Details

Autor(en) / Beteiligte

• Charitidis, Filippos T.
• Adabi, Elham
• Thalheimer, Frederic B.
• Clarke, Colin
• Buchholz, Christian J.

Titel

Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics

Ist Teil von

• Molecular therapy. Methods & clinical development, 2021-12, Vol.23, p.359-369

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2021

Quelle

EZB Free E-Journals

Beschreibungen/Notizen

• 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. [Display omitted] 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.