CRISPR Gene-Editing of Chemokine Receptors As a Novel Strategy to Redirect NK Cell Trafficking In Vivo

Allogeneic and autologous natural killer (NK) cell-based therapies have proven to be safe in patients with hematological malignancies across multiple clinical trials. However, at present, only a small percentage of patients who have received NK cell based therapy have achieved complete remissions. NK cells must first traffic into the tumor microenvironment in order to effectively lyse tumor targets in vivo. Many hematological malignancies reside in or metastasize through bone marrow (BM) compartments. Recent studies in both murine and macaque models evaluating NK cell distribution following intravenous (i.v.) infusion unexpectedly revealed preferential trafficking into liver tissue over BM niches, which for hematological malignancies is undesirable. Therefore, we evaluated surface expression and mRNA transcript abundance of critical chemotactic receptors and adhesion molecules in primary fresh and ex vivo expanded NK cells.

NK cells from six healthy donor PBMC samples were expanded for 14 days ex vivo, using two different irradiated feeder cell lines (either EBV-infected LCL or K562 cells with membrane-bound IL-21 and 41BBL). mRNA from fresh and expanded NK cells was sequenced and analyzed for differential gene expression. We observed the transcriptome of NK cells expanded with the two different feeder cell lines were similar, with only 13 differentially expressed genes. However, we observed the transcriptional landscape between fresh and expanded NK cells to be vastly different (Figure 1A). As expected, amongst the thousands of differentially expressed genes, mitotic phase transitions and DNA replication were pathways that were strongly enriched in expanded compared to fresh NK cells. Further, expanded NK cells had a robust amplification in mRNA transcription of known surface cytotoxicity and activation receptors, as well as enhanced mRNA transcription of chemotactic ligands that are implicated in bridging the innate and adaptive immune responsesincluding XCL1, XCL2, CCL5, CXCL16, and CXCL8. Remarkably, we found expanded NK cells had upregulated mRNA transcription and surface expression of CCR1, CCR5, CXCR3, and CXCR6, coupled with a substantial decrease in mRNA transcription and surface expression of CXCR4 (Figure 1B). We postulate that this particular shift in molecular expression may have the net effect of routing adoptively transferred NK cells out of the circulation into liver and other inflammatory environments, ultimately impeding cellular trafficking into the bone marrow (BM). To test this hypothesis, we used CRISPR/Cas9 to disrupt the CCR5 gene which has upregulated expression in expanded NK cells. Five days following ex-vivo expansion, NK cells were electroporated with a mix of 3 sgRNAs targeting CCR5, complexed with Cas9. Electroporated cultures harvested on day 14 maintained their proliferative capacity and had a substantial reduction in CCR5 expression (22%) compared to non-electroporated control NK cells (88%; Figure 1C). Both expanded NK cell populations were then injected i.v. into NSG mice, with blood, BM, lungs, and livers being harvested 24 hours following infusion. Disrupting CCR5 in NK cells significantly reduced cellular trafficking into the liver compared to control NK cells (p<.01). Moreover, the percentage of infused CCR5 CRISPR/Cas9 disrupted NK cells in the circulation was increased compared to control NK cells (Figure 1D).

In conclusion, these collective data reveal NK cells undergo profound transcriptional changes when cultivated ex vivo. Many chemotactic receptors that were previously unknown to be impacted by ex vivo expansion were discovered to have a shift in transcriptional regulation which would be predicted to compromise NK cell homing to the bone-marrow. Importantly, we show for the first time that CRISPR gene-editing of chemokine receptors can be used as a novel strategy to redirect NK cell trafficking in vivo, which could bolster the effectiveness of adoptive NK cell immunotherapy for hematological malignancies and other cancers.