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.     

Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-ras mRNA in human cells.

Antisense oligodeoxynucleotides targeted to Ha-ras mRNA have been designed to discriminate between the codon 12-mutated oncogene and the normal proto-oncogene. An in vitro assay using two different sources of RNase H (rabbit reticulocyte lysates and nuclear extract from HeLa cells) was used to characterize oligonucleotide binding to normal and mutated Ha-ras mRNA. Short oligonucleotides (12- or 13mers) centered on the mutation had a very high discriminatory efficiency. Longer oligonucleotides (16mers) did not discriminate efficiently between the mutated and the normal mRNA. We have tested the efficacy of dodecanucleotides to induce RNase H cleavage of the full-length mRNA, moving the target sequence from the loop to the stem region which is formed in the vicinity of mutated codon 12. The most selective oligonucleotides were centered on the mutation which is located near the junction between the loop and stem regions even though they were less efficient at inducing RNase H cleavage than those targeted to the loop region. The 12mer antisense oligonucleotide with the highest discriminatory power was selected for cell culture studies. This oligonucleotide inhibited the proliferation of a human cell line which had been transformed with the mutated Ha-ras gene (HBL100ras1) but had no effect on the parental cell line which was transfected with the vector DNA (HBL 100neo) and expressed only the normal Ha-ras gene. Growth inhibition of HBL100ras1 cells was associated with specific ablation of targeted Ha-ras mRNA as shown by RT-PCR. These results show that 'in vitro' evaluation using an RNase H assay allowed us to select an antisense oligonucleotide which elicited a selectivity towards point-mutated Ha-ras mRNA when added at 10 microM concentration to the culture medium of cells expressing wild type and mutated Ha-ras mRNA.     

Cellular proteins prevent antisense phosphorothioate oligonucleotide (SdT18) to target sense RNA (rA18): development of a new in vitro assay

There are numerous indications that the "antisense" mechanism alone cannot account for the observed effects in living cells. Despite that, interactions between antisense oligonucleotides (ASO) and cellular proteins are usually not considered. In this work, we have tested the ability of antisense phosphorothioate (SdT) oligonucleotides and natural deoxyoligonucleotides (dT) for their ability to interact with target RNA in the presence of cellular proteins. We show that the affinity for cellular proteins is an essential factor that determines the success of RNA targeting. We have used a simple nuclease digestion assay to detect RNA/ASO hybrid formation in the presence of proteins. The results show the inability of a phosphorothioate oligonucleotide (SdT18) to reach the target RNA (rA18) in vitro in the presence of proteins. However, if proteins are absent, the RNA targeting was successful, as is usual in in vitro assays. Note that the target RNA concentration exceeded physiological values by several orders of magnitude while the crude protein extract was 20-fold diluted in the reaction tube. This finding is compatible with the notion that therapeutic properties of phosphorothioates could largely derive from a so-called "aptamer" effect.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Gene targeting in mouse embryos mediated by RecA and modified single-stranded oligonucleotides

Gene targeting is a precise manipulation of endogenous gene by introduction of exogenous DNA and has contributed greatly to the elucidation of gene functions. Conventional gene targeting has been achieved through a use of embryonic stem cells. However, such procedure is often long, tedious, and expensive. This study was carried out to develop a simple procedure of gene targeting using E. coli recombinase A (RecA) and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene in mouse embryos. The single-stranded oligonucleotide to target an exon 3 of HPRT was 74 bases in length including phosphorothioate linkages at each terminus to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except a single nucleotide that induces a mismatch between an introduced oligonucleotide and endogenous HPRT gene. Endogenous repairing of such mismatch would give rise to the conversion of TAT to TAG stop codon thereby losing the function of the target gene. Before an introduction into zygotes, single-stranded oligonucleotides were bound to RecA to enhance the homologous recombination. The RecA–oligonucleotide complex was microinjected into the pronucleus of zygote. Individual microinjected embryos developed to the blastocyst stage were analyzed for the expected nucleotide conversion using polymerase chain reaction (PCR) and subsequent sequencing. The conversion of TAT to TAG stop codon was detected in three embryos among 48 tested blastocysts (6.25% in frequency). The result suggests that the gene targeting was feasible by relatively easier and direct method.     

Polymorphisms in the large subunit of human RNA polymerase II as target for allele-specific inhibition

The lack of specificity of cancer treatment causes damage to normal cells as well, which limits the therapeutic range. To circumvent this problem one would need to use an absolute difference between normal cells and cancer cells as therapeutic target. Such a difference exists in the genome of all individuals suffering from a tumor that is characterized by loss of genetic material [loss of heterozygosity (LOH)]. Due to LOH, the tumor is hemizygous for a number of genes, whereas the normal cells of the individual are heterozygous for these genes. Theoretically, polymorphic sites in these genes can be utilized to selectively target the cancer cells with an antisense oligonucleotide, provided that it can discriminate the alleles and inhibit gene expression. Furthermore, the targeted gene should be essential for cell survival, and 50% gene expression sufficient for the cell to survive. This will allow selective killing of cancer cells without concomitant toxicity to normal cells. As an initial step in the experimental test of this putative selective cancer cell therapy, we have developed a set of antisense phosphorothioate oligonucleotides which can discriminate the two alleles of a polymorphic site in the gene encoding the large subunit of RNA polymerase II. Our data show that the exact position of the antisense oligonucleotide on the mRNA is of essential importance for the oligonucleotide to be an effective inhibitor of gene expression. Shifting the oligonucleotide position only a few bases along the mRNA sequence will completely abolish the inhibitory activity of the antisense oligonucleotide. Reducing the length of the oligonucleotides to 16 bases increases the allele specificity. This study shows that it is possible to design oligonucleotides that selectively target the matched allele, whereas the expression level of the mismatched allele, that differs by one nucleotide, is only slightly affected.     

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA ‘gapmer’ oligonucleotide comprising a phosphorothioate central sequence flanked by 5′ and 3′ HNA sequences to conventional phosphorothioate oligonucleotides and to a 2′-O-methoxyethyl (2′-O-ME) phosphorothioate ‘gapmer’. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2′-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate ‘gap’) was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.     

Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides

The antisense approach is conceptually simple and elegant; to design an inhibitor of a specific mRNA, one needs only to know the sequence of the targeted mRNA and an appropriately modified complementary oligonucleotide. Of the many analogs of oligodeoxynucleotides explored as antisense agents, phosphorothioate analogs have been studied the most extensively. The use of phosphorothioate oligodeoxynucleotides as antisense agents in various studies have shown promising results. However, they have also indicated that quite often, biological effects observed could be solely or partly non-specific in nature. It is becoming clear that not all phosphorothioate oligodeoxynucleotides of varying length and base composition are the same, and important consideration should be given to maintain antisense mechanisms while identifying effective antisense oligonucleotides. In this review, I have summarized the progress made in my laboratory in understanding the specificity and mechanism of actions of phosphorothioate oligonucleotides and the rationale for designing second-generation mixed-backbone oligonucleotides.     

bFGF反义寡核苷酸增强鼠黑色素瘤B16细胞化疗药物敏感性研究

目的:研究bFGF反义硫代寡核苷酸增强肿瘤细胞对化疗药物敏感性作用。方法:设计、合成bFGF寡核苷酸,用聚乙烯亚胺(polyemyleneimine,PEI)介导bFGF反义硫代寡核苷酸转染入黑色素瘤B16细胞,MTT法检测bFGF反义硫代寡核苷酸及其与化疗药物联合处理后的细胞增殖率;半定量RT-PCR测定bFGF反义硫代寡核苷酸转染后细胞中bFGF mRNA水平;流式细胞仪分析bFGF反义硫代寡核苷酸诱导的细胞凋亡。结果:bFGF反义硫代寡核苷酸对B16细胞增殖的抑制率为64.8%,且呈剂量依赖效应。B16细胞中bFGF mRNA被bFGF反义硫代寡核苷酸显著降低,为对照细胞的57.9%,且bFGF反义硫代寡核苷酸诱导B16细胞凋亡,凋亡率为41.8%。bFGF反义硫代寡核苷酸转染能显著增强B16细胞对阿霉素、5-氟脲嘧啶及顺铂的敏感性,非特异性硫代寡核苷酸不影响阿霉素、5-氟脲嘧啶及顺铂抑制B16细胞增殖。结论:bFGF反义硫代寡核苷酸显著增强B16细胞的化疗敏感性,表明其可协同化疗药物用于治疗肿瘤。     

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells. A cell-free translation system was used to demonstrate an RNase H-dependent specific inhibition of activated ras protein synthesis. 50% inhibition was observed at a concentration of 0.5 microM of the most efficient oligonucleotide (5'-substitution with an acridine derivative and 3'-substitution by a dodecanol chain). This inhibitory effect stems from a point mutation-sensitive cleavage of the mRNA and it mirrors the growth inhibition obtained with T24 bladder carcinoma cells, which carry activated Ha-ras. The proliferation of HBL100 cells (non tumorigenic human mammary cell line) which carry two copies of normal Ha-ras was unaffected. This study shows that it is possible to design antisense agents that will inactivate the mutated oncogene but not the protooncogene which is generally essential to cell survival.     

Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.     

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.     

反义寡核苷酸药物癌泰得的定性分析方法研究

 癌泰得 (ACTCACTCAGGCCTCAGACT)为端粒酶表达抑制活性反义寡核苷酸 .为了探讨其定性检测手段 ,通过阴离子交换高效液相色谱和毛细管凝胶电泳分析方法 ,确定了该硫代寡核苷酸以及与其有关的短序列和部分未被硫代类似物的保留时间 ,并分析了不同混合物样品 .结果表明 ,阴离子交换高效液相色谱对硫代寡核苷酸骨架上的差异非常敏感 ,可很好地分离长度相同的硫代和未完全硫代类似物 ,并且随未被硫代磷酸基数目增加 ,保留时间依次缩短 .阴离子交换高效液相色谱对硫代寡核苷酸的长度不敏感 ,不能分离相差一个碱基的硫代寡核苷酸 .毛细管凝胶电泳可很好地分离长度相差一个碱基的硫代寡核苷酸 ,不能分离同长硫代和部分硫代寡核苷酸 .高效液相色谱结合毛细管凝胶电泳可有效地确定癌泰得的纯度和修饰程度 .     

A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides

Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5–15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.     

High sequence fidelity in a non-enzymatic DNA autoligation reaction.

The success of oligonucleotide ligation assays in probing specific sequences of DNA arises in large part from high enzymatic selectivity against base mismatches at the ligation junction. We describe here a study of the effect of mismatches on a new non-enzymatic, reagent-free method for ligation of oligonucleotides. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5'-end iodide with a 3'-phosphorothioate group. The data show that this ligation proceeds somewhat more slowly than ligation by T4 ligase, but with substantial discrimination against single base mismatches both at either side of the junction and a few nucleotides away within one of the oligonucleotide binding sites. Selectivities of >100-fold against a single mismatch are observed in the latter case. Experiments at varied concentrations and temperatures are carried out both with the autoligation of two adjacent linear oligonucleotides and with intramolecular autoligation to yield circular 'padlock' DNAs. Application of optimized conditions to discrim-ination of an H- ras codon 12 point mutation is demonstrated with a single-stranded short DNA target.     

Polyethylenimine but not cationic lipid improves antisense activity of 3'-capped phosphodiester oligonucleotides

Lipofectin, which is a mixture of neutral lipid with a cationic lipid, has been widely used to enhance cellular delivery of phosphorothioate, 2'-sugar-modified, and chimeric antisense oligonucleotides. Phosphodiester oligonucleotides delivered with Lipofectin usually do not elicit antisense activity probably because cationic lipid formulations do not sufficiently protect unmodified oligonucleotides from nuclease degradation. We show that a cationic polymer, polyethylenimine (PEI), improves the uptake and antisense activity of 3'-capped 20-mer and 12-mer antisense phosphodiester oligonucleotides (PO-ODN) targeted to different regions of Ha-ras mRNA and to the 3'-untranslated region (3'-UTR) of C-raf kinase. In contrast, PEI, which forms a very stable complex with the 20-mer phosphorothioate oligonucleotide (PS-ODN), does not enhance its antisense activity. Using fluorescently labeled carriers and ODN, we show that PEI-PS-ODN particles are very efficiently taken up by cells but PS-ODN is not dissociated from the carrier. Our results indicate that carrier-ODN particle size and stability and ODN release kinetics vary with the chemical nature of the ODN and the carrier being transfected into the cells. The very low cost of PEI compared with cytofectins and the increased affinity for target mRNA and decreased affinity for proteins of PO-ODN compared with PS-ODN make the use of PEI-PO-ODN very attractive.     

Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro.

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.     

Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.