Using synthetic siRNA standards, we estimated that approximately equal amounts (∼1 fmol) of the four active miRNAs were present in 25 μg of total liver RNA, suggesting that these four miRNAs were processed from the primary and precursor miRNA with similar efficiencies. In contrast,
no mature miR-UTR2 was observed (Fig. 4B), consistent with the lack of inhibition of the RLuc-HCV UTR2 reporter plasmid that was observed in the dual luciferase assays. However, when the orientation of miR-UTR1 and miR-UTR2 was reversed in HCV-miR-Cluster 2, mature miR-UTR2 (Fig. 4F), but no mature miR-UTR1 was produced (Fig. 4G), consistent with the gene silencing data using this cluster. With the ultimate goal of developing a safe and effective treatment for HCV infection, we used recombinant AAV vectors as delivery vehicles for HCV-miRNA-Cluster selleck 1. These vectors are currently being evaluated for safety in multiple gene therapy clinical trials, and thus far, no evidence of any serious safety issues have been seen,21 although careful evaluation of anti-AAV immune responses have not
always been systematically performed.22 A self-complementary (sc) AAV2 vector expressing HCV-miR-Cluster 1 (scAAV2-HCV-miR-Cluster 1) was produced because these vectors lead to higher transduction levels than traditional single-stranded AAV vectors.23 A control vector that expresses the enhanced GFP protein (scAAV2-eGFP) was also produced. To evaluate the inhibitory NVP-BKM120 price potential of the anti-HCV miRNAs on HCVcc replication, Huh-7.5 cells were Edoxaban treated with scAAV2-HCV-miR-Cluster
1 or scAAV2-eGFP at one of three doses, and 24 hours later, HCVcc was added. Using 104, 105, and 106 vg/cell of scAAV2-HCV-miR-Cluster 1, the amount of HCVcc in the supernatants decreased in a dose-dependent manner, resulting in 65%, 83%, and 88% inhibition of HCVcc replication, respectively (Fig. 5A). The decrease in HCVcc RNA levels found in the supernatants correlated with a 57%-93% decrease in the presence of intracellular genomic HCVcc RNA, as measured by northern blot (Fig. 5B). These results were confirmed using QRT-PCR to quantify intracellular HCVcc RNA (data not shown). Finally, HCVcc core protein also declined by 69%-98% as the dose of scAAV2-HCV-miR-Cluster 1 increased (Fig. 5C). Thus, four independent methods demonstrated that scAAV2-HCV-miR-Cluster 1 has the ability to inhibit bona fide HCVcc replication by up to 98%. The combined data described above demonstrate that plasmids expressing the anti-HCV miRNAs are capable of HCV gene silencing both in vitro and in vivo, and that AAV vectors expressing this cluster inhibit HCVcc replication in vitro. We were next interested in determining if the AAV vector system could efficiently deliver the miRNA cluster to liver and mediate gene silencing of RLuc-HCV reporter plasmids.