Volume 44, Issue 6, Pages 1017-1025 (June 2006) Hammerhead ribozymes with cleavage site specificity for NUH and NCH display significant anti-hepatitis C viral effect in vitro and in recombinant HepG2 and CCL13 cells  Maria-Angeles Gonzalez-Carmona, Sabine Schüssler, Matthias Serwe, Michael Alt, János Ludwig, Brian S. Sproat, Robin Steigerwald, Per Hoffmann, Maria Quasdorff, Oliver Schildgen, Wolfgang H. Caselmann  Journal of Hepatology  Volume 44, Issue 6, Pages 1017-1025 (June 2006) DOI: 10.1016/j.jhep.2005.10.022 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 NCH cleaving hammerhead ribozymes; nt positions and nature of chemical modifications are given. G5/A6/G8/G12/I15.1=ribonucleosides; Rz1293: U4=2′-O-allyl-U; Rz1425: U4=2′-amino-U; Rz1426: U4=2′-O-allyl-U; G5=2′-O-allyl-G; G12=2′-O-allyl-G. Modification of Rz1293m was identical to Rz1293. A sequence mismatch was introduced at C15.2⇒G15.2 (s. arrow). [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 1017-1025DOI: (10.1016/j.jhep.2005.10.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Comparative inhibitory activity of Rz3, ORN 328 and S-ODN 328 on HCV translation in rabbit reticulocyte lysates. Rz3 (▴), ORN 328 (●), and S-ODN 328 (♦) in varying concentrations (μM) were mixed with 10ng of a HCV-luciferase fusion RNA obtained by in vitro transcription from the BamHI linearized plasmid pT7NCRluc. Luciferase activity was measured and compared to controls to which no Rz, S-ODN and ORN had been added (100% luciferase activity). All experiments were performed at least in quadruplicate. Percent mean values and standard errors of the mean are shown. [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 1017-1025DOI: (10.1016/j.jhep.2005.10.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Inhibition of HCV translation by NCH specific ribozymes in vitro. The translation inhibition was analyzed at varying concentrations. Rz1293, Rz1425: cleavage active ribozymes; Rz1426: cleavage inactive ribozyme; Rz1437: non-specifically binding ribozyme; Rz1293m: mismatch ribozyme. [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 1017-1025DOI: (10.1016/j.jhep.2005.10.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Inhibition of HCV translation in vitro by ribozyme Rz1293 with competitor ODN. The inhibition of luciferase expression by 2μM Rz1293 or Rz1425 was analysed after addition of 10 or 20μM sense (HCVrzCI-ODN) or mismatch competitor DNA (HCVrzMM-ODN). Mean luciferase activity and standard errors of the mean are given. Dark gray bar: Rz1293 and HCVrzMM-ODN, light gray bar: Rz1293 and HCVrzCI-ODN, squared pattern: Rz1425 and HCVrzMM-ODN and striped pattern: Rz1425 and HCVrzCI-ODN. Journal of Hepatology 2006 44, 1017-1025DOI: (10.1016/j.jhep.2005.10.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Cellular assays measuring the translation inhibition of HCV. Recombinant cell lines CCL13 (a) and HepG2 (b) stably transfected with the HCV/luciferase-fusion recombinant plasmid pCMVNCRluc and 2μM (CCL13) or 0.5μM (HepG2) of ribozymes Rz1293, Rz1425, Rz1426, or Rz1437 were lysed and normalized protein amounts were used for luciferase assays. Mean luciferase activity and standard errors of the mean are given. Journal of Hepatology 2006 44, 1017-1025DOI: (10.1016/j.jhep.2005.10.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions