New Therapeutics for Extracellular Vesicles: Delivering CRISPR for Cancer Treatment.

Cancers are defined by genetic defects, which underlines the prospect of using gene therapy in patient care. During the past decade, CRISPR technology has rapidly evolved into a powerful gene editing tool with high fidelity and precision. However, one of the impediments slowing down the clinical translation of CRISPR-based gene therapy concerns the lack of ideal delivery vectors. Extracellular vesicles (EVs) are nano-sized membrane sacs naturally released from nearly all types of cells. Although EVs are secreted for bio-information conveyance among cells or tissues, they have been recognized as superior vectors for drug or gene delivery. Recently, emerging evidence has spotlighted EVs in CRISPR delivery towards cancer treatment. In this review, we briefly introduce the biology and function of the CRISPR system and follow this with a summary of current delivery methods for CRISPR applications. We emphasize the recent progress in EV-mediated CRISPR editing for various cancer types and target genes. The reported strategies for constructing EV-CRISPR vectors, as well as their limitations, are discussed in detail. The review aims to throw light on the clinical potential of engineered EVs and encourage the expansion of our available toolkit to defeat cancer.

Trichostatin D as a Novel KLF2 Activator Attenuates TNFα-Induced Endothelial Inflammation.

Krüppel-like factor 2 (KLF2) is an atherosclerotic protective transcription factor that maintains endothelial cell homeostasis through its anti-inflammatory, anti-oxidant, and antithrombotic properties. The aim of this study was to discover KLF2 activators from microbial secondary metabolites and explore their potential molecular mechanisms. By using a high-throughput screening model based on a KLF2 promoter luciferase reporter assay, column chromatography, electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) spectra, trichostatin D (TSD) was isolated from the rice fermentation of Streptomyces sp. CPCC203909 and identified as a novel KLF2 activator. Real-time-quantitative polymerase chain reaction (RT-qPCR) results showed that TSD upregulated the mRNA level of KLF2 in endothelial cells. Functional assays showed that TSD attenuated monocyte adhesion to endothelial cells, decreased vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression, and exhibited an anti-inflammatory effect in tumor necrosis factor alpha (TNFα)-induced endothelial cells. We further demonstrated through siRNA and western blot assays that the effects of TSD on monocyte adhesion and inflammation in endothelial cells were partly dependent on upregulating KLF2 expression and then inhibiting the NOD-like receptor protein 3 (NLRP3)/Caspase-1/interleukin-1beta (IL-1ß) signaling pathway. Furthermore, histone deacetylase (HDAC) overexpression and molecular docking analysis results showed that TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 activities. Taken together, TSD was isolated from the fermentation of Streptomyces sp. CPCC203909 and first reported as a potential activator of KLF2 in this study. Furthermore, TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 and attenuated endothelial inflammation via regulation of the KLF2/NLRP3/Caspase-1/IL-1ß signaling pathway.

Three new isocoumarin analogues from an endolichenic fungus Aspergillus flavus CPCC 400810.

Five isocoumarin derivatives including three new compounds, aspermarolides A-C (1-3), and two known analogues, 8-methoxyldiaporthin (4) and diaporthin (5) were obtained from the culture extract of Aspergillus flavus CPCC 400810. The structures of these compounds were elucidated by spectroscopic methods. The double bond geometry of 1 and 2 were assigned by the coupling constants. The absolute configuration of 3 was determined by electronic circular dichroism experiment. All compounds showed no cytotoxic activities against the two human cancer cells HepG2 and Hela.

Cytotoxic hexadepsipeptides and anti-coronaviral 4-hydroxy-2-pyridones from an endophytic Fusarium sp.

Three new hexadepsipeptides (1-3), along with beauvericin (4), beauvericin D (5), and four 4-hydroxy-2-pyridone derivatives (6-9) were isolated from the endophytic fungus Fusarium sp. CPCC 400857 that derived from the stem of tea plant. Their structures were determined by extensive 1D and 2D NMR, and HRESIMS analyses. The absolute configuration of hexadepsipeptides were elucidated by the advanced Marfey's method and chiral HPLC analysis. Compounds 4, and 7-9 displayed the cytotoxicity against human pancreatic cancer cell line, AsPC-1 with IC50 values ranging from 3.45 to 29.69 µM, and 7 and 8 also showed the antiviral activity against the coronavirus (HCoV-OC43) with IC50 values of 13.33 and 6.65 µM, respectively.

AAV-Containing Exosomes as a Novel Vector for Improved Gene Delivery to Lung Cancer Cells.

Lung carcinoma is the most common type of cancer and the leading cause of cancer-related death worldwide. Among the numerous therapeutic strategies for the treatment of lung cancer, adeno-associated virus (AAV)-mediated gene transfer has been demonstrated to have the potential to effectively suppress tumor growth or reverse the progression of the disease in a number of preclinical studies. AAV vector has a safety profile; however, the relatively low delivery efficacy to particular subtypes of lung carcinoma has limited its prospective clinical translation. Exosomes are nanosized extracellular vesicles secreted from nearly all known cell types. Exosomes have a membrane-enclosed structure carrying a range of cargo molecules for efficient intercellular transfer of functional entities, thus are considered as a superior vector for drug delivery. In the present study, we developed a novel strategy to produce and purify AAV-containing exosomes (AAVExo) from AAV-packaging HEK 293T cells. The cellular uptake capacity of exosomes assisted and enhanced AAV entry into cells and protected AAV from antibody neutralization, which was a serious challenge for AAV in vivo application. We tested a list of lung cancer cell lines representing non-small-cell lung cancer and small-cell lung cancer and found that AAVExo apparently improved the gene transfer efficiency compared to conventional AAV vector. Our in vitro results were supported in vivo in a lung cancer xenograft rodent model. Additionally, we evaluated the gene delivery efficiency in the presence of neutralizing antibody on lung cancer cells. The results demonstrated that AAVExo-mediated gene transfer was not impacted, while the AAV vectors were significantly blocked by the neutralizing antibody. Taken together, we established an efficient methodology for AAVExo purification, and the purified AAVExo largely enhanced gene delivery to lung cancer cells with remarkable resistance to antibody neutralization.

Aberrant PD-1 ligand expression contributes to the myocardial inflammatory injury caused by Coxsackievirus B infection.

Coxsackievirus group B (CVB) is considered as one of the most common pathogens of human viral myocarditis. CVB-induced myocarditis is mainly characterized by the persistence of the virus infection and immune-mediated inflammatory injury. Costimulatory signals are crucial for the activation of adaptive immunity. Our data reveal that the CVB type 3 (CVB3) infection altered the expression profile of costimulatory molecules in host cells. CVB3 infection caused the decrease of PD-1 ligand expression, partially due to the cleavage of AU-rich element binding protein AUF1 by the viral protease 3Cpro, leading to the exacerbated inflammatory injury of the myocardium. Moreover, systemic PD-L1 treatment, which augmented the apoptosis of proliferating lymphocytes, alleviated myocardial inflammatory injury. Our findings suggest that PD1-pathway can be a potential immunologic therapeutic target for CVB-induced myocarditis.