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Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants

Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genot...

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Autores principales: Bastone, Antonella L., Dziadek, Violetta, John-Neek, Philipp, Mansel, Friederike, Fleischauer, Jenni, Agyeman-Duah, Eric, Schaudien, Dirk, Dittrich-Breiholz, Oliver, Schwarzer, Adrian, Schambach, Axel, Rothe, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10491817/
https://www.ncbi.nlm.nih.gov/pubmed/37693949
http://dx.doi.org/10.1016/j.omtm.2023.08.017
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author Bastone, Antonella L.
Dziadek, Violetta
John-Neek, Philipp
Mansel, Friederike
Fleischauer, Jenni
Agyeman-Duah, Eric
Schaudien, Dirk
Dittrich-Breiholz, Oliver
Schwarzer, Adrian
Schambach, Axel
Rothe, Michael
author_facet Bastone, Antonella L.
Dziadek, Violetta
John-Neek, Philipp
Mansel, Friederike
Fleischauer, Jenni
Agyeman-Duah, Eric
Schaudien, Dirk
Dittrich-Breiholz, Oliver
Schwarzer, Adrian
Schambach, Axel
Rothe, Michael
author_sort Bastone, Antonella L.
collection PubMed
description Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) for pre-clinical genotoxicity prediction of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPCs) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell- and cancer-related transcriptional programs (SAGA). However, both assays present an intrinsic myeloid bias due to culture conditions. To detect lymphoid mutants, we differentiated mHSPCs to mature T cells and analyzed their phenotype, insertion site pattern, and gene expression changes after transduction with retroviral vectors. Mutagenic vectors induced a block in differentiation at an early progenitor stage (double-negative 2) compared to fully differentiated untransduced mock cultures. Arrested samples harbored high-risk insertions close to Lmo2, frequently observed in clinical trials with severe adverse events. Lymphoid insertional mutants displayed a unique gene expression signature identified by SAGA. The gene expression-based highly sensitive molecular readout will broaden our understanding of vector-induced oncogenicity and help in pre-clinical prediction of retroviral genotoxicity.
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spelling pubmed-104918172023-09-10 Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants Bastone, Antonella L. Dziadek, Violetta John-Neek, Philipp Mansel, Friederike Fleischauer, Jenni Agyeman-Duah, Eric Schaudien, Dirk Dittrich-Breiholz, Oliver Schwarzer, Adrian Schambach, Axel Rothe, Michael Mol Ther Methods Clin Dev Original Article Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) for pre-clinical genotoxicity prediction of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPCs) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell- and cancer-related transcriptional programs (SAGA). However, both assays present an intrinsic myeloid bias due to culture conditions. To detect lymphoid mutants, we differentiated mHSPCs to mature T cells and analyzed their phenotype, insertion site pattern, and gene expression changes after transduction with retroviral vectors. Mutagenic vectors induced a block in differentiation at an early progenitor stage (double-negative 2) compared to fully differentiated untransduced mock cultures. Arrested samples harbored high-risk insertions close to Lmo2, frequently observed in clinical trials with severe adverse events. Lymphoid insertional mutants displayed a unique gene expression signature identified by SAGA. The gene expression-based highly sensitive molecular readout will broaden our understanding of vector-induced oncogenicity and help in pre-clinical prediction of retroviral genotoxicity. American Society of Gene & Cell Therapy 2023-08-22 /pmc/articles/PMC10491817/ /pubmed/37693949 http://dx.doi.org/10.1016/j.omtm.2023.08.017 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Article
Bastone, Antonella L.
Dziadek, Violetta
John-Neek, Philipp
Mansel, Friederike
Fleischauer, Jenni
Agyeman-Duah, Eric
Schaudien, Dirk
Dittrich-Breiholz, Oliver
Schwarzer, Adrian
Schambach, Axel
Rothe, Michael
Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants
title Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants
title_full Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants
title_fullStr Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants
title_full_unstemmed Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants
title_short Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants
title_sort development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10491817/
https://www.ncbi.nlm.nih.gov/pubmed/37693949
http://dx.doi.org/10.1016/j.omtm.2023.08.017
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