PROJECT SUMMARY Lentiviral vectors (LVVs) are an effective gene therapy agent due to their ability to integrate into host DNA, providing stable expression of transgenes. LVVs have been under clinical development as a gene delivery system to treat diseases, including blood cancers, through the generation of chimeric antigen receptor (CAR) T- cells. The first two CAR T-cell therapies were approved for clinical use in the treatment of lymphoma or acute lymphoblastic leukemia in 2017, and since then four more CAR T-cell therapies have been approved by the Food and Drug Administration (FDA) for clinical use. Despite the success of these agents, on November 28, 2023, it was announced that all clinically approved CAR T-cell therapies were under FDA investigation due to the emergence of cellular transformation in patients receiving LVV-derived CAR T-cells, leading to cancers, and resulting in hospitalization or death. While much work has been done to define LVV integration profiles, such as the identification of preferential integration into intronic genomic elements, it is still thought to be a mostly random process. For example, LVV integration has been seen in promoters, transcription start sites, exons, and untranslated regions, resulting in differential gene expression and alternative splicing patterns when compared to untransduced controls. Standard methods for characterizing integration profiles, such as ligation-mediated PCR (LM-PCR) remain constrained by short read lengths and only identify the integration sites but provide no information regarding the proviral integrity. Short-read sequencing is limiting in situations of integration into low complexity or repeat-rich genomic areas that cannot be efficiently mapped with these data. Additionally, LM- PCR and other common methods require priming from long-terminal repeat regions; however, integrated lentiviral genomes contain large deletions including, in some cases, missing LTRs that would not be captured by current methods and may result in incomplete transgene expression from that event. To address this unmet need, we developed a novel method, single-molecule, real-time capture (SMRTcap) that leverages long reads to resolve integrated lentiviral genomes. We hypothesize that current methods for resolving LVV integration are incomplete, thereby limiting our ability to comprehensively characterize LVV integration sites, proviral integrity, and their impact on host cell biology. Using SMRTcap we will resolve LVV integrations in experimentally- generated, primary CAR T-cells, providing a complete view of integration sites, clonal expansion, and presence of intact transgenes. Using single-cell (sc-) isoform sequencing (IsoSeq) and sc-RNA seq on these same CAR T-cells, we will evaluate the influence of LVV integration on host gene expression, splicing, and isoform usage. These data will provide a comprehensive view of LVV integration into primary T cells and the impact of this event on the host genome...