THE ROLE OF ANTISENSE OLIGONUCLEOTIDES IN THE WAVE OF GENOMIC INFORMATION

Technologies which efficiently dissect gene function and validate therapeutic targets are of great value in the post-sequencing era of the human genome project. The antisense oligonucleotide approach can directly use genomic sequence information, in a relatively time and cost effective manner, to define a gene's function and/or validate it as a potential therapeutic target. Antisense oligonucleotide inhibitors of gene expression may be applied to cellular assays (in vitro) or animal models of disease (in vivo). Information generated by this approach may then direct or supplement traditional drug discovery programs, or support development of the antisense oligonucleotide inhibitor, used to validate the target, as a drug.     

Taking control of gene expression with light-activated oligonucleotides

The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.     

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.     

Antisense strategies

Antisense technology exploits oligonucleotide analogs to bind to target RNAs via Watson-Crick by hybridization. Once bound, the antisense agent either disables or induces the degradation of the target RNA. Antisense agents may also be used to alter splicing. Developing antisense technology involves the creation of a new pharmacology. The receptors, pre- and mRNAs, had never been studied before as sites for drug binding and action. The drugs, oligonucleotide analogs, had never made or tested as drugs before and no medicinal chemistry had been performed. The receptor binding mechanism, Watson-Crick hybridization had never been demonstrated as feasible to exploit from a pharmacological perspective. The post-receptor binding events were literally unknown and unexplored. During the past decade or more, substantial progress has been made in developing antisense pharmacology. A great deal has been learned about the basic mechanisms of antisense, the medicinal chemistry, the pharmacological, pharmacokinetic and toxicological properties of antisense molecules. Antisense technology has proven of great value in gene functionalization and target validation. With one drug marketed, Vitravene, and approximately 20 antisense drugs in clinical development, it appears that antisense drugs may prove of value in the treatment of a wide range of diseases. In this review, the progress is summarized, the limitations of the technology discussed and the future considered.     

Rational selection and quantitative evaluation of antisense oligonucleotides

Antisense oligonucleotides are an attractive therapeutic option to modulate specific gene expression. However, not all antisense oligonucleotides are effective in inhibiting gene expression, and currently very few methods exist for selecting the few effective ones from all candidate oligonucleotides. The lack of quantitative methods to rapidly assess the efficacy of antisense oligonucleotides also contributes to the difficulty of discovering potent and specific antisense oligonucleotides. We have previously reported the development of a prediction algorithm for identifying high affinity antisense oligonucleotides based on mRNA-oligonucleotide hybridization. In this study, we report the antisense activity of these rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gp130) in cell culture. The effectiveness of oligonucleotides was evaluated by a kinetic PCR technique, which allows quantitative evaluation of mRNA levels and thus provides a measure of antisense-mediated decreases in target mRNA, as occurs through RNase H recruitment. Antisense oligonucleotides that were predicted to have high affinity for their target proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries. This approach may aid the development of antisense oligonucleotides for a variety of applications.     

Polymorphisms in the large subunit of human RNA polymerase II as target for allele-specific inhibition

The lack of specificity of cancer treatment causes damage to normal cells as well, which limits the therapeutic range. To circumvent this problem one would need to use an absolute difference between normal cells and cancer cells as therapeutic target. Such a difference exists in the genome of all individuals suffering from a tumor that is characterized by loss of genetic material [loss of heterozygosity (LOH)]. Due to LOH, the tumor is hemizygous for a number of genes, whereas the normal cells of the individual are heterozygous for these genes. Theoretically, polymorphic sites in these genes can be utilized to selectively target the cancer cells with an antisense oligonucleotide, provided that it can discriminate the alleles and inhibit gene expression. Furthermore, the targeted gene should be essential for cell survival, and 50% gene expression sufficient for the cell to survive. This will allow selective killing of cancer cells without concomitant toxicity to normal cells. As an initial step in the experimental test of this putative selective cancer cell therapy, we have developed a set of antisense phosphorothioate oligonucleotides which can discriminate the two alleles of a polymorphic site in the gene encoding the large subunit of RNA polymerase II. Our data show that the exact position of the antisense oligonucleotide on the mRNA is of essential importance for the oligonucleotide to be an effective inhibitor of gene expression. Shifting the oligonucleotide position only a few bases along the mRNA sequence will completely abolish the inhibitory activity of the antisense oligonucleotide. Reducing the length of the oligonucleotides to 16 bases increases the allele specificity. This study shows that it is possible to design oligonucleotides that selectively target the matched allele, whereas the expression level of the mismatched allele, that differs by one nucleotide, is only slightly affected.     

Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.

A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.     

Antisense therapy in clinical oncology

Nucleic acid molecules have emerged as versatile tools with promising utility as therapeutics for human diseases. The specificity of hybridization of an antisense oligonucleotide (AS ODN) to the target mRNA makes the AS strategy attractive to selectively modulate the expression of genes involved in the pathogenesis of malignant or non-malignant diseases. One AS drug has been approved for local therapy of cytomegalovirus retinitis, and a number of AS ODN are currently tested in clinical trials including ODN that target bcl-2, survivin, and DNA methyltransferase. The clinical studies indicate that AS ODN are well tolerated and may have therapeutic activity. In this overview, we summarize therapeutic concepts, clinical studies, and new promising molecular targets to treat human cancer with AS ODN.     

Utilization of antisense oligodeoxynucleotides with embryonic tissues in culture.

Experimental embryology has long used manipulation of interacting tissues to examine questions of tissue interaction and differentiation. The potential for specific manipulation of gene expression in such tissues has made the utilization of antisense techniques desirable. However, problems with this methodology have discouraged many investigators from using this approach. Selection of target sequences for antisense oligonucleotides, delivery of oligonucleotides into cells or tissues, and the type of modification of the oligonucleotide to be used all present concerns that must be addressed. This paper describes our approach to selection of target sequence and methods of delivery and describes the synthesis of a methoxyethylamidate-modified antisense oligonucleotide that has proved useful in our studies. This approach has enabled us to explore aspects of tissue interaction in the embryonic heart that would have been difficult to explore in a genetic model.     

Validation of RNAi silencing specificity using synthetic genes: salicylic acid-binding protein 2 is required for innate immunity in plants

RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.     

Antisense imaging: And miles to go before we sleep?

Labeled oligonucleotide analogues for antisense imaging of messenger RNA (mRNA) have great potential for detection of endogenous gene expression in vivo. Successful antisense imaging may be useful for detecting cellular gene expression patterns and early molecular changes in disease. Conclusive demonstration of this technique has been hindered by formidable challenges in surmounting biological barriers and detecting low concentrations of target mRNA. Recent advances in the development of novel antisense molecules, high specific activity radiolabeling chemistry, sophisticated drug targeting technology, and complementary molecular imaging modalities make it quite possible that true antisense imaging will be realized in the near future.     

Role of Toll-like receptors in antisense and siRNA [corrected

About 25 years ago, researchers first demonstrated that a short synthetic oligodeoxynucleotide, referred to as antisense, can inhibit replication of Rous sarcoma virus through hybridization to viral RNA. Since then, several hybridization-based oligonucleotide approaches have been developed to elucidate the functions of genes and their potential as therapeutic agents. Short-interfering (si) RNA is the most recent example. To effectively inhibit gene expression, an antisense or siRNA must be resistant to nucleases, be taken up efficiently by cells, hybridize efficiently with the target mRNA and activate selective degradation of the target mRNA or block its translation without causing undesirable side effects. However, both antisense and siRNA agents have been shown to exert non-target-related biological effects including immune stimulation. Do antisense and siRNA agents work as ligands for Toll-like receptors (TLRs), a family of pathogen-associated, molecular pattern recognition receptors?     

靶向C—Raf-1基因的反义核酸抗乙型肝炎病毒活性研究

目的：通过体内外实验验证靶向c-Raf-1基因的反义核酸是否具有抑制乙型肝炎病毒（HBV）的活性。方法：设计靶向c-Raf-1基因的反义核酸,并在细胞水平进行体外抗HBV活性筛选,通过RT-PCR检测c-Raf-1基因mRNA水平的变化,通过体内药效学实验进一步验证反义核酸的抗HBV效果。结果：经体外筛选,靶向c-Raf-1基因的反义核酸Raf-3145具有相对明显的抑制HBV表面抗原（HBsAg）的作用,并可剂量依赖性地抑制c-Raf-1基因的表达;体内药效学结果显示,反义核酸Raf-3145在30 mg/kg剂量下对HBsAg的表达具有一定的抑制作用。结论：经体内外活性评价,初步确定了靶向宿主基因c-Raf-1的反义核酸具有一定的抑制HBsAg表达的活性,也进一步验证了c-Raf-1基因可以作为抗HBV药物设计的候选靶点。     

Target validation through protein-domain knockout – applications of intracellularly stable single-chain antibodies

The human genome project has delivered a large number of genes and respective proteins that await to be validated as potential drug targets. Such a complexity has made target validation the main bottleneck in today's drug development process. In addition, the majority of these new potential targets are proteins that function intracellularly. Approaches such as gene knockout, antisense RNA or RNA interference (RNAi) are currently used to validate candidate drug targets by analysing the effects of their deletion. Intrabodies (single-chain antibodies expressed within the cell) present an attractive alternative for directly modulating protein function in vivo. In particular, intrabodies can be used to target specific domains of a protein and perform so-called ‘protein-domain knockouts’, thus allowing the dissection of the varied functions of multi-domain proteins.     

Evaluating the specificity of antisense oligonucleotide conjugates. A DNA array analysis

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.     

In silico determination of potential antisense targets for human beta-globin variants

The functional characterization of available genomic sequences is the major task of the research in the post-genome era. This complex task requires an integrative approach of high-throughput systems with in vitro and in vivo models in order to have a reliable evaluation of the biological function. The oligonucleotide antisense technology is one of the most promising approaches for the investigation of gene function; the crucial point of antisense experiments is the identification of optimal target sites for hybridisation. In this paper we have applied a bioinformatic tool for the recognition of optimal antisense targets. In order to evaluate the effect of mutational events on target selection we have tested the program on a sample of human beta-hemoglobin variants. The proposed algorithm software will be integrated in a web based tool at the site: http://www.nettab.org/agewa.     

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.