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Identification and Characterization of Second-Generation Antisense Oligonucleotides
... Oligonucleotide Synthesis-Phosphorothioate deoxyoligonucleotides and 2Ј-O-(2-methyoxy)ethylphosphothioate deoxyoligonucleotides were synthesized and purified as described previously (32,33). ...
... Assay for Oligonucleotide Inhibition of PP5 Expression-A549 cells were plated in 60-mm dishes and cultured in DMEM containing 10% fetal calf serum. When the cells were about 70% confluent, they were treated with oligonucleotides as described previously (32,33). Briefly, cells were washed with DMEM. ...
... The oligonucleotides tested in the initial screen were "chimeric" oligonucleotides, containing eight central phosphorothioate oligodeoxy residues ("oligodeoxy gap") flanked by six 2Ј-methoxyethyl residues on the 3Ј-and 5Ј-ends. These modifications have been shown previously to enhance the potency of antisense oligonucleotides targeting mRNAs encoding other proteins (32,33), and the oligonucleotides tested in the initial screen were designed to target specific regions in the protein coding region or the 3Ј-untranslated region of human PP5 mRNAs (Fig. 1). Because phosphorothioate oligonucleotides have been shown to commonly act through an RNase H-dependent mRNA cleavage mechanisms in cells (35), the ability of each oligodeoxynucleotide to specifically inhibit the expression of PP5 was initially determined by Northern blot analysis probing for levels of PP5 mRNA (Fig. 1A). ...
Understanding how alterations in growth control pathways are translated into changes in the cell cycle regulatory machinery is a major challenge for understanding the development of human cancers. The ability of both tumor suppressor proteins, p53 and BRCA1, to induce the expression of p21(WAF1/Cip1) in combination with the inhibitory activity of p21(WAF1/Cip1) against cyclin-dependent kinases suggests that the regulation of p21(WAF1/Cip1) expression is an important aspect of mammalian cell cycle growth control. To elucidate the role of serine/threonine protein phosphatase type 5 (PP5) in processes regulating cell cycle progression, we developed antisense oligodeoxynucleotides targeted against PP5 (e.g. ISIS 15534) that specifically inhibit PP5 gene expression. Employing ISIS 15534, we demonstrate that the specific inhibition of PP5 gene expression has a marked antiproliferative effect on cells, characterized by induction of p21(WAF1/Cip1) and the subsequent arrest of cell growth. Investigations into the mechanisms leading to growth arrest reveal that, in the absence of PP5, the expression of p21(WAF1/Cip1) is induced in p53-competent A549 cells but not in p53 protein-deficient T-24 cells. Employing a stable cell line derived from p53-deficient human fibroblast that contains tetracycline-regulated transactivator and operator plasmids to control the expression of wild-type p53 (TR9-7 cells), we then show that the induction of p21(WAF1/Cip1), which occurs in response to the inhibition of PP5 expression, requires the p53 protein. Additional studies indicate that PP5 acts upstream of p53, influencing both the phosphorylation state and the ability of p53 to bind DNA, without causing an increase in p53 gene transcription. Together these studies suggest that PP5 is a regulatory component of a signaling pathway that affords replicating cells G1 checkpoint growth control and that it is the regulation of PP5 that, in turn, controls p53-mediated expression of p21(WAF1/Cip1) and growth arrest in this pathway. In addition, since the inhibition of PP5 gene expression has marked antiproliferative activity and the overexpression of p21(WAF1/Cip1) blocks the growth of tumor cells, these studies suggest that compounds that inhibit of PP5 gene expression may be useful in the treatment of human cancers.
... In addition, DNA enzymes do not have phosphorothioate backbones, which confer additional negative charge onto the oligomer 37 and reduce binding affinity for target mRNA (ref. 38), increase binding avidity for serum and/or cellular proteins 38 and may be toxic 39 . The sequence-specific nature of rat ED5 may preclude use of this particular DNA enzyme in other animals, given the subtle differences in Egr-1 mRNA sequences between species. ...
... In addition, DNA enzymes do not have phosphorothioate backbones, which confer additional negative charge onto the oligomer 37 and reduce binding affinity for target mRNA (ref. 38), increase binding avidity for serum and/or cellular proteins 38 and may be toxic 39 . The sequence-specific nature of rat ED5 may preclude use of this particular DNA enzyme in other animals, given the subtle differences in Egr-1 mRNA sequences between species. ...
Early growth response factor-1 (Egr-1) binds to the promoters of many genes whose products influence cell movement and replication in the artery wall. Here we targeted Egr-1 using a new class of DNA-based enzyme that specifically cleaved Egr-1 mRNA, blocked induction of Egr-1 protein, and inhibited cell proliferation and wound repair in culture. The DNA enzyme also inhibited Egr-1 induction and neointima formation after balloon injury to the rat carotid artery wall. These findings demonstrate the utility of DNA enzymes as biological tools to delineate the specific functions of a given gene, and implicate catalytic nucleic acid molecules composed entirely of DNA as potential therapeutic agents.
... This oligonucleotide is a chimeric molecule containing a uniform phosphorothioate backbone and a stretch of ten 2Ј-deoxy residues in the center of the molecule which supports RNase H-mediated cleavage. The 2Ј-deoxy core is flanked by five bases at each of the 5Ј and 3Ј ends that are MOE modified and thus convey greater resistance to exonuclease activities and higher affinity for hybridization to RNA (22)(23)(24). The cytosines within the sequence were methylated at the 5-position to reduce the immunostimulatory potential sometimes observed with oligonucleotides in rodent studies. ...
... In vivo distribution studies have shown that appreciable bone marrow uptake occurs after i.v. administration of oligonucleotide in rats (41,42) and, combined with the improved nuclease resistance of the 2Ј-MOE chemistry used in the 5Ј-and 3Ј-ends of the oligonucleotide (23,24), these data suggest that one site of action is likely to be the bone marrow. Recent evaluation of oligonucleotide uptake in various lymphoid tissues of mice by capillary gel electrophoresis analysis of i.v. ...
Chronic airway eosinophilia is associated with allergic asthma and is mediated in part by secretion of IL-5 from allergen-specific Th2 lymphocytes. IL-5 is a known maturation and antiapoptotic factor for eosinophils and stimulates release of nascent eosinophils from bone marrow into the peripheral circulation. An antisense oligonucleotide found to specifically inhibit IL-5 expression in vitro was observed to significantly reduce experimentally induced eosinophilia in vivo, in both the murine OVA lung challenge and allergic peritonitis models. Intravenous administration resulted in sequence-dependent inhibition of eosinophilia coincident with reduction of IL-5 protein levels, supporting an antisense mechanism of action. Potent suppression of lung eosinophilia was observed up to 17 days after cessation of oligonucleotide dosing, indicating achievement of prolonged protection with this strategy. Furthermore, sequence-specific, antisense oligonucleotide-mediated inhibition of Ag-mediated late phase airway hyperresponsiveness was also observed. These data underscore the potential utility of an antisense approach targeting IL-5 for the treatment of asthma and eosinophilic diseases.
... These data suggest a substantial preabsorptive barrier to successful bioavailability of PS-ODNs. Although the barriers to efficacious oral bioavailability are large, the use of appropriate ODN chemistries and formulations has done much to break them down (Dean and Griffey, 1997; Geary et al., 2001a). For example, ISIS 14725, a partially 29-MOE-modified PS-ODN, remained 50% intact after 8 hours in the gastrointestinal tract. ...
... Since RNase H is an important terminating mechanism for antisense compounds, this has led to the development of a chimeric oligonucleotide strategy. One such chimeric strategy uses 2Ј-O-(2-methoxyethyl) modifications on the 3Ј-and 5Ј-ends, and a "gapped" region remains in the center with a contiguous sequence of 2Ј-deoxy phosphorothioate nucleotides (Inoue et al., 1987;Lamond and Sproat, 1993;Monia et al., 1993;Yu et al., 1996;Dean and Griffey, 1997). This approach has led to the development of potent, pharmacologically active, specific antisense oligonucleotides, one of which is ISIS 104838. ...
The pharmacokinetics of a 2'-O-(2-methoxyethyl)-ribose modified phosphorothioate oligonucleotide, ISIS 104838 (human tumor necrosis factor-alpha antisense), have been characterized in mouse, rat, dog, monkey, and human. Plasma pharmacokinetics after i.v. administration exhibited relatively rapid distribution from plasma to tissues with a distribution half-life estimated from approximately 15 to 45 min in all species. Absorption after s.c. injection was high (80-100%), and absorption after intrajejunal administration in proprietary formulations was as high as 10% bioavailability compared with i.v. administration. Urinary excretion of the parent drug was low, with less than 1% of the administered dose excreted in urine after i.v. infusion in monkeys at clinically relevant doses (< or = 5 mg/kg). ISIS 104838 is highly bound to plasma proteins, likely preventing renal filtration. However, shortened oligonucleotide metabolites of ISIS 104838 lose their affinity to bind plasma proteins. Thus, excretion of radiolabel (mostly as metabolites) in urine (75%) and feces (5-10%) was nearly complete by 90 days. Elimination of ISIS 104838 from tissue was slow (multiple days) for all species, depending on the tissue or organ. The highest concentrations of ISIS 104838 in tissues were seen in kidney, liver, lymph nodes, bone marrow, and spleen. In general, concentrations of ISIS 104838 were higher in monkey tissues than in rodents at body weight-equivalent doses. Plasma pharmacokinetics scale well across species as a function of body weight alone. This favorable pharmacokinetic profile for ISIS 104838 provides guidance for clinical development and appears to support infrequent and convenient dose administration.
... MHT cells were cultured in DMEM supplemented with 10% fetal calf serum, streptomycin (0.1 µg/ml), and penicillin (100 U/ml). siRNA treatment was performed using Opti-MEM containing 5 µg/ml Lipofectamine 2000 at the indicated amount of siRNA for 4 h at 37°C, as described previously (21,22). Heparan sulfate, 10K dextran sulfate, chloroquine, methyl-b-cyclodextrin, Brefeldin A were purchased from Sigma (St. Louis, MI, USA). ...
Single-stranded antisense oligonucleotides (SSOs) are used to modulate the expression of genes in animal models and are being
investigated as potential therapeutics. To better understand why synthetic SSOs accumulate in the same intracellular location
as the target RNA, we have isolated a novel mouse hepatocellular SV40 large T-antigen carcinoma cell line, MHT that maintains the ability to efficiently take up SSOs over several years in culture. Sequence-specific
antisense effects are demonstrated at low nanomolar concentrations. SSO accumulation into cells is both time and concentration
dependent. At least two distinct cellular pathways are responsible for SSO accumulation in cells: a non-productive pathway
resulting in accumulation in lysosomes, and a functional uptake pathway in which the SSO gains access to the targeted RNA.
We demonstrate that functional uptake, as defined by a sequence-specific reduction in target mRNA, is inhibited by brefeldin
A and chloroquine. Functional uptake is blocked by siRNA inhibitors of the adaptor protein AP2M1, but not by clathrin or caveolin.
Furthermore, we document that treatment of mice with an AP2M1 siRNA blocks functional uptake into liver tissue. Functional
uptake of SSO appears to be mediated by a novel clathrin- and caveolin-independent endocytotic process.
... Inhibitory effect of 5-methylcytidine-modified antisense oligonucleotides on HCV gene expression. Replacement of the nucleoside cytidine with 5-methylcytidine in antisense oligonucleotides confers higher-affinity binding to target RNA while still permitting RNase H-mediated cleavage of hybrid message RNA (19). 5-Methylcytidine-modified oligonucleotides are also less immunostimulatory and can reduce host complement system activation and B-cell and natural killer cell proliferation (9,25). ...
Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. Current treatments are not curative for most infected individuals, and there is an urgent need for both novel therapeutic agents and small-animal models which can be used to evaluate candidate drugs. A small-animal model of HCV gene expression was developed with recombinant vaccinia virus vectors. VHCV-IRES (internal ribosome entry site) is a recombinant vaccinia viral vector containing the HCV 5' nontranslated region (5'-NTR) and a portion of the HCV core coding region fused to the firefly luciferase gene. Intraperitoneal injection of VHCV-IRES produced high levels of luciferase activity in the livers of BALB/c mice. Antisense oligonucleotides complementary to the HCV 5'-NTR and translation initiation codon regions were then evaluated for their effects on the expression of these target HCV sequences in BALB/c mice infected with the vaccinia virus vector. Treatment of VHCV-IRES-infected mice with 20-base phosphorothioate oligonucleotides complementary to the sequence surrounding the HCV initiation codon (nucleotides 330 to 349) specifically reduced luciferase expression in the livers in a dose-dependent manner. Inhibition of HCV reporter gene expression in this small-animal model suggests that antisense oligonucleotides may provide a novel therapy for treatment of chronic HCV infection.
... PS molecules do cause some non-specific inhibition of cell growth and have specific non-sequence dependent toxicities [17,18]. New backbone modifications creating chimaeric molecules may be as effective in protecting against nuclease activity [19] whilst reducing the non-specific toxicity (Table 1) [20,21]. ...
Curative, malignancy-specific therapy, based on the molecular pathology of each cancer subtype which causes minimal side effects to the person suffering the disease. This is a goal which has long eluded us despite an already daunting though incomplete knowledge of the basis of human disease. Although increasing dose inten- sity of chemotherapy, and improving management of supportive care for high-dose therapy has won us short- term rewards, there are many malignant diseases where the underlying biology of the disease is still the major determinant of outcome, irrespective of therapy. The boundaries of what can be achieved with the current chemotherapeutic agents are apparent and a radically new approach to targeted therapy is well overdue. How- ever, the promise of genetic therapy has been implied for over a decade without broadly filling the promise. Slowly and not always steadily, antisense therapy has been gaining ground (1). A large base of knowledge of pharmacodynamics and toxicology profiles, has been built from extensive in vivo testing and phase I and II human clinical trials. Many questions may be asked and now answered from this body of data. These include from the simplest to the most difficult questions. What is antisense therapy? What major obstacles have had to be overcome during its development and what does it hope to achieve? But perhaps the most frequently asked question is - does it work? What is antisense therapy? In its purest definition, antisense therapy is a gene targeted therapeutic approach based on a synthetic piece of single stranded DNA (oligonucleotide) which is used to bind to the complementary sequence of RNA for a specific gene. The ultimate silencing of gene expression by the exogenous administration of oligonucleotides results in gradual downregulation of the protein and loss of function of that gene. This implies the oligo- nucleotides should be completely specific in their action and the outcome should be dose dependent and propor- tional to the measured downregulation of the gene (2). Downregulation is mostly due to an intracellular enzyme, RNAse H, being activated which results in cutting of the RNA at RNA/DNA duplex sites by this enzyme (3, 4). The resultant protein downregulation is due to the loss of messenger RNA and the loss of function of that protein, is therefore attributable to a true antisense effect. They are true 'designer drugs' and have been proposed as the ultimate in drug therapy, but are they?
... Antisense Oligonucleotide Synthesis and Assay for Oligonucleotide Inhibition of PP5 Expression-Phosphorothioate deoxyoligonucleotides and 2Ј-O-(2-methoxy)ethylphosphothioate deoxyoligonucleotides were synthesized and purified as described previously (30,31). Antisense oligonucleotide-mediated inhibition of PP5 expression and Northern blot analysis were performed as described previously (28 -31). ...
The proliferation of many estrogen receptor (ER)-positive breast cancer cells depends on estradiol, and tumors arising from these cells are often responsive initially to treatment with selective ER modulators, which produce an antiestrogen effect. However, tumors that are refractory to the antiestrogenic effects of selective ER modulators often reemerge, and the prognosis for these patients is poor because of the lack of additional effective therapy. Accordingly, deciphering the cellular events associated with estrogen-dependent growth and the subsequent outgrowth of tumors with an estrogen-independent phenotype is of considerable interest. Here we show that the expression of PP5, an evolutionarily conserved Ser/Thr phosphatase that functions as an inhibitor of glucocorticoid- and p53-induced signaling cascades leading to growth suppression, is responsive to 17beta-estradiol (E(2)) in ER-positive human breast carcinoma cells (MCF-7). Northern analysis revealed that E(2)-induced PP5 expression is blocked by treatment with tamoxifen, and a consensus ER recognition element was identified in the PP5 promoter. The PP5-ER recognition element associates with human ERs and confers E(2)-induced transcriptional activation to reporter plasmids. The specific inhibition of PP5 expression ablates E(2)-mediated proliferation in MCF-7 cells without having an apparent effect on E(2)-induced expression of c-myc or cyclin D1. Thus, although critical for cell growth, PP5 likely acts either downstream or independently of c-Myc and Cyclin D1. To further characterize the role of PP5 in E(2)-regulated growth control, we constructed stable MCF-7 cell lines in which the expression of PP5 was placed under the control of tetracycline-regulated transactivator and operator plasmids. Studies with these cells revealed that the constitutive overexpression of PP5 affords E(2)-dependent MCF-7 cells with the ability to proliferate in E(2)-depleted media. Together, these studies indicate that E(2)-induced PP5 expression functions to enhance E(2)-initiated signaling cascades leading to cell division and that aberrant PP5 expression may contribute to the development of MCF-7 cells with an estrogen-independent phenotype.
... Synthesis and purification of chimeric deoxyphosphorothioate/2Ј-O-methoxyethyl base oligonucleotides were performed using an Applied Biosystems 380B automated DNA synthesizer, as described by ISIS Pharmaceuticals (37). ...
Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.
p>CD40 is a cell surface signalling molecule expressed on normal B-cells and B-cells malignancies. Activations of CD40 (via binding CD40 ligand, CD40L) in normal B-cells promotes B-cell survival and is vital for activation of the immune response. However, activation of CD40 can induce or prevent apoptosis in malignant B-cells. Furthermore, CD40-activating agents are entering clinical trails as cancer therapies.
B-cells were isolated from over twenty Non-Hodgkin’s lymphomas and non-malignant lymph nodes and the effect of CD40 ligation on apoptosis and expression of apoptosis regulators was assessed. CD40L suppressed apoptosis in all B-cell malignancies tested, accompanied by significant increases in Bcl-X<sub>L</sub> protein expression and differential regulation of Bcl-X splicing and promoters. Mcl-1 protein expression was also regulated by CD40 stimulation. Although A20, survivin, Bfl-1 and BNIP3 (a novel CD40-target gene identified in this study) were regulated by CD40 in some samples, this was not consistent. Antisense oligonucleotides to Bcl-X<sub>L</sub> and Mcl-1 demonstrated that these proteins were key for survival of lymphoma cells. NF-κB mediated both the pro-survival effects of CD40 and upregulation of Bcl-X<sub>L</sub> and Mcl-1 expression. Microarrays were developed and used to characterise CD40-induced gene expression. This identified both novel CD40-target genes and also genes that were differentially regulated in cell lines with different survival outcomes to CD40 activation e.g. SLAM and SH2D1A.
Therefore, CD40 acts as a potent survival signal for primary B-cell lymphomas leading to regulation of the key anti-apoptotic proteins Bcl-X<sub>L</sub> and Mcl-1, via activation of NF-κB. This raises concerns over forthcoming clinical trails utilising CD40 activation as a treatment for cancer but suggests that targeting Bcl-X<sub>L</sub>, Mcl-1 or NF-κB may be a useful approach for treating lymphoma.</p
Since the discovery of the structure and function of DNA over 40 years ago, the established knowledge of molecular biology has increased dramatically, and many new tools have been discovered and utilized by scientists to develop new therapeutic agents. Important tools that are used in recombinant DNA technology include restriction endonucleases (cleave DNA), DNA ligase (link DNA molecules together), and cloning vectors (place foreign DNA into an organism such as bacterial or yeast cells in order to mass produce the protein encoded by that foreign DNA). The development of hybridoma technology provided a method to produce virtually unlimited quantities of pure antibody with a single specificity. These immunoglobulins are known as monoclonal antibodies, and have provided both therapeutic and diagnostic agents. Antisense molecules are oligonucleotides which bind to the messenger RNA (mRNA) of a target gene and selectively inhibit the production of specific proteins. Potential applications for these molecules include cancer and viral and inflammatory diseases. The more recent development of the polymerase chain reaction (PCR) has provided a tool that has revolutionized diagnostic testing in diverse areas such as infectious diseases, genetic abnormalities, and cancer.
The synthesis of 1-(2-deoxy-3-O-phosphonomethyl-beta-D-erythropentofuranosyl)thymine (17) and its alpha-anomer 18 is described. Attempts to prepare 1-[2-deoxy-3-O-(pyrophosphoryl)phosphonomethyl-beta-D-erythro-pentofuranosyl]thymine (19) by an activation of the respective phosphonate 17 with 1,1'-carbonyldiimidazole (Im2CO) resulted in the quantitative formation of the corresponding pyrophosphonate derivative 21 (Scheme 2). Activation of inorganic pyrophosphate with Im2CO followed by the condensation with the phosphonates 17 and 18 afforded the desired analogues of nucleoside triphosphate 19 (35%) and its alpha-anomer 20 (27%) along with the respective pyrophosphonate derivatives 21 (37%) and 24 (38%) (Scheme 3). It was found that compounds 19 and 20 display (i) no substrate properties toward calf thymus terminal deoxynucleotidyl transferase (TDT) and AMV reverse transcriptase, and (ii) moderate substrate activity with E. coli DNA polymerase I (Klenow fragment).
The wound-healing response is involved in many diseases throughout the body, including the eye. It is a key factor in influencing
results of surgery, particularly that occurring in the treatment of the blinding disease glaucoma, where the postoperative
fibrotic response is the major determinant of outcome. Transforming growth factor-β (TGF-β) has been widely established as
a target for post-operative antifibrotic therapy in glaucoma. Strategies against TGF-β have included antibodies, antisense
phosphorothiate oligonucleotides, and naturally occurring antagonists. These have either reached preclinical or clinical trials
in the eye, but offer potential widespread applications anywhere in the body where the wound-healing response requires modulation.
The genomics revolution of the past decade has allowed the sequences of all human genes to be determined. The challenge for the future is to establish the functions of all of these genes. Antisense technology is a powerful tool for gene functionalization. This review describes a system used to test antisense oligonucleotides to over 2000 genes in multiple cell-based assays. These assays were designed to help assign gene function. The data generated from these assays is subsequently added to a searchable database that allows potential drug targets to be identified.
Unlabelled:
Fostriecin is a natural product purified from Sterptomyces extracts with antitumor activity sufficient to warrant human clinical trials. Unfortunately, difficulties associated with supply and stable drug formulation stalled further development. At a molecular level, fostriecin is known to act as a catalytic inhibitor of four PPP-family phosphatases, and reports describing the design of molecules in this class suggest derivatives targeting enzymes within the fostriecin-sensitive subfamily can be successful. However, it is not clear if the tumor-selective cytotoxicity of fostriecin results from the inhibition of a specific phosphatase, multiple phosphatases, or a limited subset of fostriecin sensitive phosphatases. How the inhibition of sensitive phosphatases contributes to tumor-selective cytotoxicity is also not clear. Here, high-content time-lapse imaging of live cells revealed novel insight into the cellular actions of fostriecin, showing that fostriecin-induced apoptosis is not simply induced following a sustained mitotic arrest. Rather, apoptosis occurred in an apparent second interphase produced when tetraploid cells undergo mitotic slippage. Comparison of the actions of fostriecin and antisense-oligonucleotides specifically targeting human fostriecin-sensitive phosphatases revealed that the suppression PP4C alone is sufficient to mimic many actions of fostriecin. Importantly, targeted suppression of PP4C induced apoptosis, with death occurring in tetraploid cells following mitotic slippage. This effect was not observed following the suppression of PP1C, PP2AC, or PP5C. These data clarify PP4C as a fostriecin-sensitive phosphatase and demonstrate that the suppression of PP4C triggers mitotic slippage/apoptosis.
Implications:
Future development of fostriecin class inhibitors should consider PP4C as a potentially important target. Mol Cancer Res; 11(8); 845-55. ©2013 AACR.
Most current therapeutic approaches focus on targeting a protein or its function after it has been translated. A therapeutic approach that is gaining interest is altering the mRNA that encodes for a disease associated protein and preventing the protein from being produced. There are two major therapeutic methods to alter mRNA levels, antisense and RNA interference (RNAi). Currently, antisense is the more developed technology and has been extensively used in vivo. As this review will mostly focus on in vivo studies, animal models or humans, it will concentrate on antisense. The lung is an attractive target for this technology as the drug can be introduced to the organ with limited systemic involvement. Inflammatory diseases of the lung, such as allergy or asthma, are potential areas in which antisense technology could be beneficial. There are many mRNA targets being investigated for treatment in inflammatory lung disease that include signaling molecules as well as receptors. Also, antisense is being investigated for treatment of lung cancers. In lung cancer, some oncogenic genes may be unique to the tumor and can be specifically targeted without affecting other cellular processes. Thus, antisense and RNAi potentially provide an alternative to current therapy for treatment of pulmonary disorders.
The capacity of DNA to bind RNA via Watson-Crick base-pairing is fundamental to antisense oligonucleotide strategies to inhibit gene expression, and is a property that has been exploited by bioengineers in the generation of catalytic molecules such as ribozymes, ribozyme subtypes, and more recently DNAzymes. This review describes the evolution of these gene-specific agents and summarizes recent efforts to inhibit smooth muscle cell growth with these molecules as candidate therapeutic tools in restenosis.
Objective: To explore and investigate the selection of effective antisense oligodeoxynuleotides
with the help of computer and RNAstructure folding software. Methods: Bcl-2 gene was used as the
target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal
secondary structure regions that were predicted free from intramolecular fold or instability of free energy.
The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including
cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry
and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed
and determined with morphonological method, electrophoresis and flowcytometry. Results: The results
showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides,
which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to
induce apoptosis of leukemia cells significantly. Conclusion: The computational prediction of antisense
efficacy is faster than other methods and more efficient, which can potentially speed the development of
sequences for both research and clinical applications.
We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides
(ASOs) in Tm, cell culture and animal experiments and compare them to their gap-matched 20-mer 2′-O-methoxyethyl (MOE) and 14-mer 2′,4′-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs
showed similar Tm and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and
14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments
where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED50 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several
extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer
cEt ASO showed the best activity in the animal experiments despite significantly lower Tm and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish
tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing
ASO pharmacology and pharmacokinetic properties in animals.
In the past decade, there has been a vast increase in the amount of gene sequence information that has the potential to revolutionize the way diseases are both categorized and treated. Old diagnoses, largely anatomical or descriptive in nature, are likely to be superceded by the molecular characterization of the disease. The recognition that certain genes drive key disease processes will also enable the rational design of gene-specific therapeutics. Antisense oligonucleotides represent a technology that should play multiple roles in this process.
Six methylene(methylimino) (MMI, Bhat et al. J. Org. Chem., 61, 8186, 1996) linked oligonucleotides a-f (* = MMI linkage; 5'-GCGT*TT*TT*TT*TT*TGCG-3') containing various combinations of 2'-O-methyl and 2'-fluoro substituent were synthesized as a model to study the global conformational change upon hybridization to the complement RNA. Fourier transform infrared (FTIR) spectroscopic technique has been used to study and compare the influence of these modifications on the solution conformation of 2'-modified MMI DNA-RNA duplexes. FTIR analysis of the single-stranded RNA (5'-CGCAAAAAAAAAACGC-3') and the modified oligonucleotides a-f showed that all sugar residues adopted a C3'-endo conformation (North-type). Stable duplexes were formed when oligonucleotides a-f were hybridized to the complement RNA. These duplexes retained the original C3'-endo conformation for all sugar residues, hallmark of an A-form of duplex. We postulate that the observed preorganization of the sugar residues and oligonucleotides containing 2'-modified MMI modifications may play an important role in both improving the recognition of RNA target and enhancing the stability of duplex formation with RNA.
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.
The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.
Despite many uncertainties concerning mechanism, synthetic single-strand antisense deoxyribonucleic acids (DNAs) are now in clinical trials for the chemotherapy of viral infections such as human immunodeficiency virus (HIV) and human papilloma virus; several cancers, including follicular lymphoma and acute myelogenous leukemia; inflammatory processes such as Crohn's disease and rheumatoid arthritis and in allergic disorders. There are approximately 10 trials, and early results are generally encouraging. Therefore, the expectation is that antisense DNAs will be important to future chemotherapy. The question considered here is whether antisense DNAs will also be important to future nuclear medicine imaging. While efforts toward developing antisense imaging are comparatively nonexistent thus far, investigations into the mechanisms of cellular transport and localization and the development of a second generation of antisense DNAs have occurred largely within the antisense chemotherapy industry. Fortunately, many of the properties of DNA for antisense imaging, such as high in vivo stability and adequate cell membrane transport, are the same as those for antisense chemotherapy. Unfortunately, interests diverge in the case of several other key properties. For example, rapid localization and clearance kinetics of the radiolabel and prolonged retention in the target are requirements unique to nuclear medicine. No doubt the development of antisense imaging will continue to benefit from improvements in the antisense chemotherapy industry. However, a considerable effort will be required to optimize this approach for imaging (and radiotherapy). The potential of specifically targeting virtually any disease or normal tissue should make this effort worthwhile.
Ischemia-reperfusion injury is an acute inflammatory process during which leukocytes are intimately involved. In this review, we summarize the current data on the leukocyte cell adhesion cascade in ischemia-reperfusion injury, focus upon studies which have demonstrated specific cell adhesion molecule interactions which mediate the leukocyte involvement in ischemia-reperfusion injury, and suggest future avenues of therapeutic interventions. The increased adhesion between activated vascular endothelium and peripheral blood leukocytes is central to the structural and the functional impairment in ischemia-reperfusion injury. Several families of adhesion molecules, namely the selectins, the intercellular adhesion molecules (ICAMs), and the integrins expressed either on the endothelium or on the leukocytes, are involved the cascade of events. Sequential and overlapping cellular interactions between the members of the three gene families of adhesion receptors result in adhesion of the leukocytes to the endothelium and extravasation at the site of ischemia. The functional importance of ICAM-1 and its beta2 integrin ligands in ischemia-reperfusion of the kidney has been demonstrated by monoclonal antibody blockade studies, in knockout mice and by treatment with antisense oligodeoxynulceotides (ODN). We have shown that antisense ODN for ICAM-1 protected the kidney against ischemic renal failure. In addition, in transplanted kidneys, ICAM-1 inhibition by antisense ODN ameliorates ischemia-reperfusion injury and prevents delayed graft function. Recent developments in antisense ODN technology make this a promising therapeutic approach, and antisense ODN treatment of donors or donor organs for ICAM-1 may be useful for the prevention of reperfusion injury in human renal transplantation and could influence acute and chronic graft function.
2',5'-Oligoadenylate (2-5A) antisense chimeric oligonucleotides were synthesized containing varying 2'-O-methyl-ribonucleotide substitution patterns in the antisense domain. The ability of these composite oligonucleotides to mediate RNase H- and RNase L-catalyzed RNA degradation showed that these two enzymes have different activation requirements.
Antisense therapeutics using synthetic oligodeoxynucleotides (ODNs) are currently being evaluated in clinical trials for cancer, inflammation, and viral diseases. These macromolecules afford a unique opportunity to treat disease at the molecular level. The specificity of these compounds is derived from the genetic code and Watson-Crick base pairing, utilizing an antisense paradigm for the inhibition of translation and the regulation of protein expression. Currently, most antisense ODNs in development contain a phosphorothioate (P=S) backbone. Additional modifications primarily involve the 2' position on the ribose or modification of the nucleotide linkages of the backbone. To date, no toxicities in animal models appear related to inhibition of the pharmacologic target, rather toxicities induced by P=S ODNs appear similar and are independent of pharmacologic target. In general, toxicities correlate well with pharmacokinetic or tissue distribution parameters. In primates, the primary acute effects are associated with complement activation and the systemic effects associated with accumulation of high concentrations of P=S ODNs in the kidneys. In rodents, the primary effect is an immune stimulation characterized by splenomegaly, lymphoid hyperplasia, and mononuclear cell infiltrates in multiple tissues. At extraordinarily high doses (15-50 times the targeted clinical doses), hepatocellular and renal tubular degeneration are evident in rodents. Second generation antisense compounds, new routes of administration, and new formulations appear to broaden and improve the application of antisense technology.
Ligand-induced glucocorticoid receptor (GR) activation has recently been linked to the inhibition of cell proliferation via the transcriptional induction of p21(WAF1/Cip1), which functions as a universal inhibitor of cyclin-dependent protein kinases. Herein, we identify a Ser/Thr protein phosphatase (PP5) that promotes cellular proliferation by inhibiting both glucocorticoid and p53-mediated signaling pathways leading to p21(WAF1/Cip1)-mediated growth arrest. The suppression of PP5 expression (1) markedly increases the association of GR with its cognate DNA-binding sequence, (2) induces GR transcriptional activity without the addition of hormone, and (3) increases dexamethasone-mediated induction of GR reporter activity to a level that is approximately 10 times greater than the maximal response obtainable in the presence of PP5. PP5 has no apparent effect on the binding of hormone to the GR, and dexamethasone-mediated growth arrest correlates with an increase in p53 phosphorylation. Comparative studies in p53-wild-type, p53-defective, and p53-deficient cell lines indicate that either (1) p53 participates in GR-mediated induction of p21(WAF1/Cip1), with the hyperphosphorylation of basal p53 induced by glucocorticoids sufficient for the propagation of an antiproliferative response when PP5 expression is inhibited, or (2) PP5 acts where p53-mediated and GR-induced signaling networks converge to regulate the transcriptional induction of p21(WAF1/Cip1). Thus, aberrant PP5 expression may have an additive effect on the development of human cancers by promoting cell proliferation via the inhibition of a GR-induced antiproliferative signaling cascade, and facilitating neoplastic transformation via the inhibition of a growth-arresting p53-mediated response that guards against genomic instability.
The epidermis is continually exposed to harmful mutagens that have the potential to cause DNA damage. To protect the skin from accumulating mutated cells, keratinocytes have developed a highly regulated mechanism of eliminating damaged cells through apoptosis. Bcl-xL is a well-described cell survival protein that when overexpressed in skin can protect keratinocytes from UV radiation-induced apoptosis. To begin to unravel the complex mechanisms that keratinocytes use to survive, we wanted to characterize the role of endogenous Bcl-xL in protecting cells from death. In this study, we describe the development and characterization of an antisense inhibitor to Bcl-xL. We show that this inhibitor reduces Bcl-xL RNA and protein in a concentration-dependent, sequence-specific manner. Furthermore, treatment of keratinocytes and epithelial cells with this inhibitor sensitizes these cells to UV-B radiation and cisplatinum treatment-induced apoptosis. Thus, these results offer direct evidence that Bcl-xL is critical in the protection of skin and epithelial cells from apoptosis and provide a basis for the role of Bcl-xL in keratinocyte and epithelial cell survival.
Recent advances in molecular cell biology have revolutionised our understanding of medical diseases and provided new and alternative strategies for developing treatments. Here we review the spectrum of molecular therapies that are either currently available or have potential application as agents that are able to modulate the wound healing response in the eye. For the purposes of this review, we define molecular therapy as the targeting of specific molecules known to be involved in the processes of wound healing. This may be at the level of either protein or gene expression.
The process of wound healing is involved in either the pathogenesis or failure of treatment of many of the major blinding or visually disabling conditions in the world today. It is implicated in scarring diseases throughout the eye, some examples of which are described below.
The conjunctival wound healing response is important in many ocular conditions such as pemphigoid, trachoma, and chronic cicatrisation, where the development of complications arises from the disruption of the ocular surface.1 It is also a major determinant of outcome following glaucoma filtration and squint surgery.2The severity and extent of clinical disease are closely related to the degree of conjunctival scarring. Another example of scarring is that occurring in the cornea after excimer laser photorefractive keratectomy (PRK), giving rise to symptoms of haze and resulting, in some cases, in a reduction of the best corrected visual acuity.3 4 The scarring condition of proliferative vitreoretinopathy (PVR) accounts for 7–10% of surgical failures in primary retinal detachment repair procedures5 6 and is responsible for producing significant visual morbidity and blindness. It is characterised by the development of fibrosing and proliferating cellular membranes on the vitreous and retinal surfaces that contract and cause irreparable tractional retinal detachments.
The complex process of wound healing …
Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of antiapoptotic to proapoptotic proteins. By manipulating levels of these proteins, cells can become sensitized to undergo apoptosis in response to chemotherapeutic agents. Alternative splicing of the bcl-x gene gives rise to two proteins with antagonistic functions: Bcl-xL, a well-characterized antiapoptotic protein, and Bcl-xS, a proapoptotic protein. We show here that altering the ratio of Bcl-xL to Bcl-xS in the cell using an antisense oligonucleotide permitted cells to be sensitized to undergo apoptosis in response to ultraviolet B radiation and chemotherapeutic drug treatment. These results demonstrate the ability of a chemically modified oligonucleotide to alter splice site selection in an endogenous gene and illustrate a powerful tool to regulate cell survival.
Nature Biotechnology journal featuring biotechnology articles and science research papers of commercial interest in pharmaceutical, medical, and environmental sciences.
The ability to inhibit gene expression is an important tool for studying the normal function of specific gene products within a cell. Antisense (AS) nucleic acids have been utilized successfully as one means of downregulating intracellular mRNA levels, through either endonucleolytic cleavage by RNase H or by blocking initiation or elongation of translation. Antisense nucleic acid is most commonly delivered in the form of short (<30 nucleotide) oligodeoxynucleotides (ODNs). These techniques have been used to show that cellular protooncogenes, especially transcription factors, are sensitive to AS inhibition, and that they function in regulating proliferation and differentiation. Translocation of the c-myc oncogene on chromosome 8 with the immunoglobulin loci on chromosome 2, 14, or 22, resulting in c-rnyc overexpression, is characteristic of Burkitt's lymphoma. In the most frequent translocation [t(8;14)] of the c-myc gene (8q24) with the Ig heavy chain gene (IgH; 14q32), two forms of the disease arise: the sporadic form in which the breakpoint is located within the gene, commonly near the first intron of the c-myc gene, and the endemic form in which the breakpoint is located outside of the gene, commonly in the 5'-untranslated region of the c-myc gene. Translocation of the c-myc gene results in its overexpression because of disruption of its transcriptional regulation. The focus of this chapter is to detail the methodology required to efficiently utilize cationic lipids for delivering phosphorothioate AS ODNs into several different lymphoma cell lines and, in particular, on the joint optimization of conditions for two cell lines acting as controls for each other during the same AS experiments.
In lower eukaryotic organisms, the loss of serine/threonine protein phosphatase type 1 (PP1) results in growth arrest after the onset of mitosis. In humans, four highly homologous isoforms of PP1 (PP1alpha, PP1delta, PP1gamma1, and PP1gamma2) have been identified. Determining the roles of these phosphatases, however, has proven difficult due to the lack of subtype-specific inhibitors. In this study, we developed chimeric antisense 2'-O-(2-methoxy)ethylphosphothioate oligonucleotides targeting human PP1gamma1 that specifically inhibit PP1gamma1 gene expression. Two potent antisense oligonucleotides (ISIS 14435 and 14439; IC(50) approximately 50 nM) were then employed to elucidate the cellular functions of PP1gamma1 during cell cycle progression. In A549 cells, the inhibition of PP1gamma1 expression resulted in a dose-dependent inhibition of cellular proliferation, with growth arrest occurring after approximately 36-48 h, when PP1gamma1 mRNA expression was inhibited by >85%. Fluorescence-activated cell sorter analysis revealed that ISIS 14435/14439-induced growth arrest was associated with an increase in the number of cells containing 4N DNA. Immunostaining of treated cells revealed that the inhibition of PP1gamma1 expression had no apparent effect on the formation of mitotic spindles. However, decreased expression was associated with the failure of cell division in a late stage of cytokinesis and the formation of dikaryons.
An in situ single-pass perfusion model was used to assess the effect of chemical modification and length on permeability and absorption of various oligonucleotides in rat intestine. Phosphorothioate oligodeoxynucleotides (PS-ODN) were compared with oligoribonucleotides with 2'-methoxyethyl (MOE) or 2'-O-methyl (OMe) modifications. A 25-mer PS-OMe-modified oligonucleotide showed relatively poor permeability in this model, as did unmodified 20-mer PS-ODN (permeability coefficient [P(eff)] = 2-8 X 10(-6)cm/sec). Modifying some or all of the oligonucleotides with 2'-MOE groups on deoxyribose and 5'-methylation of the cytosines substantially increased intestinal permeability of oligonucleotides. Both partially and fully modified PS-MOE oligonucleotides showed a (2-4)-fold increase in permeability as compared with unmodified PS-ODN. The presence of a phosphodiester backbone in MOE-modified compounds led to further increases in intestinal permeability. PS-MOE composed of 6, 8, 10, 12, 14, 16, 18, 20, and 22 nucleotides were also examined. It was found that the permeability of these oligonucleotides increased linearly with decreasing length.
Expression of the interleukin-5 receptor-alpha (IL-5Ralpha) chain is thought to play an important role in the pathogenesis of asthma and other eosinophilic diseases. With antisense oligonucleotides (ASOs) chemically modified to provide increased hybridization affinity for RNA but that do not support RNase H-mediated cleavage (2'-O-methoxyethyl-modified ASOs), we show that constitutive splicing of murine IL-5Ralpha mRNA can be modulated in cells such that individual exons may be selectively deleted from mature transcripts. Specific deletion of individual exons and redirection of alternative splicing of the IL-5Ralpha mRNA have been achieved with this approach, by targeting 3'-splice sites or exon sequences immediately downstream of an alternative splice site. ASO targeting with these strategies resulted in inhibition of mRNA and protein levels of the membrane IL-5Ralpha isoform capable of signaling IL-5-mediated growth and antiapoptotic signals to eosinophils. Membrane isoform IL-5Ralpha inhibition was coupled with an increase in expression of mRNA for the alternatively spliced soluble isoform, which binds IL-5 extracellularly and may block its function. These observations suggest the potential general therapeutic use of an antisense approach to increase expression of variant RNA transcripts and to thereby produce proteins devoid of specific functional domains that may impact disease processes, as well as its specific utility for modulating expression of a key cytokine receptor implicated in allergic inflammation.
Binding of human interleukin-5 (HuIL-5) to its membrane-anchored receptor (IL-5R) triggers multiple signaling pathways, cellular proliferation, and maturational responses, as well as protection from apoptosis. In contrast, soluble forms of the HuIL-5R have been shown to inhibit IL-5 signaling and, therefore, may represent naturally occurring negative regulators of IL-5 function. Because of the central role of IL-5 in promoting eosinophilia and airway hyperresponsiveness in animal models of asthma, antisense oligonucleotides specific either for the membrane form alone or for sequences shared between both the membrane and soluble forms of the HuIL-5Ralpha ligand binding chain were designed. The activities of these oligonucleotides were characterized in IL-5R-expressing erythroleukemic TF-1 cells. Herein we report that an antisense oligonucleotide targeted to a sequence unique to the alternatively spliced membrane-bound form of the HuIL-5Ralpha chain has been developed that selectively inhibits membrane, but not soluble, mRNA isoform expression. Both this membrane-specific oligonucleotide and an antisense oligonucleotide targeted to sequence common to both membrane and soluble isoforms were found to potently suppress cell surface IL-5Ralpha levels and IL-5-mediated cell survival by inducing apoptosis similar to IL-5 withdrawal. Thus, these oligonucleotides represent unique genetic agents with therapeutic potential for diseases with an eosinophilic component.
Duplex formation between an oligonucleotide and a strand of mRNA can effectively inhibit the expression of a specific gene
via interfering with the cellular protein synthesis process. The antisense technology (1) is regarded as a powerful tool in molecular biology. Advances in the field have also led to the development of many pharmaceutical/therapeutic
applications, with a number of oligonucleotides undergoing human clinical trials at present (2). Making the best use of this approach requires a good understanding of the subject, careful planning, a knowledge of limitations
and pitfalls, and a capacity to go beyond the basic antisense techniques.
The unique role of interleukin (IL)-5 in eosinophil production, activation, and localization makes this cytokine a prime target for therapeutic intervention in diseases characterized by a selective blood and tissue eosinophilia. In an attempt to block the effects of IL-5 on eosinophils, a strategy was developed to suppress the expression of the IL-5 receptor alpha chain (IL-5Ralpha) by antisense oligonucleotides (ASOs). IL-5Ralpha ASOs were identified which selectively and specifically suppress the expression of messenger RNA and proteins of both the membrane and the soluble form of the receptor in constitutively IL-5R-expressing murine BCL-1 cells in vitro. Moreover, these IL-5Ralpha-specific ASOs were able to selectively inhibit the IL-5-induced eosinopoesis from murine fetal liver and bone marrow cells in vitro, suggesting that these molecules may affect the development of IL-5-mediated eosinophilia in vivo. Indeed, intravenous administration of IL-5Ralpha-specific ASOs not only suppressed the bone-marrow and blood eosinophilia in mice after short-term treatment with recombinant murine IL-5 but also inhibited the development of blood and tissue eosinophilia in a ragweed-induced allergic peritonitis model. Thus, blocking the expression of IL-5Ralpha on eosinophil using ASOs may have therapeutic benefits in eosinophilic diseases such as asthma.
Despite the simplicity of the concept, almost every step in an antisense experiment poses difficulties. Finding a site that is accessible to intermolecular hybridization with complementary nucleic acids is a major problem and determines the success or failure of an antisense experiment. A major determinant of accessibility appears to be the intramolecular folding in mRNAs that renders much of the molecule inaccessible. However, owing to our poor understanding of RNA folding and the mechanisms of heteroduplex formation, theoretical methods have limited use in finding accessible sites. Such methods are unable to address two major considerations when designing an antisense reagent, i.e., which is the most accessible sequence in the target and what length of the reagent works best in terms of activity and specificity. Empirical approaches appear more successful. Of notable interest, and reviewed here, are 'global' methods based on DNA arrays and on mapping of transcripts with RNase H.
The purpose of this review is to examine whether our current knowledge of the higher order control of gene expression and nuclear organization can help us understand the mechanisms of genomic imprinting. Imprinting involves the inheritance of a silenced allele of a gene through either a paternal or maternal germline. We have approached the problem of imprinting using a model based on the dynamic attachment of chromatin loops to immobilized RNA polymerases and control elements. We have combined the information from different experimental approaches, examining primarily the IGF2-H19 locus, in an attempt to simplify the complexity of the imprinting data that has accumulated. It is hoped that a unified model may generate predictions amenable to experimental testing and contribute to the interpretation of future experiments.
To evaluate the pharmacokinetic (PK) properties of Bcl-2 antisense oligodeoxynucleotide G3139 when combined with the anthracycline anticancer drug doxorubicin (DOX) in a model of MDA435/LCC6 human breast cancer in severely compromised immunodeficient (SCID) mice.
An orthotopic model of MDA435/LCC6 solid breast tumors was developed by bilateral implantation of passaged cells in female SCID-RAG2 mice. The G3139 plasma profile was compared for two common routes of administration (i.v. or i.p.) in single and multiple dose treatment regimens of 5 mg/kg G3139 alone or with simultaneous DOX (5 mg/kg) administration. At selected times, plasma and major organs were assayed for [3H]G3139 using scintillation counting and DOX determined using HPLC. The molecular integrity of G3139 was analyzed using SDS-PAGE. The PKs of G3139 and DOX were evaluated using a two-compartment model.
G3139 administered i.v. at 5 mg/kg revealed a biexponential plasma concentration-time curve with a Cmax of 99.9 microg/ml and elimination half-lives of 0.03 h and 9.8 h, respectively, which resulted in an area under the concentration-time curve (AUC) of 15.9 microg x h/ml. G3139 administered i.p. showed a plasma absorption, distribution and elimination profile typical of this route of administration, characterized by half-lives of 0.03 h, 0.2 h and 8.9 h, respectively and a Cmax of 8.6 microg/ml. Based on AUC comparisons, the bioavailability of G3139 injected i.p. was 84% compared to i.v. administration. Subtle changes were observed in G3139 PKs after three prior i.p. doses of G3139. Specifically, a six-fold slower absorption rate, lower Cmax (6.9 microg/ml), increased Tmax (0.2 h), and an AUC of 17.4 microg x h/ml were observed, consistent with concentrations approaching saturation levels in tissue sites to which G3139 distributes. Coadministration of DOX had significant effects on the PK properties of G3139, manifested by an increased Cmax (11.2 microg/ml), higher AUC (19.7 microg x h/ml), and ninefold lower plasma clearance for single-dose G3139 administration. G3139 in plasma remained largely intact (< 17% degraded in plasma over 4 h), and increased plasma protein association occurred as a function of time. G3139 was detected in both healthy and tumor tissue after i.v. and i.p. administration. The highest tissue levels of G3139 were observed in the kidneys (40 microg/g), and low levels (< 2 microg/g) were detected in lung, heart and muscle. The rate of accumulation of G3139 in organs was dependent upon G3139 levels in plasma and the presence of coadministered DOX. Significant accumulation of G3139 was observed in solid tumors, with peak levels of approximately 5 microg G3139/g tumor, and approximately a two-to threefold tumor/muscle AUC ratio. The kinetics of G3139 accumulation in tumor tissue increased with increasing circulating G3139 concentration. The tissue distribution properties of DOX were also altered in the presence of coadministered G3139: in the presence of G3139, tumor exposure to DOX increased two-to threefold without alteration in plasma DOX PKs.
These findings indicate that drug-drug interactions between G3139 and DOX are modest and favorable in that elevated tumor DOX levels are achieved without compromising G3139 tumor uptake or significantly altering plasma drug concentrations.
Antisense oligonucleotides are short pieces of synthetic, chemically modified DNA or RNA that are designed to interact by Watson-Crick base pairing with mRNA encoding a targeted protein. During the past 20 years the technology associated with the development of antisense has improved dramatically, and emerging chemistries have made antisense oligonucleotides into powerful and versatile tools to study the function of proteins in living cells. The dramatic increase in novel genomic sequence information that has recently become available has generated enormous opportunities for the development of antisense oligonucleotides capable of altering the expression level of virtually any gene. With this will come a nearly equal opportunity to determine the role of individual proteins in a vast array of cardiovascular disease. The great specificity that these compounds exhibit in vitro suggests that they may also have an exciting future for development into therapeutics useful for the treatment of human disease. This review highlights some of the advances made in the field of antisense research, placing an emphasis on uses and proper controls.
Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.
From efforts to improve the biophysical properties of antisense oligonucleotides by incorporating backbone- or sugar-modified nucleoside analogs, 2'-O-methoxyethyl ribonucleosides 8b were identified as building blocks for a second generation of antisense oligonucleotides. Compounds
containing these modifications were demonstrated to combine the benefit of a high binding affinity to the RNA complement with a large increase in nuclease resistance, allowing the use of regular phosphodiester linkages. Chimeric oligonucleotides with 2'-O-methoxyethyl ribonucleosides, 8b,
in the wings and a central DNA-phosphorothioate window were shown to efficiently downregulate C- 'rat' kinase and PKC-a messenger-RNA in tumor cell lines resulting in a profound inhibition of cell proliferation. The same compounds were able to effectively reduce the growth of tumors in animal
models at low concentrations indicating the potential utility of these second generation antisense oligonucleotides for therapeutic applications.
The mechanism of hybrid-arrested translation by antisense oligodeoxynucleotides has been investigated with the rabbit reticulocyte lysate system. The oligonucleotides studied were directed against different regions of mouse alpha- or beta-globin mRNAs. Freshly prepared reticulocyte lysates were found to contain 1-2% of the level of RNase H in nucleated cells. This level of activity was sufficient to cleave nearly 100% of the targeted mRNA at the site of hybridization with a complementary oligodeoxynucleotide in 1 hr under conditions of active translation. Using poly(rA).oligo(dT) as a competitive inhibitor of the enzyme, hybrid arrest by oligodeoxynucleotides complementary to the sequence spanning the initiation codon or to a sequence in the coding region was found to be due entirely to cleavage of mRNA by RNase H. Hybridization of oligodeoxynucleotides adjacent to the cap site of beta-globin mRNA, but not the alpha-globin mRNA, also inhibited protein synthesis directly. Even in this case, however, cleavage of the mRNA by RNase H was the predominant pathway of inhibition.
We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.
We have used a previously described 17-mer phosphorothioate (Monia, B.P., Johnston, J.F., Ecker, D. J., Zounes, M.A., Lima, W.F., and Freier, S.M. (1992) J. Biol. Chem. 267, 19954-19962) for structure-function analysis of 2'-sugar modifications including 2'-O-methyl, 2'-O-propyl, 2'-O-pentyl, and 2'-fluoro. These modifications were analyzed for hybridization affinity to complementary RNA and for antisense activity against the Ha-ras oncogene in cells using a highly sensitive transactivation reporter gene system. Hybridization analysis demonstrated that all of the 2'-modified oligonucleotides hybridized with greater affinity to RNA than an unmodified 2'-deoxy oligonucleotide with the rank order of affinity being 2'-fluoro > 2'-O-methyl > 2'-O-propyl > 2'-O-pentyl > 2'-deoxy. Evaluation of antisense activities of uniformly 2'-modified oligonucleotides revealed that these compounds were completely ineffective in inhibiting Ha-ras gene expression. Activity was restored if the compound contained a stretch of at least five 2'-deoxy residues. This minimum deoxy length correlated perfectly with the minimum length required for efficient RNase H activation in vitro using partially purified mammalian RNase H enzyme. These chimeric 2'-modified/deoxy phosphorothioates displayed greater antisense potencies in inhibiting Ha-ras gene expression, compared with the unmodified uniform deoxy phosphorothioate. Furthermore, antisense potency correlated directly with affinity of a given 2' modification for it's complementary RNA. These results demonstrate the importance of target affinity in the action of antisense oligonucleotides and of RNase H as a mechanism by which these compounds exert their effects.
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.
The artificial control of gene expression by modified oligonucleotides (ONs) provides an exciting opportunity for the rational design of antisense ONs and, in particular, for the design of antiviral and antitumor ONs that can be used for therapeutic purposes.1 The effectiveness of antisense ONs in vitro and in vivo has been demonstrated by a first generation of phosphate-modified analogs such as phosphorothioates (PT)2 and methylphosphonates (MP).3 The initial choice of phosphate-modified ONs arose from their ease of synthesis and resistance to nuclease degradation. However, both the PT and MP modifications lead to the introduction of diastereoisomers, which results in a 2(n-1) mixture of diastereomers in the modified ONs. The presence of multiple diastereomers considerably weakens the capability of the ONs to hybridize with the target sequences. Duplexes formed between ON diesters and MP ONs of defined stereochemistry show an enhanced Tm relative to their racemic counterparts.1g Also, stereoselective automated synthesis for PT or MP is not available. Despite these stereochemical problems, both MP and PT ONs are clinical candidates at the present time.4 This is creating a need for cost-effective scale-up processes for producing MP and PT ONs. The demand is currently being met by a solid-support automated synthesis, which may not be suitable for producing drugs on a large-scale for human applications.2
The protected nucleoside analogs of 5-(1-propynyl)-2′-O-allyl-uridine and 5-(1-propynyl)-2′-O-allyl-cytidine are described. Oligonucleotides containing this modification significantly enhance double-helix formation with single-strand RNA.
A tridecamer oligodeoxynucleotide, d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to reiterated 3'- and 5'-terminal nucleotides of Rous sarcoma virus 35S RNA, is an efficient inhibitor of the translation of proteins specified by the viral RNA in the wheat embryo cell-free system. The inhibition specificity for oncornavirus RNA is greater than for rabbit reticulocyte mRNA or brome mosaic virus RNA. Other oligodeoxynucleotides of similar size have little or no specific effect on the RNA-directed translation. The tridecamer acts as a primer for the avian myeloblastosis virus DNA polymerase when Rous sarcoma virus heated 70S RNA is used as a template, offering evidence that it can hybridize to the RNA. The possible use of such an oligodeoxynucleotide hybridization competitor to inhibit Rous sarcoma virus replication is described in the preceding paper [Zamecnik, P. C. & Stephenson, M. L. (1978) Proc. Natl. Acad. Sci. USA. 75, 280--284].
Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.
To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.
The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2′-fluoro, 2′-O-methyl, 2′-O-propyl and 2′-O-pentyl nucleotides. Duplexes formed between 2′ modified oligoribonucleotides and RNA have typical A-form geometry as observed
by circular dichroism spectroscopy. Modifications, with the exception of 2′-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2′-fluoro:2′fluoro > 2′-O-propyl:2′-O-propyl > 2′-O-methyl:2′-O-methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2′-modified oligoribonucleotides was examined using snake venom phosphodiesterase
(SVPD) and nuclease S1. The stability imparted by 2′ modifications was observed to correlate with the size of the modification.
An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure,
but only for 2′ modified oligoribonucleotides and not for 2′-deoxy oligoribonucleotides.
Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.
A 20-mer phosphorothioate oligodeoxynucleotide designed to hybridize to the AUG translation initiation codon of mRNA encoding murine protein kinase C-alpha (PKC-alpha) inhibits the expression of PKC-alpha both in vitro and in vivo. In mouse C127 mammary epithelial cells, the reduction in PKC-alpha mRNA expression was both dose and time dependent. The oligodeoxynucleotide exhibited an IC50 value of 100-200 nM and reduced PKC-alpha mRNA expression for up to 48 hr. This reduction was specific for PKC-alpha versus other PKC isozymes (delta, epsilon, and zeta) and completely dependent upon oligodeoxynucleotide sequence. When administered intraperitoneally in mice, the same oligodeoxynucleotide caused a dose-dependent, oligodeoxynucleotide sequence-dependent reduction of PKC-alpha mRNA in liver, with an IC50 value of 30-50 mg/kg of body weight. Inhibition of expression was 64 +/- 11% after a single 50-mg/kg dose. The expression of PKC-delta, epsilon, and zeta mRNA was unaffected by this treatment. The oligodeoxynucleotide activity in vivo did not require the presence of cationic liposomes or any other delivery systems, although in vitro, the oligodeoxynucleotide required cationic liposomes for inhibition of PKC-alpha expression. This study demonstrates the utility of phosphorothioate oligodeoxynucleotides as specific inhibitors of gene expression in vivo after systemic administration.
"Uniformly" modified phosphodiester or phosphorothioate oligonucleotides incorporating 2'-deoxy-2'-fluoroadenosine, -guanosine, -uridine, and -cytidine, reported herein for the first time, when hybridized with RNA afforded consistent additive enhancement of duplex stability without compromising base-pair specificity. CD spectra of the 2'-deoxy-2'-fluoro-modified oligonucleotides hybridized with RNA indicated that the duplex adopts a fully A-form conformation. The 2'-deoxy-2'-fluoro-modified oligonucleotides in phosphodiester form were not resistant to nucleases; however, the modified phosphorothioate oligonucleotides were highly nuclease resistant and retained exceptional binding affinity to the RNA targets. The stabilizing effects of the 2'-deoxy-2'-fluoro modifications on RNA-DNA duplexes were shown to be superior to those of the 2'-O-methylribo substitutions. RNA hybrid duplexes with uniformly 2'-deoxy-2'-fluoro-modified oligonucleotides did not support HeLa RNase H activity; however, incorporation of the modifications into "chimeric" oligonucleotides has been shown to activate mammalian RNase H. "Uniformly" modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides afforded antisense molecules with (1) high binding affinity and selectivity for the RNA target and (2) stability toward nucleases.
The binding of peptide nucleic acids (PNAs) T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 to double-stranded DNA targets A10, A5GA4 and A2GA2GA4 was studied by nuclease S1 probing. It is found that the PNAs bind preferentially to their complementary targets, weaker to targets containing one mismatch and not to targets containing two mismatches. Using an RNA polymerase T3 in vitro transcription system, it is found that a PNA T10-LysNH2 bound downstream from the promoter causes transcription elongation arrest at the PNA binding site only when the PNA is bound to the template strand. Finally, it is shown that primer extension by Taq DNA polymerase on a single-stranded template is arrested at an occupied PNA T10 binding site. These results are discussed in relation to PNAs as potential anti-sense and anti-gene drugs.
Role of RNase H in hy¬ brid-arrested translation by antisense oligonucleotides Address reprint requests to: Nicholas M
- R Y Wälder
- J A Wälder
WÄLDER, R.Y., and WÄLDER, J.A. (1988).. Role of RNase H in hy¬ brid-arrested translation by antisense oligonucleotides. Proc. Nati. Acad. Sci. USA 85, 5011-5015. Address reprint requests to: Nicholas M. Dean ISIS Pharmaceuticals
Antisense oligonucleotide safety and efficacy for CMV re¬ tinitis in AIDS patients Se¬ quence dependent hydrolysis of RNA using modified oligonu¬ cleotide splints and RNase H
- S L Hutcherson
- A G Palestine
- H L Cantrill
- R M Lieberman
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- H Hayase
- Y Iwai
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HUTCHERSON, S.L., PALESTINE, A.G., CANTRILL, H.L., LIEBERMAN, R.M., HOLLAND, G.N., and ANDERSON, K.P. (1995). Antisense oligonucleotide safety and efficacy for CMV re¬ tinitis in AIDS patients. 35th ICAAC204. INOUE, H, HAYASE, Y., IWAI, S., and OHTSUKE, E. (1987). Se¬ quence dependent hydrolysis of RNA using modified oligonu¬ cleotide splints and RNase H. FEBS Lett. 215, 327-330.
Medicinal chemistry strategies for antisense re¬ search
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COOK, P.D. (1993). Medicinal chemistry strategies for antisense re¬ search. In: Antisense Research and Applications. S.T. Crooke and B.
Heterocyclic base modifications in nucleic acids and their applications in antisense oligonucleotides
- Y S Sanghvi
SANGHVI, Y.S. (1993). Heterocyclic base modifications in nucleic acids and their applications in antisense oligonucleotides. In: Anti-sense Research and Applications. S.T Crooke and B. Lebleu, eds. (CRC Press, Boca Raton, FL), pp. 273-288.