Cell and Genome Engineering Core

PROJECT SUMMARY/ABSTRACT Immunotherapy with chimeric antigen receptors (CAR) that engage a specific antigen and trigger immune effector cell (IEC) activity has transformed cancer therapy. This application focuses on the development and clinical implementation of engineered T-regulatory (Treg) and NK cells due to their potent tumoricidal activity with no risk of graft-versus-host disease (GVHD). The central hypothesis of this P01 s that allogeneic IECs (Tregs and NK cells) can be engineered to maximize tumor killing with fewer risks including undesirable on- target/off-tumor adverse events. Toward achieving the goals of the three Projects, Core D will provide the molecular tools and analytics crucial for optimizing the genetic engineering platforms and examining the cell products to maximize safety prior to testing in the proposed clinical trials. In addition, Core D will provide umbilical cord blood (UCB) Tregs and induced pluripotent stem cell (iPSC) derived induced CD8+ Tregs (iPSC- iTreg) for the studies outlined in Projects 1 and 2 in particular and Project 3 for comparative studies. The Core will perform molecular analyses of gene edited cell products and cell lines as a quality control. The Core integrates leading edge genetic constructs and proteins for engineering IECs with enhanced therapeutic properties as an off-the- shelf product for widespread and urgent use. Core D will produce lentiviral and adeno-associated virus (AAV) serotype 6 vectors that will be used to introduce/overexpress novel immune therapy genes in Treg, NK, and iPSC to enhance tumor eradication. Luciferase lentiviruses will be used to generate cells that can be imaged in vitro/vivo to track IECs or tumor killing over time in all three Projects. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing platform allows for target gene disruption, user defined modification (i.e., incorporation of novel exogenous sequences), and transcriptome modulation. CRISPR/Cas9 will be employed in each project for enhancing IEC activity through deletion of target genes, introduction of immune therapy candidates into user defined genomic loci, and upregulation of specific gene(s) that regulate immune responses. Core D will also serve as the hub for distributing nanobody proteins and cellular products to the Project Leaders. Nanobodies that have high affinity to leukemia antigens will be provided to the project and program leaders for application in IECs for the Projects’. This will inform project leaders as to how to best implement or modify Treg and NK cell immunotherapeutic interventions. Uniform, high-quality reagents and data generated in these studies will be integrated with clinical outcomes in collaboration with Cores A, B, and C, and will be made available to all three projects in order to maximize synergy and limit variation generated by having different laboratories generate molecular tools for each individual project(s).