反义寡核苷酸递送方法研究进展

如何将反义寡核苷酸 (AS ODNs)有效递送进入细胞是反义核酸领域面临的一大难题。近年来 ,出现了多种寡核苷酸 (ODNs)的递送方法。在培养细胞中 ,使用的递送方法包括阳离子载体包裹、特异受体的配体导向、ODNs偶联修饰、细胞膜辅助穿透以及利用逆转录病毒载体转染等 ,其应用有效增强了AS ODNs的作用效果 ,大幅度降低了AS ODNs的使用浓度 ;在体内 ,由于临床使用裸露AS ODNs连续给药能达到一定的反义效果 ,而使递送方法的研究和应用尚处于初步尝试和探索之中 ,迄今报道的递送方法有脂类和非脂类两类。     

人工诱骗子的入核分布对核因子-κB活性的调控作用

探讨了由核定位信号(NLS)多肽介导的核因子-κB(NF-κB)寡核苷酸诱骗子(ODNs decoy)进入HeLa细胞核的效率,以及对细胞核内NF-κB活性的调控作用。利用双功能交联剂(Sulfo-SMCC)共价交联末端氨基修饰的ODNs decoy和末端巯基修饰的NLS多肽,形成NLS多肽共价连接的ODNs decoy。依靠TransME转染试剂的辅助转染NLS-ODNs decoy进入HeLa细胞,用荧光显微镜观察荧光标记的NLS-ODNs在细胞内的分布。用MTT法检测HeLa细胞的活力,以凝胶迁移实验(EMSA)检测TNF-α诱导的HeLa细胞核抽提物中NF-κB的活性。结果表明,NLS多肽成功地连接到ODNs decoy上,NLS-ODNs可高效入核,入核率达到17.9%。转染NLS-ODNs进入HeLa细胞,对细胞活力无明显影响,而显著抑制核内NF-κB的活性。结果表明NLS多肽可提高ODNs decoy的入核效率,显著增强诱骗子对NF-κB活性的抑制效果。     

EGFR反义脱氧寡核苷酸及其硫代修饰片段抑制人肝癌细胞系BEL-7404细胞生长

本文旨在研究针对EGFR mRNA的反义寡核苷酸片段对BEL-7404细胞EGFR基因表达及其对细胞生长的影响。合成了互补于EGFR基因5′起始编码区的21聚脱氧寡核苷酸(ODNs)。实验结果显示,3.2μmol/L ODNs明显抑制BEL-7404细胞生长,[~3H]TdR掺入抑制试验显示其对细胞DNA合成的抑制作用表现剂量依赖性;密度扫描分析显示ODNs处理6、24h后,肝癌细胞EGFR mRNA分别下降10.5%和14.3%,ODNs处理4天后,细胞EGFR含量下降37.4%,同时观察了同一长度、针对同一靶基因区域的硫代磷酸型反义寡核苷酸对BEL-7404细胞的作用,表明3.2μmol/L ODNs在作用30h内,细胞DNA合成抑制率达62.1%,但此后渐下降,而SODNs在作用96h后达相应的抑制率(此后作用仍持续),提示S-ODNs在体外培养体系中有较好的稳定性。结果表明,针对EGFR基因的反义脱氧寡核苷酸片段可通过部分封闭EGFR基因表达而抑制BEL-7404细胞的生长,而硫代修饰型比未修饰型反义核酸片段具有更好的稳定性。     

Egr-1的诱骗性寡脱氧核苷酸抑制大鼠颈总动脉损伤后MMP-2的表达

目的观察局部转染早期生长反应因子-1(early growth response factor-1,Egr-1)的特异诱骗寡脱氧核苷酸(decoy oligodeoxynucleotides,decoy ODNs)对球囊损伤颈总动脉后基质金属蛋白酶-2(MMP-2)蛋白表达的影响及内膜增生的情况,初步探讨Egr-1,decoy ODNs抑制球囊损伤后内膜增生的机制。方法 96只健康雄性Wistar大鼠,随机分为4组,分别为假手术组、对照组、杂码组和诱骗组,每组24只。除假手术组外均应用2F球囊导管行颈总动脉球囊损伤术,术中采用转染试剂FuGENE6介导的Egr-1decoy ODNs转染至损伤后大鼠血管中,与假手术组、对照组、杂码组相比较。术后3、7、14、21d每组处死6只动物。应用HE染色和免疫组织化学方法观察大鼠颈总动脉球囊损伤后内膜增生情况和MMP-2蛋白的表达及转染Egr-1decoy ODNs后对它们的影响。结果 (1)、内膜损伤后3d内膜增厚不明显,7d内膜开始增厚,14、21d时内膜明显增厚。(2)、在假手术组近腔面中膜可见MMP-2有少量散在阳性表达;在对照组及杂码组动脉损伤后3d,在近腔面中膜,有少量阳性表达,与假手术组相比,阳性表达指数上升。7d时在新生内膜和靠近新生内膜处中膜表达明显,14d后表达逐渐下降。(3)转染decoy ODNs治疗后,在各个时间点内膜增厚程度减轻,MMP-2蛋白表达减少,与对照组比较差异有显著性(P<0.01)。结论血管球囊损伤后,内膜7d开始增生,14d、21d增生更明显,而MMP-2在7d时表达明显,之后逐渐下降,Egr-1decoy ODNs能抑制MMP-2的表达,从而减轻血管损伤后内膜的增生。     

《微生物学杂志》2003年总目录

题目·研究报告·大肠杆菌murB和birA基因的转录通读现象单组分妥布霉素产生菌的快速筛选部分肝病患者和健康人血清中HBV基因型分析类胡萝卜素产生菌№5205的分类鉴定CpG ODNs对肿瘤细胞端粒酶活性及细胞周期和凋亡的影响     

外源E2F和CArG顺式元件对血管平滑肌细胞表型相关基因表达的影响

利用转录因子“诱骗”策略 ,阻断血管平滑肌细胞 (VSMC)表型特异基因和增殖相关基因的反式激活 ,揭示VSMC表型转化和增殖之间的关系 .电泳迁移率改变分析结果表明 ,相当于分化型VSMC特异表达基因共有顺式元件CArG和细胞增殖相关基因共有顺式元件E2F的双股寡核苷酸(ODNs)可分别与从分化型和去分化型VSMC中提取的核蛋白特异性结合 ,形成DNA 蛋白质复合物 .Northern杂交结果显示 ,导入VSMC中的CArGODN可使平滑肌α肌动蛋白 (α actin)表达活性降低 ,肌丝数量减少 ,明显抑制转染细胞的再分化过程 .去分化型VSMC被E2FODN转染后 ,增殖相关基因c myc表达受到抑制 ,细胞增殖速率减慢 ,去分化表型特征减弱 .结果提示 ,E2F和CArG调控元件分别对VSMC增殖和分化起重要调节作用 ,并证实VSMC表型转化与增殖是两个密切相关但不完全相同的细胞事件 .     

PPENK-MIDGE-NLS基因载体的构建及其在活体大鼠的表达

增加体内免疫细胞合成分泌内源性阿片肽,可对心肌缺血再灌注损伤产生保护作用。基因治疗是增加内源性脑啡肽(Enkephalin,ENK)的一种有前景的研究方向,但是以往的病毒、质粒等载体受到其自身免疫原性的限制并存在基因重组、原癌基因激活、抗细菌蛋白抗体产生以及基因表达改变等诸多问题。本研究采用非病毒、非质粒的微量免疫原定义的基因表达(Minimalistic immunological defined gene expression,MIDGE)方法构建前脑啡肽原(Preproenkephalin,PPENK)PPENK-MIDGE-NLS基因载体能克服病毒和质粒载体的上述缺点。用PCR技术扩增大鼠前脑啡肽原(Preproenkephalin,PPENK)外显基因,产物插入p EGFP-N1质粒,双酶切质粒获得包含启动子、目的基因、RNA稳定序列的线性载体,两端以不受外切酶作用的发夹样脱氧寡核苷酸序列(Oligodesoxynucleotides,ODNs)封闭。为保证载体的入核和表达效率,载体的一端连接了核定位序列(Nuclear localization sequence,NLS)。流式细胞术和激光共聚焦检测其转染效率,Western blotting检测组织内前脑啡肽蛋白的表达。结果显示,PPENK-MIDGE-NLS能够转染大鼠活体细胞并表达前脑啡肽蛋白,增加载体的量能够在一定范围内提高转染效率。研究结果表明该载体可能成为预防和治疗心肌缺血再灌注损伤的新方法。     

c-erbB2与小鼠体外受精及作用机理研究

目的和方法：用免疫组织化学的方法观察ErbB2在小鼠睾丸、附睾、卵巢、输卵管、卵母细胞－卵丘细胞复合物及精子细胞上的分布。用不同浓度反义c-erbB2寡脱氧核苷酸（c-erbB2 ASODNs)与精子和卵母细胞－卵丘细胞复合物共孵育,观察其对小鼠体外受精率的影响,并进一步探讨其机制。结果：在附睾的小管上皮细胞的管腔面,卵丘细胞和精子的细胞膜上有ErbB2蛋白分布。c-erbB2 ASODNs呈剂量依赖方式抑制小鼠体外受精率。小鼠体外受精率在空白对照组、低、中、高浓度c-erbB2 ASODNs组、无义tat ODNs组分别为38．3％、19．6％、10．7％、5．0％、33．8％,同时伴有精子细胞上ErbB2免疫组化染色明显下降,中、高c-erbB2 ASODNs浓度组卵丘细胞贴壁生长受抑制。GABA和db cAMP均能增加精子小鼠体外受精率,两者均部分逆转c-erbB2 ASODNs对受精的抑制作用,但对精子细胞上ErbB2免疫组化染色均无明显影响,而Verapamil能抑制小鼠体外受精率,并协同c-erbB2 ASODNs的抑制作用,且使精子细胞膜上ErbB2免疫组化染色明显下降。结论：c-erbB2 ASODNs与受精密切相关,阻断Ca^2 内流后能通过抑制精子的c-erbB2表达,从而抑制小鼠体外受精,而GABA、dbcAMP可能不是通过c-erbB2来影响受精过程。     

c-myb对小鼠体外受精的影响及作用机制的分析

目的: 本文采用免疫组织化学方法观察Myb转录因子家族成员c-myb蛋白在小鼠精子和卵母细胞-卵丘复合物中的分布.方法: 建立小鼠体外受精模型,用不同浓度反义c-myb寡脱氧核苷酸(c-myb ASODNs)与精子、卵母-卵丘细胞复合物共孵育观察其对小鼠体外受精率的影响,并进一步探讨c-myb ASODNs对小鼠体外受精率的影响机制.结果: 在颗粒细胞胞核和精子的头部有c-myb蛋白分布.c-myb ASODNs呈剂量依赖性抑制小鼠体外受精率(小鼠体外受精率在空白组、低、中、高浓度c-myb ASODNs组、无义tat ODNs组分别为34.97%、30.89%、20.14%、16.68%、34.47% ).GABA、P4、Verapamil和dbcAMP与c-myb ASODNs共同作用时,GABA、P4、dbcAMP三者均逆转c-myb ASODNs对受精的抑制作用,(小鼠体外受精率在空白组、中浓度c-myb ASODNs组、GABA、P4、Verapamil和dbcAMP组、分别为34.81%、22.96%、40.83%、39.12%、7.463% 、40.61%),GABA、P4、和dbcAMP三者对精子中的c-myb蛋白免疫组化染色阳性的细胞百分率无明显影响.Verapamil能抑制小鼠体外受精,并协同c-myb ASODNs的抑制作用,且使精子中的c-myb蛋白免疫组化染色阳性的细胞百分率明显下降.结论: c-myb ASODNs与受精密切相关, Verapamila可能通过调节精子中的c-myb表达,从而抑制小鼠体外受精,而GABA、P4和dbcAMP可能不是通过c-myb来影响受精过程.     

Achieving antisense inhibition by oligodeoxynucleotides containing N(7)-modified 2'-deoxyguanosine using tumor necrosis factor receptor type 1.

Antisense oligodeoxynucleotides (ODNs) are being explored as therapeutic agents for the treatment of many disorders including viral infections, cancers, and inflammatory disorders. In addition, antisense technology can be of great benefit to those attempting to assign function to the multitude of new genes being uncovered in the genomics initiative. However, the demonstration that the gene-regulating effects produced by antisense-designed ODNs are attributable to an antisense mechanism of action requires carefully designed experimentation. Critical to the assignment of an antisense mechanism of action is the availability of nuclease-stable ODNs, inside cells, that have a high binding affinity with the target mRNA and modulate gene functions in a sequence-dependent manner. To help us achieve a goal of sequence-specific antisense activity we designed antisense ODNs containing C(5)-propyne-modified 2'-deoxyuracil and N(7)-propyne-modified 7-deaza-2'-deoxyguanosine bases and partially modified (phosphorothioate) internucleoside linkages. These modified ODNs were found to have enhanced binding affinity to their target mRNA sequences as well as reduced sequence-independent side effects. We used these ODNs to specifically inhibit p55 tumor necrosis factor receptor type 1 expression and tumor necrosis factor alpha-mediated functions in culture assays.     

Fundamental differences in the equilibrium considerations for siRNA and antisense oligodeoxynucleotide design

Both siRNA and antisense oligodeoxynucleotides (ODNs) inhibit the expression of a complementary gene. In this study, fundamental differences in the considerations for RNA interference and antisense ODNs are reported. In siRNA and antisense ODN databases, positive correlations are observed between the cost to open the mRNA target self-structure and the stability of the duplex to be formed, meaning the sites along the mRNA target with highest potential to form strong duplexes with antisense strands also have the greatest tendency to be involved in pre-existing structure. Efficient siRNA have less stable siRNA–target duplex stability than inefficient siRNA, but the opposite is true for antisense ODNs. It is, therefore, more difficult to avoid target self-structure in antisense ODN design. Self-structure stabilities of oligonucleotide and target correlate to the silencing efficacy of siRNA. Oligonucleotide self-structure correlations to efficacy of antisense ODNs, conversely, are insignificant. Furthermore, self-structure in the target appears to correlate with antisense ODN efficacy, but such that more effective antisense ODNs appear to target mRNA regions with greater self-structure. Therefore, different criteria are suggested for the design of efficient siRNA and antisense ODNs and the design of antisense ODNs is more challenging.     

Antisense hairpin loop oligonucleotides as inhibitors of expression of multidrug resistance-associated protein 1: their stability in fetal calf serum and human plasma

Multidrug resistance-associated protein (MRP1) is a transmembrane pump protein responsible for the efflux of chemotherapeutic drugs, an important cause of anticancer treatment failure. Trying to circumvent MRP-mediated resistance we designed and synthesized hairpin loops forming antisense oligodeoxyribonucleotides (ODNs), both phosphodiesters (PO-ODNs) and their phosphorothioate analogues (PS-ODNs), to reduce the protein expression by targeting its mRNA in a sequence specific manner. Melting temperature measurements as well as polyacrylamide gel electrophoresis supported the preferential formation of a secondary structure, which was expected to protect ODNs against 3'-exonuclease degradation. ODNs and PS-ODNs designed in this work were successfully tested as antisense inhibitors of the expression of MRP1 in the leukaemia HL60/ADR cell line. Foreseeing the necessity to perform clinical studies with such ODNs we investigated their stability against the 3'-exonuclease activity of fetal calf serum and human plasma. Under the conditions, corresponding to physiological ones, we observed high stability of hairpin loop forming ODNs, especially those containing longer (e.g. 7 base pair) stems. Comparative studies on the stability of chemically unmodified hairpin loop forming ODNs and their PS-counterparts indicated that endonuclease activity did not play any important role in the process of their nucleolytic degradation. Our studies provide strong evidence for high stability of chemically unmodified hairpin loop ODNs, making them an attractive alternative to phosphorothioate analogues commonly used in antisense strategy.     

Potent inhibitory activity of chimeric oligonucleotides targeting two different sites of human telomerase

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.     

Differential distribution of oligodeoxynucleotides in developing organs with epithelial-mesenchymal interactions.

The use of antisense oligodeoxynucleotides (ODNs) to inhibit gene expression is a usefull method for determining protein function and has potential therapeutic applications. However, there is still great variability in the successfull application of antisense technology to individual systems. In order to assess the ability of different cell types to take up ODNs, developing embryonic tissues were cultured in vitro in the presence of fluoresceine labelled, phosphorothioate substituted ODNs. The distribution of ODNs in individual cell populations was assayed by fluorescent microscopy and the tissue sections were counterstained for epithelial basement membrane formation. High intracellular levels of ODNs were observed in all mesenchymal cells of the lung, salivary gland, kidney, ovary and testis. However, a significant decrease in ODN levels was observed with the formation of new epithelium in kidney and gonads, whereas mature epithelial cells in all tissues had no detecable levels of ODNs. The ability to inhibit gene expression in mesenchymal cells, but not in epithelial cells, was consistent with the distribution pattern of labeled ODNs. These results may indicate a general resistance of epithelial cells to take up ODNs in culture and bear directly on the ability of ODNs to affect gene expression in complex organs with epithelial components.     

Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours

We have shown recently that downregulation of the androgen receptor (AR), one of the key players in prostate tumor cells, with short antisense oligodeoxynucleotides (ODNs) results in inhibition of prostate tumor growth. Particularly with regard to an application of these antisense drugs in vivo, we now investigated the usefulness of microbubble-enhanced ultrasound to deliver these ODNs into prostate cancer cells.

Our short antisense AR ODNs were loaded onto the lipid surface of cationic gas-filled microbubbles by ion charge binding, and delivered into the cells by bursting the loaded microbubbles with ultrasound. In vitro experiments were initially performed to show that this kind of delivery system works in principle. In fact, transfection of prostate tumor cells with antisense AR ODNs using microbubble-enhanced ultrasound resulted in 49% transfected cells, associated with a decrease in AR expression compared to untreated controls. In vivo, uptake of a digoxigenin-labelled ODN was found in prostate tumour xenografts in nude mice following intratumoral or intravenous injection of loaded microbubbles and subsequent exposure of the tumour to ultrasound, respectively. Our results show that ultrasound seems to be the driving force of this delivery system. Uptake of the ODN was also observed in tumors after treatment with ultrasound alone, with only minor differences compared to the combined use of microbubbles and ultrasound.     

Antisense oligodeoxynucleotides: useful tool for search and assessment of new targets for anti-malarial drugs.

We investigated about targeting for new antimalarial drugs using antisense (AS) oligodeoxynucleotides (ODNs). Synthetic nuclease-resistant ODNs (phosphorothioate (PS) ODNs and ODNs containing 4'alpha-C-(2-aminoethyl)thymidines (4'-amino ODNs)) which target mitochondrial succinate dehydrogenase (SDH) iron-sulfur subunit (IP), had antimalarial activity (EC50; about 1.0 microM). Furthermore we showed that intra-parasitic SDH IP mRNA levels, which were detected using quantitative RT-PCR assay, were decreased 13% of control after the 24 h expose to SDH IP AS. From the results, we conclude that SDH has potential as the target for novel antimalarials, and AS ODNs is effective for search and assessment of targets for new antimalarial drugs.     

Electroporation enhances c-myc antisense oligodeoxynucleotide efficacy.

Obtaining high transfection efficiencies and achieving appropriate intracellular concentrations and localization are two of the most important barriers to the implementation of gene targeted therapy. The efficiency of endogenous uptake of oligodeoxynucleotides (ODNs) varies from cell type to cell type and may be a limiting factor of antisense efficacy. The use of electroporation to obtain high intracellular concentrations of a synthetic ODN in essentially 100% of viable cells is described. It is also shown that the transfected ODNs initially localize to the nucleus and remain there for at least 48 hours. The cellular trafficking of electroporated ODNs is shown to be an energy dependent process. Targeting of the c-myc proto-oncogene of U937 cells by electroporation of phosphorothioate-modified ODNs results in rapid and specific suppression of this gene at ODN concentrations much lower than would otherwise be required. This technique appears to be applicable to a variety of cell types and may represent a powerful new investigate tool as well as a promising approach to the ex vivo treatment of hematologic disorders.     

Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo

Oligonucleotide (ODN) therapy is a powerful tool for modulation of gene expression in vivo. With advances in ODN chemistry and progress in formulation development, ODNs are becoming widely acceptable drugs. This review summarizes the current status and future trend of the in vivo application of ODN therapeutics, especially antisense ODNs. Here, we review the current understanding of the tissue/organ distribution and cellular uptake of ODN drugs administered parenterally or nonparenterally to intact animals. The problems and advantages inherent in the use of different delivery methods for the treatment of particular diseases are discussed in detail. Emphasis is placed on the most widely studied ODN analogs, the phosphorothioates (PS). Lessons learned from antisense PS studies have broad implications for ODN therapeutics in general.