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Status |
Public on Oct 01, 2021 |
Title |
Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single cell transcriptomics |
Organism |
Homo sapiens |
Experiment type |
Expression profiling by high throughput sequencing
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Summary |
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 VSV-LV and the CD8-targeted CD8-LV. LV exposed human donor PBMC 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 for 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 above 99%. Beyond that, the 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.
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Overall design |
Analysis of lentiviral vector generated products
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Contributor(s) |
Charitidis FT, Adabi E, Thalheimer FB, Clarke C, Buchholz CJ |
Citation(s) |
- Charitidis FT, Adabi E, Thalheimer FB, Clarke C et al. Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics. Mol Ther Methods Clin Dev 2021 Dec 10;23:359-369. PMID: 34729382
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Submission date |
Sep 28, 2021 |
Last update date |
Dec 14, 2022 |
Contact name |
Christian J. Buchholz |
E-mail(s) |
christian.buchholz@pei.de
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Organization name |
Paul Ehrlich Institut
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Street address |
Paul-Ehrlich-Strasse 51-59
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City |
Langen |
State/province |
Hessen |
ZIP/Postal code |
63225 |
Country |
Germany |
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Platforms (1) |
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Samples (3) |
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Relations |
BioProject |
PRJNA766855 |
SRA |
SRP339084 |