Core B. Quantifying outcomes of genome modification Core B will track the nature and consequences of genome modifications carried out in Projects 1, 3 and 4, and use these data to design improved reagents for CART genome modification. Core B will analyze several types of genome modification as dictated by the needs of each project. Integration of lentiviral vectors necessarily disrupts the host cell locus at the site of integration. We and others have found that insertional mutagenesis by vectors used to deliver CARs can be associated with altered cell growth, which has resulted in clinical adverse events, but also clinically favorable clonal expansions that have bolstered therapeutic effects. The core will use these data to identify genes and pathways to manipulate to optimize CART expansion and persistence. Vector integration also marks each transduced cell uniquely, allowing tracing of the partitioning of descendant cells, information useful in tracking outcomes and understanding mechanisms of possible adverse events. Cleavage of the host genome using targeted nucleases is also an effective means of achieving genome modification; however, off-target cleavage and associated deletions are safety concerns. The core will monitor off-target cleavage using our iGUIDE technology. Lastly, in protocols where multiple sgRNA/CAS9 complexes cleave multiple targets in a single cell, nuclease cleavage at multiple sites can result in rejoining of DNA ends in a ?swapped? fashion, causing chromosomal translocations. Core B will monitor molecular outcomes in each of these categories to allow optimization of reagents, assessment of safety, and scoring of cell function.
