In normal cells, tumor necrosis factor-alpha (TNF-alpha) activates caspase 8 in both mitochondrion-dependent and mitochondrion-independent apoptotic pathways. It is believed that these two pathways converge, with resultant activation of effector caspases, such as caspase 6 and caspase 7. However, the precise mechanism of the activation of caspases 6 and 7 remains unknown. In this study, in order to focus on the mitochondrion-dependent pathway, we employed MCF7 human breast carcinoma cells, which do not have a functional mitochondrion-independent (caspase 3-dependent) pathway. We specifically targeted the transcript of Bid, a proapoptotic facilitator that is a substrate of caspase 8 in the mitochondrial pathway. In the TNF-alpha-treated MCF7 cells that expressed Bid-targeted ribozymes, the release of cytochrome c and the activation of caspase 9, but not of caspase 8, was delayed. Furthermore, the proteolysis of procaspase 7 was also delayed in Bid ribozyme-expressing cells. Because MCF7 cells are caspase 3 deficient, the direct cross-talk between caspase 8 and caspase 3 does not take place. Therefore, it became clear for the first time that caspase 9 by itself can activate caspase 7 in the absence of the caspase 3-dependent pathway in TNF-alpha-induced apoptosis by the use of specific ribozymes.
The proliferative response of primary B cells to CpG oligonucleotides (ODN) involves induction of nuclear activation promoting-1 (AP-1) transcription factor. AP-1 subunits c-Fos, Fos-B, Jun-B, and Jun-D, but not Fra-1 or Fra-2, were all induced by CpG ODNs in B cells within 30 minutes of stimulation, followed by c-Jun at 1-2 hours. c-Jun reached maximum at 6 hours. By 40 hours, Jun-B and Jun-D became dominant. Synthetic ODNs containing a single guanosine triplet/tetrad appropriately distanced from the 5' pyrimidine-rich unit, which inhibit CpG-driven cell cycle entry and apoptosis protection, blocked AP-1 induction by stimulatory ODNs when they were added simultaneously. After 30 minutes of stimulation, adding inhibitor no longer affected AP-1 at 6 hours. No AP-1 subunits escaped ODN inhibition. In a cell line transfected with an AP-1-beta-galactosidase reporter construct, CpG ODN-induced AP-1 transcriptional activity was prevented by inhibitory ODN, but lipopolysaccharide (LPS)-induced AP-1 activity was not. These data suggest that inhibitory ODNs block the CpG ODN-driven signaling pathway at a site proximal to AP-1 induction.
Short interfering RNA (siRNA)-mediated knockdown of deleterious endogenous transcripts has potential applications for the treatment of hereditary diseases. In situations where the mutant and wildtype transcripts cannot be discriminated from one another by siRNAs, it may be necessary to simultaneously carry out gene replacement with a modified form of the target RNA that is resistant to siRNA activity. To test this possibility, we have taken advantage of a potent siRNA that knocks down EGFP mRNA. In this system, wild-type EGFP expression is suppressed by the siRNA, whereas an EGFP construct with codon modifications in the target region that is otherwise fully functional is not downregulated. When expression of the wild-type message is inhibited, EGFP expression can be simultaneously restored by transfecting these cells with the codon-modified version of EGFP. These studies provide a detailed methodology and system for testing this strategy with RNA interference (RNAi).
Antisense and Nucleic Acid Drug DevelopmentVol. 13, No. 1 Claude Hélène, Ph.D.Tula Ester Behmoaras and Jean-Jacques ToulméTula Ester BehmoarasSearch for more papers by this author and Jean-Jacques ToulméSearch for more papers by this authorPublished Online:8 Jul 2004https://doi.org/10.1089/108729003764097287AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Claude Hélène, Ph.D.." , 13(1), pp. 7l–8lFiguresReferencesRelatedDetails Volume 13Issue 1Feb 2003 To cite this article:Tula Ester Behmoaras and Jean-Jacques Toulmé.Claude Hélène, Ph.D..Antisense and Nucleic Acid Drug Development.Feb 2003.7l-8l.http://doi.org/10.1089/108729003764097287Published in Volume: 13 Issue 1: July 8, 2004PDF download
ISIS 2302, an antisense phosphorothioate oligonucleotide (ODN) targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, is currently being evaluated for treatment of patients with Crohn's disease. From data collected in phase II clinical studies with ISIS 2302, validated population pharmacokinetic and exposure-response models were developed and used to simulate the plasma exposure and clinical response results for a proposed phase III trial design involving 100 patients treated with active drug and 50 patients treated with placebo. Simulated results of 1000 replications of the trial were calculated for various proposed dosing regimens. Overall, the simulated results indicated that a fixed dose regimen (250-400 mg, depending on patient sex and total body weight) given three times weekly provides both desirable ISIS 2302 plasma exposure and a high rate of clinical response in this patient population. However, the simulated results also suggest that inclusion of a larger number of patients than projected may be necessary to provide a desirable probability of study success (i.e., >80%), regarding demonstration of statistically significant differences between the active treatment and placebo groups for the primary clinical response measure (CCR rate).
Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.
Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.
RNA interference appears to be a potentially powerful tool for studies of genes of unknown function. However, differences in efficacy at different target sites remain problematic when small interfering RNA (siRNA) is used as an effector. Similar problems are associated with attempts at gene inactivation using antisense oligonucleotides (ODNs) and ribozymes. We performed a comparative analysis of the suppressive effects of three knockdown methods, namely, methods based on RNA interference (RNAi), antisense ODNs, and ribozymes, using a luciferase reporter system. Dose-response experiments revealed that the IC(50) value for the siRNA was about 100-fold lower than that of the antisense ODN. Our results provide useful information about the positional effects in RNAi, which might help to improve the design of effective siRNAs.
Antisense oligonucleotides (ODNs) are powerful tools with which to determine the consequences of the reduced expression of a selected target gene, and they may have important therapeutic applications. Methods for predicting optimum antisense sites are not always effective because various factors, such as RNA-binding proteins, influence the secondary and tertiary structures of RNAs in vivo. To overcome this obstacle, we have attempted to engineer an antisense system that can unravel secondary and tertiary RNA structures. To create such an antisense system, we connected the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, to an antisense sequence so that helicase-binding hybrid antisense ODN would be produced in cells. We postulated that this modification would enhance antisense activity in vivo, with more frequent hybridization of the antisense ODN with its targeting site. Western blotting analysis demonstrated that a hybrid antisense ODN targeted to the bcl-2 gene suppressed the expression of this gene more effectively than did the antisense ODN alone. Our results suggest that the effects of antisense ODNs can be enhanced when their actions are combined with those of RNA helicases.
A recently developed antisense splicing assay was used to determine the relative activities of 2'-O-methoxyethoxy (2'-MOE) phosphorothioate oligonucleotides containing base modifications. In the assay, RNase H-inactive oligonucleotides are used to block aberrant splicing and restore correct splicing of an Enhanced Green Fluorescence Protein (EGFP) reporter pre-mRNA stably expressed in HeLa cells. Thus, the extent of EGFP upregulation is proportional to the antisense activity of the tested molecule. The base modifications included C-5 propynyl analogs of uridine and cytidine and phenoxazine and G-clamp analogs of cytosine. Base-modified 2'-MOE oligonucleotides were delivered to the HeLa EGFP-654 test cells by cationic lipid transfection or scrape-loading or without any delivery method (free uptake). When delivered with a cationic lipid, the G-clamp and phenoxazine oligomers showed increases in activity over the unmodified 2'-MOE parent compound. However, when delivered by scrape-loading or without a delivery method, the unmodified oligomer performed best. The results suggest that base modifications do not enhance the free uptake activity of RNase H inactive 2'-MOE oligomers.
Transfection of full-length antisense cDNA is used frequently to achieve stable downregulation of gene expression. However, screening for clones that express the antisense mRNA is complicated by the presence of endogenous sense mRNA. Thus, clones usually are screened for downregulation of the target protein by Western blotting, which can be time consuming. Here, we used strand-specific RT-PCR to identify antisense-expressing clones, which can then be screened for protein downregulation. This approach allows earlier identification of potentially useful clones and cuts down on the number of clones to be screened by Western blotting.
The retroviruses, including the human pathogens HIV-1 and HIV-2, are diploid inasmuch as they encapsidate two copies of their RNA genome. Prior to or during encapsidation, two copies of full-length genomic RNA recognize and stably bind each other in a process called dimerization. RNA structures within the viral genome promote dimerization in both HIV-1 and HIV-2 and are located in the 5'-untranslated leader region. Inhibition of dimerization by mutation of these RNA signals has been demonstrated to drastically reduce viral infectivity and replication kinetics and, thus, represents a potential target for antiretroviral therapy. In this study, we identified sites in HIV-2 leader region RNA that are functionally accessible to hybridization with oligonucleotides (ODNs) by reverse transcription with random ODN libraries (RT-ROL). We then tested specific ODNs directed against these regions for their efficacy in inhibiting RNA dimerization in vitro. We determined that of several hybridization-competent ODNs, only two were very effective in inhibiting RNA dimerization. Both of these ODNs were complementary to viral RNA at the primer binding site (PBS). These results identify regions with high accessibility to ODN binding on HIV-2 RNA and help to map the region(s) essential for dimerization within the viral RNA.
The antisense inhibitor poly-2'-O-(2,4-dinitrophenyl)-5'-GGCUGCGUGCCUCCUCACUGG (antisense poly-DNP RNA-21) has been synthesized by in vitro transcription followed by chemical derivatization. Its base sequence is complementary to that of nucleotides 110-130 in the mRNA of the regulatory RIalpha subunit of PKA (RIalpha/PKA), which is overexpressed in MCF-7 breast cancer cells and A549 lung cancer cells. The bioavailable and RNase-resistant antisense poly-DNP RNA-21 was found to inhibit cell growth with 50% inhibitory concentration (IC50) values of 0.05 nM in MCF-7 cells and 4 nM in A549 cells. The control 21-nt RNAs with the same poly-DNP oligonucleotide (ODN) platform but with scrambled, sense, or mismatched base sequence are inactive. Treatment of MCF-7 cells with antisense poly-DNP RNA-21 abolishes both the steady-state concentration of RIalpha mRNA and the synthesis of RIalpha protein. At sufficiently high concentration, antisense poly-DNP RNA-21 selectively kills the targeted cancer cells by inducing apoptosis. The observed sequence specificity and extremely low IC50 values of antisense poly-DNP RNA-21 suggest that it is a promising candidate for in vivo testing as an effective anticancer agent.
We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea.
Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.
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.
Inclusion of C-5 propynyl pyrimidines in phosphorothioate antisense oligonucleotides (ASOs) has been shown to significantly increase their potency for inhibiting gene expression in vitro. This increased potency is believed to be the result of enhanced binding affinity to target RNA. Our results show that C-5 propynyl pyrimidine-modified oligonucleotides caused an increase in the melting temperature (T(m)) of both oligodeoxynucleotides (ODNs) and 2'-O-(2-methoxy)ethyl (2'-MOE)-modified oligonucleotides. The in vitro data show a moderate increase in potency for an antisense oligodeoxynucleotide containing C-5 propynyl pyrimidines targeting the murine PTEN (MMAC1) transcript. Second-generation 2'-MOE chimeric ASOs containing C-5 propynyl pyrimidines showed no improvement in potency in PTEN target reduction in vitro or in vivo compared to their nonpropyne-modified parent. These results suggest that increasing affinity for target RNA beyond that achieved with the 2'-MOE modification does not further increase potency in cell-based assays. To evaluate whether this observation held true for in vivo applications, we evaluated both compounds in mice. We were unable to establish a dose-response relationship with C-5 propynyl pyrimidine-modified ODNs because of severe toxicity. The toxicity was characterized by mortality in animals receiving 50 mg/kg and an increase in infiltrating cells and apoptotic cells in livers of mice receiving 20 mg/kg. C-5 propynyl pyrimidine-modified chimeric oligonucleotides exhibited decreased hepatotoxicity compared with C-5 propynyl-modified ODNs but did not exhibit an increase in potency compared with unmodified chimeric oligonucleotides. The hepatotoxicity could be further limited if incorporation of propynyl pyrimidines was restricted to 2'-MOE nucleosides.
Phosphorodiamidate morpholino oligomers (PMO) are uncharged antisense molecules that bind complementary sequences of RNA, inhibiting gene expression by preventing translation or by interfering with pre-mRNA splicing. The techniques used to deliver PMO into cultured cells have been mostly mechanical methods. These delivery methods, although useful, have limitations. We investigated the ability of the HIV Tat peptide (pTat) and other cationic peptides to deliver PMO into cultured cells. Fluorescence was seen in 100% of HeLa cells treated with pTat-PMO-fluorescein conjugate. pTat-PMO conjugate targeted to c-myc mRNA downregulated c-myc reporter gene expression with an IC50 of 25 microM and achieved nearly 100% inhibition. pTat-PMO conjugate targeted to a mutant splice site of beta-globin pre-mRNA dose-dependently corrected splicing and upregulated expression of the functional reporter gene. Neither unconjugated PMO nor unconjugated pTat caused antisense activities. However, compared with mechanically mediated delivery, pTat-mediated PMO delivery required higher concentrations of PMO (>10 microM) to cause antisense activity and caused some toxicity. Most pTat-PMO conjugate was associated with cell membranes, and internalized conjugate was localized in vesicles, cytosol, and nucleus. The other three cationic peptides are much less effective than pTat. pTat significantly enhances delivery of PMO in 100% of cells assayed. pTat-mediated delivery is a much simpler procedure to perform than other delivery methods.
Oligodeoxyribonucleotides (ODNs) are currently employed to switch-off genes selectively routinely in the laboratory practice. The drawback of ODN application is that they have been often reported to elicit non-antisense effects by different mechanisms. Recently, it has been shown that double-stranded DNA oligonucleotides (30-mers) with protruding ends activate p53 in a cell-free system. In a previous work, we described that simple addition to the culture medium of heterogeneous DNA combined with cationic lipids culminated in inhibition of mitosis and induction of apoptosis. Here, we report that the same effects are achieved by lipotransfecting cultured cells with phosphorodiester ODNs (30-mers). Such effects of ODN were mediated by a non-antisense mechanism that required the wild-type form of the p53 oncosuppressor protein and was dependent on ODN concentration. Mitosis inhibition and apoptosis induction appeared to be determined by the 3' and 5' free ends of ODNs, which activated p53 independently from their sequence. Most probably, this mechanism is analogous to that evoked by genotoxic agent-induced DNA damage or by lipotransfecting cells with heterogeneous DNA.
