Designed Hammerhead Ribozymes Research Articles

Inhibition of miR-21 in glioma cells using catalytic nucleic acids.

Despite tremendous efforts worldwide, glioblastoma multiforme (GBM) remains a deadly disease for which no cure is available and prognosis is very bad. Recently, miR-21 has emerged as a key omnipotent player in carcinogenesis, including brain tumors. It is recognized as an indicator of glioma prognosis and a prosperous target for anti-tumor therapy. Here we show that rationally designed hammerhead ribozymes and DNAzymes can target miR-21 and/or its precursors. They decrease miR-21 level, and thus silence this oncomiR functions. We demonstrated that anti-miRNA catalytic nucleic acids show a novel terrific arsenal for specific and effective combat against diseases with elevated cellular miR-21 content, such as brain tumors.

Ribozyme-mediated cleavage of the human survivin mRNA and inhibition of antiapoptotic function of survivin in MCF-7 cells.

Survivin is a new member of the inhibitor of apoptosis protein (IAP) family that is implicated in the control of cell proliferation and the regulation of cell life span. This protein is selectively expressed in most human carcinomas but not in normal adult tissues. To down-regulate a human survivin expression as a strategy for cancer gene therapy, we designed two hammerhead ribozymes (RZ-1, RZ-2) targeting human survivin mRNA. RZ-1 and RZ-2 efficiently cleaved the human survivin mRNA at nucleotide positions +279 and +289, which was identified by in vitro cleavage assay using in vitro transcribed ribozymes and truncated survivin mRNA substrate. To investigate the function of the ribozymes in cells, the sequences of the ribozymes were cloned into replication-deficient adenoviral vector and transferred to breast cancer cell, MCF-7. The infection with adenovirus encoding the ribozymes resulted in a significant reduction of survivin mRNA (74% and 73%, respectively) and protein. As revealed by nuclear condensation/ fragmentation and flow cytometry analysis, inhibition of survivin gene by ribozymes increased apoptosis and sensitivity induced by etoposide or serum starvation. Our results suggest that the designed hammerhead ribozymes against survivin mRNA are good candidates for feasible gene therapy in the treatment of cancer.

Hammerhead ribozyme targeting human platelet-derived growth factor A-chain mRNA inhibited the proliferation of human vascular smooth muscle cells

Platelet-derived growth factor (PDGF) A-chain contributes to the pathogenesis of cardiovascular proliferative diseases, such as hypertensive vascular disease, atherosclerosis, and re-stenosis of an artery after angioplasty. To develop a ribozyme against human PDGF A-chain mRNA as a gene therapy for human arterial proliferative diseases, we designed and synthesized a 38-base hammerhead ribozyme to cleave human PDGF A-chain mRNA at the GUC sequence at nucleotide 591. In the presence of MgCl 2, synthetic hammerhead ribozyme to human PDGF A-chain mRNA cleaved the synthetic target RNA to two RNA fragments at a predicted size. Doses of 0.01–1.0 μM hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited angiotensin II (Ang II) and transforming growth factor (TGF)-β 1-induced DNA synthesis in vascular smooth muscle cells (VSMC) from human in a dose-dependent manner. One micromolor of hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited Ang II-induced PDGF A-chain mRNA and PDGF-AA protein expressions in VSMC from humans. These results indicate that the designed hammerhead ribozyme to human PDGF A-chain mRNA effectively inhibited growth of human VSMC by cleaving the PDGF A-chain mRNA and inhibiting the PDGF-AA protein expression in human VSMC. This suggests that the designed hammerhead ribozyme to PDGF A-chain mRNA is a feasible gene therapy for treating arterial proliferative diseases.

Potential of Ribozymes against Deoxycytidine Kinase to Confer Drug Resistance to Cytosine Nucleoside Analogs

Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs.

Hammerhead Ribozyme Cleavage of Apolipoprotein B mRNA Generates a Truncated Protein

Target substrate-specific hammerhead ribozyme cleaves the specific mRNA and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To modulate apoB gene expression, we designed hammerhead ribozymes targeted at AUA(6665) and GUA(6679) of apoB mRNA, designated RB16 and RB15, respectively, and investigated their effects on apoB mRNA in HepG2 cells. The results demonstrated that RB15 and RB16 ribozyme RNAs cleaved apoB RNA efficiently in vitro. Both ribozymes, RB15 and RB16, were used to construct recombinant adenoviral vectors, designated AvRB15 and AvRB16, respectively, for in vivo gene transfer. HepG2 cells were infected with 2 x 10(5) plaque-forming units of AvRB15 for 5, 10, 15, and 24 h. An RNase protection assay showed that the expression of the RB15 transcript was time-dependent; it increased approximately 300-fold from 5 to 24 h. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site of apoB mRNA was confirmed by sequencing. Importantly, the levels of apoB mRNA in HepG2 cells decreased approximately 80% after AvRB15 infection. Pulse/chase experiments on HepG2 cells treated with AvRB15 and AvRB16 demonstrated that ribozyme cleavage produced a truncated protein that was secreted at a density of 1. 063-1.210 g/ml. The cleavage activity of RB15 on apoB mRNA was more efficient than that of RB16. Moreover, pulse/chase experiments in HepG2 cells treated with AvRB15 revealed that most of the truncated apoB protein was degraded intracellularly. We conclude that hammerhead ribozyme targeted at GUA(6679) of apoB mRNA cleaves apoB mRNA, results in decreased apoB mRNA levels, and generates a truncated apoB of the expected size in vivo. Thus, the therapeutic application of ribozyme in regulating apoB production holds promise.

Cleavage of viral RNA and inhibition of viral translation by hepatitis C virus RNA-specific hammerhead ribozyme in vitro

Background/Aims: A hammerhead ribozyme has been used as a new way to suppress specific gene expression. We designed hammerhead ribozymes directed against hepatitis C virus RNA, and investigated their cleavage efficiency and inhibitory effect on viral translation in vitro.Methods: Three hammerhead ribozymes bearing different cleavage sites in the core region of hepatitis C virus RNA (genotype 1b) were designed in this study. Ribozymes and the target hepatitis C virus RNA were synthesized by in vitro transcription. The cleavage efficiency was evaluated by the ribozyme cleavage assay. The inhibitory effect of the ribozyme on viral translation was further studied by the viral translation inhibition assay.Results: All ribozymes specifically cleaved the target RNA of 1217 bases at a physiological temperature in a dose-dependent manner, with the specific cleavage increasing with a longer incubation period. The target RNA was cleaved most efficiently by the ribozyme with the cleavage site located nearest to the initiation codon. In the viral translation inhibition assay, all ribozymes showed a significant inhibitory effect on viral translation. The ribozyme with the cleavage site located farthest from the initiation codon blocked viral translation most efficiently, and demonstrated almost 70 to 80% inhibition. For ribozymes with the T7 transcription terminator sequence, both the target RNA cleavage and the inhibition of viral translation tended to be achieved less efficiently by ribozymes with T7 terminator than by those without it.Conclusions: These findings suggest that ribozyme-mediated hepatitis C virus RNA cleavage may serve as a new strategy in the treatment of hepatitis C virus infection.

Detection of hammerhead ribozyme-mediated cleavage and reduced expression of LacZ′ mRNA in E. coli

Hammerhead ribozymes have been shown to specifically suppress the expression of target genes in various cells, but their in vivo cleavage products have seldom been directly detected. A hammerhead ribozyme sequence was designed to cleave the phosphodiester bond just 3′ to the GUC of SalI site of M13mp18. The ribozyme was inserted some base pairs upstream of the target region without disrupting the reading frame of the lacZ′ gene without introducing any translational stop codons. More than 90% RNAs synthesized in vitro were cleaved at the expected site after 1-h incubation in the presence of 10 mM nagnesium ion at 37 and 50°C. Inclusion of the designed ribozyme sequence was also shown to suppress the expression of the fused lacZ′ gene in E. coli cells. When the cells were infected by the ribozyme-containing phage, they remained colourless in the presence of X-gal, and the cellular β-galactosidase activity was reduced by more than 90%. Insertion of the same ribozyme sequence in reverse orientation showed little effect on β-galactosidase activity. Furthermore, a primer extension by reverse transcriptase revealed a cleavage product that resulted from cleavage of LacZ′ mRNA at the targeted site as designed. Thus, our data demonstrated that the designed hammerhead ribozyme cleaves and reduces the expression of a fused LacZ′ mRNA in E. coli cells.

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels.

Inhibition of c-Ha-ras gene expression by hammerhead ribozymes containing a stable C(UUCG)G hairpin loop.

Catalytic RNAs recognize specific sequences of RNA and cleave at a specific site. In this study, we designed hammerhead ribozymes with a thermodynamically stable loop of the sequence 5'C(UUCG)G3' to prevent the aggregation of ribozymes with hammerhead structures. The cleavage activities of these ribozymes were examined using a synthetic pentadecamer with the sequence for the c-Ha-ras mRNA mutated at codon 12 (GGU-->GUU). For in vivo studies, we constructed a plasmid which expressed a highly active ribozyme targeted against the mutated c-Ha-ras mRNA. When this ribozyme-encoding gene and the activated c-Ha-ras gene were cotransfected into NIH3T3 cells, morphologically normal cells were obtained. We also determined that the expression of the c-Ha-ras gene was inhibited in these cells. These results show that ribozymes containing this stable hairpin loop are useful for the regulation of specific gene expression in vivo.

Ribozymes designed to inhibit transformation of NIH3T3 cells by the activated c-Ha-ras gene

We have designed hammerhead ribozymes that cleave c-Ha-ras mRNA mutated at codon 12 (GGU→GUU). Plasmids containing the ribozyme-encoding genes were expressed under the control of the long terminal repeats of Rous sarcoma virus in NIH3T3 cells transfected with the activated c-Ha-ras gene. These ribozymes were found to inhibit formation of foci (by about 50%) by cleaving the oncogene mRNA, rather than by hybridizing to it. Furthermore, when the activated c-Ha-ras gene was cotransfected with the ribozyme-encoding gene, three morphologically flat colonies were found and isolated. We also found that expression of c-Ha-ras was suppressed in cells containing ribozymes.