Genome-Wide profiling of viral vector integration in clinical CAR T products using comprehensive methodologies

Abstract

Background & Aim Chimeric antigen receptor (CAR)-engineered T cells have become an important cancer immunotherapy and the clinical application of these cells is increasing rapidly. For clinical CAR T-cell therapies, autologous T-cells are genetically modified to express a specific/bispecific chimeric antigen receptor. These CAR T-cells have high specificity and affinity for antigens expressed on the surface of target cells. At present, T-cells are genetically modified by introduction of the receptor by gamma- and lentiviral vectors, which integrate into the cell genome and is subsequently expressed. However, as transduction efficiency and the number of integration events increases, the risk of insertional mutagenesis also increases. In order to maintain the safety and effectiveness of clinical CAR T-cells, it is essential to monitor the insertion efficiency, accurately assess insertion sites and determine the effects of vector instertion on the CAR T-cell transcriptome profile. Methods, Results & Conclusion We developed a comprehensive methodology for evaluating the safety and effectiveness of CAR T-cells, utilizing droplet digital PCR to measure vector copy number, DNA-seq to measure vector insertion sites and RNA-seq to assess gene expression profiles. These assays were applied to 38 clinical CAR T products (18 gamma- and 20 lentiviral vector). We verified that ddPCR can reliably measure the average number of vector copies integrated into the CAR T-cell genome. CAR T products with a detectable number of vector copies were subjected to DNA-seq. The results showed that DNA-seq accurately revealed integration site maps in the whole genome and found some loci that were integration hot spots. Moreover, we found gamma-retroviral vectors resulted in more integration events than lentiviral vectors. Combining the RNA-seq and DNA-seq data revealed that most integration events within coding genes did not result in a change in gene expression. We did noticed that some gene loci both have an integration event and changed gene expression. Further studies will focus on these finding and the impact on CAR T-cell clinical outcomes. In summary, we established a series of methods to detect vector copy number, insertion sites and gene expression profiles in clincial CAR T products. These methods will be useful for exploring the risk of insertional mutagenesis of viral vector in CAR T products, the signficance of specific integration sites, and facilates improved safety in development of CAR T cell therapies.