When E-tagged SpCas9 was mixed with ArgNPs, self-assembled superstructures were generated via carbo ylate-guanidinium binding

When E-tagged SpCas9 was mixed with ArgNPs, self-assembled superstructures were generated via carbo ylate-guanidinium binding. of this system is required for cancer progression.1 One such mechanism is the generation of dont eat me signals by the cancer cells, preventing phagocytosis by macrophages 2 The avoidance signal of perhaps greatest interest is CD47,3 a cell surface protein overexpressed by most cancer cells. Interaction between cancer cell CD47 receptors and macrophage signal regulatory protein- (SIRP-) is sufficient to bypass phagocytosis even if phagocytic signals are present (Figure 1). CD47:SIRP- binding leads to activation of SIRP- via phosphorylation of its immune-receptor tyrosine-based Atosiban inhibition motifs on the cytoplasmic tail,4 resulting in binding and activation of Src homology phosphatase-1 (SHP-1) and SHP-2.5 As a result, phagocytosis is blocked by preventing accumulation of Mouse monoclonal to PR myosin-IIA at the phagocytic synapse. This inhibitory mechanism of CD47:SIRP- binding is evident in a wide range of cancer-initiated malignancies making it a promising therapeutic target.6 Open in a separate window Figure 1. (a) Prevention of cancer cell phagocytosis by CD47:SIRP- interaction (b) Genomic editing using CRISPR-Cas9 machinery (c) Cell-based immunotherapy through elimination of CD47:SIRP- interaction by knocking out SIRP- using nanoparticle-mediated delivery of CRISPR-Cas9/sgRNA and resulting phagocytosis of cancer cell by SIRP– macrophage Recently, strategies have been developed to block the interaction of CD47 with SIRP-. Use of an anti-CD47 monoclonal antibody has shown efficacy in preclinical studies with different human cancers both in vitro and in mouse xenotransplantation models.2,7C8,9,10,11 An engineered SIRP- variant, CV1,12 has been used as an antibody adjuvant and shown to facilitate macrophage-mediated phagocytosis in tumor models with increased tumor penetration and low toxicity. However, overexpression of CD47 by cancer cells leads Atosiban to a large antigen sink in the employment of antibody-based strategies that both reduces bioavailability and increases the potential for toxicity to normal cells.7,13 An alternative to blocking CD47 is the targeting of SIRP-. Studies have shown that anti-SIRP- antibodies significantly enhanced antibody-mediated killing of tumor cells by phagocytes in vitro. Nevertheless, due to their large size, penetration of the antibodies into solid tumors was a major limitation for therapeutic efficacy. SIRP–targeting agents must have sufficient tumor penetration to interact with and block tumor infiltrating macrophages,14,15 to be a viable therapeutic approach. 16 Recently, we have developed nanomaterial platforms for the delivery of biologics. These scaffolds can simultaneously transport proteins and nucleic acids directly to the cytosol through a membrane fusion mechanism. In our approach, we have delivered the complete CRISPR/Cas9 machinery (Figure 1b) by engineering Cas9 protein to facilitate association with cationic arginine-coated gold nanoparticles (ArgNPs).17 This strategy has demonstrated ~90% delivery efficiency along with ~30% gene editing efficiency. Here, we use the same system to knock out SIRP- in macrophages to turn off this dont eat me signal and enable phagocytosis of cancer cells (Figure 1c), thus providing a strategy for cancer immunotherapy.18,19 RESULTS AND DISCUSSION Delivery of CRISPR-Cas9 protein and subsequent knockout of SIRP- gene. Recently, the CRISPR-Cas9 nuclease system has emerged as a powerful tool for genome editing.20 It is a two-component system consisting of sgRNA and Cas9 nuclease (generally derived from Streptococcus pyogenes, or SpCas9) for generating sequence-specific targeted mutations in the genome.21 This targeted modification of the genome is permanent and can be passaged to offspring cells. In our previous research,17,22 we have demonstrated CRISPR/Cas9 mediated gene editing by engineering Cas9 protein to facilitate association with cationic ArgNPs. We inserted a peptide tag containing glutamic acids (E-tags) at the N-terminus of Cas9 protein derived from S. pyogenes (SpCas9) and appended a nuclear localization signal (NLS) tag23 at the C terminus to enhance nuclear accumulation (Figure 2a-d). When E-tagged SpCas9 was mixed with ArgNPs, self-assembled superstructures were generated via carbo ylate-guanidinium binding. Atosiban We found that E20-tag provided the most efficient delivery of SpCas9 into the cytosol and nucleus in multiple cell lines, including the RAW 264.7 model macrophage cell line. Therefore, we engineered SpCas9, first, by introducing an E-20 tag at the N-terminus of the protein so that the protein could self-assemble with cationic ArgNPs. After engineering and purifying CasE20 protein, we fabricated nanoassemblies with CasE20-sgRNA (Cas9-ribonucleoprotein, hereafter, referred to as Cas9-RNP) and ArgNPs. These nanoassemblies were incubated with RAW264.7 cells in cell culture media. Delivery efficiency was Atosiban monitored by using Cas9E20.

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