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Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the cHS4 chromatin insulator.

ABSTRACT: Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by evidence of vector-mediated genotoxicity involving the activation of cellular oncogenes flanking sites of vector integration. We report here that the rate of gammaretroviral vector genotoxicity can be significantly reduced by addition of the cHS4 chromatin insulator, based on two complementary approaches for assessing vector-mediated genotoxicity. One approach involves the direct, genomewide assessment of cellular gene dysregulation using panels of transduced cell clones and genomic microarrays, whereas the other involves the functional assessment of malignant transformation using a factor-dependent cell line. Both assays are robust and quantitative, and indicate the cHS4 chromatin insulator can reduce vector-mediated genotoxicity approximately sixfold (ranged three to eight fold). These approaches also provide a means for assessing various aspects of vector-mediated genotoxicity, including the overall rate of cellular gene dysregulation, the potential influence of vector provirus over large genomic distances, and the involvement of oncogenic pathways in vector-mediated malignant transformation.

SUBMITTER: Li CL

PROVIDER: S-EPMC2835102 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the cHS4 chromatin insulator.

Li Chang Long CL Xiong Ding D Stamatoyannopoulos George G Emery David W DW

Molecular therapy : the journal of the American Society of Gene Therapy 20090224 4

Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by evidence of vector-mediated genotoxicity involving the activation of cellular oncogenes flanking sites of vector integration. We report here that the rate of gammaretroviral vector genotoxicity can be significantly reduced by addition of the cHS4 chromatin insulator, based on two complementary approaches for assessing vector-mediated genotoxicity. One approach involves the direct, genomewide asse ...[more]

PMID: 19240697

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Project description:Genotoxicity is a critical component of a comprehensive toxicological profile. The Tox21 Program used five quantitative high-throughput screening (qHTS) assays measuring some aspect of DNA damage/repair to provide information on the genotoxic potential of over 10?000 compounds. Included were assays detecting activation of p53, increases in the DNA repair protein ATAD5, phosphorylation of H2AX, and enhanced cytotoxicity in DT40 cells deficient in DNA-repair proteins REV3 or KU70/RAD54. Each assay measures a distinct component of the DNA damage response signaling network; >70% of active compounds were detected in only one of the five assays. When qHTS results were compared with results from three standard genotoxicity assays (bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus), a maximum of 40% of known, direct-acting genotoxicants were active in one or more of the qHTS genotoxicity assays, indicating low sensitivity. This suggests that these qHTS assays cannot in their current form be used to replace traditional genotoxicity assays. However, despite the low sensitivity, ranking chemicals by potency of response in the qHTS assays revealed an enrichment for genotoxicants up to 12-fold compared with random selection, when allowing a 1% false positive rate. This finding indicates these qHTS assays can be used to prioritize chemicals for further investigation, allowing resources to focus on compounds most likely to induce genotoxic effects. To refine this prioritization process, models for predicting the genotoxicity potential of chemicals that were active in Tox21 genotoxicity assays were constructed using all Tox21 assay data, yielding a prediction accuracy up to 0.83. Data from qHTS assays related to stress-response pathway signaling (including genotoxicity) were the most informative for model construction. By using the results from qHTS genotoxicity assays, predictions from models based on qHTS data, and predictions from commercial bacterial mutagenicity QSAR models, we prioritized Tox21 chemicals for genotoxicity characterization.

Dataset Information

Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the cHS4 chromatin insulator.

Publications

Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the cHS4 chromatin insulator.

Similar Datasets

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