CRISPRed Macrophages for Cell-Based Cancer Immunotherapy

Abstract

We present here an integrated nanotechnology/biology strategy for cancer immunotherapy that uses arginine nanoparticles (ArgNPs) to deliver CRISPR-Cas9 gene editing machinery into cells to generate SIRP-α knockout macrophages. The NP system efficiently codelivers single guide RNA (sgRNA) and Cas9 protein required for editing to knock out the "don't eat me signal" in macrophages that prevents phagocytosis of cancer cells. Turning off this signal increased the innate phagocytic capabilities of the macrophages by 4-fold. This improved attack and elimination of cancer cells makes this strategy promising for the creation of "weaponized" macrophages for cancer immunotherapy.

Figure 1.

(a) Prevention of cancer cell phagocytosis by CD47:SIRP-α interaction (b) Genomic editing using CRISPR-Cas9 machinery (c) Cell-based immunotherapy through elimination of CD47:SIRP-α interaction by knocking out SIRP-α using nanoparticle-mediated delivery of CRISPR-Cas9/sgRNA and resulting phagocytosis of cancer cell by SIRP-α- macrophage

Figure 2.

(a) Engineering Cas9 by introducing an E20-tag at the N-terminus and nuclear localization signal (NLS) at the C-terminus (b) Chemical structure of ArgNPs (c) Schematic showing formation of nanoassembly by Cas9E20-RNP and ArgNPs (d) Delivery of Cas9E20 via membrane fusion mechanism. Fusion of nanoassemblies to the cell membrane facilitates direct release of the protein payload into cytoplasm, bypassing endosomes (e) Endogenous SIRP-α gene locus showing the CRISPR-Cas9 target site (f) Cytoplasmic/nuclear delivery of FITC- labelled SpCas9E20 into RAW264.7 cells; (Cell nuclei stained with Hoechst 33342)(g) Delivery of Cas9E20-RNP to target SIRP-α gene in RAW264.7 cells resulted in efficient gene editing, as determined by indel (insertion and deletion) assay: Lane 1: Cas9E20-RNP:ArgNPs, Lane 2: cells only and Lane 3: Marker (the bottom one is 200 bp and each increase in fragment size is 100 bp). Indel efficiency is given in percentages. (h) Fluorescence histogram from flow cytometry analysis on SIRP-α- RAW264.7 cells and RAW264.7 cells; RAW264.7 cells: green histogram; SIRP-α- RAW264.7 cells purple histogram

Figure 3.

(a) Flow cytometry plots of PE anti-mouse F4/80 antibody-stained and unstained RAW264.7 cells, PE anti-mouse F4/80 antibody-stained and unstained SIRP-α- RAW264.7cells, GFP+/GFP- U2OS cells, co-culture of PE anti-mouse F4/80 antibody-stained RAW264.7 cells and U2OS-GFP+ cells and co-culture of PE anti-mouse F4/80 antibody-stained SIRP-α- RAW264.7 cells and U2OS-GFP+ cells (b) Percentage of phagocytosis of U2OS-GFP+ by SIRP-α- RAW264.7 cells and RAW264.7 cells (c) Confocal images showing phagocytosis or no phagocytosis of U2OS cells; U2OS cells (green) labelled with pHrodo Green AM Intracellular pH Indicator. Scale bar: 20 µm (d) Representative images U2OS-GFP+ cells internalized by PE-F4/80 labelled SIRP-α- RAW264.7 cells measured by Imaging flow cytometry. The negative control (no internalization) is a representative PE-F4/80 labelled SIRP-α- RAW264.7 cell without internalized U2OS-GFP+ cells. Scale bar: 10 µm.