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Investigating a new generation of ribozymes in order to target HCV.

ABSTRACT: For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV) have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes) that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with which to target HCV (+) RNA strands. These ribozymes resulted from the coupling of a specific on/off adaptor (SOFA) to the ribozyme domain derived from the Hepatitis Delta Virus (HDV). The former switches cleavage activity "on" solely in the presence of the desired RNA substrate, while the latter was the first catalytic RNA reported to function naturally in human cells, specifically in hepatocytes. In order to maximize the chances for success, a step-by-step approach was used for both the design and the selection of the ribozymes. This approach included the use of both bioinformatics and biochemical methods for the identification of the sites possessing the greatest potential for targeting, and the subsequent in vitro testing of the cleavage activities of the corresponding SOFA-HDV ribozymes. These efforts led to a significant improvement in the ribozymes' designs. The ability of the resulting SOFA-HDV ribozymes to inhibit HCV replication was further examined using a luciferase-based replicon. Although some of the ribozymes exhibited high levels of cleavage activity in vitro, none appears to be a potential long term inhibitor in cellulo. Analysis of recent discoveries in the cellular biology of HCV might explain this failure, as well as provide some ideas on the potential limits of using nucleic acid-based drugs to control the propagation of HCV. Finally, the above conclusions received support from experiments performed using a collection of SOFA-HDV ribozymes directed against HCV (-) strands.

SUBMITTER: Levesque MV

PROVIDER: S-EPMC2835756 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Investigating a new generation of ribozymes in order to target HCV.

Lévesque Michel V MV Lévesque Dominique D Brière Francis P FP Perreault Jean-Pierre JP

PloS one 20100310 3

For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV) have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes) that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with ...[more]

PMID: 20224783

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Project description:BackgroundHepatitis C virus (HCV) genomic variability is a major challenge to the generation of a prophylactic vaccine. We have previously shown that HCV specific T-cell responses induced by a potent T-cell vaccine encoding a single strain subtype-1b immunogen target epitopes dominant in natural infection. However, corresponding viral regions are highly variable at a population level, with a reduction in T-cell reactivity to these variants. We therefore designed and manufactured second generation simian adenovirus vaccines encoding genomic segments, conserved between viral genotypes and assessed these for immunogenicity.MethodsWe developed a computer algorithm to identify HCV genomic regions that were conserved between viral subtypes. Conserved segments below a pre-defined diversity threshold spanning the entire HCV genome were combined to create novel immunogens (1000-1500 amino-acids), covering variation in HCV subtypes 1a and 1b, genotypes 1 and 3, and genotypes 1-6 inclusive. Simian adenoviral vaccine vectors (ChAdOx) encoding HCV conserved immunogens were constructed. Immunogenicity was evaluated in C57BL6 mice using panels of genotype-specific peptide pools in ex-vivo IFN-ϒ ELISpot and intracellular cytokine assays.ResultsChAdOx1 conserved segment HCV vaccines primed high-magnitude, broad, cross-reactive T-cell responses; the mean magnitude of total HCV specific T-cell responses was 1174 SFU/106 splenocytes for ChAdOx1-GT1-6 in C57BL6 mice targeting multiple genomic regions, with mean responses of 935, 1474 and 1112 SFU/106 against genotype 1a, 1b and 3a peptide panels, respectively. Functional assays demonstrated IFNg and TNFa production by vaccine-induced CD4 and CD8 T-cells. In silico analysis shows that conserved immunogens contain multiple epitopes, with many described in natural HCV infection, predicting immunogenicity in humans.ConclusionsSimian adenoviral vectored vaccines encoding genetic segments that are conserved between all major HCV genotypes contain multiple T-cell epitopes and are highly immunogenic in pre-clinical models. These studies pave the way for the assessment of multi-genotypic HCV T-cell vaccines in humans.

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Investigating a new generation of ribozymes in order to target HCV.

Publications

Investigating a new generation of ribozymes in order to target HCV.

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