Conjugation of an antisense oligodeoxynucleotide to ribonuclease h results in sequence-specific cleavage and intracellular inhibition of HCV gene expression.
A recombinant E. coli ribonuclease H (RNase H) was chemically coupled to an antisense oligodeoxynucleotide (ODN) against the 5'-noncoding region (5'-NCR) of the hepatitis C virus. Purity of the conjugates was confirmed by sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a band corresponding to approximately 23 kDa. Conjugate function was tested by the cleavage of a HCV RNA transcript including the 5'-NCR and core region and showed HCV sequence-specific cleavage by the appearance of an expected approximately 1000 nt fragment of RNA. Cleavage was not seen by RNase H alone, or ODN alone. Delivery studies using (32)P- and (125)I-labeling showed that while RNAse H failed to enter cells, the conjugate was efficiently taken into the cells. To assess intracellular effects, a cell line, Huh-7/CMV-NCRCDeltaluc, which expresses HCV mRNA (nt 1-585) fused to a marker gene, was transfected with the conjugate. Reporter gene expression was suppressed by 51.2% with the conjugate compared to only 39.7% by ODN alone, 35.8% by a mixture of RNase H plus ODN, and not at all by RNase H alone. In conclusion, the RNase H-ODN conjugate effectively cleaved an HCV transcript in vitro and inhibited expression of an HCV-marker fusion construct in a liver-derived cell line.
Fukuma
Toshiko
T
Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Farmington 06030, USA.
Walton
Cherie M
CM
Wu
Catherine H
CH
Wu
George Y
GY
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D007553:Isotope Labeling; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000494:pharmacology*; D016914:Ribonuclease H / Q000737:chemistry* / Q000494:pharmacology*; D014158:Transcription, Genetic
D016376:Oligonucleotides, Antisense; D008156:Luciferases; D016914:Ribonuclease H