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Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078460/ https://www.ncbi.nlm.nih.gov/pubmed/32201711 http://dx.doi.org/10.1016/j.omtm.2020.02.003 |
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author | Kunz, Alexander Gern, Ulrike Schmitt, Anita Neuber, Brigitte Wang, Lei Hückelhoven-Krauss, Angela Michels, Birgit Hofmann, Susanne Müller-Tidow, Carsten Dreger, Peter Schmitt, Michael Schubert, Maria-Luisa |
author_facet | Kunz, Alexander Gern, Ulrike Schmitt, Anita Neuber, Brigitte Wang, Lei Hückelhoven-Krauss, Angela Michels, Birgit Hofmann, Susanne Müller-Tidow, Carsten Dreger, Peter Schmitt, Michael Schubert, Maria-Luisa |
author_sort | Kunz, Alexander |
collection | PubMed |
description | Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration. |
format | Online Article Text |
id | pubmed-7078460 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
spelling | pubmed-70784602020-03-20 Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients Kunz, Alexander Gern, Ulrike Schmitt, Anita Neuber, Brigitte Wang, Lei Hückelhoven-Krauss, Angela Michels, Birgit Hofmann, Susanne Müller-Tidow, Carsten Dreger, Peter Schmitt, Michael Schubert, Maria-Luisa Mol Ther Methods Clin Dev Article Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration. American Society of Gene & Cell Therapy 2020-02-20 /pmc/articles/PMC7078460/ /pubmed/32201711 http://dx.doi.org/10.1016/j.omtm.2020.02.003 Text en © 2020 The Author(s) http://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 | Article Kunz, Alexander Gern, Ulrike Schmitt, Anita Neuber, Brigitte Wang, Lei Hückelhoven-Krauss, Angela Michels, Birgit Hofmann, Susanne Müller-Tidow, Carsten Dreger, Peter Schmitt, Michael Schubert, Maria-Luisa Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients |
title | Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients |
title_full | Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients |
title_fullStr | Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients |
title_full_unstemmed | Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients |
title_short | Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients |
title_sort | optimized assessment of qpcr-based vector copy numbers as a safety parameter for gmp-grade car t cells and monitoring of frequency in patients |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078460/ https://www.ncbi.nlm.nih.gov/pubmed/32201711 http://dx.doi.org/10.1016/j.omtm.2020.02.003 |
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