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

Emily R Levy,Robert N Reger,Joseph A Clara,Richard W Childs,David S J Allan

doi:10.3390/cancers13040872

Abstract

Simple SummaryAdoptive 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. 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.

Highlights

Clinical trials targeting both hematologic malignancies and solid tumors have shown that adoptive therapy with allogeneic and autologous natural killer (NK) cells is safe, with clinically meaningful tumor responses being observed in multiple studies [1,2,3]
We performed a detailed analysis using flow cytometry and RNA-seq to characterize phenotypic and gene transcription changes associated with ex vivo expansion of NK cells using two feeder lines utilized for clinical use: K562.mbIL-21.41BBL and EBVLCLs (Figure S1A)
We have previously shown with macaque and murine models that after i.v. infusion of expanded NK cells, the majority of infused NK cells are taken up by liver tissue [18,25]; the second aim of our study was to mitigate this sequestration by disrupting chemokine receptor type 5 (CCR5) in expanded NK cells by clustered regularly interspaced short palindromic repeats (CRISPR) gene editing

Summary

Introduction

Clinical trials targeting both hematologic malignancies and solid tumors have shown that adoptive therapy with allogeneic and autologous natural killer (NK) cells is safe, with clinically meaningful tumor responses being observed in multiple studies [1,2,3]. Ex vivo activation and expansion of primary NK cells through contact with irradiated feeder cells is commonly utilized to produce large numbers of highly cytotoxic NK cells for clinical use. Modifications to the K562 cell line to promote NK cell expansion ex vivo include induced expression of membrane-bound (mb) interleukin (IL)-15 or IL-21, along with the costimulatory molecule 41BB ligand [7,8,9,10]. As a mechanism to link innate and adaptive immune responses, NK cells produce various chemokines and cytokines, such as CCL3, CCL5, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), and tumor necrosis factor- 1 alpha (TNF-α), to recruit and activate alternate immune cells to sites of infection or the tumor microenvironment [17]

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