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bioRxiv · 10.1101/2024.02.14.580371

Virus-free CRISPR knock-in of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells

Shankar, K.·Zingler-Hoslet, I.·Shi, L.·Katta, V.·Russell, B. E.·Tsai, S. Q.·Capitini, C. M.·Saha, K.
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Abstract

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2+ human melanoma cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2+ melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E+ solid tumors.

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Shankar, K., Zingler-Hoslet, I., Shi, L., Katta, V., Russell, B. E., Tsai, S. Q., Capitini, C. M., Saha, K.. 2024-02-14. Virus-free CRISPR knock-in of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells. https://doi.org/10.1101/2024.02.14.580371

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