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

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. Overall design: Analysis of lentiviral vector generated products