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Richard S. Geary - One of the best experts on this subject based on the ideXlab platform.
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treatment with volanesorsen a 2 o methoxyethyl modified antisense Oligonucleotide Targeting apoc3 mrna does not affect the qtc interval in healthy volunteers
Nucleic Acid Therapeutics, 2020Co-Authors: Lynnetta Watts, Richard S. Geary, Ewa Karwatowskaprokopczuk, Eunju Hurh, Veronica J Alexander, Kristin Balogh, Louis Odea, Sotirios TsimikasAbstract:The aim of this study was to assess the effect of volanesorsen on the corrected QT (QTc) interval. This thorough QT study enrolled 52 healthy male and female subjects who were randomized at a singl...
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effect of dose and plasma concentration on liver uptake and pharmacologic activity of a 2 methoxyethyl modified chimeric antisense Oligonucleotide Targeting pten
Biochemical Pharmacology, 2009Co-Authors: Richard S. Geary, Eric E Swayze, John Matson, Ed Wancewicz, Megan Pearce, Andrew M Siwkowski, Frank BennettAbstract:Abstract The role of dose and plasma concentration on liver tissue uptake and resulting antisense pharmacology using a chemically modified antisense Oligonucleotide (ASO) Targeting PTEN was assessed in mice. A single bolus s.c. dose of 60 mg/kg in mice showed a time-dependent reduction in liver PTEN mRNA that was maximal at 48–72 h and returned to near control levels by 20 days after administration. These pharmacodynamics are in good agreement with liver concentrations of ASO and are consistent with slow elimination (t1/2 = 8 days) of the PTEN ASO from Balb/C mouse liver. As expected, highest ASO concentrations in liver resulted from the s.c. slow infusion at all doses tested. Unexpectedly, the liver EC50 for the 24-h s.c. slow infusion was approximately twofold higher than the two bolus routes of administration. Based on plasma concentration analysis it appears that 1–2 μg/mL ASO plasma concentration is a threshold that, if exceeded, results in robust antisense effects and below which there is reduced or complete loss of antisense pharmacology in liver even though bulk uptake in the organ is improved. Co-administration of a nonsense ASO competed for liver uptake, but unexpectedly increased pharmacodynamic response for the active Oligonucleotide (ISIS 116847) supporting inhibition of a nonproductive bulk uptake pathway while simultaneously improving productive uptake (pharmacodynamics). This competition effect was similar whether the nonsense Oligonucleotide was co-administered with ASO or administered up to 24 h prior to active ASO injection.
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Lack of Pharmacokinetic Interaction of Mipomersen Sodium (ISIS 301012), a 2′-—O-Methoxyethyl Modified Antisense Oligonucleotide Targeting Apolipoprotein B-100 Messenger RNA, with Simvastatin and Ezetimibe
Clinical Pharmacokinetics, 2009Co-Authors: Rosie Z. Yu, Richard S. Geary, Jo Ann D. Flaim, Gina C. Riley, André A. Vanvliet, Diane L. Tribble, Mark K WedelAbstract:Background and objectives Mipomersen sodium (ISIS 301012) is a 20-mer phosphorothioate antisense Oligonucleotide that is complementary to human apolipoprotein B-100 (apoB-100) messenger RNA and subsequently reduces translation of ApoB-100 protein, the major apolipoprotein of very low-density lipoprotein, intermediate-density lipoprotein and low-density lipoprotein (LDL). Mipomersen sodium is currently being studied in phase II/III clinical studies to determine its clinical utility as add-on therapy to HMG-CoA reductase inhibitors or other lipid-lowering agents in subjects with hypercholesterolaemia. The aim of this study was to characterize the pharmacokinetic interactions of mipomersen sodium with simvastatin and ezetimibe. Another aim was to evaluate the ability of mipomersen sodium to inhibit major cytochrome P450 (CYP) isoenzymes in vitro . Methods In a phase I clinical study, ten healthy subjects per cohort received a single oral dose of simvastatin 40 mg or ezetimibe 10 mg followed by four 2-hour intravenous doses of mipomersen sodium 200 mg over an 8-day period, with simvastatin 40 mg or ezetimibe 10 mg being administered again with the last dose of mipomersen sodium. Mipomersen sodium pharmacokinetic profiles were assessed following the first dose (mipomersen sodium alone) and the last dose (mipomersen sodium in combination with simvastatin or ezetimibe). Plasma samples for measurement of simvastatin, simvastatin acid, and free and total ezetimibe concentrations were collected at various timepoints following their first and last oral dosing. A comparative pharmacokinetic analysis was performed to determine if there were any effects resulting from coadministration of mipomersen sodium with these lipid-lowering drugs. In addition to the clinical pharmacokinetic analysis, the ability of mipomersen sodium to inhibit the major CYP isoform enzymes (namely CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was evaluated in cryo-preserved human hepatocytes in vitro . Results The area under the plasma concentration-time curve (AUC) from 0 to 24 hours (AUC24), maximum plasma concentration and apparent elimination half-life values of mipomersen sodium were similar when administered alone and in combination with oral simvastatin or oral ezetimibe. The 90% confidence intervals of the geometric least squares means ratios (%Reference) of the mipomersen sodium AUC24 values were 93.6, 107 when administered together with simvastatin, and 92.4, 111 when administered with ezetimibe. Therefore, there were no large deviations outside the default no-effect boundaries (80–125%) for total exposure (the AUC) of mipomersen sodium in combination with either simvastatin or ezetimibe. Similarly, large deviations outside the default no-effect boundaries were not observed for simvastatin, simvastatin acid, or free and total ezetimibe exposure in combination with mipomersen sodium. In cryo-preserved human hepatocytes, mipomersen sodium exhibited no cytotoxicity. Significant cell uptake was demonstrated by analysing cell-associated concentrations of mipomersen sodium. All evaluated enzyme activities had
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lack of pharmacokinetic interaction of mipomersen sodium isis 301012 a 2 o methoxyethyl modified antisense Oligonucleotide Targeting apolipoprotein b 100 messenger rna with simvastatin and ezetimibe
Clinical Pharmacokinectics, 2009Co-Authors: Richard S. Geary, Jo Ann D. Flaim, Gina C. Riley, André A. Vanvliet, Diane Tribble, Mark K WedelAbstract:Background and objectives Mipomersen sodium (ISIS 301012) is a 20-mer phosphorothioate antisense Oligonucleotide that is complementary to human apolipoprotein B-100 (apoB-100) messenger RNA and subsequently reduces translation of ApoB-100 protein, the major apolipoprotein of very low-density lipoprotein, intermediate-density lipoprotein and low-density lipoprotein (LDL). Mipomersen sodium is currently being studied in phase II/III clinical studies to determine its clinical utility as add-on therapy to HMG-CoA reductase inhibitors or other lipid-lowering agents in subjects with hypercholesterolaemia. The aim of this study was to characterize the pharmacokinetic interactions of mipomersen sodium with simvastatin and ezetimibe. Another aim was to evaluate the ability of mipomersen sodium to inhibit major cytochrome P450 (CYP) isoenzymes in vitro.
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plasma protein binding of an antisense Oligonucleotide Targeting human icam 1 isis 2302
Oligonucleotides, 2006Co-Authors: Tanya A Watanabe, Richard S. Geary, Arthur A LevinAbstract:In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate Oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN Targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.
Arthur A Levin - One of the best experts on this subject based on the ideXlab platform.
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58 final results randomized double blind placebo controlled safety anti viral proof of concept study of miravirsen an Oligonucleotide Targeting mir 122 in treatment naive patients with genotype 1 chronic hcv infection
Journal of Hepatology, 2012Co-Authors: H W Reesink, H L A Janssen, S Zeuzem, E Lawitz, M Rodrigueztorres, Keyur Patel, A Chen, C Davis, B King, Arthur A LevinAbstract:58 FINAL RESULTS – RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED SAFETY, ANTI-VIRAL PROOF-OF-CONCEPT STUDY OF MIRAVIRSEN, AN Oligonucleotide Targeting MIR-122, IN TREATMENT-NAIVE PATIENTS WITH GENOTYPE 1 CHRONIC HCV INFECTION H.W. Reesink, H.L.A. Janssen, S. Zeuzem, E. Lawitz, M. Rodriguez-Torres, K. Patel, A. Chen, C. Davis, B. King, A. Levin, M.R. Hodges. Department of Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands; Department of Medicine, J.W. Goethe University Hospital, Frankfurt/Main, Germany; Alamo Medical Research, San Antonio, TX, Fundacion de Investigacion, San Juan, PR, Duke University, Durham, NC, Santaris Pharma, San Diego, CA, USA E-mail: h.w.reesink@amc.nl
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plasma protein binding of an antisense Oligonucleotide Targeting human icam 1 isis 2302
Oligonucleotides, 2006Co-Authors: Tanya A Watanabe, Richard S. Geary, Arthur A LevinAbstract:In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate Oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN Targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.
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pharmacokinetics and pharmacodynamics of an antisense phosphorothioate Oligonucleotide Targeting fas mrna in mice
Journal of Pharmacology and Experimental Therapeutics, 2001Co-Authors: Hong Zhang, Richard S. Geary, Mark J Graham, Lilit Masarjian, Kristina M Lemonidis, Rosanne M Crooke, Nicholas M Dean, Arthur A LevinAbstract:ISIS 22023 is a modified phosphorothioate antisense Oligonucleotide Targeting murine Fas mRNA. Treatment of mice with ISIS 22023 reduced Fas expression in liver in a concentration-dependent and sequence-specific manner, which completely protected mice from fulminant death induced by agonistic Fas antibody. In this study, we characterized the relationships in mice between total dose administered, dose to the target organ, and ultimately, the intracellular concentration within target cell types to the pharmacologic activity of ISIS 22023. After subcutaneous injection, ISIS 22023 distributed to the liver rapidly and remained in the liver with the t 1/2 ranging from 11 to 19 days, depending on dose. There were apparent differences in patterns of uptake and elimination in different types of liver cells. Oligonucleotide appeared within hepatocytes rapidly, whereas the peak concentrations in Kupffer cells were delayed until 2 days after dose administration. Hepatocytes cleared Oligonucleotide the most rapidly, whereas Kupffer cells appeared to retain Oligonucleotide longer. The reduction of Fas mRNA levels (pharmacodynamic response) paralleled the increase of Oligonucleotide concentration in mouse liver with maximum mRNA reduction of 90% at 2 days after a single 50 mg/kg subcutaneous administration. Moreover, the pharmacodynamics of ISIS 22023 correlated better with the pharmacokinetics in hepatocytes, supporting the concept that the presence of Oligonucleotide in target cells results in reductions in mRNA and, ultimately, pharmacologic activity. These results provide a comprehensive understanding of the kinetics of an antisense drug at the site of action and demonstrate that the reductions in mRNA induced by this antisense Oligonucleotide correlate with its concentrations in cell targets.
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comparison of pharmacokinetics and tissue disposition of an antisense phosphorothioate Oligonucleotide Targeting human ha ras mrna in mouse and monkey
Journal of Pharmaceutical Sciences, 2001Co-Authors: Richard S. Geary, Janet M Leeds, Tanya Watanabe, Max N Moore, J R Fitchett, John Matson, Todd Burckin, Michael V Templin, Arthur A LevinAbstract:Abstract The plasma pharmacokinetics and tissue disposition of ISIS 2503 were studied in mice following single and multiple bolus intravenous (iv) injections of 1–50 mg/kg, and in monkeys following single and multiple 2‐h iv infusions of 1–10 mg/kg and bolus iv injections of 1 mg/kg of ISIS 2503. ISIS 2503 and its metabolites were measured in plasma, urine, and tissues using solid‐phase extraction followed by capillary gel electrophoresis (CGE). In both species, the plasma clearance of ISIS 2503 was characterized by rapid distribution to tissues, and to a lesser extent, metabolism. The plasma clearance in mice was at least two‐fold more rapid than in monkeys at equivalent doses. The plasma disposition ( t 1/2 ) increased with dose. The highest concentrations of Oligonucleotide were consistently observed in the kidney and liver in both species. At equivalent doses, tissue concentrations in monkeys were much higher than tissue concentrations in mice. Urinary excretion of total Oligonucleotide was a minor elimination pathway in both species at doses
Nigel J Gooderham - One of the best experts on this subject based on the ideXlab platform.
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a triple helix forming Oligonucleotide Targeting genomic dna fails to induce mutation
Mutagenesis, 2012Co-Authors: Reshat Reshat, Catherine C Priestley, Nigel J GooderhamAbstract:Purine tracts in duplex DNA can bind Oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming Oligonucleotides (TFOs) Targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for Oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO Targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in Oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.
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abstract 5413 is a non conjugated triple helix forming Oligonucleotide Targeting the genomic hprt locus capable of sequence specific mutagenesis
Cancer Research, 2011Co-Authors: Reshat Reshat, Catherine C Priestley, Mick D Fellows, Michael R Odonovan, Nigel J GooderhamAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oligonucleotide strands have been shown to be capable of binding to duplex DNA in a sequence specific manner to form a triple-helix or triplex structure. Such phenomenons have been shown to induce mutation, as demonstrated using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). These studies have engendered concern from the European Medicines Agency (EMEA) in regard to biotechnology derived pharmaceuticals such that antisense Oligonucleotides may also form triplex structures in a sequence specific manner at genomic DNA with potentially mutagenic effects. In this study, we have examined the mutagenic potential of a non-conjugated triplex forming Oligonucleotide (TFO27) Targeting the genomic hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mutagenicity of TFO27 was assessed using the human lymphoblastoid TK6 cell line through 6-thioguanine resistance (6TGr). Electrophoretic mobility shift assays were used to demonstrate triplex formation by TFO27 at the target motif at sub-nanomolar concentrations. As expected, a control Oligonucleotide, SCR27, failed to form a triplex at the target motif. Subsequently, a range of transfection facilitators were evaluated for optimal delivery of a fluorescently labelled Oligonucleotide probe. In preliminary experiments treating 3×106 cells, TFO27 appeared to produce a dose dependent increase in 6TGr mutants. Furthermore, TFO27 failed to induce mutation at the non-targeted thymidine kinase (TK) locus suggesting locus specificity for its mode of action. Moreover, SCR27 failed to induce mutation at both loci. However, TFO27 failed to induce mutation at the HPRT locus in experiments treating 10×106 cells. Increasing the Oligonucleotide concentration had no effect on the number of 6TGr recovered. These contradictory findings are difficult to explain, but emphasise the importance of using appropriate target cell numbers in mutation assays. Overall, our data would suggest that triplex formation could occur in cells but chromatin structure, intranuclear pH, ion concentrations and nucleases are all potential barriers that probably influence the dynamics of triplex formation and stability. In view of these factors, we suggest that studies of the mutagenic potential of triplex formation are better performed at genomic loci rather than extra-chromosomal test systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2011-5413
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abstract 6 a triple helix forming Oligonucleotide Targeting a genomic locus is capable of sequence specific mutagenesis in human lymphoblastoid tk6 cells
Cancer Research, 2010Co-Authors: Reshat Reshat, Catherine C Priestley, Nigel J GooderhamAbstract:Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC It has been reported that DNA triplex formation induces mutagenesis as determined using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). Triplex mediated mutagenesis has been shown to include point mutations, deletions, small insertions and homologous recombination. To the best of our knowledge, no study has successfully examined the mutagenic potential of a non-conjugated triplex-forming Oligonucleotide (TFO) Targeting a genomic sequence. In this study, we have designed a TFO that targets the hemizygous hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, in the human lymphoblastoid TK6 cell line, and assessed mutagenicity through resistance to 6-thioguanine. Our TFO, TFO27, has been designed to form a triplex with a purine tract in exon 3 of the HPRT gene. Triplex formation at the target motif was shown to occur at nanomolar concentrations, confirmed by Electrophoretic Mobility Shift Assays. A scrambled Oligonucleotide, SCR27, failed to form a triplex at the target motif. A range of transfection reagents were evaluated for facilitated delivery of TFO27, and resulted in variable cellular toxicity. Transfection of high concentrations of Oligonucleotide, with acceptable levels of cytotoxicity, resulted in HPRT mutation with TFO27 but not SCR27. Similar experiments failed to result in mutation at the non-targeted thymidine kinase (TK) locus, suggesting locus specificity for the mode of action of TFO27. The target specificity and sequence context of these mutagenic events is being determined to establish the mechanism of mutation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 6.
H W Reesink - One of the best experts on this subject based on the ideXlab platform.
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58 final results randomized double blind placebo controlled safety anti viral proof of concept study of miravirsen an Oligonucleotide Targeting mir 122 in treatment naive patients with genotype 1 chronic hcv infection
Journal of Hepatology, 2012Co-Authors: H W Reesink, H L A Janssen, S Zeuzem, E Lawitz, M Rodrigueztorres, Keyur Patel, A Chen, C Davis, B King, Arthur A LevinAbstract:58 FINAL RESULTS – RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED SAFETY, ANTI-VIRAL PROOF-OF-CONCEPT STUDY OF MIRAVIRSEN, AN Oligonucleotide Targeting MIR-122, IN TREATMENT-NAIVE PATIENTS WITH GENOTYPE 1 CHRONIC HCV INFECTION H.W. Reesink, H.L.A. Janssen, S. Zeuzem, E. Lawitz, M. Rodriguez-Torres, K. Patel, A. Chen, C. Davis, B. King, A. Levin, M.R. Hodges. Department of Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands; Department of Medicine, J.W. Goethe University Hospital, Frankfurt/Main, Germany; Alamo Medical Research, San Antonio, TX, Fundacion de Investigacion, San Juan, PR, Duke University, Durham, NC, Santaris Pharma, San Diego, CA, USA E-mail: h.w.reesink@amc.nl
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58 FINAL RESULTS – RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED SAFETY, ANTI-VIRAL PROOF-OF-CONCEPT STUDY OF MIRAVIRSEN, AN Oligonucleotide Targeting MIR-122, IN TREATMENT-NAIVE PATIENTS WITH GENOTYPE 1 CHRONIC HCV INFECTION
Journal of Hepatology, 2012Co-Authors: H W Reesink, H L A Janssen, S Zeuzem, E Lawitz, Keyur Patel, A Chen, C Davis, B King, Maribel Rodriguez-torres, A. LevinAbstract:58 FINAL RESULTS – RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED SAFETY, ANTI-VIRAL PROOF-OF-CONCEPT STUDY OF MIRAVIRSEN, AN Oligonucleotide Targeting MIR-122, IN TREATMENT-NAIVE PATIENTS WITH GENOTYPE 1 CHRONIC HCV INFECTION H.W. Reesink, H.L.A. Janssen, S. Zeuzem, E. Lawitz, M. Rodriguez-Torres, K. Patel, A. Chen, C. Davis, B. King, A. Levin, M.R. Hodges. Department of Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands; Department of Medicine, J.W. Goethe University Hospital, Frankfurt/Main, Germany; Alamo Medical Research, San Antonio, TX, Fundacion de Investigacion, San Juan, PR, Duke University, Durham, NC, Santaris Pharma, San Diego, CA, USA E-mail: h.w.reesink@amc.nl
Reshat Reshat - One of the best experts on this subject based on the ideXlab platform.
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a triple helix forming Oligonucleotide Targeting genomic dna fails to induce mutation
Mutagenesis, 2012Co-Authors: Reshat Reshat, Catherine C Priestley, Nigel J GooderhamAbstract:Purine tracts in duplex DNA can bind Oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming Oligonucleotides (TFOs) Targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for Oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO Targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in Oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.
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abstract 5413 is a non conjugated triple helix forming Oligonucleotide Targeting the genomic hprt locus capable of sequence specific mutagenesis
Cancer Research, 2011Co-Authors: Reshat Reshat, Catherine C Priestley, Mick D Fellows, Michael R Odonovan, Nigel J GooderhamAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oligonucleotide strands have been shown to be capable of binding to duplex DNA in a sequence specific manner to form a triple-helix or triplex structure. Such phenomenons have been shown to induce mutation, as demonstrated using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). These studies have engendered concern from the European Medicines Agency (EMEA) in regard to biotechnology derived pharmaceuticals such that antisense Oligonucleotides may also form triplex structures in a sequence specific manner at genomic DNA with potentially mutagenic effects. In this study, we have examined the mutagenic potential of a non-conjugated triplex forming Oligonucleotide (TFO27) Targeting the genomic hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mutagenicity of TFO27 was assessed using the human lymphoblastoid TK6 cell line through 6-thioguanine resistance (6TGr). Electrophoretic mobility shift assays were used to demonstrate triplex formation by TFO27 at the target motif at sub-nanomolar concentrations. As expected, a control Oligonucleotide, SCR27, failed to form a triplex at the target motif. Subsequently, a range of transfection facilitators were evaluated for optimal delivery of a fluorescently labelled Oligonucleotide probe. In preliminary experiments treating 3×106 cells, TFO27 appeared to produce a dose dependent increase in 6TGr mutants. Furthermore, TFO27 failed to induce mutation at the non-targeted thymidine kinase (TK) locus suggesting locus specificity for its mode of action. Moreover, SCR27 failed to induce mutation at both loci. However, TFO27 failed to induce mutation at the HPRT locus in experiments treating 10×106 cells. Increasing the Oligonucleotide concentration had no effect on the number of 6TGr recovered. These contradictory findings are difficult to explain, but emphasise the importance of using appropriate target cell numbers in mutation assays. Overall, our data would suggest that triplex formation could occur in cells but chromatin structure, intranuclear pH, ion concentrations and nucleases are all potential barriers that probably influence the dynamics of triplex formation and stability. In view of these factors, we suggest that studies of the mutagenic potential of triplex formation are better performed at genomic loci rather than extra-chromosomal test systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2011-5413
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abstract 6 a triple helix forming Oligonucleotide Targeting a genomic locus is capable of sequence specific mutagenesis in human lymphoblastoid tk6 cells
Cancer Research, 2010Co-Authors: Reshat Reshat, Catherine C Priestley, Nigel J GooderhamAbstract:Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC It has been reported that DNA triplex formation induces mutagenesis as determined using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). Triplex mediated mutagenesis has been shown to include point mutations, deletions, small insertions and homologous recombination. To the best of our knowledge, no study has successfully examined the mutagenic potential of a non-conjugated triplex-forming Oligonucleotide (TFO) Targeting a genomic sequence. In this study, we have designed a TFO that targets the hemizygous hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, in the human lymphoblastoid TK6 cell line, and assessed mutagenicity through resistance to 6-thioguanine. Our TFO, TFO27, has been designed to form a triplex with a purine tract in exon 3 of the HPRT gene. Triplex formation at the target motif was shown to occur at nanomolar concentrations, confirmed by Electrophoretic Mobility Shift Assays. A scrambled Oligonucleotide, SCR27, failed to form a triplex at the target motif. A range of transfection reagents were evaluated for facilitated delivery of TFO27, and resulted in variable cellular toxicity. Transfection of high concentrations of Oligonucleotide, with acceptable levels of cytotoxicity, resulted in HPRT mutation with TFO27 but not SCR27. Similar experiments failed to result in mutation at the non-targeted thymidine kinase (TK) locus, suggesting locus specificity for the mode of action of TFO27. The target specificity and sequence context of these mutagenic events is being determined to establish the mechanism of mutation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 6.
