Conjugation of an Antisense Oligodeoxynucleotide to Ribonuclease H Results in Sequence-Specific Cleavage and Intracellular Inhibition of HCV Gene Expression

摘要

A recombinant E. coli ribonuclease H (RNase H) was chemically coupled to an antisense oligodeoxy-nucleotide (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 approximatley 23 kDa. Conjugate function was tested by the cleavage of a HCV RNA transcript including the 5'-NCR and core region and showed HCV seuqnce-specific cleavage by the appearance of an expected approx 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 intracellualr 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-market fusion construct in a liver-derived cell line.