Inhibition of hepatitis C virus by an M1GS ribozyme derived from the catalytic RNA subunit of Escherichia coli RNase P

Abstract

BackgroundHepatitis C virus (HCV) is a human pathogen causing chronic liver disease in about 200 million people worldwide. However, HCV resistance to interferon treatment is one of the important clinical implications, suggesting the necessity to seek new therapies. It has already been shown that some forms of the catalytic RNA moiety from E. coli RNase P, M1 RNA, can be introduced into the cytoplasm of mammalian cells for the purpose of carrying out targeted cleavage of mRNA molecules. Our study is to use an engineering M1 RNA (i.e. M1GS) for inhibiting HCV replication and demonstrates the utility of this ribozyme for antiviral applications.ResultsBy analyzing the sequence and structure of the 5′ untranslated region of HCV RNA, a putative cleavage site (C67-G68) was selected for ribozyme designing. Based on the flanking sequence of this site, a targeting M1GS ribozyme (M1GS-HCV/C67) was constructed by linking a custom guide sequence (GS) to the 3′ termini of catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli through an 88 nt-long bridge sequence. In vitro cleavage assays confirmed that the engineered M1GS ribozyme cleaved the targeted RNA specifically. Moreover, ~85% reduction in the expression levels of HCV proteins and >1000-fold reduction in viral growth were observed in supernatant of cultured cells that transfected the functional ribozyme. In contrast, the HCV core expression and viral growth were not significantly affected by a “disabled” ribozyme (i.e. M1GS-HCV/C67*). Moreover, cholesterol-conjugated M1GS ribozyme (i.e. Chol-M1GS-HCV/C67) showed almost the same bioactivities with M1GS-HCV/C67, demonstrating the potential to improve in vivo pharmacokinetic properties of M1GS-based RNA therapeutics.ConclusionOur results provide direct evidence that the M1GS ribozyme can function as an antiviral agent and effectively inhibit gene expression and multiplication of HCV.

Ribozymes as Therapeutic Agents against Infectious Diseases

“…Ribozymes can be modified to improve their stability and resistance towards nucleases. Some modifications include using locked nucleic acids (LNAs) [109], cholesterol [83], nanoparticles [110], or low-molecular-weight polyethyleneimine [111]. Modifications to the ribozyme tertiary structure or interactions can improve their stability.…”

Enteroviruses: Classification, diseases they cause, and approaches to development of antiviral drugs

“…Approaches associated with designing or searching for antiviral agents whose action is directed against IRESs are being extensively developed. The following com pounds are considered as such preparations or a basis for their development: complementary oligonucleotides [131], peptide nucleic acids [130], locked nucleic acids [130], morpholines [134,135], short RNA hairpins [133,136,137], small interfering RNAs [133,136,137], RNA aptamers, ribozymes [138,139], DNAzymes [140,141], peptides [142,143], and low molecular weight inhibitors [141 148].…”

RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences

“…Two M1GS constructs were made, M1GS-HCV/C67 with a linker sequence between the M1 RNA and the 13-nucleotide GS, and M1GS-HCV/C67* without a linker sequence. In vitro , the linker sequence appeared to be required for efficient cleavage of the genomic RNA of HCV [ 58 ]. To improve the pharmacokinetic properties of M1GS-HCV/C67, cholesterol was conjugated to its 5'-end, which did not diminish the cleavage activity.…”

“…Both M1GS-HCV/C67 and Cholesterol-M1GS-HCV/C67 resulted in an 85% reduction in HCV core protein expression, as well as a more than 1000-fold reduction of viral growth in transfected Huh7.5.1 cells. These results indicate that the M1GS approach allows the efficient inhibition of HCV growth, that a linker between the M1 and GS parts may be crucial for activity, and that the cholesterol modification does not reduce its antiviral activity and therefore might be used to improve the kinetics for M1GS ribozymes [ 58 ].…”