Figure 1.

Highly efficient method for CD38 KO in ex vivo expanded NK cells. (A) Ex vivo expansion and CRISPR/Cas9 gene editing protocol. NK cells were isolated from healthy donor PBMCs and cocultured with irradiated LCL feeder cells and IL-2 for 1 week. Expanded NK cells were electroporated using precomplexed Cas9 and CD38-targeting gRNA. Edited NK cells were then expanded further in IL-2 containing media and analyzed for KO efficiency, phenotype, and function 7 days after CRISPR/Cas9 editing. CD38^WT NK cells were unmanipulated. (B) Representative histograms of relative expression of CD38 in CD38^WT and CD38^KO NK cells from one NK cell donor. (C) Pooled data showing relative expression of CD38 in CD38^WT and CD38^KO NK cells (n=8 donors). (D) Ex vivo fold expansion of CD38^KO NK cells compared with unedited CD38^WT NK cells (n=8 donors). (E) Assessment of off-target activity of the CD38-targeting gRNA/Cas9 RNP, shown are percentage of indels detected in CD38^KO NK cells at the CD38 on-target site as well as five in-silico-predicted off-target sites, as analyzed using the on-line ICE tool (n=5 donors). (F) Expression of a panel of NK cell surface markers examined by flow cytometry in CD38^KO and CD38^WT NK cells (n=6 donors). (G) NK cell degranulation (measured by CD107a expression), and intracellular IFN-γ and TNF-α expression examined by flow cytometry of CD38^KO NK cells compared with CD38^WT NK cells following coculture with K562 cells and Raji cells with and without rituximab (RITUX) (n=6 donors). Statistics determined with the Student’s t-test, two tailed, ****p<0.0001. Differences between CD38^WT and CD38^KO NK cells in panels F and G were not significant. ICE, inference of CRISPR Edits; KO, knockout; WT, wild type; NK, natural killer; PBMC, peripheral blood mononuclear cell; LCL, lymphoblastoid cell line.