Ribozyme inhibition of alphavirus replication

A model system to examine the expression and antiviral activity of trans-acting ribozymes in mammalian cells has been developed and evaluated. Hairpin ribozymes were engineered to cleave a specific site, identified by a combinatorial activity-based selection method, within genomic and subgenomic RNA species of Sindbis virus. Transiently transfected cells expressed moderate levels of ribozyme (approximately 50,000 molecules/cell) with predominant nuclear localization and a short half-life (23 min). Stable cell lines expressed ribozymes at modest levels (approximately 2,000 molecules/cell). Ribozyme-mediated RNA cleavage activity was detected in cell extracts. Clonal cell lines were challenged with recombinant Sindbis virus, and viral replication was examined using plaque formation and green fluorescent protein assays. Significant inhibition of viral replication was observed in cells expressing the active antiviral ribozyme, and lower levels of inhibition in control cells expressing inactive or irrelevant ribozymes. These findings are consistent with a model in which inhibition of viral replication occurs via ribozyme cleavage of viral RNAs, suggesting that ribozymes may represent useful antiviral agents.     

A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

Design and specificity of hammerhead ribozymes against calretinin mRNA.

We obtained a partial sequence of mouse calretinin mRNA from cDNA clones, and designed hammerhead ribozymes to cleave positions within it. With a view to optimising hammerhead ribozymes for eliminating the mRNA in vivo, we varied the length and sequence of the three duplex 'arms' and measured the cleavage of long RNA substrates in vitro at 37 degrees C (as well as 50 degrees C). Precise cleavage occurred, but it could only go to completion with a large excess of ribozyme. The evidence suggests that the rate-limiting step with a large target is not the cleavage, but the formation of the active ribozyme: substrate complex. The efficiency varied unpredictably according to the target site, the length of the substrate RNA, and the length of the ribozyme; secondary structure in vitro may be responsible. We particularly investigated the degree of sequence-specificity. Some mismatches could be tolerated, but shortening of the total basepairing with the substrate to less than 14 bp drastically reduced activity, implying that interaction with weakly-matched RNAs is unlikely to be a serious problem in vivo. These results suggest that specific and complete cleavage of a mRNA in vivo should be possible, given high-level expression of a ribozyme against a favourable target site.     

Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs.     

Antiviral ribozymes

Catalytic RNAs are a genetic property not only of some particular viroids or viruses, but also are more common naturally among eukaryotes and even prokaryotes than earlier expected. However, the major interest in ribozymes results from their potential for development of “tailor-made” cDNA constructions designed to be transcribed into catalytic RNAs that will recognize by hybridization and destroy by specific cleavage their cellular or viral RNA targets. The efficiency of an antiviral ribozyme is determined by both the accessibility and sequence conservation of the target region, as well as the design of the ribozyme: its type, size, and composition of flanking sequences; expression rates; and cellular compartment localization. Until now the most frequently selected viral target is the human immunodeficiency virus, where an up to a 10⁴-fold inhibition in its progeny production has been achieved. Although the first generation ribozymes focused on improvements in basic design and expression rates, more recently the efficiency of antiviral catalytic activity has been increased by employing polyribozymes and/or multitarget ribozymes, as well as special constructions to enhance the cellular co-compartmentation of the ribozyme with its viral RNA target.     

UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.     

Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo.

The subclass of catalytic RNAs termed ribozymes cleave specific target RNA sequences in vitro. Only circumstantial evidence supports the idea that ribozymes may also act in vivo. In this study, ribozymes with a hammerhead motif directed against a target sequence within the mRNA of the neomycin phosphotransferase gene (npt) were embedded into a functional chimeric gene. Two genes, one containing the ribozyme and the other producing the target, were cotransfected into plant protoplasts. Following in vivo expression, a predefined cleavage product of the target mRNA was detected by ribonuclease protection. Expression of both the ribozyme gene and the target gene was driven by the CaMV 35S promoter. Concomitant with the endonucleolytic cleavage of the target mRNA, a complete reduction of NPT activity was observed. An A to G substitution within the ribozyme domain completely inactivates ribozyme-mediated hydrolysis but still shows a reduction in NPT activity, albeit less pronounced. Therefore, the reduction of NPT activity produced by the active ribozyme is best explained by both hydrolytic cleavage and an antisense effect. However, the mutant ribozyme--target complex was more stable than the wildtype ribozyme--target complex. This may result in an overestimation of the antisense effect contributing to the overall reduction of gene expression.     

Ribozymes for specific inhibition of mRNA function in the nematode Caenorhabditis elegans.

Nine different hammerhead ribozymes were designed for three specific sites of unc-22 mRNA in C. elegans, which carry the common catalytic core and 12, 16 or 20 flanking nucleotides for base pairing with the mRNA, and tested for cleavage of short substrate RNA in vitro. All the ribozymes cleaved the substrate RNA catalytically at 37 degrees C and the activities at 37 degrees C were higher for all the ribozymes than those at 20 degrees C, the nematode growth temperature. Plasmids carrying each of a few different promoters and lacZ reporter gene were prepared and tested in the nematode as a test of vectors for the expression of ribozymes in vivo.     

Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1.

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.     

Effective inhibition of human cytomegalovirus gene expression and growth by intracellular expression of external guide sequence RNA

RNase P complexed with external guide sequence (EGS) represents a novel nucleic-acid-based gene interference approach to modulate gene expression. In this study, a functional EGS RNA was constructed to target the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin. The EGS RNA was shown to be able to direct human RNase P to cleave the target mRNA sequence efficiently in vitro. A reduction of approximately 75%-80% in the mRNA and protein expression levels of both CSP and assemblin and a reduction of 800-fold in viral growth were observed in human cells that expressed the functional EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS that carried nucleotide mutations that precluded RNase P recognition. The action of the EGS is specific as the RNase P-mediated cleavage only reduces the expression of the CSP and assemblin but not other viral genes examined. Further studies of the antiviral effects of the EGS indicate that the expression of the functional EGS has no effect on HCMV genome replication but blocks viral capsid maturation, consistent with the notion that CSP and assemblin play essential roles in HCMV capsid formation. Our study provides the first direct evidence that EGS RNAs effectively inhibit HCMV gene expression and growth. Moreover, these results demonstrate the utility of EGS RNAs in gene therapy applications, including the treatment of HCMV infection by inhibiting the expression of virus-encoded essential proteins.     

Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences.

Conventionally designed ribozymes may be unable to cleave RNA at sites which are inaccessible due to secondary structure. In addition, it may also be difficult to specifically target a conventionally designed ribozyme to some chimeric RNA molecules. Novel approaches for ribozyme targeting were developed by using the L6 bcr-abl fusion RNA as a model. Using one approach, we successfully directed ribozyme nucleation to a site on the bcr-abl RNA that is distant from the GUA cleavage site. These ribozymes bound to the L6 substrate RNA via an anchor sequence that was complementary to bcr sequences. The anchor was necessary for efficient cleavage as the anchor minus ribozyme, a conventionally designed ribozyme, was inefficient at catalyzing cleavage at this same site. The effect of anchor sequences on catalytic rates was determined for two of these ribozymes. Ribozymes generated by a second approach were designed to cleave at a CUU site in proximity to the bcr-abl junction. Both approaches have led to the development of a series of ribozymes specific for both the L6 and K28 bcr-abl chimeric RNAs, but not normal abl or bcr RNAs. The specificity of the ribozyme correlated in part with the ability of the ribozyme to bind substrate as demonstrated by gel shift analyses. Secondary structure predictions for the RNA substrate support the experimental results and may prove useful as a theoretical basis for the design of ribozymes.     

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. This enzyme is a ribonucleoprotein complex for tRNA processing. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). EGSs, which are RNAs derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P and M1 ribozyme. When covalently linked with a guide sequence, M1 can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which cleaves any target RNAs that base pair with the guide sequence. Studies have demonstrated efficient cleavage of mRNAs by M1GS and RNase P complexed with EGSs in vitro. Moreover, highly active M1GS and EGSs were successfully engineered using in vitro selection procedures. EGSs and M1GS ribozymes are effective in blocking gene expression in both bacteria and human cells, and exhibit promising activity for antimicrobial, antiviral, and anticancer applications. In this review, we highlight some recent results using the RNase P-based technology, and offer new insights into the future of using EGS and M1GS RNA as tools for basic research and as gene-targeting agents for clinical applications.     

Effect of ribozyme against transforming growth factorbeta1 on biological character of activated HSCs

Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would provide a potential therapeutic approach for liver fibrosis.