Antisense Reagents Research Articles

Morphant technology in model developmental systems.

Morpholino phosphorodiamidate oligonucleotides (morpholinos; MOs) are synthetic DNA analogues with some highly favorable properties as in vivo gene targeting tools (Summerton, 1999). MOs have a morpholine ring in lieu of the standard ribose sugar moiety and contain a neutral charge backbone (Fig. 1a). With high affinity for RNA, MOs serve as excellent antisense reagents, although they do not recruit RNAseH, and thus efficacy is achieved through nonclassical antisense approaches. MOs also display little toxicity and have no known cellular binding proteins or nucleases (reviewed in Summerton, 1999). MOs were developed as potential therapeutics (Arora et al., 2000; Qin et al., 2000) and are now being used for functional genomics applications (see Ekker, 2000 for review). The alteration of transcriptional processing of newly synthesized (i.e., zygotic) transcripts is one approach for MO-based gene targeting (Fig. 1b, c). Binding of an antisense MO to one potential intron/exon junction can cause the splicing machinery to effectively skip an entire exon. This strategy, pioneered by Kole and colleagues for therapeutic applications, can be used for the activation of genes if the skipped exon contains a missense or nonsense mutation (Schmajuk et al., 1999). More recent work by Draper and colleagues (2001) has shown its effectiveness for gene inactivation in zebrafish embryos. This method has one significant advantage because the effectiveness at splice alteration can be readily monitored using reverse transcriptase polymerase chain reaction or other, standard RNA analysis techniques. Once the genomic sequence of the zebrafish and other relevant developmental biology systems becomes available (Duyk and Schmitt, 2001), this targeting strategy should become much more amenable to broader use. Translational inhibition is another successful major MO gene targeting strategy (Fig. 2). In this approach, an antisense MO selected against the leader sequence or nearby bases can bind and sterically inhibit scanning of the mRNA by the 40S ribosomal subunit. The resulting double-stranded RNA:MO complex may mimic doublestranded regulatory sites often found in 59 untranslated regions (UTR) of mRNAs. MOs have been shown to invade even native secondary structure due to MO’s high affinity for RNA (Summerton, 1999). Efficacy appears restricted to target sites within the leader and sequences surrounding the start codon (Fig. 2c; Summerton, 1999); a bound MO does not appear capable of altering activity of the ribosomal complex once translation has initiated. Thus, only a small fraction of the transcribed RNA sequences bound by MOs within a cell will result in a deleterious effect on gene function (Summerton, 1999). This mechanism of targeting has been successfully used and published in a variety of systems for gene “knockdown” studies (Table 1). For zebrafish and sea urchin embryos, this is the first viable sequence-specific gene inactivation method. MOs are also an effective tool for Xenopus, chicken and Drosophila melanogaster embryos (Table 1; N. Patel, personal communication; Fig. 3d). The potential impact of this technology is perhaps greatest in systems not amenable to standard genetic approaches. MOs readily function against both zygotic and maternal transcripts for translational inhibition (Table 1). Work with a novel member of the BMP receptor gene family, Alk8 (Bauer et al., 2001), demonstrates one advantage of MO targeting for assigning gene function over conventional zygotic screens. The loss-of-function, zygotic null mutation of Alk8 shows a significantly weaker phenotype than a loss-of-function of BMP ligands because of the translation of functional protein from maternal message pools. In contrast, targeting Alk8 using MOs inhibited translation of both maternal and zygotic message pools, yielding a loss-of-function phenotype of the receptor that was indistinguishable from that of loss-of-function of the ligand. MO targeting has the potential to target a larger pool of genes required for early embryogenesis than would readily be found using conventional zygotic mutagenesis techniques.

Using oligonucleotide scanning arrays to find effective antisense reagents.

Antisense oligonucleotides (AONs) are synthetic deoxyribonucleic acids, typically between 15–25 nucleotides, that can bind to complementary sites in a mRNA and inhibit translation. AONs show promise as therapeutics to many diseases caused by abnormal or unwanted expression of a gene, such as cancers. The first antisense oligonucleotide-based drug (Vitravene™: ISIS Pharmaceuticals) was introduced in 1998 to treat cytomegalovirus (CMV) retinitis in AIDS patients: several others are in advanced stages of clinical trials for viral diseases and cancer. Antisense oligonucleotides are also a useful tool in the functional analysis of genes.

Microarrays for basic studies of nucleic acid hybridization and selection of antisense reagents

Microarrays for basic studies of nucleic acid hybridization and selection of antisense reagents

Antisense properties of peptide nucleic acids.

PNA is a nucleic acid analog with an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units (figure 1). The purine or pyrimidine bases are linked to the each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid (4). PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules (5-7). The uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes. The non-natural character of the PNA makes PNA oligomers highly resistant to protease and nuclease attacks (8). These properties of PNA oligomers suggest that they could potentially serve as efficient antisense or antigene reagents. Indeed, peptide nucleic acids have been applied to block protein expression on the transcriptional (9) and translational level (10,11), and microinjected PNA oligomers demonstrate a strong antisense effect in intact cells (12). However, contrary to the "normal" nucleic acid analogs, PNA oligomers are not efficiently delivered into the cytoplasm of the cell, and until recently this has hindered the application of PNA oligomers as antisense reagents. In this work we summarize some recent achievements on PNA antisense application, especially these concerned with whole cell or tissue delivery of the PNA.

A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons.

A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.

Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Rules for Ribozymes

The selective inactivation of genes, in a tissue-specific or temporally controlled manner, is now an important requirement for the analysis of nervous system development and function. Hammerhead ribozymes--catalytic RNA enzymes that specifically bind to and then cleave target RNAs--may provide a way to meet this requirement, particularly for organisms in which gene inactivation by homologous recombination is not feasible. However, ribozyme application has to some extent been hampered by limited knowledge as to the base-pairing accessibility of RNA target sites in vivo. In an attempt to circumvent this limitation, we have used a computer program based on free energy minimization to predict secondary structures for 128 RNA sequences for which corresponding ribozymes or antisense oligonucleotides have been synthesized, tested, and reported in the literature. A comparative analysis of the predicted structures of these targets with the reported efficacy of their corresponding antisense reagents has allowed us to formulate a set of rules for the rational choice of hammerhead ribozyme flanking arms and cleavage sites.

Chapter 14 Using Antisense Technology to Study Mitosis

This chapter discusses the application of antisense techniques for the study of cell division in tissue culture systems. The chapter presents useful guidelines for the practical and cost-effective use of antisense reagents in the small lab as a complimentary technique for the functional analysis of specific gene products. The application of antisense reagents to the study of mitosis is technically simple but the mechanism by which these reagents operate is exceedingly complex and variable. Antisense reagents interfere with the transcriptional and translational regulation of gene expression and antisense reagents in the cytoplasm can inhibit gene expression at the level of translation. Antisense-induced protein depletion has been successfully employed to identify redundant functions for mitotic proteins in vivo. Both methoxyethylphosphoramidate and phosphorothioate end-capped linkages were used to synthesize oligos against Xklp1 and a centrosomal kinesin-related protein Xklp2. Antisense-induced depletion has also been used to confirm the nonmitotic function of a mitotic midzone motor CHO1/MKLP1.

Selecting effective antisense reagents on combinatorial oligonucleotide arrays.

An array of 1,938 oligodeoxynucleotides (ONs) ranging in length from monomers to 17-mers was fabricated on the surface of a glass plate and used to measure the potential of oligonucleotide for heteroduplex formation with rabbit beta-globin mRNA. The oligonucleotides were complementary to the first 122 bases of mRNA comprising the 5' UTR and bases 1 to 69 of the first exon. Surprisingly few oligonucleotides gave significant heteroduplex yield. Antisense activity, measured in a RNase H assay and by in vitro translation, correlated well with yield of heteroduplex on the array. These results help to explain the variable success that is commonly experienced in the choice of antisense oligonucleotides. For the optimal ON, the concentration required to inhibit translation by 50% was found to be five times less than for any other ON. We find no obvious features in the mRNA sequence or the predicted secondary structure that can explain the variation in heteroduplex yield. However, the arrays provide a simple empirical method of selecting effective antisense oligonucleotides for any RNA target of known sequence.

Inhibition of herpes simplex virus replication by antisense oligo-2'-O-methylribonucleoside methylphosphonates.

Antisense oligonucleoside methylphosphonates complementary to the 12 nucleotides found at the intron/exon junction of the splice acceptor site of herpes simplex virus type 1 (HSV-1) immediate early mRNAs 4 and 5 were synthesized. The methylphosphonate oligomers contained either 2'-deoxyribose nucleosides, d-OMPs, or 2'O-methylribose nucleosides, mr-OMPs. At 37 degrees C, the affinity of the mr-OMP for a complementary 12-mer RNA target was approximately four times higher than that of the corresponding d-OMP as measured by a constant activity gel electrophoresis mobility shift assay. An mr-OMP whose sequence contained two mismatched bases did not bind to the RNA target under these conditions. The mr-OMP also showed improved ability to inhibit HSV-1 replication in HSV-1 infected Vero cells in culture. Thus the IC50 of the mr-OMP was five times less than that of the d-OMP. No inhibition was observed by the mismatched mr-OMP, and no inhibition of herpes simplex virus type 2 (HSV-2) replication was observed with any of the oligomers. These results demonstrate a direct correlation between oligomer binding affinity and antisense activity in cell culture and suggest that oligo-2'-O-methylribonucleoside methylphosphonates are promising candidates for development of effective antisense reagents.

Distribution of phosphodiester and phosphorothioate oligonucleotides in rat brain after intraventricular and intrahippocampal administration determined by in situ hybridization

The distribution and stability of exogenously administered oligonucleotides (oligos) are important variables determining the potential utility of antisense oligos as agents for modifying gene expression within a given brain region in vivo. In the present study, phosphodiester (PO) and phosphorothioate (PS) oligos antisense with respect to a recently cloned rat hsp70 sequence were localized in rat brain following intraventricular and intrahippocampal administration using an in situ hybridization detection method. Unlabeled PO and PS oligos were dissolved in artificial cerebrospinal fluid and infused under stereotaxic control using a syringe pump. At various intervals after administration frozen brain sections were collected on gelatin-coated slides and hybridized with 35S-labeled probe consisting of the corresponding phosphodiester sense sequence. After intraventricular administration the unmodified PO oligo exhibited a limited and strictly periventricular distribution. In contrast the PS oligo showed significant penetration into and accumulation within brain, with extensive uptake in ipsilateral striatum and dorsal hippocampus, as well as in midline periventricular structures. Both oligos remained detectable for at least two days after administration. Following intrahippocampal injection the PO oligo was rapidly lost from the injection site, with detectable signal persisting only along the hippocampal fissure at 24 h. The PS oligo exhibited a more diffuse initial distribution as well as greater stability. While there was no indication of specific accumulation in the major hippocampal neuron layers through 24 h, there was some indication of selective localization in neuronal soma by 48 h. These results confirm that the relative instability of unmodified oligos may severely limit their utility as antisense reagents in brain in vivo. While PS oligos show more widespread distribution than PO oligos after intraventricular infusion, even these do not detectably accumulate in cortex and other structures without immediate access to the ventricular space under the dosing conditions employed here. The hybridization approach used in these studies should prove to be of general use in verifying the targeting of specific brain structures with antisense oligos by various routes of administration.

Cell-specific delivery of bacteriophage-encapsidated ricin A chain.

We have used covalent coupling of deglycosylated ricin A chain (RAC) to the assembly initiation/translational repression RNA stem-loop (TR) of the bacteriophage MS2 to direct encapsulation of the toxin in bacteriophage capsids. Multiple copies of the TR-RAC conjugate can be incorporated into single capsid shells. The resultant particles can then be directed to specific cells by receptor-mediated endocytosis (RME) of complexes formed with anti-MS2 coat protein antibodies or by further covalent modification of the capsids by addition of human transferrin molecules. The results suggest that bacteriophage encapsulation and targeting is an efficient way to deliver toxins in a cell-specific fashion. The system may have widespread application in the field of targeted drug delivery, including antisense reagents.

Ribozyme mimics as catalytic antisense reagents.

Viral and fungal infections and some cancers may be described as diseases that are characterized by the expression of certain unwanted proteins. They could be termed induced genetic disorders, with induction provided by mutation or infection. A comprehensive method to inactivate injurious genes based on their nucleic acid sequences has the potential to provide effective antiviral and anticancer agents with greatly reduced side effects. We describe a chemical approach to such gene-specific pharmaceutical agents. Our initial efforts have been to develop new chemical reagents that can carry out catalytic destruction of specific mRNA sequences. We chose hydrolysis as a chemical means of destruction, because hydrolysis is compatible with living cells. Our sequence-specific catalytic RNA hydrolysis reagents may be described as functional ribozyme mimics. Reactivity is provided by small-molecule catalysts, such as metal complexes. Specificity is provided by oligonucleotide probes. Here we report initial results on the sequence-specific, hydrolytic cleavage of mRNA from the HIV gag gene, using a ribozyme mimic. The reagent is composed of a terpyridylCu(II) complex for cleavage activity and an oligonucleotide for sequence specificity.

The influence of the target structure on the efficiency of alkylation of single-stranded DNA with the reactive derivatives of antisense oligonucleotides

Site-directed alkylation of three oligonucleotide targets: 41-mer (hairpin structure), 22-mer (loop part of this hairpin) and 10-mer (part of the loop) with 5'-p-(N-2-chloroethyl-N-methylamino)benzylamides of oligonucleotides complementary to the loop region was studied. Thermodynamic parameters of the interaction were estimated using the dependence of the limit modification extent on the reagent concentration at several temperatures. The stability of the complex increases significantly in the set: 302-mer carrying above hairpin, 41-mer, 22-mer, the data for 22-mer and 10-mer being nearly identical. This indicates significant influence of the loop supporting structure on the interaction with antisense reagents.

Oligonucleoside methylphosphonates as antisense reagents.

Oligonucleoside methylphosphonates contain nonionic internucleotide bonds that resist degradation by cellular nucleases and allow the oligomers to be taken up intact by mammalian cells in culture. Antisense methylphosphonate oligomers targeted against cellular or viral mRNA initiation codon or coding regions or against precursor mRNA splice sites effectively and specifically inhibit mRNA expression in cells. The efficacy of antisense methylphosphonate oligomers can be enhanced by derivatization with functional groups that allow the oligomer to covalently cross-link with its targeted mRNA. These oligonucleotide analogs will be useful tools for studying and controlling gene expression and are also promising candidates for development as therapeutic agents.