An overview of sugar-modified oligonucleotides for antisense therapeutics

Among the multitude of chemical modifications that have been described over the past two decades, oligonucleotide analogs that are modified at the 2'-position of the furanose sugar have been especially useful for improving the drug-like properties of antisense oligonucleotides (ASOs). These modifications bias the sugar pucker towards the 3'-endo-conformation and improve ASO affinity for its biological target (i.e., mRNA). In addition, antisense drugs incorporating 2'-modified nucleotides exhibit enhanced metabolic stability, and improved pharmacokinetic and toxicological properties. Further conformational restriction of the 2'-substituent to the 4'-position of the furanose ring yielded the 2',4'-bridged nucleic acid (BNA) analogs. ASOs containing BNA modifications showed unprecedented increase in binding affinity for target RNA, while also improved nuclease resistance, in vitro and in vivo potency. Several ASO drug candidates containing 2'-modified nucleotides have entered clinical trials and continue to make progress in the clinic for a variety of therapeutic indications.     

反义技术研究进展

反义技术利用DNA或RNA分子通过Watson Crick碱基配对原则与目的基因的mRNA互补结合 ,通过各种机制使其降解或抑制其编码蛋白的翻译 ,从而抑制目的基因的表达。与基因敲除(geneknockout)等功能缺失性研究方法相比 ,反义技术具有投入少 ,周期短 ,操作简单等优点 ,因此受到了广泛的关注。对几种常用反义技术的研究进展及存在的问题进行概述。     

Antisense protein kinase A RI alpha-induced tumor reversion: portrait of a microarray

Antisense oligonucleotides can selectively block disease-causing genes due to the specificity of the Watson-Crick base-pairing mechanism of action. A genome-wide view of antisense technology is illustrated via protein kinase A RI alpha antisense. Complementary DNA microarray analysis of the RI alpha antisense-induced expression profile shows the up- and down-regulation of clusters of coordinately expressed genes that define the molecular portrait of a reverted tumor cell phenotype. This global view broadens the horizons of antisense technology; it advances the promise of antisense beyond a single target gene to the whole cell and the whole organism. Along with recent rapid advances in oligonucleotide technologies-including new chemical and biological understanding of more sophisticated nucleic acid drugs-oligonucleotide-based gene silencing offers not only an exquisitely specific genetic tool for exploring basic science but also an exciting possibility for treating and preventing cancer and other diseases.     

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.     

Thermodynamic and Kinetic Characterization of Antisense Oligodeoxynucleotide Binding to a Structured mRNA

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit β-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.     

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.     

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.     

软件预测和MAST技术筛选mRNA反义核酸靶点的比较

基因mRNA的结构靶点筛选是反义核酸药物研发的一个难题 .兔 (Oryctolaguscuniculus) β珠蛋白基因mRNA的结构靶位点通过运用MAST技术筛选获得 ,和计算机软件RNAstructure3 71模拟分析的位点进行了比较 ,也和寡核苷酸微阵列杂交技术筛选获得的靶点结果 (M .Natalie ,1 997)进行了比较 ,显示 :据MAST技术获得的兔 β珠蛋白基因 2个反义核酸结合靶位点 ,和用RNAstructure3 71软件给出的模拟分析的 2个靶位点相同 ,且它们与寡核苷酸微阵列杂交技术的结果完全一致 .运用MAST技术筛选获得绿色荧光蛋白 (GFP)mRNA有 4个结构靶位点 ,体外分析表明这 4个靶位点均有效 ,其中有 3个与RNAstructure3 71软件分析的靶点相同 ,但计算机模拟推荐的结构靶位点较多 ,而且随着基因长度增加确认靶位点的难度增大 ,获得的靶位点还需要实验验证 ,计算机软件模拟分析对实验筛选靶点、设计反义核酸有辅助价值 .MAST方法能筛选各种长度基因mRNA的全部可及位点和准确给定核苷酸的起止位置以供设计反义核酸 ,具有简单快捷的优点 ,将能为反义核酸设计起重要作用 .     

The therapeutic potential of antisense oligonucleotides

Specific inhibition of gene expression by antisense agents provides the basis for rational drug discovery based on molecular targets. Due to the specificity of Watson-Crick base-pair hybridization, antisense oligodeoxynucleotides have been used extensively in attempts to inhibit gene expression in both in vitro and in vivo models. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials against diseases including AIDS and cancer. Although the precise mechanism of action of these drugs has not been clarified, these oligodeoxynucleotides offer considerable promise as novel molecular therapeutics. We review the recent attempts to harness the therapeutic potential of these oligodeoxynucleotides and appraise the near-term prospects for antisense technology.     

反义RNA技术在花色育种中的应用

反义RNA技术是用反义RNA链去抑制靶基因的活性,从而达到对目的基因调控的一项分子生物学技术.该项技术应用于观赏植物的花色育种已有16年的历史并且取得了一定的成就.到目前为止,已经利用该技术对14种花卉花色形成过程中的3大类基因进行了正义和反义导入,获得了花色改变的转基因植株.本文简要回顾了反义RNA技术的产生与发展,并在介绍花色形成的分子生物学的基础上,综述了国际园艺育种中利用反义RNA技术调控花色基因表达的研究进展,以期为花色改良的分子育种提供参考资料.     

反义RNA 技术在花色育种中的应用

反义RNA技术是用反义RNA链去抑制靶基因的活性, 从而达到对目的基因调控的一项分子生物学技术。该项技术应用于观赏植物的花色育种已有16年的历史并且取得了一定的成就。到目前为止, 已经利用该技术对14种花卉花色形成过程中的3大类基因进行了正义和反义导入, 获得了花色改变的转基因植株。本文简要回顾了反义RNA技术的产生与发展, 并在介绍花色形成的分子生物学的基础上, 综述 了国际园艺育种中利用反义RNA技术调控花色基因表达的研究进展, 以期为花色改良的分子育种提供参考资料。     

Inhibition of dengue virus by novel, modified antisense oligonucleotides.

Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex.     

Effects of RNA secondary structure on cellular antisense activity

The secondary and tertiary structures of a mRNA are known to effect hybridization efficiency and potency of antisense oligonucleotides in vitro. Additional factors including oligonucleotide stability and cellular uptake are also thought to contribute to antisense potency in vivo. Each of these factors can be affected by the sequence of the oligonucleotide. Although mRNA structure is presumed to be a critical determinant of antisense activity in cells, to date little direct experimental evidence has addressed the significance of structure. In order to determine the importance of mRNA structure on antisense activity, oligonucleotide target sites were cloned into a luciferase reporter gene along with adjoining sequence to form known structures. This allowed us to study the effect of target secondary structure on oligonucleotide binding in the cellular environment without changing the sequence of the oligonucleotide. Our results show that structure does play a significant role in determining oligonucleotide efficacy in vivo. We also show that potency of oligonucleotides can be improved by altering chemistry to increase affinity for the mRNA target even in a region that is highly structured.     

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.     

Molecular and cellular barriers limiting the effectiveness of antisense oligonucleotides

Antisense oligonucleotides present a powerful means to inhibit expression of specific genes, but their effectiveness is limited by factors including cellular delivery, biochemical attack, and poor binding to target. We have developed a systems model of the processes required for an antisense oligonucleotide to enter, gain access to its target mRNA, and exert activity in a cell. The model accurately mimics observed trends in antisense effectiveness with the stability of the oligonucleotide backbone and with the affinity/kinetics of binding to the mRNA over the time course of inhibition. By varying the model parameters within the physically realizable range, we note that the major molecular and cellular barriers to antisense effectiveness are intracellular trafficking, oligonucleotide-mRNA binding rate, and nuclease degradation of oligonucleotides, with a weaker dependence on total cellular uptake than might be expected. Furthermore, the model may serve as a predictive tool to design and test strategies for the cellular use of antisense oligonucleotides. The use of integrated mathematical modeling can play a significant role in the development of antisense and related technologies.     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Summary Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress within vivo gene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissuesin vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages forin vivo applications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumventedin vivo, which is possible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCMin vivo. When labeled with a radioisotope (e.g.,¹²⁵I or¹¹¹In), the antisense chimeric peptide provides imaging of gene expression in the brainin vivo in a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow forin vivo imaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Anti-cholinesterase hybrids as multi-target-directed ligands against Alzheimer’s disease (1998–2018)

Alzheimer’s disease (AD) is a genetically complex, progressive and irreversible neurodegenerative disorder of the brain which involves multiple associated etiological targets. The complex pathogenesis of AD gave rise to multi-target-directed ligands (MTDLs) principle to combat this dreaded disease. Within this approach, the design and synthesis of hybrids prevailed greatly because of their capability to simultaneously target the intertwined pathogenesis components of the disease. The hybrids include pharmacophoric hybridization of two or more established chemical scaffolds endowed with the desired pharmacological properties into a single moiety. In AD, the primary foundation of medication therapy and drug design strategies includes the inhibition of cholinesterase (ChE) enzymes. Hence the development of ChE inhibition based hybrids is the central choice of AD medicinal chemistry research. To illustrate the progress of ChE inhibition based hybrids and novel targets, we reviewed the medicinal chemistry and pharmacological properties of the multi-target molecules published since 1998-December 2018. We hope that this article will allow the readers to easily follow the evolution of this prominent medicinal chemistry approach to develop a more efficient inhibitor.     

Thermodynamics of RNA-RNA duplexes with 2- or 4-thiouridines: implications for antisense design and targeting a group I intron

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.     

Oligonucleotide therapy

Rapid progress in oligonucleotide therapeutics has continued over the past year as major programs established in the past four years have grown and begun to be productive. Important advances were reported in the medicinal chemistry of oligonucleotides and in understanding their pharmacodynamic properties. Significant progress was made in understanding the pharmacokinetic and toxicologic properties of first generation analogs, particularly phosphorothioates and one oligonucleotide, ISIS 2105, entered clinical trials. Additionally, combinatorial approaches designed to identify oligonucleotides that may bind to a variety of targets were reported.