Phosphorothioate-modified Oligodeoxynucleotides Research Articles

Phosphorothioate-modified CpG oligodeoxynucleotide (CpG ODN) induces apoptosis of human hepatocellular carcinoma cells independent of TLR9.

Toll-like receptors (TLRs) expressed on cancer cells are closely associated with tumor development. In this study, we investigated the biological functions of the TLR9 ligand, CpG oligodeoxynucleotide (CpG ODN), on TLR9 expressed in the cytoplasm of hepatocellular carcinoma (HCC) cells. In vitro, human HCC cell lines were transfected with phosphorothioate-modified oligodeoxynucleotides TLR9 agonist OND M362 and its negative control ODN M362 ctrl, which inhibited the proliferation of HCC cells by inducing apoptosis without altering the cell cycle. Interestingly, ODN M362 and ODN M362 Ctrl displayed a similar proapoptotic effect on HCC, possibly related to phosphorothioate modification of the structure of CpG ODN. Although both of them resulted in the upregulation of the TLR9 receptor, their effect on HCC apoptosis was independent of TLR9. They also upregulated inflammatory cytokines, but did not activate the NF-κB signaling pathway. Finally, the activities of ODN M362 and ODN M362 Ctrl were demonstrated in nude mice inoculated with HCC cells. These findings suggest that the phosphorothioate-modified TLR9 agonist ODN M362, and its control, elicit antitumor activity in HCC cells and may serve as a novel therapeutic target for HCC therapy.

Nucleolin antisense oligodeoxynucleotides induce apoptosis and may be used as a potential drug for nasopharyngeal carcinoma therapy

Nucleolin (C23, NCL) mRNA was up-regulated in nasopharyngeal carcinoma (NPC) cells compared to that of normal nasomucosal (NNM) cells using a cDNA microarray approach. The level of nucleolin protein was also up-regulated in 13 NPC cell lines, 30 biopsy specimens and nine other cancer cell lines compared to five NNM cells or normal stromal cells, which were analyzed using immunoblotting or immunohistochemistry. We transfected nucleolin antisense oligodeoxynucleotides (phosphorothioate-modified oligodeoxynucleotides; S-ODNs) into NPC-TW01 cells to knockdown nucleolin expression to evaluate the function of nucleolin in cancer cells. Nucleolin knockdown induced NPC cells but not NNM cells to undergo apoptosis. Furthermore, treatment of NPC-TW01 xenograft tumors with nucleolin antisense oligodeoxynucleotides suppressed the growth of xenograft tumors without obvious side effects. Therefore, we suggest that nucleolin may be a potential cancer therapeutic target and that nucleolin antisense oligodeoxynucleotides may be used as a potential drug for therapy in NPC.

Automated synthesis of phosphorus–sulfur analogs of nucleic acids—25 years on: potential therapeutic agents and proven utility in biotechnology

Replacement of non-bridging oxygen by sulfur in one or more internucleotide linkages in synthetic oligodeoxynucleotides (ODNs) was first fully automated in 1984. The present account highlights how ready access to these nuclease-resistant, phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) and chemically related compounds, notably phosphorothiolates, has enabled remarkably broad advances across basic science, medical research, and biotechnology. These developments exemplify how an initially narrow-scope chemical reaction—in this case, automated sulfurization of an internucleoside phosphite—can give rise to diverse applications far beyond initial expectations.

Spermine Moiety Attached to the C-5 Position of Deoxyuridine Enhances the Duplex Stability of the Phosphorothioate DNA/Complementary DNA and Shows the Susceptibility of the Substrate to RNase H

Novel phosphorothioate-modified oligodeoxynucleotides (S-ODNs) containing a deoxyuridine derivative bearing a spermine moiety at the C-5 position were synthesized. The study of the thermal stability and the thermodynamic stability showed that the modified S-ODNs have been able to form the stable duplexes with the complementary DNA. It was also found that the duplex composed of the modified S-ODN and its complementary RNA strand is the substrate for Escherichia coli RNase H, and the cleavage of the RNA strand by the enzyme was almost similar as in the case of the unmodified one.

Imaging Cerebral Gene Transcripts in Live Animals

To circumvent the limitations of using postmortem brain in molecular assays, we used avidin-biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15-20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and beta-actin mRNA (SPION-cfos and SPION-beta-actin, respectively) (14-22 nm). The Stern-Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 x 10(6)/m. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (MRI) to monitor gene transcription, and demonstrated that these complexes at 40 mug of Fe per kilogram of body weight were retained at least 1 d after intracerebroventricular infusion into the left ventricle of C57Black6 mice. SPION retention measured by MRI as T(2)* or R(2)* maps (R(2)* = 1/T(2)*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T(2)* signal in the right cortex and striatum; retention of SPION-cfos and SPION-beta-actin positively correlated with c-fos and beta-actin mRNA maps obtained from in situ hybridization. Histological examination showed that intracellular iron oxide and FITC-sODN correlated positively with in vivo MR signal reduction. Furthermore, in animals that were administered SPION-cfos and amphetamine (4 mg/kg, i.p.), retention was significantly elevated in the nucleus accumbens, striatum, and medial prefrontal cortex of the forebrain. Control groups that received SPION-cfos and saline or that received a SPION conjugate with a random-sequence probe and amphetamine showed no retention. These results demonstrated that SPION-sODN conjugates can detect active transcriptions of specific mRNA species in living animals with MRI.

DNA Motifs Suppressing TLR9 Responses

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythematosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleotides (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.

NF-κB-dependent regulation of tumor necrosis factor-α gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-α (TNF-α). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-α gene expression, TNF-α promoter activity, IκB degradation, and NF-κB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-α promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.

Modulation of allergic response in nasal mucosa by antisense oligodeoxynucleotides for IL-4

Background: The cytokine IL-4 is highly involved in TH2 inflammation, such as that seen in allergic rhinitis. IL-4 can induce IgE synthesis and eotaxin. Recent studies have shown that stimulation of allergic nasal tissue with ragweed allergen can induce local IL-4 mRNA production. Objective: We set out to determine whether IL-4 antisense phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) could inhibit IL-4 production and downstream events of IL-4. Methods: Nasal mucosa biopsy specimens were obtained from patients with seasonal ragweed allergic rhinitis out of season, incubated ex vivo with or without PS-ODNs, and challenged with ragweed. FITC-labeled oligonucleotides were used to determine tissue uptake. By using immunocytochemistry, IL-4–, IL-13–, eotaxin 1–, and IFN-γ–producing cells were enumerated, and by using in situ hybridization, ϵ germline transcript RNA– and IL-4 mRNA–positive cells were examined. Results: The antisense PS-ODN was taken up by the tissue, and preincubation of the tissue with the IL-4 antisense PS-ODN caused a decrease in allergen-induced IL-4 mRNA and a decrease in the amount of IL-4 immunoreactivity (n = 7, P < .001). PS-ODNs had inhibitory effects on allergen-induced ϵ germline transcript RNA expression (n = 7, P < .001) and mucosa eotaxin 1 immunoreactivity (n = 7, P < .05). In contrast, the PS-ODNs increased the amount of IFN-γ immunoreactivity (n = 7, P < .05), suggesting a nonspecific mechanism for reduced synthesis of IgE and eotaxin. Conclusions: Our results show that the IL-4 antisense PS-ODN effectively inhibits IL-4, IgE synthesis, and eotaxin, principal mediators of allergic inflammation, suggesting that PS-ODNs might offer a possible topical treatment for allergic rhinitis. (J Allergy Clin Immunol 2003;111:580-6.)

Is antisense an appropriate nomenclature or design for oligodeoxynucleotides aimed at the inhibition of HIV-1 replication?

We have evaluated the specificity and the variation in activity against human immunodeficiency virus (HIV) infection of antisense oligodeoxynucleotides (ODNs) with regard to factors such as dose-response range, number and choice of experimental controls, backbone modifications of the ODNs, type of cell infection, length of assays, and delivery approach. The highest level of inhibition was achieved in our long-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB0 and treated with free phosphorothioate-modified ODNs (PS-ODNs). The highest level of specificity was observed in our short-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB) and treated with free PS-ODNs. the highest potency (IC50 level) was observed in our short-term chronic-infection model with (DLS)-delivered ODNs in which the DLS delivery improved the ODN activity up to 106 times compared to the activity of free ODNs. Thus, the near blocking of HIV replication obtained when using PS-ODNs appears because of the addition of extracellular and/or membranar effects. The higher efficacy of PS-ODNs compared to unmodified ODNs, when both are delivered with the DLS system, was demonstrated solely in our short-term assay with MOLT-3 cells. Important variations in the level of sequence specificity were observed and depended on the type of control used and the type of cell assay employed. It seems that all 3 groups of control-tested, random, sense sequence, and non-antisense T30177 ODNs might have distinct activity and, consequently, different modes of action in inhibiting HIV replication. Our data buttress the notion that the contribution of the sequence-specific mediated mode of action is minor compared to the other mechanisms involved in ODN antiviral activity.

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.

Local intracoronary administration of antisense oligonucleotide against c-myc for the prevention of in-stent restenosis: Results of the randomized investigation by the thoraxcenter of antisense dna using local delivery and ivus after coronary stenting (ITALICS) trial

ObjectivesThis study was designed to determine whether antisense oligodeoxynucleotides (ODN) directed against the nuclear proto-oncogene c-myc could inhibit restenosis when given by local delivery immediately after coronary stent implantation. BackgroundFailure of conventional pharmacologic therapies to reduce the incidence of coronary restenosis after percutaneous revascularization techniques has prompted interest in the use of agents that target intracellular central regulatory mechanisms. MethodsEighty-five patients were randomly assigned to receive either 10 mg of phosphorothioate-modified 15-mer antisense ODN or saline vehicle by intracoronary local delivery after coronary stent implantation. The primary end point was percent neointimal volume obstruction measured by computerized analysis of electrocardiogram-gated intravascular ultrasound (IVUS) at six-month follow-up. Secondary end points included clinical outcome and quantitative coronary angiography analysis. ResultsAnalysis of follow-up IVUS data was performed on 77 patients. In-stent volume obstruction was similar between groups (44 ± 16% and 46 ± 14%, placebo vs. ODN; p = 0.57; 95% confidence interval: −1.13 to 0.85). Minimum luminal diameter increased from 0.84 ± 0.36 and 0.90 ± 0.45 (p = 0.55) to 2.70 ± 0.37 and 2.80 ± 0.37 (p = 0.28) after stent implantation, which decreased to 1.50 ± 0.61 and 1.50 ± 0.53 (p = 0.98) by six months, yielding similar loss indexes (placebo vs. ODN, respectively). There were no differences in angiographic restenosis rates (38.5 and 34.2%; p = 0.81; placebo vs. ODN) or clinical outcome. ConclusionsTreatment with 10 mg of phosphorothioate-modified ODN directed against c-myc does not reduce neointimal volume obstruction or the angiographic restenosis rate in this patient population.

Persistent activation of nuclear factor-kappaB by interleukin-1beta and subsequent inducible NO synthase expression requires extracellular signal-regulated kinase.

The role of extracellular signal-regulated kinase (ERK) was studied in the signaling pathway by which interleukin-1beta (IL-1beta) increases the expression of inducible NO synthase (iNOS) in rat vascular smooth muscle cells. IL-1beta induced a rapid and transient activation of nuclear factor-kappaB (NF-kappaB), followed by a prolonged activation of NF-kappaB that was required to induce iNOS expression. Either PD98059 or U0126, selective inhibitors of ERK activation, did not influence IL-1beta-induced early activation but effectively reduced the prolonged activation of NF-kappaB and significantly reduced IL-1beta induction of iNOS. Transfection with antisense, but not sense, phosphorothioate-modified oligodeoxynucleotides directed toward ERK also reduced IL-1beta-induced prolonged NF-kappaB activation and iNOS expression. IkappaBbeta, but not IkappaBalpha degradation, induced by IL-1beta was markedly attenuated when ERK activation was inhibited and could be partially responsible for the persistent NF-kappaB activation. These data suggest that ERK activity is required for persistent NF-kappaB activation by IL-1beta that is necessary for iNOS gene expression.

Inhibition of human colon cancer cell growth by antisense oligodeoxynucleotides targeted at basic fibroblast growth factor.

Basic fibroblast growth factor has been shown to be mitogenic in colon cancer cell lines. In human malignant melanoma cells, antisense oligodeoxynucleotides targeted against basic fibroblast growth factor messenger RNA significantly inhibit cell growth. However, the efficacy of such an antisense oligodeoxynucleotide strategy has not been evaluated for colon cancer cells. To investigate whether basic fibroblast growth factor can stimulate the growth of HT-29 human colon cancer cells and whether antisense oligodeoxynucleotides can inhibit growth of these cells at baseline. Western blotting analyses were used to confirm the presence of basic fibroblast growth factor protein in this cell line. Cell growth was assessed after 2, 4 and 6 days of treatment by cell counting using the trypan blue exclusion method. Phosphorothioate-modified oligodeoxynucleotides (10 microM) were used, complementary to codon 60 of the basic fibroblast growth factor messenger RNA. Cationic liposomes (DOTAP) were used to enhance the cellular uptake of the oligodeoxynucleotides. Western blotting demonstrated the presence of basic fibroblast growth factor protein in this cell line. Basic fibroblast growth factor (1-40 ng/mL) dose-dependently stimulated cell growth and peak values were obtained at a dose of 20 ng/mL. By contrast, antisense oligodeoxynucleotide treatment significantly inhibited cell growth compared with the sense oligodeoxynucleotide-treated cells (P=0.007). This inhibition was reversed by the addition of basic fibroblast growth factor, 20 ng/mL. Treatment targeted against basic fibroblast growth factor messenger RNA inhibits growth of HT-29 human colon cancer cells. This finding may provide a rationale for the therapeutic use of antisense oligodeoxynucleotides targeted at basic fibroblast growth factor for the treatment of colon cancer.

Phosphorothioate Backbone Modification Modulates Macrophage Activation by CpG DNA

Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.

Reversible Downregulation of Protein Kinase A during Olfactory Learning Using Antisense Technique Impairs Long-Term Memory Formation in the Honeybee,Apis mellifera

In this study, we examined the role of cAMP-dependent protein kinase (PKA) in associative olfactory learning of the honeybee, Apis mellifera. In the bee, specific interference with molecules to clarify their role in a certain behavior is difficult, because genetic approaches, such as mutants or transgenic animals, are not feasible at the moment. As a new approach in insects in vivo, we report the use of short antisense oligonucleotides. We show that phosphorothioate-modified oligodeoxynucleotides complementary to the mRNA of a catalytic subunit of PKA directly injected into the bee brain cause a reversible and specific downregulation of both the amount of the catalytic subunit and of PKA activity by 10-15%. The amounts of the regulatory subunit of PKA, as well as PKC, are not affected. The slight "knockdown" of PKA activity during the training procedure, a classical olfactory conditioning of the proboscis extension reflex, neither affects acquisition nor memory retention 3 or 6 hr after training. However, it causes an impairment of long-term memory retention 24 hr after training. Downregulation of PKA 3 hr after training has no detectable effect on memory formation. We conclude that PKA contributes to the induction of a long-term memory 24 hr after training when activated during learning. Second, we demonstrate that the antisense technique is feasible in honeybees in vivo and provides a new and powerful tool for the study of the molecular basis of learning and memory formation in insects.

Interruption of activin A autocrine regulation by antisense oligodeoxynucleotides accelerates liver tumor cell proliferation.

Administration of activin A, a member of the transforming growth factor-beta superfamily inhibits hepatocyte proliferation in vitro and reduces liver mass in vivo. However, a role of endogenous activin A in local growth modulation has not been established in any system. The aim of this study was to examine the production of activin A in the human hepatoma cell line HLF and to explore a possible autocrine role of activin as a cell growth inhibitor by blocking production of endogenous activin using antisense oligodeoxynucleotides. Administration of exogenous activin A suppressed HLF cell growth, and immunoreactive activin A was shown to be produced in the cells at confluency by Western blotting analysis. Cells were exposed to phosphorothioate-modified oligodeoxynucleotides, synthesized with antisense or randomly shuffled base sequences of activin betaA subunit messenger RNA, under serum-free conditions. Uptake of the oligodeoxynucleotides into the cells was confirmed by use of fluorescein isothiocyanate-labeled oligodeoxynucleotides. Administration of antisense oligodeoxynucleotides reduced activin A production as confirmed by both competitive PCR and Western blotting. Activin betaA antisense oligodeoxynucleotides significantly increased cell proliferation compared with controls. These findings are consistent with the existence of an autocrine role of activin A as an inhibitor of hepatocyte proliferation.

CAMP Response Element-Binding Protein in the Amygdala Is Required for Long- but not Short-Term Conditioned Taste Aversion Memory

In conditioned taste aversion (CTA) organisms learn to avoid a taste if the first encounter with that taste is followed by transient poisoning. The neural mechanisms that subserve this robust and long-lasting association of taste and malaise have not yet been elucidated, but several brain areas have been implicated in the process, including the amygdala. In this study we investigated the role of amygdala in general, and the cAMP response element-binding protein (CREB) in the amygdala in particular, in CTA learning and memory. Toward that end, we combined antisense technology in vivo with behavioral, molecular, and histochemical analysis. Local microinjection of phosphorothioate-modified oligodeoxynucleotides (ODNs) antisense to CREB into the rat amygdala several hours before CTA training transiently reduced the level of CREB protein during training and impaired CTA memory when tested 3-5 d later. In comparison, sense ODNs had no effect on memory. The effect of antisense was not attributable to differential tissue damage and was site-specific. CREB antisense in the amygdala had no effect on retrieval of CTA memory once it had been formed, and did not affect short-term CTA memory. We propose that the amygdala, specifically the central nucleus, is required for the establishment of long-term CTA memory in the behaving rat; that the process involves long-term changes, subserved by CRE-regulated gene expression, in amygdala neurons; and that the amygdala may retain some CTA-relevant information over time rather than merely modulating the gustatory trace during acquisition of CTA.

Critical issues in the antisense inhibition of brain gene expression in vivo: Experiences targetting the 5-HT 1A receptor

There have been many recent reports of receptor down-regulation in the brain by antisense oligodeoxynucleotides (ODNs) administered in vivo. However, the literature is inconsistent regarding the experimental criteria that are necessary or sufficient to demonstrate a true antisense effect. Here we review some of the critical conceptual and methodological issues. We highlight the problems of specificity and toxicity encountered in our attempts to down-regulate the 5-HT 1A receptor using a phosphorothioate-modified ODN. We also present preliminary data suggestive of a decreased hippocampal 5-HT 1AR expression induced by the antisense ODN, but it is a reduction which is of limited extent and which does not provide unequivocal evidence for an antisense-mediated effect. We conclude that antisense ODNs are not yet suitable as tools for routine in vivo neuropharmacological use, although they show considerable promise.

Identification of an oligodeoxynucleotide sequence motif that specifically inhibits phosphorylation by protein tyrosine kinases.

Protein tyrosine kinases (PTKs) have central roles in cellular signal transduction. We have identified a sequence motif (CGT[C]GA) in phosphorothioate-modified oligodeoxynucleotides (ODNs) that specifically inhibits the enzymatic activity of recombinant or immunoprecipitated PTK in vitro. Hexamer ODNs containing this motif block both substrate and autophosphorylation of at least four different PTKs but have no apparent effect on the enzymatic activity of a serine/threonine protein kinase. These data suggest possible new applications for ODNs and have implications for the design and interpretation of experiments using antisense or triplex ODNs.

Sequence-independent Effects of Phosphorothioated Oligonucleotides on Synaptic Transmission and Excitability in the Hippocampus In Vivo

Antisense oligodeoxynucleotides (ODNs) have the potential to be a powerful tool for regulating gene expression and mRNA translation in spatially and temporally restricted domains. Prior to investigating the effects of antisense ODNs on hippocampal long-term potentiation, we investigated whether there are any non-specific effects of ODNs on perforant path synaptic transmission in the dentate gyrus of both pentobarbital-anaesthetized and awake, freely moving rats. Single injections of phosphorothioated antisense ODNs (4 nmol) to the immediate early gene zif/268 caused a rapid (within minutes) and long-lasting (>24 hr) profound depression of the perforant path evoked field potentials. This depressive effect was due to the phosphorothioate modification since a depression was not seen with unmodified antisense ODNs, relative to saline controls. Furthermore, the effect was not sequence-specific since modified sense ODNs caused the same degree of depression. The depression caused by the modified antisense ODNs was dose-dependent and specific to synaptic transmission, since antidromic population spikes elicited by mossy fibre stimulation were relatively unaffected compared to the orthodromic responses. A second unexpected side-effect of the modified ODNs was cellular hyperexcitability, such that bursts of epileptiform spikes in the EEG occurred both spontaneously and as a result of synaptic stimulation. While the mechanism of the synaptic depression remains unknown, these results indicate that phosphorothioate-modified ODNs exert profound non-specific effects on synaptic transmission in the hippocampus, that have the potential to seriously compromise any corresponding behavioural or electrophysiological studies. © 1997 Published by Elsevier Science Ltd. All rights reserved.