Structural modifications of antisense oligonucleotides

Antisense oligonucleotides are efficient tools for the inhibition of gene expression in a sequence specific way. Natural oligonucleotides are decomposed rapidly in biological systems, which strongly restrict their application. In contrast, artificial oligonucleotides are designed to be more stable against degradation than the target mRNA, which results in a catalytic effect of the drug. Modification of the phosphate linkage has been the first successful strategy for antisense drug developments and Fomivirsene the first antisense drug in therapy. The launch of Fomivirsene has resulted in a revolutionary spin off to antisense research leading to a second generation of antisense oligonucleotides, which are stable against oligonucleotide cleaving enzymes. Among these, oligonucleotides bearing an alkoxy substituent in position 2' were the most successful ones. The third generation of antisense oligonucleotides contains structure elements, which enhance the antisense action. Zwitterionic oligonucleotides show remarkable results, first, because the stability against ribozymes is largely increased, and secondly, because the electrostatic repulsion between the anionic sense and the zwitterionic antisense cords is minimized. Promising new target molecules in antisense research are oligonucleotide chim?res, which enhance the antisense action (chim?res with intercalators, chelators or polyamines) or enable an application as sequence specific detectors (chim?res with biotin, fluorescein or radioligands).     

Antisense oligonucleotides as drugs for HIV treatment

Nowadays, several million people suffer from AIDS and more than 100 million people are forecasted to be infected with HIV. Among new drugs used to stop HIV virus infection, antisense oligodeoxynucleotides (ODNs) are under investigation and several biotechnology companies are currently developing antisense drugs. Antisense ODNs are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder. This review describes some of the clinical patents in the field of HIV treatments by antisense ODNs. These prior attempts at targeting HIV have largely focused on the nature of the chemical modification used in the ODN moiety. Although each of the described inventions have reported some degree of success in inhibiting some function of the virus, a general therapeutic scheme to target HIV has not yet been found.     

Organ distribution and stability of phosphorothioated oligodeoxyribonucleotides in mice.

Results of recent studies in our laboratory have suggested a potential role for antisense oligodeoxyribonucleotides (oligo(dN)s) as therapeutic agents in the treatment of human hepatitis B virus infection. As a first step towards assessing the potential utility of oligo(dN) in therapy, we have examined the organ distribution, stability and toxicity of a phosphorothioated oligo(dN) (S-oligo) of 20 nucleotides in length which was administered to mice via different routes. Among the various organs analysed, the liver retained the highest amount of S-oligo (1.3-2 per cent of the total injection) at the peak time (10-30 min) regardless of the route of injection. However, the S-oligo appeared to be degraded in the liver to about 40 per cent of its original length within 30 min of injection, presumably by the action of 3' exonucleases. Injection of doses of up to 5 mg kg-1 of S-oligo had no apparent toxic effects on the mice.     

多胺胆固醇缀合物纳米粒作为反义核苷酸传递系统的研究

目的研究多胺胆固醇缀合物传递反义核苷酸的能力。方法通过对自制脂质体的载药量、红细胞毒性、M3骨髓瘤细胞的转染实验。结果自制脂质体具有比常规脂质体良好的载药量,并且对红细胞毒性相对偏小,对M3骨髓瘤细胞具有一定的转染能力。结论自制的脂质体具有一定的应用前景。     

Clinical trials of a new class of therapeutic agents: antisense oligonucleotides

Antisense oligodeoxynucleotides (ODNs) are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder whose genetic aetiology is well known. For this reason, researchers thought that if antisense drugs proved to be so specific there would be no side effects. However, toxicity-related problems arose in initial animal studies of antisense drugs in the early 1990s and since then companies have been using these compounds cautiously. In order to be useful therapeutically, an ODN must (a) exhibit reasonable stability in the physiological environment, (b) be taken up and retained in adequate quantities by the target cells, (c) specifically bind target mRNA with high affinity, (d) have an acceptable therapeutic ratio, free of unwanted toxic and non-specific side effects and (e) be easily synthesised in sufficient quantities to allow clinical use. Most of these criteria have already been met by ODNs recently used in this way. This review describes certain therapeutic applications of antisense techniques currently under investigation in oncology, haematopathology and inflammatory diseases.     

c-myc反义寡核苷酸对人肝癌细胞SMMC-7721端粒酶活性的影响

目的 :探讨c -myc反义基因治疗肿瘤的机制。方法:将正义 (sense)、反义 (antisense)、错配 (mismatch)的c -myc寡核苷酸作用于体外培养的人肝癌细胞SMMC -7721 ,分别应用免疫组化、RT -PCR、PCR -ELISA等方法 ,观察对c -myc蛋白表达、端粒酶亚单位及活性表达的影响。结果:10μmol/L浓度的反义c -myc寡核苷酸作用于SMMC -772124h、48h后 ,相应的c -myc蛋白、端粒酶催化亚单位hTERTmRNA及其端粒酶活性与空白对照组相比 ,差别有统计学意义(P<0.01);而正义组和错配组与空白对照组相比 ,差别无统计学意义(P>0.05)。结论:c -myc反义寡核苷酸可能通过抑制端粒酶活性而抑制肿瘤细胞增殖。     

c-myc反义寡核苷酸对大鼠脑胶质瘤细胞凋亡及增殖作用

目的　探讨c myc反义寡核苷酸 (c mycASON)对大鼠脑胶质瘤细胞凋亡及增殖作用的影响。方法　立体定向法将C6胶质瘤细胞接种于大鼠右侧尾壳核建立鼠脑胶质瘤模型 ,采用人工合成的c mycASON和c myc正义寡核苷酸 (c mycSON)注射于瘤区。按接种后给药的种类、浓度及给药间隔时间将动物随机分成ASON 1组、2组、3组 ;SON 1组、2组、3组 ;生理盐水 1组、2组 (NS1组、2组 )。电镜观察凋亡细胞 ,流式细胞仪检测细胞凋亡率及细胞周期的变化。结果　 (1)反义组出现较多的凋亡细胞 ,正义组与生理盐水组凋亡细胞少。 (2 )各治疗组均显示凋亡峰 ,ASON 1组、2组、3组细胞凋亡率分别为 (18 8± 1 3) %、(2 0 3± 1 4 ) %、(17 8± 1 1) % ,各反义组与各正义组、各生理盐水组分别比较差异有显著性 (P <0 0 5 )。 (3)反义组肿瘤细胞在细胞周期G0 /G1期所占比例较大 ,各反义组差异无显著性 (P >0 0 5 ) ;各反义组与各正义组、各生理盐水组分别比较差异有显著性 (P <0 0 1)。结论　c myc反义寡核苷酸能诱导鼠脑胶质瘤细胞凋亡和抑制细胞增殖。     

定量构效关系方程对靶向蛋白激酶 C-α m RNA的反义药物的活性预测(英文)

以往研究对 34个针对蛋白激酶 C- α m RNA二级结构设计的反义药物 (AS- ODNs)进行了定量构效关系 (QSAR)分析并得到 QSAR方程 .本研究再次设计 1 0个 AS- ODNs验证 QSAR方程对药物活性预测的可靠性 .结果显示其中 4个 AS- ODNsIC50 值明显低于阳性对照 ISIS352 1 (P<0 .0 5) . 8个AS- ODNs与 ISIS352 1的实测 IC50 与 QSAR方程预测相符 ,实测与预测 IC50 之间相关系数为 0 .76(P<0 .0 5) .两个 AS- ODNs,AP1 2 61 (2 0 )和 AP0 1 86(2 0 )的实测 IC50 与预测不符 .结果提示 QSAR方程一定程度上反映了 AS- ODNs活性与靶结构的关系 ,对预测反义药物生物活性有益 .但 QSAR方程难以解释的其他因素需进一步研究以优化反义药物设计     

The group of Professor Xinjing Tang has made progress in the study of cyclic antisense oligonucleotides as microRNA inhibitors

The group of Professor Xinjing Tang has made progress in the study of cyclic antisense oligonucleotides as microRNA inhibitors.     

反义寡核苷酸治疗乳腺癌研究进展(英文)

乳腺癌是一类与多基因相关的恶性肿瘤,有些痛基因如HER-2(c-erbB-2,Neu),bcl-2/6cl-xL,蛋白激酶A(PKA),运铁蛋白受体基因(TfR gene)等的过度表达对乳腺癌的预后有明显影响,有证据表明抑制上述过度表达基因能明显改善乳腺痛的治疗效果。近年,反义治疗这种能抑制特定癌基因过度表达的有效手段被应用于乳腺癌的治疗。研究表明,在多数情况下,反义寡核苷酸(ON)能在mRNA或蛋白水平抑制目的基因的表达,有些反义ON在体和离体对乳腺癌细胞系或动物乳腺癌异植肿瘤均显示出令人鼓舞的治疗效果。此外,反义ON与常规化疗药物合用也能产生协同的抗肿瘤作用。反义ON与化疗药物合用可能在不远的将来是治疗乳腺癌的最佳方法之一。     

定量构效关系方程对靶向蛋白激酶C-α mRNA的反义药物的活性预测

以往研究对34个针对蛋白激酶C-α mRNA二级结构设计的反义药物（AS-ODNs）进行了定量构效关系（QSAR）分析并得到QSAR方程. 本研究再次设计10个AS-ODNs验证QSAR方程对药物活性预测的可靠性．结果显示其中4个AS-ODNs IC₅₀值明显低于阳性对照ISIS3521（P<0.05）. 8个AS-ODNs与ISIS3521的实测IC₅₀与QSAR方程预测相符,实测与预测IC₅₀之间相关系数为0.76(P<0.05）. 两个AS-ODNs,AP1261(20)和AP0186(20)的实测IC₅₀与预测不符. 结果提 示QSAR方程一定程度上反映了AS ODNs活性与靶结构的关系,对预测反义药物生物活性有益. 但QSAR方程难以解释的其他因素需进一步研究以优化反义药物设计.     

The applications of synthetic oligonucleotides to molecular biology

Over the last few years, the chemical synthesis of DNA in vitro has become a routine technique. The ready availability of synthetic DNA has revolutionized molecular biological research and has enabled new research approaches to be attempted that had not been possible previously. The most commonly used approaches can be divided into three broad areas: the cloning and manipulation of genes, the diagnosis of diseases by probing gene structure, and the specific in vitro mutagenesis of DNA for structure/function studies. In this review, each of these three areas is discussed through specific applications from published work. This review is not intended to be comprehensive either in its scope or in its documentation of published results. Rather, it is meant to present some of the most commonly used applications of oligonucleotides in molecular biology through representative examples.     

含（3′→5′）亚甲基缩醛的胸苷二聚体和三聚体的简便合成

胸苷的3′-O-（甲硫甲基）缩醛（2）与N-碘代丁二酰亚胺（NIS）和二苯基次膦酸反应得到3′-O-（二苯磷酰氧）甲基缩醛（3c) ,在三甲基硅三氟甲磺酸酯（TMSOTf)的条件下,后者与3′一位保护的胸苷（5）缩合,得到（3′→5′）甲缩醛连接的胸苷二聚体（T-T）（6）,同样的,3c与3′-位保护的二聚体（T-T）（11）缩合,可得到三聚体（T-T-T）（12）。     

Therapeutic applications of aptamers

Aptamers constitute a new class of oligonucleotides that have gained therapeutic importance. With the approval of the first aptamer drug, pegaptanib, interest in this class of oligonucleotides, often referred to as ‘chemical antibodies’, has increased. This article discusses aptamers in relation to other oligonucleotide molecules such as antisense nucleotides, short inhibitory sequences, ribozymes and so on. The development of pegaptanib is looked at from the point of view of the challenges faced in converting aptamers into therapeutic molecules. Cases of other aptamers, which show promise as drugs, are discussed in slightly greater detail. Comparison with antibodies and small molecules, which have hitherto held monopoly in this area, is also made.     

新型c-Myc反义硫代磷酸寡脱氧核苷酸在小鼠体内的药动学

目的 :研究新筛选合成的c Myc基因反义硫代磷酸寡脱氧核苷酸 (antisensephosphorothioateoligodeoxynucleotidestoc Myc ,c MycAS PS ODN )在小鼠体内的药动学、组织分布和排泄情况。方法 :给小鼠按 5 ,10 ,2 0mg·kg- 13个剂量静脉注射3H c MycAS PS ODN后 ,液闪仪测定不同时间点血浆药物浓度 ;按 10mg·kg- 1静脉注射3H c MycAS PS ODN ,测定不同时间组织器官中3H c MycAS PS ODN的含量和其在粪、尿中排泄率 ,药动学统计程序拟合分析。结果 :静脉注射后 3种剂量血浆药物浓度 时间曲线均符合二室分布模型 ,3种剂量下的AUC之间有明显的线性关系 ;多数组织药物分布浓度 2h达高峰 ,肝、脾、肾、肺和骨髓中药物浓度较高 ,2 4h尿、粪总排泄率 82 %。结论 :c MycAS PS ODN在体内分布快 ,除脑、生殖腺等亲脂性组织外 ,其他组织含量高 ;主要通过尿液排泄。     

Submicron Cationic Emulsions as a New Delivery System for Oligonucleotides

Purpose. The main purpose of the present study was to investigate submicron emulsions as potential oligonucleotide (ON) delivery system. Methods. Submicron emulsions containing various concentrations of stearylamine (SA) were prepared by microfluidization. After association with model oligothymidylates, these emulsions were characterized in terms of particle size, -potential, association efficiency and release upon dilution. The interactions between ON and SA were investigated by partitioning studies between water and oily phases, with ON of three different lengths (pdT₁₆, pdT₃₀, pdT₅₀). The stability of pdT₁₆ in the presence of nucleases was evaluated by incubation in cell culture medium supplemented with 10% of foetal calf serum. Results. The ON association efficiency was much higher with emulsions containing SA (E_SA) than with control emulsions (E₀), whatever the ON length. In addition, E_SA was shown to protect ON against degradation for up to 3 hours in culture medium. ON and SA were able to form ion-pairs and the resulting complex was found to be insoluble both in water and in oil. Zeta potential was maintained constant when increasing the ON concentration, until flocculation occurred (up to 250M in the case of pdT₁₆ for example). This has been explained by the presence of SA in excess, soluble in the oily core of droplets, able to migrate towards the interface and replacing SA neutralized in ion-pairs. Conclusions. E_SA appears to be a valuable system for delivery of ON and might even be improved by selecting an oily phase in which the SA/ON complex would be soluble.Affiliated with the David R. Bloom Center for Pharmacy at the Hebrew University of Jerusalem.     

纯合子型家族性高胆固醇血症治疗药物-Mipomersen sodium

Mipomersen sodium是一种apo B-100合成的寡核苷酸抑制剂,是一种具有全新作用机制的降胆固醇药,临床上用于纯合子型家族性高胆固醇血症的辅助治疗。文中对Mipomersen sodium的作用机制、药效学、药代动力学、药物相互作用、临床评价和安全性等进行综述。     

Nucleic acid drugs in the clinic

As a number of diseases are caused, or accompanied, by abnormal gene expression an understandable temptation to modulate the expression of the abnormal gene’s mRNA or protein to restore proper functioning of the cellular machinery has arisen. In addition, as many traditional therapeutic interventions are accompanied by serious side effects (because the cell killing they cause is not specific to the tumors they are intended to treat) there is a natural desire to design drugs with a very targeted mode of action so as to minimize these side effects. The motivation for developing tumor-specific therapies has now become so strong and so pervasive that we are now truly entering an era of targeted therapeutics. One of the major breakthroughs in the field of targeted therapies, and an example that many are hoping to duplicate, has been the development and successful introduction into the clinic of the first small-molecule inhibitor of the bcr-abl tyrosine kinase – imatinib. Indeed, the spectacular success of imatinib has rapidly led to the development of second-generation inhibitors, which are now either approved themselves or are in advanced clinical trials. Monoclonal antibodies have also found their way to the bedside and are being used widely to treat malignant and non-malignant diseases. In the present treatment climate, dominated as it is by small-molecule drugs and antibodies, one could wonder whether alternative approaches, such as RNA or gene-targeted nucleic acid-based drugs, are still needed. For reasons discussed in the body of this review, many colleagues believe that there is still a place for nucleic acid-based therapeutics. Here, the reason for believing that this is true is reviewed in the context of nucleic acid drug development and the early clinical experience with these new medicines.     

Tumour-associated macrophages targeted transfection with NF-κB decoy/mannose-modified bubble lipoplexes inhibits tumour growth in tumour-bearing mice

Abstract

Tumour-associated macrophages (TAM) exhibit an M2 phenotype that promotes tumour progression, and conversion of M2 TAM toward a tumouricidal M1 phenotype is a promising anti-cancer therapy. As NF-κB is a key regulator of macrophage polarization, we developed an in vivo TAM-targeting delivery system that combines mannose-modified bubble liposomes/NF-κB decoy complexes (Man-PEG bubble lipoplexes) and ultrasound (US) exposure. We investigated the effects of NF-κB decoy transfection on TAM phenotype in solid tumour-bearing mice. Post-transfection tumour growth and survival rates were also recorded. Th2 cytokine (IL-10) level in TAM was significantly lower by NF-κB decoy transfection using Man-PEG bubble lipoplexes and US exposure, while Th1 cytokine levels (IL-1β, TNF-α and IL-6) were significantly higher when compared with controls. In addition, mRNA levels of vascular endothelial growth factor, matrix metalloproteinase-9 and arginase were significantly lower in TAM post-NF-κB decoy transfection. Importantly, TAM-targeted NF-κB decoy transfection inhibited tumour growth and prolonged survival rates in mice. Therefore, TAM-targeted NF-κB decoy transfection using Man-PEG bubble lipoplexes and US exposure may be an effective approach for anti-cancer therapy based on TAM phenotypic conversion from M2 toward M1.