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.
OBJECTIVE
Four different ribozymes (Rz) targeting the hepatitis C virus (HCV) 5'-non-coding region (NCR) at nucleotide (nt) positions GUA 165 (Rz1), GUC 270 (Rz2), GUA 330 (Rz3) and GCA 348 (Rz1293) were compared for in vitro cleavage using a 455 nt HCV RNA substrate. The GUA 330 (Rz3) and GCA 348 (Rz1293) ribozymes, both targeting the HCV loop IV region, were found to be the most efficient, and were further analyzed in an in vitro translation system.


METHODS
For this purpose RNA transcribed from a construct encoding a HCV-5'-NCR-luciferase fusion protein was used. Cleavage-inactive (Rz1426), mismatch (Rz1293m) or unrelated ribozymes (Rz1437) were synthesized as controls for Rz-1293. HCV specificity was analysed by competition experiments using sense and mismatch oligodeoxynucleotides HCVrzCI and HCVrzMM, respectively.


RESULTS
A chemically modified nuclease-resistant variant of the GCA 348 cleaving ribozyme was selected for cell culture experiments using recombinant HepG2 or CCL13 cell lines stably transfected with a HCV-5'-NCR-luciferase target construct.


CONCLUSIONS
This ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in vivo and may be suitable for the development of novel optimized hammerhead structures.
Gonzalez-Carmona
Maria-Angeles
MA
Department of Medicine I, University of Bonn, Bonn, Germany.
Schüssler
Sabine
S
Serwe
Matthias
M
Alt
Michael
M
Ludwig
János
J
Sproat
Brian S
BS
Steigerwald
Robin
R
Hoffmann
Per
P
Quasdorff
Maria
M
Schildgen
Oliver
O
Caselmann
Wolfgang H
WH
D000998:Antiviral Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D016174:Hepacivirus / Q000187:drug effects*; D006801:Humans; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000187:drug effects; D016337:RNA, Catalytic / Q000494:pharmacology*; D012367:RNA, Viral / Q000187:drug effects*; D020506:Untranslated Regions / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D016337:RNA, Catalytic; D012367:RNA, Viral; D020506:Untranslated Regions; C427204:hammerhead ribozyme