Chimeric antigen receptor T-Cell (CAR-T) immuno-therapies have been transformative solutions to treat cancer patients. As most CAR-T therapies rely on the introduction of CAR into host cells using lentiviral vectors followed by re-introducing the modified T-cell back into patients, the quality of CAR-T is extensively regulated. Key safety and efficacy attributes such as transduction efficiency and transgene copy number, or viral vector copy number (VCN), needs to be accurately measured. Yet conventional methods for measuring gene transfer lack the resolution and representation to truly reflect sample composition and either report a population average (bulk) or involve laborious and time-consuming clonal outgrowth.
Mission Bio has developed an end-to-end solution from panel design to data analysis for single-cell targeted DNA sequencing to interrogate transgenes. We applied this single-cell protein + DNA multi-omic VCN workflow to analyze a bi-cistronic CD19 and CD22 CAR-T product. Our VCN analysis confirmed the distribution of VCN in the product closely adhered to the expected Poisson distribution throughout the expansion, suggesting a low risk of biased functional clonal expansion favoring cells with high VCN. Additionally, we quantitatively measured surface protein expression for lineage assignment. This revealed differential transduction percentages and VCN distribution patterns across cell lineages (e.g., CD4+ and CD8+ T cells), providing insights into factors potentially influencing treatment outcomes.
