Flow cytometry was performed on a FACSCalibur flow cytometer

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 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 Cobicistat 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 replication, whereas the LDM4676-type ASO targets the HCV genome itself. Combinations of drugs with different mechanisms of action have been key for the successful treatment of chronic infections caused by viruses capable of rapidly developing drug resistance. It is also likely that despite recent progress in the development of orally deliverable oligonucleotide drugs, a subcutaneous injection will remain the main delivery method for ASOs or their combinations. Therefore, the long half-life of ASOs, allowing once-a-week administration, represents an important property of such compounds. Correspondingly, increases in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713490 the serum half-life, resulting from the insertion of 8-oxo-dG residues, may represent another important benefit of modified ASO compounds. In this study, multiple obstacles that are commonly encountered in the development of new and efficient ASOs were addressed using unconventional and efficient approaches. For the first time, several highly accessible ASO target sequences in the heavily structured coding region of the RNA genome of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19711918 HCV were revealed. RNAi-based screening represents an efficient and 20 / 25 8-oxo-dG Modified LNA ASO Inhibit HCV Replication reliable general method for ASO target site selection. This study also provides an important set of findings concerning the use of naturally occurring, minimally modified nucleobases in ASOs. In contrast to nucleobases that contain bulky modifications, the 8-oxo-dG residues reduced the Tm of ASO:RNA duplexes and had no negative effects on RNase H-mediated degradation of RNA strands in ASO:RNA duplexes. Instead, 8-oxo-dG residues facilitated cleavage by RNase H at multiple positions within the target region. Furthermore, the incorporation of 8-oxo-dG residues increased the stability of ASOs in human serum. These effects, possibly combined with other properties of modified nucleobases, outweigh the negative effects on the overall Tm of the ASO. This enabled us to obtain modified LNA/DNA gapmer oligonucleotides with EC50 values similar to their non-modified counterparts but capable of almost completely inhibiting HCV replication in replicon cell lines at higher concentrations. ~~ ~~ The identification of prognostic determinants of non-small cell lung cancer is an important goal in both clinical trials and routine practice. In clinical trials, prognostic co-variables must be taken into account in survival analyses. For instance, in a randomized trial, the statement that a difference in survival is related to t