Specific inhibition of macrophage TNF-alpha expression by in vivo ribozyme treatment.

The overproduction of the cytokine TNF-alpha is associated with inflammatory and autoimmune diseases. We have developed a means to block TNF-alpha production with ribozymes directed against TNF-alpha mRNA to selectively inhibit its production in vitro and in vivo. Following cationic lipid-mediated delivery to peritoneal murine macrophages in culture, anti-TNF-alpha ribozymes were more effective inhibitors of TNF-alpha secretion than catalytically inactive ribozyme controls. Inhibition of TNF-alpha secretion was proportional to the concentration of ribozyme administered, with an IC50 of approximately 10 nM. After i.p. injection of cationic lipid/ribozyme complexes, elicited macrophages accumulated approximately 6% of the administered ribozyme. The catalytically active ribozyme suppressed LPS-stimulated TNF-alpha secretion by approximately 50% relative to an inactive ribozyme control without inhibiting secretion of another proinflammatory cytokine produced by macrophages, IL-1alpha. Ribozyme-specific TNF-alpha mRNA degradation products were found among the mRNA extracted from macrophages following in vivo ribozyme treatment and ex vivo stimulation. Thus, catalytic ribozymes can accumulate in appropriate target cells in vivo; once in the target cell, ribozymes can be potent inhibitors of specific gene expression.     

A cis and trans adenine-dependent hairpin ribozyme against Tpl-2 target

Ribozymes are catalytic RNAs that possess the property of cutting an RNA target via site-specific cleavage after sequence-specific recognition. Ribozymes can moreover cleave multiple substrate molecules. An increasing number of studies show that ribozymes are particularly well adapted tools against cancer, silencing or down-regulating gene expression at the RNA level. We have constructed an adenine-dependent hairpin ribozyme that cleaves the sequence at nucleotides A(225)(downward arrow)G(226) relative to the start codon of translation of the Tpl-2 kinase mRNA; this serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in breast cancer. An adenine-dependent hairpin ribozyme 1 (ADHR1) was previously isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure. Switch on/switch off ribozymes are particularly useful since high amounts of stable ribozyme can be produced in the absence of adenine and the ribozyme specifically cleaves its target in the presence of adenine. The ADHR1 target sequence was replaced by a sequence derived from the Tpl-2 kinase mRNA. The resulting Tpl-2 ribozyme is active in cis cleavage: kinetic studies have been performed as a function of Mg2+ concentration, adenine concentration, as well as at different pH and with various cofactors. Finally, the Tpl-2 ribozyme was shown to cleave its target in trans successfully. These findings demonstrate that a potential therapeutic ribozyme can be produced by simple sequence modification.     

Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes

We have developed a library of hairpin ribozyme genes that can be delivered and expressed in mammalian cells with the purpose of identifying genes involved in a specific phenotype. By applying the appropriate phenotypic selection criteria in tissue culture, we can enrich for ribozymes that knock down expression of an unknown gene or genes in a particular pathway. Once specific ribozymes are selected, their target binding sequence is used to identify and clone the target gene. We have applied this technology to identify a putative tumor suppressor gene that has been activated in HF cells, a nontransformed revertant of HeLa cells. Using soft agar growth as the selection criteria for gain of transformation, we have isolated ribozymes capable of triggering anchorage-independent growth. Isolation of one of these ribozymes, Rz 568, led to the identification and cloning of the human homologue of the Drosophila gene ppan, a gene involved in DNA replication, cell proliferation, and larval development. This novel human gene, PPAN, was verified as the biologically relevant target of Rz 568 by creating five additional "target validation" ribozymes directed against additional sites in the PPAN mRNA. Rz 568 and all of the target validation ribozymes reduced the level of PPAN mRNA in cells and promoted anchorage-independent growth. Exogenous expression of PPAN in HeLa and A549 tumor cells reduced their ability to grow in soft agar, underscoring its role in regulating anchorage-dependent growth. This study describes a novel method for gene discovery where the intracellular application of hairpin ribozyme libraries was used to identify a novel gene based solely on a phenotype.     

Ribozymes targeted to stearoyl-ACP delta9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves.

Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein (Delta9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in Delta9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize.     

专一地切割苜蓿夜蛾核多角体病毒多角体蛋白mRNA的ribozyme的设计与性质

本文针对苜蓿夜蛾Autographa californica核多角体病毒的多角体蛋白mRNA设计并合成了两个ribozyme。体外实验结果表明它们都能准确、高效地切割体外转录得到的mRNA片段。实验表明,在一定的范围内,切割百分率随着温度的升高、时间的延长和ribozyme浓度的提高而增加。Ca~(2+)、Mn~(2+)可以代替Mg~(2+)作为ribozyme切割反应所必需的二价金属离子。     

Characterization in vitro and in vivo of hammerhead ribozymes directed against murine tumor necrosis factoralpha.

A hammerhead ribozyme directed against murine TNFalpha (mTNFalpha) mRNA has been constructed. In vitro studies showed that this ribozyme was released from the parent molecule by flanking cis-acting hammerhead and hairpin ribozymes. This same anti-mTNFalpha ribozyme specifically cleaved both synthetically derived substrate RNA and mTNFalpha mRNA within a pool of total cellular RNA. Endogenous delivery of this anti-mTNFalpha ribozyme via the self-cleaving cassette reduced mTNFalpha mRNA and protein levels in lipopolysaccharide (LPS)-stimulated, stably transfected murine macrophage RAW 264.7 cells. When complexed to liposomes and exogenously delivered to mouse peritoneal macrophages, the same ribozyme, with and without the cis-acting ribozymes, reduced mTNFalpha protein levels. However, an irrelevant ribozyme delivered in an identical fashion was also effective at reducing mTNFalpha protein levels. These data suggest that anti-mTNFalpha ribozymes can be constructed which efficiently cleave mTNFalpha mRNA, but irrelevant RNA/liposome complexes also effectively limit TNFalpha mRNA expression and can mimic functional ribozyme activity under in vitro conditions.     

Ribozymes

The ability to alter genes in order to regulate their expression has become an undeniable reality. This can be performedin vitro and in cells, and the possibility of treating diseases and even preventing them now exists through such gene manipulation. A particularly intriguing form of manipulation that has been investigated for just over a decade is one that involves the use of ribozymes. These are short segments of RNA that form complementary base-pairing with mRNA. However, it is their enzymatic properties that set them apart from other antisense RNA molecules and allow them to cleave and destroy mRNA in a very specific manner. The ribozyme then dissociates from the cleaved substrate RNA, and repeatedly hybridizes to and cleaves additional substrate RNA molecules. Problems being addressed as this technology evolves involve optimization of ribozyme: substrate binding efficiencies and their effective transmission into cells. This article points out the origin of ribozymes, analyzes and summarizes the current strategies for designing ribozymes, and outlines a basic procedure for ribozyme development.     

BCR3/ABL2核酶的设计、构建及对K562细胞增殖与集落形成的抑制作用和凋亡诱导作用

利用计算机模拟设计合成了针对 K5 62细胞致癌融合 bcr3/abl2 m RNA的锤头状核酶 .该核酶以融合点附近 UUC为识别切割三联体 ,在核酶的 3′端增加一段 T7噬菌体终止子序列 .用基因克隆结合体外转录的方法 ,肯定了核酶的体外切割活性 .进而将核酶基因克隆到 p CEP4真核细胞高效表达载体上 ,利用脂质体 Lipofectin AMINE介导的转染技术将核酶与核酶基因导入靶细胞 ,从抑制靶细胞 K5 62的增殖与集落形成及引起靶细胞凋亡等方面验证了核酶在细胞水平上对融合基因 bcr3/abl2 m RNA的特异切割作用 ,并观察到了 T7噬菌体终止子序列对核酶切割效率的增强影响 .     

Inhibition of luciferase expression by synthetic hammerhead ribozymes and their cellular uptake.

Two synthetic hammerhead ribozymes, one unmodified and the other with 2"-modifications and four phosphorothioate groups, targeting a single GUA site in the luciferase mRNA, were compared for their inhibition of gene expression in cell cultureand their cellular uptake was also analysed. A HeLa X1/5 cell line stably expressing luciferase, under an inducible promoter, was treated with these ribozymes by liposome-mediated transfection to determine their activity.Luciferase expression in cells was inhibited to approximately 50% with little difference between the unmodified and the 2"-modified ribozyme. A similar degree of inhibition was observed with two catalytically inactive ribozymes, indicating that inhibition was mainly due to an antisense effect. A ribozyme carrying a cholesterol moiety, applied to the cells without carrier, showed no inhibition. Northern blotting indicated a similar amount of cellular uptake of all ribozymes. The unmodified ribozyme was essentially evenly distributed between cytoplasm and nucleus, whereas a higher proportion of the phosphorothioate-containing ribozyme was observed in the nucleus. Fluorescence microscopy, including confocal microscopy using 5"-fluorescein-labelled ribozymes, showed that the unmodified and 2"-modified ribozymes were present in the cytoplasm and in the nucleus to a similar extent, whereas the fluorescence of the phosphorothioate-containing ribozyme was much stronger in the nucleus. Both ribozymes inhibited luciferase expression to a comparable degree, suggesting that the ribozyme in the nucleus did not contribute significantly to the inhibition. Ribozymes with a cholesterol moiety were predominantly trapped in the cell membrane, explaining their inability to interfere with gene expression.     

Selection of the most potent specific on/off adaptor-hepatitis delta virus ribozymes for use in gene targeting

The Hepatitis Delta Virus (HDV) ribozyme, which is well adapted to the environment of the human cell, is an excellent candidate for the future development of gene-inactivation systems. On top of this, a new generation of HDV ribozymes now exists that benefits from the addition of a specific on/off adaptor (specifically the SOFA-HDV ribozymes) which greatly increases both the ribozyme's specificity and its cleavage activity. Unlike RNAi and hammerhead ribozymes, the designing of SOFA-HDV ribozymes to cleave, in trans, given RNA species has never been the object of a systematic optimization study, even with their recent use for the gene knockdown of various targets. This report aims at both improving and clarifying the design process of SOFA-HDV ribozymes. Both the ribozyme and the targeted RNA substrate were analyzed in order to provide new criteria that are useful in the selection of the most potent SOFA-HDV ribozymes. The crucial features present in both the ribozyme's biosensor and blocker, as well as at the target site, were identified and characterized. Simple rules were derived and tested using hepatitis C virus NS5B RNA as a model target. Overall, this method should promote the use of the SOFA-HDV ribozymes in a plethora of applications in both functional genomics and gene therapy.