Treatments of glioblastoma (GBM) have not been very effective, largely due to the inefficiency of drugs in penetrating the blood brain barrier (BBB). In this study, we investigated the potential of exosome-coated doxorubicin (DOX)-loaded nanoparticles (ENP DOX) in BBB penetration, inducing immunogenic cell death (ICD) and promoting survival of GBM-bearing mice. DOX-loaded nanoparticles (NP DOX) were coated with exosomes prepared from mouse brain endothelial bEnd.3 cells. ENP DOX cellular uptake was examined. Penetration of ENP DOX through the BBB was tested in an in vitro transwell system and a GBM mouse model. The effects of ENP DOX in inducing apoptosis and ICD were assessed. Finally, the efficacy of ENP DOX in the treatment of GBM-bearing mice was assessed. ENP DOX was taken up by bEnd.3 cells and could penetrate the BBB both in vitro and in vivo. In vitro, END DOX induced apoptosis and ICD of glioma GL261 cells. Systemic administration of ENP DOX resulted in maturation of dendritic cells, activation of cytotoxic cells, altered production of cytokines, suppressed proliferation and increased apoptosis of GBM cells in vivo and prolonged survival of GBM-bearing mice. Our findings indicate that ENP DOX may be a potent therapeutic strategy for GBM which warrants further investigation in clinical application. K E Y W O R D S blood brain barrier, doxorubicin, exosome-coated doxorubicin-loaded nanoparticle, glioblastoma, immunogenic cell death 1 | INTRODUCTION Glioblastoma (GBM) is a highly aggressive brain tumor with an extremely poor prognosis and a small rate (4%-5%) or 5-year survival. 1 Current treatments of GBM include surgeries, radiotherapy, and/or chemotherapy. These treatments not only cause severe side effects, but also only slightly improve the overall median survival (only 15 months) and 5-year survival rate. 2 Although many therapeutic strategies targeting have been developed, their application in the clinic for treatment GBM has been largely impeded due to the lack of safe and efficient drug delivery system that delivers drugs to tumor location. 3 Recent research findings suggest that, in various cancer types including GBM, human immune response has significant potential in promoting immune mediated tumor eradication and improving long term survival. 4 Recent studies have shown that anthracyclines, such as doxorubicin (DOX), not only induce apoptosis of tumor cells, but also

CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N‐myristoylation is an irreversible co/post‐translational modification that results in the covalent attachment of the myristoyl‐group to the N‐terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell‐cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N‐terminus of Src kinase was a favourable substrate for N‐myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N‐terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA‐eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV‐G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV‐G encapsulating Cas9/sgRNA‐eGFP showed up to 42% eGFP knock out efficiency with limited off‐target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome‐editing‐based gene therapy.

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The RNA-guided CRISPR-associated Cas9 proteins have been widely applied in programmable genome recombination, base editing or gene regulation in both prokaryotes and eukaryotes. SpCas9 from Streptococcus pyogenes is the most extensively engineered Cas9 with robust and manifold functionalities. However, one inherent limitation of SpCas9 is its stringent 5′-NGG-3′ PAM requirement that significantly restricts its DNA target range. Here, to repurpose SpCas9 as a universal gene repressor, we generate and screen variants of the deactivated SpCas9 (SpdCas9) with relaxed 5′-CAT-3′ PAM compatibility that can bind to the start codon ATG of almost any gene. Stepwise structure-guided mutations of the PAM-interacting residues and auxiliary PAM-proximal residues of the SpdNG (5′-NG-3′ PAM) create a PAM-flexible variant SpdNG-LWQT that preferentially accommodates 5′-NRN-3′ PAMs. SpdNG-LWQT is demonstrated to be effective in gene repression with the advantage of customizable sgRNA design in both Escherichia coli and Saccharomyces cerevisiae. This work validates the feasibility of purposeful PAM expansion of Cas9 towards signature PAMs and establishes a universal SpdCas9-based gene repressor.

Glioblastoma (GBM) is the most common and malignant brain tumor in adults. Recently, programmed death‐1/programmed death‐ligand 1 (PD‐1/PD‐L1) checkpoint blockades have been applied for GBM treatment. However, the mechanism of PD‐L1 upregulation in GBM is still unclear. COP9 signalosome 6 (CSN6) is crucial for maintaining the protein stabilization in cancer cells. In this study, we applied human GBM specimens and cell lines to investigate whether the EGFR‐ERK pathway regulates CSN6 for PD‐L1 upregulation. Data from The Cancer Genome Atlas dataset showed that high expression of EGFR, CSN6, and PD‐L1 in patients with glioma was associated with poor prognosis. In 47 human GBM specimens, high expression of PD‐L1 was associated with low amount of CD8+ T cell infiltration as well as the poor prognosis of patients. CSN6 was positively correlated with EGFR and PD‐L1 expression in human GBM specimens. We treated two GBM cell lines (U87 and U251) with epidermal growth factor (EGF) in vitro, and found EGF‐upregulated p‐EGFR, p‐ERK, CSN6, and PD‐L1 expression in GBM cells. PD98059, the ERK blocker, inhibited upregulations of CSN6 and PD‐L1 in EGF‐treated cells. Inhibition of CSN6 by small interfering RNA decreased PD‐L1 expression but also increased CHIP expression in GBM cells. When the cells were treated with EGF and cycloheximide (CHX), a protein synthesis inhibitor, EGF‐reduced CHX‐induced CSN6 and PD‐L1 turnover in GBM cells. Furthermore, CSN6‐mediated downregulation of PD‐L1 was inhibited by MG132, a proteasome inhibitor in U87 cells. Thus, these results suggest that the EGFR‐ERK pathway may upregulate CSN6, which may inhibit PD‐L1 degradation and subsequently maintain PD‐L1 stability in GBM.