化学修饰小干扰RNA作为治疗药物的研究进展

化学修饰为小干扰RNA（siRNA）治疗面临的诸多挑战提供了解决方法。此综述考察现有的各种siRNA修饰方法,包括RNA和双链siRNA结构的各个方面。然后考察化学修饰siRNA的应用,重点关注其作用的专一性（消除免疫反应和杂交依赖性的脱靶作用）和转运方法,同时对酶稳定性和效价也进行了讨论。     

低剂量小分子干扰RNA制剂

经历了RNAi作为潜在治疗药物的最初兴奋之后,人们已认识到开发基于RNAi的药物存在很多障碍。首先,未经修饰的小分子干扰RNA(siRNA在体内高度不稳定,因此需要找到在体内具有更高稳定性、更高效价和更长的作用时间的siRNA。目前已有多种化学修饰的siRNA进入临床试验,结果表明制药     

熊果酸与白藜芦醇缀合物的合成及其抗肿瘤活性评价

目的 设计、合成熊果酸与白藜芦醇缀合物,并对其抗肿瘤活性进行研究。方法 以熊果酸为原料,通过酯化反应合成了熊果酸-白藜芦醇缀合物,并采用MTT法评价缀合物的体外抗肿瘤活性。结果 合成了12个熊果酸-白藜芦醇缀合物,其结构经¹H-NMR和MS进行确证。初步的生物活性结果表明,目标缀合物对乳腺癌细胞MCF-7和MDA-MB-231均有抑制活性,其中缀合物13b-c和15b-c对MCF-7的抑制活性强于熊果酸、白藜芦醇及阳性药物他莫昔芬。此外,目标缀合物对正常的MCF-10A细胞没有毒性。结论 熊果酸结构中引入白藜芦醇结构单元可提高其抗肿瘤活性。     

绿原酸缀合物HA-CGA的抗菌活性研究

目的 合成透明质酸(HA)-绿原酸(CGA)的水溶性缀合物HA-CGA并对其结构进行表征，并比较该缀合物与单体药物CGA的抗菌活性。方法 将HA溶于水中，用1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)将其活化，然后加入溶于DMF的CGA进行酯化反应，最后将反应溶液在水中透析除杂，冻干，得到缀合物HA-CGA，并采用核磁共振氢谱法、红外光谱法对HA-CGA结构进行表征；采用平板计数法来比较CGA和HA-CGA对大肠埃希菌、金黄色葡萄球菌等2种细菌的抑制作用。结果CGA和HA-CGA对2种细菌均有不同程度的抑制作用，其中HA-CGA抗菌活性强于CGA。结论 通过HA对CGA的结构进行修饰，增加了CGA的水溶性，进一步提高了CGA的抗菌性能。     

RNA干扰技术应用新进展

RNA干扰(RNAi)是由小分子双链RNA引发的转录后基因沉默现象。作为一种具有革命性的基因转录后调控新技术,正在被广泛应用于功能基因组研究的多种领域。它不仅是基因功能研究的有力工具,而且为特异性基因治疗提供了新的技术手段和应用前景,因而RNAi技术正日益成为后基因组时代基因功能和调控分析的有力工具。     

齐墩果酸-阿司匹林缀合物促骨形成效果研究

目的 探讨齐墩果酸-阿司匹林缀合物对血清素合成的抑制活性以及促骨形成活性.方法 采用高效液相色谱和ELISA试剂盒测试齐墩果酸-阿司匹林缀合物对RBL-2H3细胞中血清素合成的关键酶色氨酸羟化酶-1(TPH-1)的抑制率及含量.构建去势大鼠骨质疏松症模型 (osteoporosis in ovariectomized rats,OVX),并将骨质疏松大鼠随机分为齐墩果酸齐墩果酸-阿司匹林缀合物组、齐墩果酸和阿司匹林物理混合组、齐墩果酸组、阿司匹林物组、空白对照组 (OVX组)组、假手术组 (sham组)和阳性药物对照组,每组5只,给药35 d后采用高效液相色谱测试血清和小肠中血清素水平.采用MTT法测试缀合物对成骨细胞促进活性.结果 在RBL-2H3细胞中,齐墩果酸-阿司匹林缀合物对于TPH-1的抑制呈剂量依赖性,抑制率为11.9%～87.2%,其中缀合物3在10 μmol/L浓度下展现出了最高抑制率 (87.2%),并能较空白组 (216 ng/ml)显著地降低TPH-1含量(65 ng/ml),甚至略强于阳性药物组(69 ng/ml).与假手术组 (sham组)相比,大鼠摘除卵巢 (OVX组)后血清和小肠中血清素的水平明显增高,而经过给缀合物后的大鼠血清和小肠中血清素的水平却明显降低,其中缀合物3降低血清素的活性或较齐墩果酸组和阿司匹林物组的2.3倍以上;此外,齐墩果酸-阿司匹林缀合物能够促进乳小鼠成骨细胞增殖.结论 本研究为开发齐墩果酸类抗骨质疏松类药物提供了新的思路.     

RNA干扰技术的研究进展

RNA干扰是一项新的分子生物学技术,是外源和内源性双链RNA在生物体内诱导同源靶基因的mRNA特异性降解,从而导致转录后基因沉默的现象。尽管RNA干扰发现的时间较短,但由于其具有操作简单、成本低、特异性高和高效性等特点,因而发展迅速,目前对于它的机制已有初步的了解,同时RNA干扰在功能基因组学以及疾病的基因干预治疗方面也有一定的进展。该文就RNA干扰技术的历史、作用机制、生物学意义及应用作简要概述。     

以二硫键连接尿嘧啶核苷C-5位肽缀合物的合成

为了方便快捷地制备嘧啶核苷-肽缀合物,我们发展了一种一釜合成的方法。从尿嘧啶核苷出发,经过4步制备了关键中间体5-乙酰巯亚甲基-2′,3′-二-O-异亚丙基尿苷（4）。在酸性条件下,脱除化合物4的乙酰基,同时游离的巯基与PySS-R（8,12,15,Py=2-吡啶基,R=氨基酸或肽）反应形成新的二硫键,即形成了尿苷与氨基酸或肽以二硫键连接的缀合物（9,13,2）。     

熊果酸衍生物与查耳酮缀合物的合成及抗肿瘤活性研究

目的 设计、合成喹喔啉熊果酸-查耳酮缀合物.方法 以喹喔啉熊果酸为先导化合物,将查耳酮通过酯化反应拼接到喹喔啉熊果酸得到了一系列喹喔啉熊果酸-查耳酮缀合物,并通过MTT法测试其抗癌活性.结果 合成了6个喹喔啉熊果酸-查耳酮缀合物,其结构均通过1H NMR,13C NMR和HRMS加以确认.初步的生物活性结果表明,这些缀合物对MCF-7、PC-3、GBC-823和KB细胞均有抑制活性,尤其是对MCF-7细胞的抑制活性与熊果酸相比均有提高,其中化合物5(IC50=14.2μmol·L-1),6(IC50=15.3μmol·L-1)和7(IC50=10.6μmol·L-1)对MCF-7细胞的抑制活性甚至强于临床上应用的药物他莫昔芬(IC50=15.9μmol·L-1).同时,这些缀合物对正常的乳腺上皮细胞MCF-10A没有毒性.结论 本研究为开发高效、低毒的的熊果酸衍生物提供了信息和依据.     

Therapeutic potential of chemically modified siRNA: Recent trends

Small interfering RNAs (siRNAs) are one of the valuable tools to investigate the functions of genes and are also used for gene silencing. It has a wide scope in drug discovery through in vivo target validation. siRNA therapeutics are not optimal drug‐like molecules due to poor bioavailability and immunogenic and off‐target effects. To overcome the challenges associated with siRNA therapeutics, identification of appropriate chemical modifications that improves the stability, specificity and potency of siRNA is essential. This review focuses on the various chemical modifications and their implications in siRNA therapy.     

多种siRNA的抗肿瘤作用研究

目的研究短干扰RNA(short interfering RNA,siRNA)的抗肿瘤作用.方法采用MTT法检测siR-NA的生长抑制作用,H&E染色法观察细胞形态变化,TUNEL标记法检测核DNA断裂,Western Blot法分析蛋白表达水平变化和流式细胞法检测细胞周期分布变化.结果siRNA-Bcl2,siRNA-MDM2,siRNA-CDK2和siRNA-HRas可以明显抑制人乳腺癌MCF-7等肿瘤细胞的生长;siRNA-MDM2和siRNA-Bcl2可以引起MCF-7细胞染色质凝集;siRNA-Bcl2,siRNA-MDM2,siRNA-CDK2和siRNA-HRas均可以明显降低靶基因的表达水平,同时诱导MCF-7细胞发生染色体DNA断裂;siRNA-MDM2还可以导致MCF-7细胞发生G1期阻滞.结论siRNA可以明显抑制肿瘤细胞的生长,降低靶基因的表达水平,并诱导肿瘤细胞凋亡和周期阻滞,提示siRNA可能发展成为一类高效特异的新型抗肿瘤药物.     

靶向Hmga2基因的siRNA药物筛选及抗肿瘤作用

HMGA2是一种结构性转录因子,在肺癌等多种肿瘤中过表达,是肿瘤治疗的候选靶点之一。RNAi技术作为一种经济、有效的基因沉默工具,是肿瘤治疗的新手段。本文以Hmga2基因为靶点,设计并合成了5条siRNA(HMGA2 siRNA1-5),通过MTT、平板克隆、Transwell、流式细胞术等手段,研究其对肺癌细胞(NCI-H446和A549)增殖、克隆形成、迁移和凋亡等能力的影响。结果表明,HMGA2 siRNA1、3、5对肺癌细胞的各项性能具有不同程度的影响,其中HMGA2 siRNA5尤为明显。HMGA2 siRNA5主要通过沉默Hmga2基因影响细胞性能,与其干扰素效应关系不大。本文筛选获得了可有效沉默Hmga2基因的siRNA,其在体外具有良好的抗肺癌活性,是具有一定潜力的肿瘤基因治疗候选药物。     

Amphiphilic small peptides for delivery of plasmid DNAs and siRNAs

Although various delivery systems for nucleic acids have been reported, development of an efficient and non‐toxic delivery carrier is still a key subject for gene therapy. To find new efficient delivery carriers for nucleic acids, we synthesized amphiphilic peptides composed of a guanidino group, an oleyl group, and a cysteine. We prepared both linear and branched types of peptides and found that the linear peptides were superior to the branched peptides as nucleic acid carriers. Our study also suggested that the intermolecular cysteine disulfides might allow the linear peptides to form the optimal particle sizes with nucleic acids for cellular uptake. The incorporation of a benzoyl group to the linear peptide gave rise to smaller, less suitable particle size with plasmid DNA, which greatly reduced the efficiency of plasmid DNA delivery. On the other hand, the benzoyl modification maintained the optimal particle size with siRNA, and interestingly it significantly enhanced the siRNA delivery. The higher efficiency is because the hydrophobicity from the benzoyl group might assist in interacting with the hydrophobic cell membrane. This demonstrates that a small structural change can modulate the preference of the carriers. Our study may provide an insight designing efficient delivery carriers.     

Chemically modified siRNA: tools and applications

Chemical modification provides solutions to many of the challenges facing siRNA therapeutics. This review examines the various siRNA modifications available, including every aspect of the RNA structure and siRNA duplex architecture. The applications of chemically modified siRNA are then examined, with a focus on specificity (elimination of immune effects and hybridization-dependent off-target effects) and delivery. We also discuss improvement of nuclease stability and potency.     

Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

Coxsackievirus B3 (CVB3) is a common pathogen of myocarditis. We previously synthesized a siRNA targeting the CVB3 protease 2A (siRNA/2A) gene and achieved reduction of CVB3 replication by 92% in vitro. However, like other drugs under development, CVB3 siRNA faces a major challenge of targeted delivery. In this study, we investigated a novel approach to deliver CVB3 siRNAs to a specific cell population (e.g. HeLa cells containing folate receptor) using receptor ligand (folate)-linked packaging RNA (pRNA) from bacterial phage phi29. pRNA monomers can spontaneously form dimers and multimers under optimal conditions by base-pairing between their stem loops. By covalently linking a fluorescence-tag to folate, we delivered the conjugate specifically to HeLa cells without the need of transfection. We further demonstrated that pRNA covalently conjugated to siRNA/2A achieved an equivalent antiviral effect to that of the siRNA/2A alone. Finally, the drug targeted delivery was further evaluated by using pRNA monomers or dimers, which carried both the siRNA/2A and folate ligand and demonstrated that both of them strongly inhibited CVB3 replication. These data indicate that pRNA as a siRNA carrier can specifically deliver the drug to target cells via its ligand and specific receptor interaction and inhibit virus replication effectively.     

Fluorine‐18 labelling of small interfering RNAs (siRNAs) for PET imaging

Small interfering RNAs (siRNAs), a class of macromolecules constituted by the association of two single‐stranded ribonucleic acids of short sequences, have been labelled with the positron‐emitter fluorine‐18 (T_1/2: 109.8 min). The strategy involves (1) prosthetic conjugation of a single‐stranded oligonucleotide with [¹⁸F]FPyBrA (N‐[3‐(2‐[¹⁸F]fluoropyridin‐3‐yloxy)‐propyl]‐2‐bromoacetamide) followed by (2) formation of the target duplex by annealing with the complementary sequence, therefore, permitting parallel and combinatorial preparation of [¹⁸F]siRNAs. Pure fluorine‐18‐labelled siRNAs (0.55–1.11 GBq, specific activity: 74–148 GBq/µmol at EOB) could be obtained within 165 min starting from 37.0 GBq of starting [¹⁸F]fluoride (1.5–3.0%, non‐decay‐corrected isolated yields). Copyright © 2007 John Wiley & Sons, Ltd.     

Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum

Small interfering RNAs (siRNA), RNA duplexes of approximately 21 nucleotides, offer a promising approach to specifically degrade RNAs in target cells by a process termed RNA interference. Insufficient in vivo-stability is a major problem of a systemic application of siRNAs in humans. The present study demonstrated that RNAse A-like RNAses degraded siRNAs in serum. The susceptibility of siRNAs towards degradation in serum was strongly enhanced by local clustering of A/Us within the siRNA sequence, i.e. regions showing low thermal stability, most notably at the ends of the molecule, and by 3'-overhanging bases. Importantly, inhibition of RNAse A family enzymes prevented the degradation and loss of silencing activity of siRNAs in serum. Furthermore, the degradation of siRNAs was considerably faster in human than in mouse serum, suggesting that the degradation of siRNAs by RNAse A family enzymes might be a more challenging problem in a future therapeutic application of siRNAs in humans than in mouse models. Together, the present study indicates that siRNAs are degraded by RNAse A family enzymes in serum and that the kinetics of their degradation in serum depends on their sequence. These findings might be of great importance for a possible future human therapeutic application of siRNAs.     

Exotoxin conjugates and their therapeutic uses

Patent Summary

A recombinant vaccine, composed of an in vitro protein fusion between a bacterial toxin and a protein to raise an immune response, is described. This single protein can carry several domains which will allow the correct processing and presentation to T lymphocytes and thus elicit a strong immune response.

The structure of the Pseudomonas exotoxin A contains several domains. One of these enables the toxin to be internalised into cells through endocytosis via receptors on the cell surface then translocate out of the resultant endosome into the cellular compartment where endogenous proteins are processed for presentation by Class I MHC proteins. The ADP-ribosylation domain of the toxin is deleted to prevent it killing the cells and the protein coding region of the protein of interest is fused to the translocation domains. In this example the protein is fused to residues 57 to 68 of the matrix protein of influenza A, an epitope known to bind to MHC HLA2. Other antigenic epitopes from HIV, papilloma virus, cytomegalovirus, Epstein-Barr virus, rotavirus, respiratory syncytial virus, tumours and parasites can be presented to T cells in this way.