Engineering Human Circulating Monocytes/Macrophages by Systemic Deliverable Gene Editing.

Delivery of plasmid DNA to transfect human primary macrophages is extremely difficult, especially for genetic engineering. Engineering macrophages is imperative for the treatment of many diseases including infectious diseases, cancer, neurological diseases, and aging. Unfortunately, plasmid does not cross the nuclear membranes of terminally differentiated macrophages to integrate the plasmid DNA (pDNA) into their genome. To address this issue, we have developed a core-shell nanoparticle (NP) using our newly created cationic lipid to deliver the anti-inflammatory cytokine IL-4 pDNA (IL-4pDNA-NPs). Human blood monocyte-derived macrophages (MDM) were effectively transfected with IL-4pDNA-NPs. IL-4pDNA-NPs were internalized in MDM within 30 minutes and delivered into the nucleus within 2 hours. Exogenous IL-4 expression was detected within 1 - 2 days and continued up to 30 days. Functional IL-4 expression led to M2 macrophage polarization in vitro and in an in vivo mouse model of inflammation. These data suggest that these NPs can protect pDNA from degradation by nucleases once inside the cell, and can transport pDNA into the nucleus to enhance gene delivery in macrophages in vitro and in vivo. In this research, we developed a new method to deliver plasmids into the nucleus of monocytes and macrophages for gene-editing. Introducing IL-4 pDNA into macrophages provides a new gene therapy solution for the treatment of various diseases.

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization.

Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.