Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
| Autor: | Carsten Müller-Tidow, Anita Schmitt, Michael Schmitt, Maria-Luisa Schubert, Susanne Hofmann, Alexander Kunz, Angela Hückelhoven-Krauss, Birgit Michels, Ulrike Gern, Brigitte Neuber, Lei Wang, Peter Dreger |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
lcsh:QH426-470 Genetic enhancement Computational biology Article 03 medical and health sciences single-copy gene 0302 clinical medicine vector copy number Single copy gene Genetics Medicine Good manufacturing practice In patient lcsh:QH573-671 Molecular Biology CAR T cells business.industry lcsh:Cytology Chimeric antigen receptor Genetically modified organism lcsh:Genetics qPCR 030104 developmental biology Real-time polymerase chain reaction 030220 oncology & carcinogenesis Molecular Medicine Car t cells business CAR T cell monitoring |
| Zdroj: | Molecular Therapy. Methods & Clinical Development Molecular Therapy: Methods & Clinical Development, Vol 17, Iss, Pp 448-454 (2020) |
| ISSN: | 2329-0501 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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