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Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC ( m RNA A AV-Sleeping-Beauty J oint E ngineering of S table T herapeutic I mmune C ells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo . This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly reveal the functional genetic landscape underlying critical NK cell characteristics against cancer, we perform perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)- CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes, and perform four independent in vivo tumor infiltration screens in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator that emerges from both screen and single cell analyses, shows both in vitro and in vivo efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells. Differential gene expression analysis reveals that CALHM2 knockout reshapes cytokine production, cell adhesion, and signaling pathways in CAR- NKs. These data directly and systematically map out endogenous factors that naturally limit NK cell function in the TME to offer a broad range of cellular genetic checkpoints as candidates for future engineering to enhance NK cell-based immunotherapies.