Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics
| العنوان: | Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics |
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| المؤلفون: | Frederic B. Thalheimer, Filippos T. Charitidis, Colin Clarke, Elham Adabi, Christian J. Buchholz |
| المصدر: | Molecular therapy, 23:359-369 Molecular Therapy: Methods & Clinical Development, Vol 23, Iss, Pp 359-369 (2021) Molecular Therapy – Methods & Clinical Development Molecular Therapy. Methods & Clinical Development Molecular Therapy: Methods & Clinical Development, Vol 24, Iss, Pp 207-209 (2022) |
| بيانات النشر: | Elsevier BV, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | scRNA sequencing, QH426-470, CAR T cells, CD19, Viral vector, Transduction (genetics), Gene expression, Genetics, Molecular Biology, chimeric antigen receptor, QH573-671, biology, lentiviral vector, transduction, biology.organism_classification, CD8-LV, Chimeric antigen receptor, CAR, CD8A, Cell biology, Vesicular stomatitis virus, VSV-LV, biology.protein, Molecular Medicine, Original Article, CAR-T-Zell-Therapie, Cytology, CD8 |
| الوصف: | Quantifying gene expression in individual cells can substantially improve our understanding about complex genetically engineered cell products such as chimeric antigen receptor (CAR) T cells. Here we designed a single-cell RNA sequencing (scRNA-seq) approach to monitor the delivery of a CD19-CAR gene via lentiviral vectors (LVs), i.e., the conventional vesicular stomatitis virus (VSV)-LV and the CD8-targeted CD8-LV. LV-exposed human donor peripheral blood mononuclear cells (PBMCs) were evaluated for a panel of 400 immune response-related genes including LV-specific probes. The resulting data revealed a trimodal expression for the CAR and CD8A, demanding a careful distribution-based identification of CAR T cells and CD8+ lymphocytes in scRNA-seq analysis. The fraction of T cells expressing high CAR levels was in concordance with flow cytometry results. More than 97% of the cells hit by CD8-LV expressed the CD8A gene. Remarkably, the majority of the potential off-target cells were in fact on-target cells, resulting in a target cell selectivity of more than 99%. Beyond that, differential gene expression analysis revealed the upregulation of restriction factors in CAR-negative cells, thus explaining their protection from CAR gene transfer. In summary, we provide a workflow and subsetting approach for scRNA-seq enabling reliable distinction between transduced and untransduced cells during CAR T cell generation. Graphical abstract Buchholz and coworkers describe a process for a nanowell-based scRNA-seq pipeline, thereby making use of a CD8-specific lentiviral vector (LV) for identification and analysis of CAR T cells. Exposure to CD8-LV or VSV-LV and CAR expression altered the transcriptomes of individual lymphocytes. Upregulation of restriction factors correlated with protection from transduction. |
| تدمد: | 2329-0501 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::fdddea4b7818b4668fcb6364cb06bcc2 https://doi.org/10.1016/j.omtm.2021.09.019 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....fdddea4b7818b4668fcb6364cb06bcc2 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 23290501 |
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