The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.

BackgroundImmunotherapy using checkpoint inhibitors, especially PD-1/PD-L1 inhibitors, has now evolved into the most promising therapy for cancer patients. However, most of these inhibitors are monoclonal antibodies, and their large size may limit their tumor penetration, leading to suboptimal efficacy. As a result, there has been a growing interest in developing low-molecular-weight checkpoint inhibitors.MethodsWe developed a novel biopanning strategy to discover small peptide-based anti-PD-L1 inhibitors. The affinity and specificity of the peptides to PD-L1 were examined using various assays. Three-dimensional (3D) spheroid penetration study was performed to determine the tumor penetration capability of the peptides. Anti-tumor activity of the peptides was evaluated in mice bearing CT26 tumor cells.ResultsWe discover several anti-PD-L1 peptide inhibitors to block PD-1/PD-L1 interaction. The peptides exhibit high affinity and specificity to human PD-L1 protein as well as PD-L1-overexpressing human cancer cells MDA-MB-231 and DU-145. Molecular docking studies indicate that the peptide CLP002 specifically binds to PD-L1 at the residues where PD-L1 interacts with PD-1. The peptide also blocks the CD80/PD-L1 interaction, which may further enhance the immune response of tumor-infiltrating T cells. Compared to antibody, the peptide CLP002 exhibits better tumor penetration in a 3D tumor spheroid model. The peptide CLP002 restores proliferation and prevents apoptosis of T cells that are co-cultured with cancer cells. The peptide CLP002 also inhibits tumor growth and increases survival of CT26 tumor-bearing mice.ConclusionsThis study demonstrated the feasibility of using phage display to discover small peptide-based checkpoint inhibitors. Our results also suggested that the anti-PD-L1 peptide represents a promising low-molecular-weight checkpoint inhibitor for cancer immunotherapy.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0705-y) contains supplementary material, which is available to authorized users.

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Insulin like growth factor II receptor (IGFIIR) is a transmembrane protein overexpressed in activated hepatic stellate cells (HSCs), which are the major target for the treatment of liver fibrosis. In this study, we aim to discover an IGFIIR-specific aptamer that can be potentially used as a targeting ligand for the treatment and diagnosis of liver fibrosis. Systematic evolution of ligands by exponential enrichment (SELEX) was conducted on recombinant human IGFIIR to identify IGFIIR-specific aptamers. The binding affinity and specificity of the discovered aptamers to IGFIIR and hepatic stellate cells were studied using flow cytometry and Surface Plasmon Resonance (SPR). Aptamer-20 showed the highest affinity to recombinant human IGFIIR protein with a Kd of 35.5 nM, as determined by SPR. Aptamer-20 also has a high affinity (apparent Kd 45.12 nM) to LX-2 human hepatic stellate cells. Binding of aptamer-20 to hepatic stellate cells could be inhibited by knockdown of IGFIIR using siRNA, indicating a high specificity of the aptamer. The aptamer formed a chimera with an anti-fibrotic PCBP2 siRNA and delivered the siRNA to HSC-T6 cells to trigger silencing activity. In Vivo biodistribution study of the siRNA-aptamer chimera also demonstrated a high and specific uptake in the liver of the rats with CCl4-induced liver fibrosis. These data suggest that aptamer-20 is a high-affinity ligand for antifibrotic and diagnostic agents for liver fibrosis.