Replication-defective retroviral vectors are central to CAR T cell manufacturing, enabling stable gene transfer for therapeutic applications. However, ensuring safety and efficacy requires precise assessment of key parameters, including transduction efficiency, vector copy number, and integration site distribution. Current analyses methods depend on multiple bulk assays, which are resource-intensive, costly, and often lack single-cell resolution.
We developed a high-throughput, droplet-based single-cell assay that integrates these analyses into a single streamlined workflow. In proof-of-concept experiments using a cell line with known integration sites, our method successfully detected integration events across more than 10,000 individual cells.
This platform provides a cost-effective and comprehensive solution for monitoring CAR T cell products, enabling direct linkage between integration sites and clonal behaviour at single-cell resolution. Such insights are critical for improving CAR T safety and understanding clonal expansion, persistence, and exhaustion in patients.