re of RNAs inside the cell. Experiments with an in vitro-synthesized 
fragment of HCV RNA and with the HCV replicon cell line confirmed that modified LNA/DNA gapmer oligonucleotides use an antisense mode of action. Danoprevir web Although the insertion of three 8-oxo-dG residues into the LNA/DNA gapmer ASO did not reduce the EC50, it somewhat increased the inhibition of virus replication at concentrations exceeding the EC50. In part, this effect can be attributed 19 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication to the increased stability of modified compounds in a biologically relevant environment. Modified ASOs, where LNA residues are dispersed over the length of the compound, were found to lack an antiviral effect. Consistent with previous studies, MixLD4676 and other similarly designed ASOs were unable to trigger RNase H-mediated degradation of the RNA strand in ASO:RNA duplexes. These data, similar to those published by Laxton and co-workers, highlighted the importance of RNase H-mediated cleavage for the antiHCV activity of ASOs. RNase H-mediated RNA degradation also depends on the ability of an ASO to form a duplex with its target. Using a short target RNA molecule, duplex formation was shown to be fast, and, as expected, its efficiency correlated with the ASO Tm. Somewhat unexpectedly, the initial speed of degradation of pre-formed ASO:RNA duplexes depended little, if at all, on the modifications introduced into the ASO. Instead, there was a clear correlation between the efficiency of ASO:RNA duplex formation and the efficiency of RNase H-mediated cleavage of FR3131 RNA, suggesting that in the in vitro RNA cleavage experiment, the efficiency and speed of ASO:RNA duplex formation was the rate-limiting step. However, due to different conditions, including differences in the specificity and abundance of RNase H enzymes in living human cells, the possibility cannot be excluded that the in vivo activity of ASOs does not necessarily correlate with its binding to small model substrates. Indeed, human cells have two different RNase H enzymes. Although the human RNase H1 shares many enzymatic properties with the bacterial enzyme, there are differences. Human RNase H1 binds to A-type RNA:DNA duplexes with much greater PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19702757 activity than bacterial RNase H and displays a strong positional preference for cleavage, i.e., it cleaves between 8 and 12 nucleotides from the 50 -RNA-30 -DNA terminus of the duplex. Therefore, it would be interesting to study whether the presence of 8-oxo-dG residues affects the cleavage specificity of human RNase H enzymes. If this indeed is the case, then such modifications might be particularly useful for constructing ASOs that target viruses PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19705070 that rapidly develop resistance to siRNAs or ASOs against RNAs with pre-existing variations in the target site. Although LDM4676 displayed high activity in different in vitro assays, its value as a potential HCV inhibitor critically depends on its in vivo performance. However, such studies are hampered by the lack of low-cost small-animal models and by the high costs of ASOs containing LNA bases, 8-oxo-dG residues and phosphorothioate modifications in its backbone. However, should such types of compounds be highly active in vivo, they could contribute to the development of ASO-based HCV treatments. Miravirsen, the first experimental drug of this type, has already been successfully used in clinical trials. However, miravirsen targets an important cofactor of HCV genome expression and repli
