OSWG Recommendations for Genotoxicity Testing of Novel Oligonucleotide-Based Therapeutics

Berman, Cindy L.; Barros, Scott; Galloway, Sheila M.; Kasper, P.; Oleson, Frederick B.; Priestley, Catherine; Sweder, Kevin; Schlosser, Michael; Sobol, Zhanna

doi:10.1089/nat.2015.0534

Cited by 40 publications

(35 citation statements)

References 49 publications

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“…As described above, multiple cellular and physiological challenges further limit the likelihood of triplex formation by antisense oligonucleotides. In fact, a recent publication by the subcommittee of the OSWG on genotoxicity testing of therapeutic oligonucleotides, issued recommendations on genotoxicity testing of clinical candidate oligonucleotides where triplex formation and resultant mutagenesis are considered highly unlikely (Berman et al, 2016). Taken together with our data reported here using a G-rich TFO optimally designed to bind to a triplex target site, further supports the statements in the OSWG report on the low likelihood of off-target triplex-induced mutagenesis by antisense oligonucleotides.…”

Section: Page 16 Of 35 Toxicological Sciencessupporting

confidence: 87%

“…However, the potential mutagenic risk from genomic interaction of general antisense oligonucleotides is unlikely as these oligonucleotides tend not to have optimal homology or long stretches of purine/pyrimidine to support triplex formation. This was recently supported by a publication by the Oligonucleotide Safety Working Group (OSWG) on the genotoxicity testing of therapeutic oligonucleotides in the clinic (Berman et al, 2016).…”

Section: Page 2 Of 35 Toxicological Sciencesmentioning

confidence: 96%

“…The first step involves sequence analysis of the oligonucleotide for the presence of continuous purine nucleotides. It is unlikely that an antisense oligonucleotide with a long purine stretch especially guanines (>10 nucleotides) would be considered for development (Berman et al, 2016). However if it is necessary to consider such a sequence, it will be important to employ a bioinformatics search engine to screen for potential triplex susceptible regions in the human genome sequence with uninterrupted oligopyrimidine-oligopurine tracts (typically 10-30 nucleotide long) as potential off-target triplex forming sequences.…”

Section: Page 17 Of 35 Toxicological Sciencesmentioning

confidence: 99%

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The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions

et al. 2016

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Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo. Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analysed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures.Subsequent assessment of mutant frequency under physiological conditions, by pretransfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.

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Section: Page 16 Of 35 Toxicological Sciencessupporting

confidence: 87%

Section: Page 2 Of 35 Toxicological Sciencesmentioning

confidence: 96%

Section: Page 17 Of 35 Toxicological Sciencesmentioning

confidence: 99%

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The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions

et al. 2016

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“…Finally, triplex formation efficiency was described to be less than optimal under physiological conditions. Thus, the likelihood of triplex formation in vivo has been deemed to be of low concern and will not be in the focus of this publication (Berman et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…The relevance of genetic toxicity testing of ONs was discussed among other topics. Recently, the group published a compiled set of available genotoxicity data performed with different types of ONs and discussed theoretical modes of action that may cause DNA damage (Berman et al, ). Importantly, all of the assay results for the most represented chemistries have been negative.…”

Section: Introductionmentioning

confidence: 99%

Locked nucleic acid (LNA): Based single‐stranded oligonucleotides are not genotoxic

Guérard

Zeller

Koller

et al. 2017

Environ and Mol Mutagen

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Over the last decade, single stranded oligonucleotides (ON) have gained increased attention as a new drug modality. Because the assessment of genotoxicity risk during early development of pharmaceuticals is essential, we evaluated the potential of locked nucleic acids (LNA)-ONs to induce DNA damage in L5178Y tk cells both with the mouse lymphoma assay (MLA) and the micronucleus test (MNT). Further, the MLA was performed to assess gene and chromosome mutation over 3 and 24h (± metabolic activation). In addition, the MNT was performed to assess, in addition, a potential aneugenic liability. None of the experiments demonstrated a genotoxic effect for the five tested LNA-ONs. We further show data from four proprietary LNA-ONs tested in standard genotoxicity assays in vitro and partially in vivo, which were all negative. In addition, cellular and nuclear uptake of LNA-ONs in L5178Y tk cells was demonstrated. Based on the results presented here as well as in the literature about other representatives of this class, we consider LNA-ONs as generally not DNA reactive and question whether genotoxicity testing of this class of ONs should be required. This is in line with recent recommendation made by the OSWG that extensively assessed the genotoxicity of oligonucleotides. Environ. Mol. Mutagen. 58:112-121, 2017. © 2017 Wiley Periodicals, Inc.

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Occupational Toxicology in the Pharmaceutical Industry

Dolan

Bercu

Graham³

et al. 2023

Patty's Toxicology

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Much of the activity in the pharmaceutical industry has its origins in the fine chemicals industry of the late nineteenth century. At that time, advances in chemistry and medicine led to the manufacture of pharmacologically active small molecules to produce desirable biological effects to treat diseases and illnesses at relatively low doses. Advances in biology and chemistry in the past quarter century have led to an increase in innovation resulting in a variety of novel modalities, especially recombinant proteins, antibody‐drug conjugates, cell and gene therapies, peptides and polypeptides, oligonucleotides, and vaccines. The roles and responsibilities of occupational toxicologists in the pharmaceutical industry have expanded dramatically in the twenty‐first century, commensurate with the changing regulatory requirements and treatment modalities. The occupational toxicologists' roles have become increasingly multidisciplinary and the scope of their involvement has expanded to encompass diverse aspects of worker safety as well as patient safety, and to include involvement throughout a molecule's development, from early development through postcommercialization.

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OSWG Recommendations for Genotoxicity Testing of Novel Oligonucleotide-Based Therapeutics

Cited by 40 publications

References 49 publications

The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions

The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions

Locked nucleic acid (LNA): Based single‐stranded oligonucleotides are not genotoxic

Occupational Toxicology in the Pharmaceutical Industry

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