Genome editing is a powerful technique for delineating complex signaling circuitry and enhancing the functionality of immune cells for immunotherapy. Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency. Here, we describe an integrated platform that streamlines feeder-free ex vivo expansion of cryopreserved primary human NK cells and nonviral genome editing by the nucleofection of CRISPR-Cas9 ribonucleoproteins (Cas9 RNPs). The optimized Cas9 nucleofection protocol allows efficient and multiplex gene knockout in NK cells while preserving high cell viability and negligible off-target effects. Cointroduction of a DNA template also enables in-frame gene knock-in of an HA affinity tag and a gfp reporter across multiple loci. This work demonstrates the advantages and flexibility of working with cryopreserved NK cells as potential off-the-shelf engineered therapeutic agents.
Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhanced cytotoxicity and could mediate antibody-dependent cellular cytotoxicity against hard to kill cancer cell lines. Our genome editing platform is straightforward and robust for both functional studies and therapeutic engineering of NK-92 cells.
Vilse/Arhgap39 is a Rho GTPase activating protein (RhoGAP) and utilizes its WW domain to regulate Rac/Cdc42-dependent morphogenesis in Drosophila and murine hippocampal neurons. However, the function of Vilse in mammalian dendrite architecture and synaptic plasticity remained unclear. In the present study, we aimed to explore the possible role of Vilse in dendritic structure and synaptic function in the brain. Homozygous knockout of Vilse resulted in premature embryonic lethality in mice. Changes in dendritic complexity and spine density were noticed in hippocampal neurons of Camk2a-Cre mediated forebrain-specific Vilse knockout (VilseΔ/Δ) mice. VilseΔ/Δ mice displayed impaired spatial memory in water maze and Y-maze tests. Electrical stimulation in hippocampal CA1 region revealed that the synaptic transmission and plasticity were defected in VilseΔ/Δ mice. Collectively, our results demonstrate that Vilse is essential for embryonic development and required for spatial memory.
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