The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Richard M Walmsley - One of the best experts on this subject based on the ideXlab platform.
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The ‘BlueScreen HC’ Assay as a decision making test in the Genotoxicity assessment of flavour and fragrance materials
Toxicology in Vitro, 2015Co-Authors: Sylvain Etter, Nicholas Billinton, Richard M Walmsley, Louise Birrell, P. A. Cahill, Heather Scott, Benjamin SmithAbstract:Abstract The Genotoxicity of a library of 70 flavour and fragrance substances having a high proportion of in vivo and/or carcinogenicity test data has been assessed using the GADD45a-GLuc ‘BlueScreen HC’ Genotoxicity Assay, with and without exogenous metabolic activation. There are only limited Genotoxicity and carcinogenicity study data for compounds in this applicability domain, but this study allowed the following conclusions: (i) The BlueScreen HC results are highly predictive of positive results from regulator-required in vitro Genotoxicity Assays for the test set of materials; the moderate negative predictivity of BlueScreen HC from the in vitro test set of material is mainly due to the high rate of false positive in regulatory in vitro mammalian tests. (ii) BlueScreen HC negative results are predictive of negative in vivo results and provide a specific prediction of in vivo Genotoxicity Assay results. (iii) In this applicability domain, which comprises a large proportion of relatively low molecular weight molecules, a 1 mM testing limit maintains the sensitivity of the Assay, and increases specificity. (iv) The predictive capacity and specificity to in vivo genotoxins and carcinogens, coupled to a microplate format with low compound requirement supports further investigation of the BlueScreen HC Assay as a useful tool in prioritizing the assessment of new F&F materials and in filling data gaps on materials with no or limited regulatory test data for Genotoxicity.
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analysis of 75 marketed pharmaceuticals using the gadd45a gfp greenscreen hc Genotoxicity Assay
Mutagenesis, 2009Co-Authors: Paul W Hastwell, Thomas W Webster, Matthew Tate, Nicholas Billinton, Anthony M Lynch, James Harvey, Robert W Rees, Richard M WalmsleyAbstract:: The GADD45a-GFP (GreenScreen HC) reporter Assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens. In this study, a collection of 75 marketed pharmaceuticals were tested in the Assay. Compounds in the collection represent a broad range of chemical structures, pharmacologies and therapeutic indications, including neoplasia and viral infection where positive Genotoxicity results are often associated with the pharmacological activity. Based on the results of this study, two main conclusions can be drawn: (i) the GreenScreen HC is more predictive of in vivo Genotoxicity (88%) and genotoxic carcinogenicity (93%) data than the any of the other regulatory in vitro Genotoxicity Assay and (ii) no compounds were uniquely positive in the GADD45a-GFP Assay. This analysis therefore provides additional evidence to support the use of the GADD45a-GFP Assay as an effective tool either in early genotoxic liability identification or non-clinical safety assessment of candidate pharmaceuticals during development.
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development and validation of a higher throughput screening approach to Genotoxicity testing using the gadd45a gfp greenscreen hc Assay
Journal of Biomolecular Screening, 2009Co-Authors: Andrew W. Knight, Louise Birrell, Richard M WalmsleyAbstract:There is a pressing need to develop rapid yet accurate screening Assays for the identification of genotoxic liability and for early hazard assessment in drug discovery. The GADD45a-GFP human cell-based Genotoxicity Assay (GreenScreen HC) has been reformatted to test 12 compounds per 96-well microplate in a higher throughput, automated screening mode and the protocol applied to the analysis of 1266 diverse, pharmacologically active compounds. Testing from a fixed starting concentration of 100 µM and over 3 serial dilutions, the hit rates for Genotoxicity (7.3%) and cytotoxicity (33%) endpoints of the Assay have been determined in a much wider chemical space than previously reported. The degree of interference from color, autofluorescence, and low solubility has also been assessed. The Assay results have been compared to an in silico approach to Genotoxicity assessment using Derek for Windows software. Where carcinogenicity data were available, GreenScreen HC demonstrated a higher specificity than in silico...
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high specificity and high sensitivity Genotoxicity assessment in a human cell line validation of the greenscreen hc gadd45a gfp Genotoxicity Assay
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2006Co-Authors: Paul W Hastwell, Thomas W Webster, James Harvey, Robert W Rees, Li Leng Chai, Kevin J Roberts, Richard M WalmsleyAbstract:The battery of genetic toxicity tests required by most regulatory authorities includes both bacterial and mammalian cell Assays and identifies practically all genotoxic carcinogens. However, the relatively high specificity of the Salmonella mutagenicity Assay (Ames test) is offset by the low specificity of the established mammalian cell Assays, which leads to difficulties in the interpretation of the biological relevance of results. This paper describes a new high-throughput Assay that links the regulation of the human GADD45a gene to the production of Green Fluorescent Protein (GFP). A study of 75 well-characterised genotoxic and non-genotoxic compounds with diverse mechanisms of DNA-damage induction (including aneugens) reveals that the Assay responds positively to all classes of genotoxic damage with both high specificity and high sensitivity. The current micro-well Assay format does not include metabolic activation, but a separate low-throughput protocol demonstrates a successful proof-of-principle for an S9 metabolic activation Assay with the model pro-mutagen cyclophosphamide. The test should be of value both as a tool in the selection of candidate compounds for further development, where additional data may be required because of conflicting information from the in vitro test battery, or in product development areas where the use of animals is to be discontinued. As a microplate Assay however, it has the qualities of high throughput and low compound use that will facilitate its application in early screening for genotoxic liability.
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Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP Genotoxicity Assay
Mutagenesis, 2005Co-Authors: L Walsh, Paul W Hastwell, Nicholas Billinton, P. O. Keenan, Andrew W. Knight, Richard M WalmsleyAbstract:The yeast (Saccharomyces cerevisiae) RAD54-GFP DNArepair reporter Assay (GreenScreen Assay, GSA) can beused for early Genotoxicity screening in drug discovery.During the initial validation of this preregulatory Assay,a subset of known genotoxic compounds that did not givereproducibly clear positive GSA results was identified. Cellpermeability, inherent drug resistance mechanisms, meta-bolic activation and compound solubility were identified aspossible barriers to the detection of specific compounds. Inthis study three types of modification to the existing Assayprotocol were explored in order to address these possibil-ities: (i) modification of the reporter host strain by deletionof genes involved in cell wall integrity or with productsfunctioning as efflux pumps (PDR5, ERG6, SNQ2, YOR1);(ii) expression in the host yeast of human phase I metabolicactivation genes and (iii) variation in the test solvent systemfor compounds with poor aqueous solubility. The modifica-tions described and the Assay results presented show howthe Assay may be tailored to suit specific classes of testcompound in a more analytical mode. Improvements inAssay sensitivity were seen in the detection of some geno-toxins using yeast cell wall mutants and those expressinghuman cytochrome P450 genes.IntroductionThe authors have previously reported the construction andassessment of a yeast-based Genotoxicity Assay system (Green-Screen Assay or GSA) utilizing a DNA damage repair reporter(1). In the reporter strain, there is a replicative plasmid con-taining the promoter of the DNA damage inducible yeastRAD54 gene fused to a gene encoding green fluorescent protein(GFP; 2–5). The principle behind the GSA is that induction ofthe RAD54 promoter, owing to DNA damage, results in theproduction of the extremely stable GFP by the yeast cells,which is readily detected by its fluorescence when illuminatedwith blue light. Following overnight exposure of the yeastculture to a test substance, both fluorescence and light absorb-ance measurements are recorded. The level of GFP fluores-cence induction gives a measure of the Genotoxicity of thesubstance. The extent of growth inhibition resulting fromexposure to the test substance (relative total growth, RTG), isalso determined by comparing the extent of proliferation oftreated cells with that of untreated cells. This provides a meas-ure of cytotoxicity. The Assay is performed on a series ofdilutions of the compound, allowing calculation of the lowesteffective concentration (LEC) for each toxicity measurement.RAD54 encodes a structural element of the homologousrecombinational repair pathway and the Rad54 protein exhibitsa double-stranded DNA-dependent ATPase activity that facil-itates recombinational repair, mediated by the homologouspairing and DNA strand exchange protein Rad51 (6–9). Theentire genome is the target for DNA damage whereas the wholecell is the target for toxicity. This contrasts with reverse muta-tion Assays which detect DNA damage at a genetic locus, suchas the Salmonella HIS operon in the Ames test (10).The GSA has been developed for use in 96-well microplateformat. The full regulatory battery of Genotoxicity tests (11)comprising in vitro and in vivo tests for the evaluation ofmutations and chromosomal damage are very low throughput,time consuming and compound hungry. As a consequence, thetesting is not done early in discovery; it would be too expensivefor large numbers of compounds. The speed, high throughputand low compound requirement of GSA made it an attractivescreening tool earlier in drug discovery. To assess its value, apreliminary screening validation study of the Assay was carriedout using a panel of 102 compounds (1). This study demon-strated that the GSA detects a different but overlapping spec-trum of compounds to bacterial Genotoxicity Assays. It wasconcluded that the combination of GSA with an in silicostructure–activity relationship (SAR) screen and possibly ahigh throughput bacterial screen, would provide an effectivepreview of the regulatory battery of Genotoxicity tests andthus make a valuable contribution to the early selection ofcompounds going forward in drug discovery.The original validation study (1) set out to discover the rangeof compounds detected as genotoxic with a single definitiveprotocol. This was an important criterion, as screening testsper se cease to be screening tests if they have to be run indifferent, compound dependent formats. All compounds weretested in a phosphate-buffered medium containing 1% DMSO.The Assays were carried out in the absence of exogenousmetabolic activation (i.e. without the addition of rat liver homo-genate S9 fraction). Under these set conditions, the screeningvalidation study demonstrated that the Assay was sensitive to abroad spectrum of mutagens and notably the clastogens. Asexpected, a number of typical eukaryotic drug targets wereidentified including topoisomerases (etoposide), the mitoticspindle apparatus (colchicine) and DNA polymerases (aphidi-colin). Furthermore, it showed that the metabolic capacity ofyeast cells is sufficient to identify several compounds thatrequire metabolic activation by rat liver S9 in bacterial geno-toxicity Assays (e.g. neutral red, 2-amino-4-nitrophenol, pro-flavin hemisulfate). The existence of more complex metabolicactivation in yeast was suggested by the detection of cimetidine[that most likely acts through conversion to nitrosocimetidine
George Stamatoyannopoulos - One of the best experts on this subject based on the ideXlab platform.
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3 safety assessment of sin lvs harboring chromatin insulators in the sensitive cdkn2a in vivo Genotoxicity Assay show enhancer blocking activity of specific insulator sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Odile Cohenhaguenauer, Luigi Naldini, Jack Lenz, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.
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3. Safety Assessment of SIN LVs Harboring Chromatin Insulators in the Sensitive Cdkn2a-/- In Vivo Genotoxicity Assay Show Enhancer-Blocking Activity of Specific Insulator Sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Luigi Naldini, Jack Lenz, Odile Cohen-haguenauer, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.
Daniela Cesana - One of the best experts on this subject based on the ideXlab platform.
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3. Safety Assessment of SIN LVs Harboring Chromatin Insulators in the Sensitive Cdkn2a-/- In Vivo Genotoxicity Assay Show Enhancer-Blocking Activity of Specific Insulator Sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Luigi Naldini, Jack Lenz, Odile Cohen-haguenauer, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.
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3 safety assessment of sin lvs harboring chromatin insulators in the sensitive cdkn2a in vivo Genotoxicity Assay show enhancer blocking activity of specific insulator sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Odile Cohenhaguenauer, Luigi Naldini, Jack Lenz, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.
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Genotoxicity Assay for gene therapy vectors in tumor prone Cdkn2a⁻/⁻ mice.
Methods in Enzymology, 2012Co-Authors: Eugenio Montini, Daniela CesanaAbstract:Integrative viral vectors are able to efficiently transduce hematopoietic stem progenitor cells allowing stable transgene expression in the entire hematopoietic system upon transplant in conditioned recipients. For these reasons, integrative vectors based on γ-retroviruses and lentiviruses have been successfully used in gene therapy clinical trials for the treatment of genetic diseases, especially blood disorders. However, in different γ-retroviral-based clinical trials, vector integration into the host cell genome triggered oncogenesis by a mechanism called insertional mutagenesis. Thus, a thorough reassessment of the safety of available gene transfer systems is a crucial outstanding issue for the whole gene therapy field. Sensitive preclinical models of vector Genotoxicity are instrumental to achieve a more detailed understanding of the factors that modulate the risks of insertional mutagenesis. Here, we will describe the methodologies used to address the mutagenesis risk of vector integration using a murine in vivo Genotoxicity Assay based on transduction and transplantation of tumor-prone hematopoietic stem and progenitor cells.
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the genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of hsc gene therapy
Journal of Clinical Investigation, 2009Co-Authors: Eugenio Montini, Daniela Cesana, Fabrizio Benedicenti, Manfred Schmidt, Francesca Sanvito, Cynthia C Bartholomae, Marco Ranzani, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio PonzoniAbstract:γ-Retroviral vectors (γRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo Genotoxicity Assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between γRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of Genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of γRVs. By comparing the Genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as γRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by γRVs. This integration-site bias was intrinsic to γRVs, as it was also observed for SIN γRVs that lacked Genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate Genotoxicity.
Fabrizio Benedicenti - One of the best experts on this subject based on the ideXlab platform.
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3 safety assessment of sin lvs harboring chromatin insulators in the sensitive cdkn2a in vivo Genotoxicity Assay show enhancer blocking activity of specific insulator sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Odile Cohenhaguenauer, Luigi Naldini, Jack Lenz, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.
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3. Safety Assessment of SIN LVs Harboring Chromatin Insulators in the Sensitive Cdkn2a-/- In Vivo Genotoxicity Assay Show Enhancer-Blocking Activity of Specific Insulator Sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Luigi Naldini, Jack Lenz, Odile Cohen-haguenauer, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.
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the genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of hsc gene therapy
Journal of Clinical Investigation, 2009Co-Authors: Eugenio Montini, Daniela Cesana, Fabrizio Benedicenti, Manfred Schmidt, Francesca Sanvito, Cynthia C Bartholomae, Marco Ranzani, Lucia Sergi Sergi, Alessandro Ambrosi, Maurilio PonzoniAbstract:γ-Retroviral vectors (γRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo Genotoxicity Assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between γRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of Genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of γRVs. By comparing the Genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as γRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by γRVs. This integration-site bias was intrinsic to γRVs, as it was also observed for SIN γRVs that lacked Genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate Genotoxicity.
Monica Volpin - One of the best experts on this subject based on the ideXlab platform.
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3 safety assessment of sin lvs harboring chromatin insulators in the sensitive cdkn2a in vivo Genotoxicity Assay show enhancer blocking activity of specific insulator sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Odile Cohenhaguenauer, Luigi Naldini, Jack Lenz, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.
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3. Safety Assessment of SIN LVs Harboring Chromatin Insulators in the Sensitive Cdkn2a-/- In Vivo Genotoxicity Assay Show Enhancer-Blocking Activity of Specific Insulator Sequences
Molecular Therapy, 2015Co-Authors: Monica Volpin, Daniela Cesana, Andrea Calabria, Erika Tenderini, Fabrizio Benedicenti, Koyel Mitra, Luigi Naldini, Jack Lenz, Odile Cohen-haguenauer, George StamatoyannopoulosAbstract:Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo Genotoxicity Assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced Genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo Genotoxicity Assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape Genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications.