Lentiviral vector (LV) has been widely adopted as an efficient vehicle to deliver transgenes into cells due to its long-term efficacy. However, the semi-random integration of LV has raised safety concerns due to its potential to trigger tumorigenesis during CAR-T therapy. To mitigate this issue, characterization of vector integration on clonal expansion after gene therapy has become a crucial practice to monitor the activity of viral vectors on in vivo selection of patient clones. Here, we developed a single-cell resolution, lentiviral vector integration site assay to survey the co-occurrence of specific integration sites and somatic genomic variants. Based on a set of LV transduced cell lines with known integration sites validated by orthogonal data, a targeted panel was designed to cover both the 5’ and 3’ ends of each integration site with predefined integration orientations. Samples include negative control and LV transduced cell lines with known vector copy numbers ranging from one to four copies. Using chemistry optimized for LTR junctions, we demonstrate the capability to quantitatively detect individual cells harboring specific vector integration sites and longitudinal tracking of cell clones with different vector copy numbers. Furthermore, besides amplicons targeting integration sites, in the same reaction, another set of internal vector amplicons (10-plex) and human genome amplicons (99-plex) are included to estimate vector copy number (VCN) and characterize somatic variants in the cell population. Taken together, a high throughput single-cell multi-omics platform enabled us to simultaneously identify somatic variants along with vector integration events in individual cells, providing both potential functional mutation identification and clonal tracking capabilities. The development of single-cell lentiviral vector integration sites and clonal tracking assay provides a unique opportunity to better study longitudinally CAR-T cell clonal dynamics and lead to a more effective therapeutic agent.
