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.     

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.     

Antisense technology in molecular and cellular bioengineering

Antisense technology is finding increasing application not only in clinical development, but also for cellular engineering. Several types of antisense methods (e.g. antisense oligonucleotides, antisense RNA and small interfering RNA) can be used to inhibit the expression of a target gene. These antisense methods are being used as part of metabolic engineering strategies to downregulate enzymes controlling undesired pathways with regard to product formation. In addition, they are beginning to be utilized to control cell phenotype in tissue engineering constructs. As improved methods for antisense effects that can be externally regulated emerge, these approaches are likely to find increased application in cellular engineering applications.     

Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta (TGF-beta) type II receptor

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.     

Expression in yeast of antisense RNA to ADE1 mRNA

The utility of antisense RNA as a means of regulating gene expression in yeast has been explored by inserting into a high copy number yeast expression vector an ADE1 gene fragment in such an orientation so as to produce antisense RNA in vivo which could hybridize to natural ADE1 mRNA. Northern blotting analysis of total cellular RNA extracted from transformed yeast cells confirmed the presence of high levels of antisense RNA to ADE1 mRNA within cells. However the high level of expression of antisense RNA did not result in production of Ade- cells.     

基于RNA的抗HIV-1基因治疗方法研究进展

艾滋病自发现以来在全球范围内迅速蔓延,危害性极高,目前广泛采用的高效抗逆转录病毒疗法(HAART)虽能够显著提高HIV-1感染者生活质量,但存在着价格昂贵,耐药和副作用的问题经常会导致HAART治疗的中断。要获得长期持续的抗病毒治疗效果还有待于研发新的抗病毒药物和治疗方法。近年来随着分子生物技术、干细胞研究、纳米技术等相关技术的发展,关于抗HIV-1基因治疗方法的研究受到了广泛关注。主要针对基于RNA的抗HIV-1基因治疗方法,包括反义RNA、核酶、RNA诱饵以及RNA干扰技术在抗HIV-1基因治疗方面进行综述。研究表明,以RNA为基础的抗HIV-1基因治疗方法有望成为传统治疗方法的一种有效辅助手段。     

Masked antisense: a molecular configuration for discriminating similar RNA targets

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These ‘masked’ antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR–ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.     

反义RNA及其在植物基因工程领域的应用

随着反义RNA的发现及对其研究的深入,反义RNA技术已被广泛应用于基因调控的研究中。本介绍了反义RNA的概念,并就反义RNA的作用机理和在植物基因工程领域的应用进行了综述。其作用机理包括：在原核生物中反义RNA与引物RNA前体及mRNA分子5′的不同区域进行互补,从而抑制其复制、转录和翻译;在其核生物中反义RNA影响mRNA前体拼接、转移及mRNA分子5′和3′正常修饰。在植物基因工程领域,反义RNA主要应用于抑制果实成熟、抗病、作为反向筛选标记基因、控制花色、控制淀粉合成、控制油料种子中脂肪酸的合成、控制雄性不育等方面。     

在鱼腥藻7120中建立反义glnA系统

应用反义技术对鱼腥藻7120切的内源glnA基因的表达进行调控,首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶（GS）的基因glnA中取得部分结构基因片段,与表达质粒载体pRL-439及穿梭质粒载体pDC－8相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒pDC－AM,然后应用三亲接合转移法把它转入鱼腥藻对7120.通过新霉素筛选,酶谱鉴定,斑点杂交,质粒的交叉转化以及内源glnA基因表达的GS活性分析,GS相关的胞外泌氨分析及所获藻株的形态学变化,证明已在鱼腥藻7120中建立了人工反义glnA基因的品系。     

Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle.

We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.     

反义RNA的作用机理及其在植物基因工程领域的应用

本文综述了反义RNA在原核生物和真核生物中的作用机理,并系统地介绍了反义RNA技术在植物基因过程中的研究进展。     

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.     

Regulation of plant gene expression by antisense RNA.

Regulation of gene expression by antisense RNA was first discovered as a naturally-occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down-regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that 'sense' genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA-RNA interaction.