143. Durable Expression of TT-034 in Cynomolgus Monkey Hepatic and Cardiac Tissues Without Long-Term Adverse Effects on Endogenous MicroRNA Levels

Background: The hepatitis C virus (HCV) is an attractive target for RNA interference (RNAi) based therapeutics because its genome consists of a single, positive stranded RNA and its replication occurs strictly within the cytoplasm. TT-034 is a DNA-directed RNAi (ddRNAi) based gene therapy product for the treatment of chronic HCV infection and is currently in a phase I/IIa clinical trial. TT-034 is comprised of a self-complementary recombinant adeno-associated virus (AAV) serotype 8 vector for transduction of hepatic tissues and is systemically administered as a single intravenous dose. Inside the hepatocyte, TT-034 uses the cell's own transcriptional machinery to continuously express three independent short hairpin RNAs (shRNAs) that simultaneously target the 5' UTR and NS5B regions of the HCV genome. Results: Following a single administration of up to 3.75E12 vg/kg of TT-034, biodistribution analyses demonstrated that greater than 90% of the quantified vector is found in the Cynomolgus monkey liver tissues. In situ hybridization analyses demonstrated almost 100% transduction of the hepatocytes when dosed at 1.25E12 vg/kg. Furthermore, qPCR data revealed that shRNA levels persisted for the duration of the 180-day experiment, demonstrating the durability of expression following a single injection. We examined the impact of continuous TT-034 expression on endogenous miRNAs in liver and cardiac tissues because previous reports have suggested that high expression of shRNA may cause global dysregulation of endogenous microRNA (miRNA) levels within cells. Non-human primates were dosed with saline control or either 1.00E11, 1.00E12, 1.00E13 vg/kg of TT-034. Expression profiles of mature miRNAs were generated from liver biopsies taken 15 days after TT-034 administration. Additionally, samples from liver and heart tissues were collected 60 and 180 days after administration. Approximately 260 different miRNAs were reliably detected in each sample type and used for the analyses. The day 15 liver samples showed significant differences in the expression levels of 27 miRNAs. When analyzing all 260 miRNAs, the day 60 and day 180 liver samples showed no significant statistical differences from the control group at any dose. In the heart tissues, no significant statistical differences between the TT-034 treated group and the control group were noted at any timepoint. Conclusions: These data suggest that a single, intravenous infusion of TT-034 results in almost complete transduction of Cynomolgus monkey hepatocytes. Furthermore, TT-034 results in durable expression of three anti-HCV shRNAs. These doses of TT-034 do not cause long-term perturbation of endogenous miRNA levels.