Introducing Antisense Oligodeoxynucleotides into Paramecium via Electroporation

ABSTRACT A method utilizing electroporation to deliver antisense oligodeoxynucleotides into Paramecium tetraurelia has been developed. For these studies antisense oligonucleotides directed to different regions of the calmodulin mRNA were used. It was found that a pulse delivered at 150-250 V (375-625 V/cm field strength) for 3.9-4.2 ms using a 275 μF capacitor with resistance set at 13 Ohms was sufficient to achieve measurable incorporation of fluorescently-labeled oligodeoxynucleotides in up to 95% of the cells treated. Optimal parameters included using oligodeoxynucleotides of at least 12 bases in length with a 3' blocking group at a dose of around 10 μM. In addition, multiple oligodeoxynucleotides directed to the same target mRNA resulted in at least a 10-fold reduction in the dose of oligodeoxynucleotide required to achieve the desired effects. Taken together, these results indicate that the use of antisense oligodeoxynucleotides can be an easy and useful method for linking genes to specific functions in Paramecium tetraurelia. Finally, this report discusses how different 3' blocking groups and the use of combinations of oligodeoxynucleotides directed to different regions of the same target mRNA can help address concerns about specificity.     

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy

Oligodeoxynucleotides modified at both 5'- and 3'-ends with inverted thymidine (5'-,3'-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5'-,3'-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5'-,3'-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5'-,3'-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5'-,3'-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.     

Enhanced Antisense Effects Resulting from an Improved Streptolysin-O Protocol for Oligodeoxynucleotide Delivery into Human Leukaemia Cells

Abstract

An improved protocol for delivering oligodeoxynucleotides into the cytoplasm and nucleus of human cells in culture using streptolysin O is described. The new procedure permitted reduced target protein expression to result from antisense suppression of target mRNA levels.     

Selective inhibition of normal murine myelopoiesis "in vitro" by a Hox 2.3 antisense oligodeoxynucleotide.

Multiple homeobox genes are expressed in haematopoietic cell lineages and their expression is cell-type specific. Thus we hypothesized that certain homeobox genes may play an important role in the process of haematopoiesis. To prove that issue, normal murine bone marrow cells were stimulated with appropriate Colony Stimulating Factors in the presence of mouse homeobox gene (Hox 2.3) sense or antisense oligodeoxynucleotides and the effects on the haematopoietic colony formation were examined. Treatment of the cells to Hox 2.3 antisense oligodeoxynucleotides led to a selective inhibition of myeloid colony formation, both in size and in numbers, but without significant effect on erythroid and megakaryocytic haematopoiesis. Exposure to Hox 2.3 sense oligodeoxynucleotides (no-oligomers), had no such effect. It was further showed that inhibition of myelopoiesis by Hox 2.3 antisense oligodeoxynucleotides was dependent on the differentiation stage of target cells. These findings demonstrated that Hox 2.3 gene plays a critical role in regulating normal murine myelopoiesis.     

Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells

We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2′-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies.     

Antisense inhibition of the renin-angiotensin system in brain and peripheral organs

Antisense inhibition is a method of attenuating the target at the gene expression level. There are two main groups of molecular tools for this goal. The first includes the use of short synthetic stretches of DNA-antisense oligodeoxynucleotides. The second tool is the use of vectors (plasmids or viruses) containing the gene of interest subcloned in the antisense orientation, which in the cells produces the antisense RNA. Both antisense DNA and RNA can bind to the complementary sense mRNA and interfere with its translation. Effects are usually short lasting (days) for oligodeoxynucleotides and longer lasting (weeks or months) for vectors. In this article we briefly describe techniques of antisense inhibition in the context of the renin-angiotensin system.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide

Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense bcl-2 oligodeoxynucleotides enhanced the sensitivity of leukemia cells to arsenic trioxide. A combination of arsenic trioxide with antisense bcl-2 oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced bcl-2 protein expression in K562 and NB4 leukemic cells more significantly than either arsenic trioxide or the oligodeoxynucleotides on their own (P<0.01). Thus, bcl-2 antisense oligodeoxynucleotides increase the sensitivity of leukemic cells to arsenic trioxide. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.     

Antisense oligodeoxynucleotide and ribozyme design

The overwhelming advances of the last few years in the field of nucleic acid-based technologies laid the basis for the development of this new technology as a frontier method not only to combat diseases and infections but also to study gene function. The development of antisense strategies has generated considerable expectations in the neurosciences and, in particular, behavioral neurobiology. Antisense application in the brain has become a technology with tremendous impact, especially for determining the molecular pathways and substrates of behavior of an organism controlled by independent stimuli. The antisense agents, either oligodeoxynucleotides or ribozymes, interfere in the genetic flow of information from DNA via RNA to protein. According to the literature it seems clear that appropriately modified antisense compounds successfully and stably bind to their target ribonucleic acid molecules. This antisense binding leads to a decrease in the corresponding protein levels. If the targeted protein exerts detrimental effects on the cell or tissue, its reduction should be beneficial from a therapeutic point of view. If the investigator wants to study the function of a specific gene product the selective and transient downregulation of the corresponding target protein will help in functional analysis. In the following article I describe the chemical nature of the antisense oligodeoxynucleotides and some of the most commonly used derivatives and give some guidelines on antisense construction and application. The possible mode of action is discussed, as is expansion of the oligonucleotide-based application to ribozyme-mediated gene inhibition. Finally, problems that may be encountered during antisense application are discussed.     

Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells.

We have previously demonstrated, in vitro, that phosphodiester and phosphorothioate antisense oligodeoxynucleotides could direct ribonuclease H to cleave non-target RNA sites and that chimeric methylphosphonodiester/phosphodiester analogue structures were substantially more specific. In this report we show that such chimeric molecules can promote point mutation-specific scission of target mRNA by both Escherichia coli and human RNases H in vitro. Intact human leukaemia cells 'biochemically microinjected' with antisense effectors demonstrated efficient suppression of target mRNA expression. It was noted that the chimeric methylphosphonodiester/phosphodiester structures showed single base discrimination, whereas neither the phosphodiester nor phosphorothioate compounds were as stringent. Finally, we show that the antisense effects obtained in intact cells were due to endogenous RNase H activity.     

Poly(propylacrylic acid) enhances cationic lipid-mediated delivery of antisense oligonucleotides

The use of antisense oligodeoxynucleotides (ODNs) to inhibit the expression of specific mRNA targets represents a powerful technology for control of gene expression. Cationic lipids and polymers are frequently used to improve the delivery of ODNs to cells, but the resulting complexes often aggregate, bind to serum components, and are trafficked poorly within cells. We show that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improves the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. In characterization studies, ODN complexation with DOTAP/ODN was maintained even when substantial amounts of PPAA were added. The formulation also exhibited partial protection of phosphodiester oligonucleotides against enzymatic digestion. In Chinese hamster ovary (CHO) cells, incorporation of PPAA in DOTAP/ODN complexes improved 2- to 3-fold the cellular uptake of fluorescently tagged oligonucleotides. DOTAP/ODN complexes containing PPAA also maintained high levels of uptake into cells upon exposure to serum. Addition of PPAA to DOTAP/ODN complexes enhanced the antisense activity (using GFP as the target) over a range of PPAA concentrations in both serum-free, and to a lesser extent, serum-containing media. Thus, PPAA is a useful adjunct that improves the lipid-mediated delivery of oligonucleotides.     

L-PhGPx expression can be suppressed by antisense oligodeoxynucleotides

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) directly reduces hydroperoxides of phospholipid and cholesterol to their corresponding alcohols. There are two forms of PhGPx: L-PhGPx localizes in mitochondria and S-PhGPx in cytosol. Antisense oligodeoxynucleotides can inhibit specific protein expression. We tested the hypothesis that antisense oligodeoxynucleotides could be designed to inhibit PhGPx expression and thereby sensitize cells to lipid peroxidation induced by singlet oxygen. We chose P4 cells, a cell line established from L-PhGPx cDNA transfected MCF-7 cells, as our cell model. Lipid peroxidation was induced by singlet oxygen generated by Photofrin and visible light. We found that the antisense oligodeoxynucleotide (5' GCCGAGGCTCATCGCGGCGG 3') was effective in suppressing L-PhGPx mRNA, PhGPx protein, and activity. This antisense oligodeoxynucleotide did not interfere with S-PhGPx. When cells were exposed to singlet oxygen, lipid hydroperoxides were produced in the cells. L-PhGPx was able to remove these hydroperoxides; this removal was inhibited by antisense treatment. The inhibition of L-PhGPx by the antisense oligodeoxynucleotides also resulted in increased membrane damage as measured by trypan blue dye exclusion. These data demonstrate that PhGPx expression can be manipulated by antisense techniques.     

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.     

Use of Sequence-Specific Antisense Oligodeoxynucleotides to Determine the Protozoan Parasite Antigen Recognized by Nonspecific Cytotoxic Cells

The antigen on the protozoan parasite Tetrahymena pyriformis recognized by catfish nonspecific cytotoxic cells (NCC) is a 46- to 48-kDa protein referred to as NKTag. The complete cDNA-derived amino acid sequence of NKTag has been obtained. The antigenic determinant of NKTag corresponding to the NCC binding site has been determined with synthetic peptides in target cell competition experiments. To more directly characterize the mechanism of parasite:effector cell interaction, we applied NKTag sequence-specific antisense oligodeoxynucleotides to Tetrahymena in vitro. NKTag mRNA translation by Tetrahymena was blocked by specific antisense (AS) oligodeoxynucleotides. 5′-3′ sense (S) oligodeoxynucleotide sequences were synthesized corresponding to the first 17 N-terminal amino acids of NKTag (in addition to −2 untranslated codons plus the start codon). Complimentary AS oligodeoxynucleotides were likewise synthesized. To determine the optimum in vitro conditions for AS treatment, we tested parasites at various phases of their growth cycle for the effects of a single AS treatment. At 9 h post-AS treatment (during the linear phase of the growth curve), maximum reduction in membrane expression of NKTag was observed. Eighty-five percent of Tetrahymena were positive for expression of NKTag at 0 time post-AS treatment versus 13% positive at 9 h. Membrane expression of AS-treated parasites returned to normal levels by 24 h post-treatment. In cold target inhibition experiments, the reduced NKTag expression by Tetrahymena at 9 h AS treatment was confirmed by observing a complete inability (compared with S-treated parasites) to compete with IM-9 cells for binding with NCC. These data demonstrated a unique experimental in vitro system to define the antigen determinant on target cells responsible for recognition by cytotoxic effector cells that participate in innate immune responses. Received: 14 June 1999 / Accepted: 4 September 1999     

A monoclonal antibody extends the half-life of an anti-HIV oligodeoxynucleotide and targets it to CD4+ cells.

An approach was sought to increase the half-life and target cell specificity of antisense oligodeoxynucleotides (oligos). A monoclonal antibody (MAb) was derived from mice immunised with an oligo complementary to a region (1-20) of the HIV genome. This MAb exerts a protective effect on the oligo from the degradation induced by plasma exonucleases in vitro and in vivo. Moreover the anti-oligo MAb dissociates from the oligo in the presence of its complementary sequence to allow hybridization of the two complementary strands. To direct the oligo to CD4+ cells the anti-oligo MAb was cross-linked to an anti-CD4 MAb. The heteroaggregate determines a 5-fold increase in the cellular membrane binding of the oligo to CD4+ lymphocytes. These findings suggest a new approach to enhancing the therapeutic action and the target specificity of antisense oligodeoxynucleotides useful for the selective inhibition of HIV replication in vivo.     

Properties of isonucleotide-incorporated oligodeoxynucleotides and inhibition of the expression of spike protein of SARS-CoV

Antisense oligonucleotides are recognized to be very efficient tools for the inhibition of gene expression in a sequence specific way. For the discovery of a novel efficient way to modify oligonucleotides, a series of single isonucleotide-incorporated antisense oligodeoxynucleotides have been synthesized, in which an isonucleotide was introduced at different positions of the sequences. The binding behaviors of modified oligodeoxynucleotides to the complementary sequence were studied by UV, CD, and molecular dynamics simulation. The results showed that although the incorporated isonucleotides at certain positions of the sequence interfere with the binding ability to a different extent, B-form duplexes were maintained and the binding abilities of the 3'-end-modified duplexes were better than the corresponding mismatched duplexes. The digestion of modified oligodeoxynucleotides by snake venom phosphodiesterase showed that an isonucleotide strongly antagonizes hydrolysis. The DNA/RNA hybrid formed by a modified oligodeoxynucleotide and its target RNA could activate RNase H. The 3'-end-modified antisense oligodeoxynucelotides inhibited S-glycoprotein expression of SARS-CoV at the mRNA levels in insect Sf9 cells. This study indicated the possibility of designing a novel and effective antisense oligodeoxynucleotide by incorporating an isonucleotide at the 3'-end of the sequence.     

Oligodeoxynucleotide analogs with 5'-linked anthraquinone

We report here the synthesis of novel 5'-linked oligodeoxynucleotides, both normal phosphodiester and phosphorothioate analogs, in which a covalently attached group at the 5'-terminus is an anthraquinone. These compounds represent a new class of antisense compounds in which the base sequence of the oligodeoxynucleotide serves to deliver a nuclease-resistant reactive drug-like molecule to a cellular target nucleic acid (mRNA or DNA).     

比较NRP-1反义寡核苷酸与VEGFR-2反义寡核苷酸对人胃癌SGC7901细胞增殖和凋亡的影响

目的:研究比较神经纤毛蛋白1(NRP-1)反义寡核苷酸(ASODN)与血管内皮生长因子受体2(VEGFR-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖活性及凋亡水平的影响。 方法:分别及同时将不同浓度经硫代磷酸化修饰的NRP-1 ASODN 和 VEGFR-2 ASODN 转染入人胃癌SGC7901细胞,逆转录-聚合酶链反应(RT-PCR)检测NRP-1基因和VEGFR-2 基因mRNA的转录水平;MTT比色法测量细胞的增殖活性;流式细胞仪测量细胞的凋亡水平。 结果:转染NRP-1 ASODN和VEGFR-2 ASODN后,人胃癌SGC7901细胞NRP-1基因和VEGFR-2 基因mRNA的转录水平均出现降低;NRP-1 ASODN和VEGFR-2 ASODN对SGC7901细胞有明显抑制增殖和促进凋亡的作用,且随着ASODN浓度升高而增强;分别转染时其作用无显著差别,联合转染时其作用明显增强。结论:NRP-1 ASODN和VEGFR-2 ASODN可抑制人胃癌SGC7901细胞 NRP-1基因和VEGFR-2 基因mRNA的转录水平及细胞增殖活性,促进细胞凋亡;与分别转染相比,两者联合转染作用明显增强。     

Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.