Catalytic Antisense RNA Research Articles

Inhibition of HIV-1 Replication and Dimerization Interference by Dual Inhibitory RNAs

The 5’-untranslated region (5’UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5’ UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5’UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.

Therapeutic applications of catalytic antisense RNAs (ribozymes).

Ribozymes have progressed from an intriguing subject of scientific study to therapeutic agents for the potential treatment of a fatal and devastating viral infection. Despite this rapid road to clinical trials, there are many unexplored avenues that should be examined to improve the intracellular effectiveness of ribozymes. Since ribozymes are RNA molecules, the cellular rules governing RNA partitioning and stability can be applied to these molecules to make them more effective therapeutic agents. Future, successful therapeutic ribozyme applications will depend upon increasing our knowledge of RNA metabolism and movement, and applying this knowledge to the design of ribozymes. This chapter discusses experimental approaches towards this goal as well as recent progress in the application of a pair of hammerhead ribozymes for the clinical treatment of HIV1 infection.

Inhibition of HIV-1 replication by RNA targeted against the LTR region

The use of small RNA molecules able to effect gene inactivation has emerged as a powerful method of gene therapy. These small inhibitory RNAs are widely used for silencing malignant cellular and viral genes. We have assayed a series of inhibitory RNAs named catalytic antisense RNAs, consisting of a catalytic domain, hairpin or hammerhead ribozyme, and an antisense domain. The aim of the present study was to evaluate the effect of these inhibitory RNAs on HIV-1 replication. A series of expression vectors has been constructed for the intracellular synthesis of inhibitory RNAs, differing in the promoter that drives their synthesis. These inhibitory RNAs were designed to act at two possible cleavage sites in the long terminal repeat (LTR) region and the TAR domain was chosen as a target for the antisense domain. We have evaluated the effects of different inhibitory RNAs in HIV replication via changes in p24 antigen levels. Mobility shift assays have been used to check the binding capacity of inhibitory RNAs. Catalytic antisense RNA designed to target the LTR region of HIV-1 inhibited viral replication in an eukaryotic cell environment by more than 90%. The conventional hairpin and hammerhead ribozymes, however, failed to inhibit viral replication. The data provide preliminary evidence of a new class of inhibitory RNAs that can be used to block HIV replication. The results clearly show the importance of the ex vivo antisense effect in the inhibition achieved. A good correlation was found between the in vitro binding efficiency of the inhibitor RNA to the HIV-1 LTR and the inhibition of viral replication.

HIV-1 TAR as anchoring site for optimized catalytic RNAs.

Ribozymes have a great potential for developing specific gene silencing molecules. One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target. Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence. We have characterized catalytic antisense RNA hybrid molecules composed of a hammerhead ribozyme and a stem-loop antisense domain. A series of artificial RNA substrates containing the TAR-RNA stem-loop and a target for the hammerhead ribozyme were constructed and challenged with a catalytic antisense RNA carrying the TAR complementary stem-loop. The catalytic antisense RNA cleaves each of these substrates significantly more efficiently than the parental hammerhead ribozyme. Deletion of the TAR domain in the substrate abolishes the positive effect. These results suggest that the enhancement is due to the interaction of both complementary stem-loop motifs. A similar improvement was corroborated when targeting the LTR region of HIV-1 with either hammerhead- and hairpin-based catalytic antisense RNAs. Our results indicate that the TAR domain can be used as an anchoring site to facilitate the access of ribozymes to their specific target sequences within TAR-containing RNAs. Finally, we propose the addition of stable stem-loop motifs to the ribozyme domain as a rational way for constructing catalytic antisense RNAs.

Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions.

Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.

Episomal expression of a hammerhead ribozyme directed against plum pox virus

Two related antisense RNAs directed against plum pox virus (PPV) were expressed episomally in Nicotiana clevelandii by infection with recombinant potato virus X (PVX). One recombinant PVX expressed an ordinary PPV antisense RNA of about 400 nucleotides, while the other expressed a related antisense RNA that carried the catalytic domain of a hammerhead ribozyme. Inoculation with the latter recombinant PVX resulted in the accumulation of ribozyme RNA that was catalytically active when tested in vitro with a PPV substrate RNA. Plants that had been inoculated with recombinant PVX viruses, expressing either PPV-directed antisense or ribozyme sequences or GUS RNA as a control, were challenged with PPV by a sequential second inoculation. In plants that expressed PPV antisense sequences, the appearance of PPV disease symptoms was delayed for 3–5 days. Quantification of PPV 1 week after inoculation showed that the protective effect by the episomally expressed catalytic antisense RNA was stronger than that of the ordinary antisense RNA. However, eventually all plants tested accumulated comparable titers of PPV.

Kinetic selection of HPV 16 E6/E7-directed antisense nucleic acids: anti-proliferative effects on HPV 16-transformed cells.

The E6/E7-coding sequences of the human papillomavirus type 16 (HPV 16) were probed for kinetic accessibility in vitro by pools of catalytic antisense RNA. Only long-chain complementary RNA and very few antisense sequences with a 3' portion complementary to a 10 nt window within unspliced and spliced E6-coding target sequences showed fast annealing with k(ass) values of up to 10(4) M-1s-1 indicating that the majority of E6/E7 RNA sequences are unfavourable targets for antisense inhibitors and ribozymes. Fast-annealing antisense oligodeoxyribonucleotides directed against the window of 10 nt inhibited cell proliferation of HPV 16-transformed SiHa cells but not slow-annealing antisense species. Antisense RNA of several hundred nucleotides in length also showed significant anti-proliferative activity. Biological effects of antisense oligodeoxyribonucleotides were specific for the antisense sequence, could only be found in HPV-positive but not in HPV-negative cell lines, and were related to decreased levels of E7 protein and E6/E7-specific transcripts. This work suggests that HPV 16 E7/E6 sequences exhibit a low accessibility for antisense oligonucleotides. This can be overcome, however, by exploiting the relationship between fast annealing of antisense species and their increased efficacy in human cells.

Length variation of helix III in a hammerhead ribozyme and its influence on cleavage activity.

The previously described HIV-1 directed hammerhead ribozyme 2as-Rz12 can form with its target RNA 2s helices I and III of 128 and 278 base pairs (bp). A series of derivatives was made in which helix III was truncated to 8, 5, 4, 3, and 2 nucleotides (nt). These asymmetric hammerhead ribozymes were tested for in vitro cleavage and for inhibition of HIV-1 replication in human cells. Truncation of helix III to 8 bp did not affect the in vitro cleavage potential of the parental catalytic antisense RNA 2as-Rz12. Further truncation of helix III led to decreased cleavage rates, with no measurable cleavage activity for the 2 bp construct. All catalytically active constructs showed complex cleavage kinetics. Three kinetic subpopulations of ribozyme-substrate complexes could be discriminated that were cleaved with fast or slow rates or not at all. Gel purification of preformed ribozyme-substrate complexes led to a significant increase in cleavage rates. However, the complex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the comparable antisense RNA on HIV-1 replication.

The antisense sequence of the HIV-1 TAR stem-loop structure covalently linked to the hairpin ribozyme enhances its catalytic activity against two artificial substrates.

This work is an in vitro study of the efficiency of catalytic antisense RNAs whose catalytic domain is the wild-type sequence of the hairpin ribozyme, derived from the minus strand of the tobacco ringspot virus satellite RNA. The sequence in the target RNA recognized by the antisense molecule was the stem-loop structure of the human immunodeficiency virus-1 (HIV-1) TAR region. This region was able to form a complex with its antisense RNA with a binding rate of 2 x 10(4) M(-1)s(-1). Any deletion of the antisense RNA comprising nucleotides of the stem-loop resulted in a decrease in binding rate. Sequences 3' of the stem in the sense RNA also contributed to binding. This stem-loop TAR-antisense segment, covalently linked to a hairpin ribozyme, enhanced its catalytic activity. The highest cleavage rate was obtained when the stem-loop structure was present in both ribozyme and substrate RNAs and they were complementary. Similarly, an extension at the 5'-end of the hairpin ribozyme increased the cleavage rate when its complementary sequence was present in the substrate. Inclusion of the stem-loop at the 3'-end and the extension at the 5'-end of the hairpin ribozyme abolished the positive effect of both antisense units independently. These results may help in the design of hairpin ribozymes for gene silencing.

A Helix 1-Extended Hairpin Ribozyme Exhibits Altered Cleavage BehaviorIn Vitro

The catalytic domain of a hairpin ribozyme was incorporated at the 3'-end of a 254-base antisense RNA directed against the RNA of human immunodeficiency virus type 1 (HIV-1), generating a hairpin ribozyme with a largely extended helix 1. In parallel, a catalytic antisense RNA based on a hammerhead ribozyme was directed toward the same cleavage motif in the HIV-1 target. Both ribozymes were expected to create identical cleavage products. Cleavage analysis in vitro confirmed that the hammerhead ribozyme delivered the expected cleavage products. However, the helix 1-extended hairpin ribozyme catalyzed additional RNA cleavage at several unexpected sites, which were mapped. Some of the 3' cleavage products had other nucleotides than G at their 5'-terminus, indicating that the helix 1-extended hairpin ribozyme was able to cleave bonds other than NpG+1. Inspection of the sequence context of the different cleavage sites suggested that unconventional helices 2 in combination with an asymmetric loop A consisting of up to 32 unpaired nucleotides in the substrate strand were formed. A second variant of a helix 1-extended hairpin ribozyme that differed in two nucleotides gave consistent results.

PCR-based construction of long hammerhead ribozymes.

AbstractWith the advent of antisense technology, there has been much interest in the use of long RNAs expressed in vivo to inhibit the expression of target genes. More recently, there have been numerous reports that the incorporation of either hairpin or hammerhead ribozyme motifs (catalytic antisense) into such RNAs increases their effectiveness (1–3). This section will describe a generally applicable, simple, PCR-based method to construct catalytic antisense RNA containing the hammerhead catalytic core that will cleave the target of interest at a GUC sequence. All discussion will focus on the hammerhead motif, although, in principle, the hairpin motif could be incorporated instead.KeywordsMicrocentrifuge TubeHammerhead RibozymeHairpin MotifFinal InsertIncomplete DenaturationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

In Vitro characterization of hammerhead ribozymes against perforin and fas-Ligand RNA

Viroids, the smallest infectious agents endowed with autonomous replication, are tiny single-stranded circular RNAs (∼250 to 400 nt) without protein-coding ability that, despite their simplicity, infect and often cause disease in herbaceous and woody plants of economic relevance. To mitigate the resulting losses, several strategies have been developed, the most effective of which include: firstly, search for naturally resistant cultivars and breeding for resistance, secondly, induced resistance by pre-infection with mild strains, thirdly, ribonucleases targeting double-stranded RNAs and catalytic antibodies endowed with intrinsic ribonuclease activity, fourthly, antisense, and sense, RNAs, fifthly, catalytic antisense RNAs derived from hammerhead ribozymes, and sixthly, hairpin RNAs and artificial small RNAs for RNA interference. The mechanisms underpinning these strategies, most of which have been implemented via genetic transformation, together with their present results and future potential, are the subject of this review.

Degradation of Transgene RNA in Virus-resistant Transgenic Plants Expressing Catalytic Antisense RNA against Virus Genome.

Degradation of Transgene RNA in Virus-resistant Transgenic Plants Expressing Catalytic Antisense RNA against Virus Genome.

Therapeutic antisense and ribozymes

The regulation of expression of genetic information by complementary pairing of sense and antisense nucleic acid strands has been termed 'antisense', and is a mechanism used throughout nature to regulate gene expression. It is now possible to design antisense DNA oligonucleotides, or catalytic antisense RNAs (ribozymes) which can pair with and functionally inhibit the expression of any single stranded nucleic acid in a sequence specific fashion. This high degree of specificity has made them attractive candidates for therapeutic agents. These molecules have the potential for the treatment of a wide variety of diseases. The recent development of retroviral as well as other viral and non-viral based delivery schemes make the clinical use of these molecules a virtual certainty.

Influence of chromosomal position and copy number of a white-directed ribozyme gene on the suppression of eye pigmentation in Drosophila melanogaster.

Different strains of transgenic Drosophila melanogaster carrying one, two, or three copies of a heat-shock promoter 70 (hsp70)-driven catalytic antisense RNA gene, directed against the white gene, were investigated for the expression level of ribozyme RNA. It was found that the steady-state concentrations of the hammerhead ribozyme were proportional to the copy number of the genes and that the suppressive effect on eye pigment accumulation was dosage dependent. In a further experiment, a D. melanogaster strain, deficient in eye pigmentation caused by a deletion of the white gene, was used for P element-mediated germline transformation: the transposon used contained the hsp70-driven, white-directed ribozyme gene and, on the same DNA, the mini-white gene under its own promoter. The spatial coupling of the transcription of ribozyme and target RNA resulted in more effective ribozyme-mediated inhibition of eye pigmentation under heat-shock conditions. These effects were dependent on the chromosomal integration site of the transposon.

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed.

The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes.

Several catalytic antisense RNAs directed against different regions of the genomic or antigenomic RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting in imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted in alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.

Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1.

Attenuation of white gene expression in transgenic Drosophila melanogaster: possible role of a catalytic antisense RNA.

We have transformed Drosophila melanogaster with a DNA construct encoding a catalytic antisense RNA directed against the primary white gene RNA transcript. Total RNA isolated from transformed flies was shown to catalyze the specific cleavage of in vitro transcribed substrate RNA, indicating the expression of a functional ribozyme. Moreover, transgenic lines carrying homozygous copies of this construct depict a further clear-cut reduction in eye pigmentation when present in a genetic background that has a priori reduced levels of white gene expression.

Catalytic antisense RNA (ribozymes): their potential and use as anti-HIV-1 therapeutic agents.

Catalytic RNAs were first discovered as natural processes in several biological systems 1,2,3.Their discovery is one of the landmark accomplishments of modern science since it was the first demonstration that an informational molecule could also possess enzymatic activity, and hence changed our views on the possible origins of life. The discovery and characterization of the reactions carried out by RNA molecules has facilitated the development of an entirely distinct approach to antiviral therapy, the site specific, cleavage of viral RNAs mediated by catalytic, anti-sense ribozymes. We are using ribozymes as a novel antiviral strategy. In the following paragraphs, we weill describe the development of anti-sense ribozymes with the primary emphasis being their application as anti-HIV-1 therapeutic agents.