RNA-Seq Analysis Reveals CCR5 as a Key Target for CRISPR Gene Editing to Regulate In Vivo NK Cell Trafficking

SIMPLE SUMMARY: Adoptive immunotherapy utilizing ex vivo expanded natural killer (NK) cells is being explored in the clinical and preclinical settings to treat hematological tumors. Previous work has shown that a large fraction of ex vivo expanded NK cells traffic into the liver following i.v. infusion. In this manuscript, Levy et al. show that ex vivo expansion of NK cells alters the mRNA transcription and surface expression of several chemokine receptors. The observed shift in chemotactic receptor expression may compromise the homing of infused cells into sites where hematological tumors reside, such as bone marrow, lymph nodes, and peripheral blood, by promoting preferential trafficking into liver tissue. Here we demonstrate clustered regularly interspaced short palindromic repeats (CRISPR) gene abrogation of C-C chemokine receptor type 5 (CCR5) as a novel strategy that reduces the trafficking of adoptively transferred ex vivo expanded NK cells into liver tissue and increases NK cell presence in the circulation. ABSTRACT: A growing number of natural killer (NK) cell-based immunotherapy trials utilize ex vivo expansion to grow and activate allogenic and autologous NK cells prior to administration to patients with malignancies. Recent data in both murine and macaque models have shown that adoptively infused ex vivo expanded NK cells have extensive trafficking into liver tissue, with relatively low levels of homing to other sites where tumors often reside, such as the bone marrow or lymph nodes. Here, we evaluated gene and surface expression of molecules involved in cellular chemotaxis in freshly isolated human NK cells compared with NK cells expanded ex vivo using two different feeder cells lines: Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) or K562 cells with membrane-bound (mb) 4-1BB ligand and interleukin (IL)-21. Expanded NK cells had altered expression in a number of genes that encode chemotactic ligands and chemotactic receptors that impact chemoattraction and chemotaxis. Most notably, we observed drastic downregulation of C-X-C chemokine receptor type 4 (CXCR4) and upregulation of C-C chemokine receptor type 5 (CCR5) transcription and phenotypic expression. clustered regularly interspaced short palindromic repeats (CRISPR) gene editing of CCR5 in expanded NK cells reduced cell trafficking into liver tissue and increased NK cell presence in the circulation following infusion into immunodeficient mice. The findings reported here show that ex vivo expansion alters multiple factors that govern NK cell homing and define a novel approach using CRISPR gene editing that reduces sequestration of NK cells by the liver.