48, 3.65 and 10.44 copies per cell respectively. Variability in transduction was noted at the higher dose of 4.00E11 vg/kg, with the two subjects yielding 17.74 and 1.01 copies per cell. qPCR analysis of the three anti-HCV shRNAs confirms concomitant, dose dependent expression. In the hand curated samples from the chimeric mouse model (u003e93% purity), a dose of 1.25E11 vg/kg yielded 1.1 or 1.3 DNA copies per cell while the 4.00E11 vg/kg dose resulted in 1.9 and 5.5 copies DNA per cell. Dynabead-enriched human hepatocytes (u003e99%) resulted in a considerable drop in DNA copy levels: the 1.25E11 vg/kg dose averaged 0.35 copies per cell while the 4.00E11 vg/kg resulted in 0.85 copies per cell. The lowered DNA levels in the chimeric mouse model led to a concomitant reduction in shRNA expressed from the hand curated tissues. Likewise, shRNA expression was reduced even further in enriched human hepatocytes.Conclusion: Our data suggests that residual mouse hepatocytes present in the chimeric livers are transduced with the scAAV8 vector more efficiently than human hepatocytes and results in lower overall transduction as compared to human clinical samples. Thus, while these models can serve as a surrogate to assess the activity of gene therapy constructs against functions of normal human liver, the doses required for optimal activity may be modestly higher than required in the human clinical setting.