In Situ Polymerization for Manufacture of Multifunctional Delivery Systems for Transcellular Delivery of Nucleic Acids.

Electrostatic self-assembly between negatively charged nucleic acids and cationic materials is the basis for the formulation of the delivery systems. Nevertheless, structural disintegration occurs because their colloidal stabilities are frequently insufficient in a hostile biological environment. To overcome the sequential biological barriers encountered during transcellular gene delivery, we attempted to use in situ polymerization onto plasmid DNA (pDNA) with a variety of functional monomers, including N-(3-aminopropyl)methacrylate, (aminopropyl)methacrylamide hydrochloride, 1-vinylimidazole, and 2-methacryloyloxyethylphosphorylcholine and N,N'-bis(acryloyl) cystamine. The covalently linked monomers could polymerize into a network structure on top of pDNA, providing excellent structural stability. Additionally, the significant proton buffering capacity of 1-vinylimidazole is expected to aid in the release of pDNA payloads from acidic and digestive endolysosomes. In addition, the redox-mediated cleavage of the disulfide bond in N,N'-bis(acryloyl)cystamine allows for the selective cleavage of the covalently linked network in the cytosolic microenvironment. This is due to the high intracellular level of glutathione, which promotes the liberation of pDNA payloads in the cell interiors. The proposed polymerization strategies resulted in well-defined nanoscale pDNA delivery systems. Excellent colloidal stabilities were observed, even when incubated in the presence of high concentrations of heparin (10 mg/mL). In contrast, the release of pDNA was confirmed upon incubation in the presence of glutathione, mimicking the intracellular microenvironment. Cell transfection experiments verified their efficient cellular uptake and gene expression activities in the hard-transfected MCF-7 cells. Hence, the polymerization strategy used in the fabrication of covalently linked nonviral gene delivery systems shows promise in creating high-performance gene delivery systems with diverse functions. This could open new avenues in cellular microenvironment engineering.

Gene therapy by virus-like self-spooling toroidal DNA condensates for revascularization of hindlimb ischemia.

Peripheral arterial diseases (PAD) have been reported to be the leading cause for limb amputations, and the current therapeutic strategies including antiplatelet medication or intervene surgery are reported to not clinically benefit the patients with high-grade PAD. To this respect, revascularization based on angiogenetic vascular endothelial growth factor (VEGF) gene therapy was attempted for the potential treatment of critical PAD. Aiming for transcellular delivery of VEGF-encoding plasmid DNA (pDNA), we proposed to elaborate intriguing virus-like DNA condensates, wherein the supercoiled rigid micrometer-scaled plasmid DNA (pDNA) could be regulated in an orderly fashion into well-defined nano-toroids by following a self-spooling process with the aid of cationic block copolymer poly(ethylene glycol)-polylysine at an extraordinary ionic strength (NaCl: 600 mM). Moreover, reversible disulfide crosslinking was proposed between the polylysine segments with the aim of stabilizing these intriguing toroidal condensates. Pertaining to the critical hindlimb ischemia, our proposed toroidal VEGF-encoding pDNA condensates demonstrated high levels of VEGF expression at the dosage sites, which consequently contributed to the neo-vasculature (the particularly abundant formation of micro-vessels in the injected hindlimb), preventing the hindlimb ischemia from causing necrosis at the extremities. Moreover, excellent safety profiles have been demonstrated by our proposed toroidal condensates, as opposed to the apparent immunogenicity of the naked pDNA. Hence, our proposed virus-like DNA condensates herald potentials as gene therapy platform in persistent expressions of the therapeutic proteins, and might consequently be highlighted in the management of a variety of intractable diseases.

A Novel Cuproptosis-Related Gene Signature Predicts Prognosis in Papillary Thyroid Carcinoma Patients.

BACKGROUND: Cuproptosis, a novel form of cell death mediated by protein lipoylation, is intricately linked to mitochondrial metabolism. However, the clinical association of cuproptosis- related genes (CRGs) in thyroid cancer remains unclear. In this study, we performed a systematic investigation on the differential expression and genetic alterations of CRGs in papillary thyroid cancer (PTC) and constructed a CRG signature to predict the prognosis of PTC patients. METHODS: We integrated the data of The Cancer Genome Atlas (TCGA) database and analyzed the expression of 10 CRGs in PTC. CRG signature was constructed using univariate Cox regression and the least absolute shrinkage and selection operator (LASSO) Cox regression. In addition, the signature-related molecular features were validated by a combination of functional enrichment, Cox regression, and immune infiltration analysis. Independent validation cohort and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the expression of differentially expressed CRG (CDKN2A). RESULTS: Thyroid cancer patients could be divided into two subtypes (high and low CRG score groups). We found that the overall survival (OS) of patients was lower in the high CRG score group (HCSG) than in the low CRG score group (LCSG) (P < 0.001). The area under the curve (AUC) values for 3 years, 5 years, and 8 years were 0.872, 0.941, and 0.976, respectively. Cox regression analysis indicated that the CRG score could serve as an independent prognostic indicator for PTC. Functional enrichment analysis indicated that the CRG prognostic signature was also associated with the tumor immune microenvironment. In HCSG, the immune suppression cell score was significantly higher than in LCSG. In addition, we identified the expression of CRG (CDKN2A) by qRT-PCR, and the results aligned with the TCGA database. CONCLUSION: Our CRG signature demonstrates excellent predictive capabilities for the prognosis of PTC patients. CRGs may play an important role in tumorigenesis and could be used to predict the immunotherapy efficacy of PTC.

Chk1 inhibition-induced BRCAness synergizes with olaparib in p53-deficient cancer cells.

Although targeting DNA-damage repair by inhibition of PARP exhibits weak or modest single-agent activity due to the existence of functional BRCA1/2 alleles, PARP inhibitors have been gradually applicable in BRCA-proficient cancers. Checkpoint kinase 1 (Chk1) inhibition selectively disrupts homologous recombination (HR)-mediated DNA repair and confers synthetic lethality in p53-deficient tumors, we therefore aim at expounding the chemopotentiating effects of Chk1 inhibition on PARPi in BRCA-proficient and p53-deficient cancer cells. Initially, BRCA wild-type, p53-null cells including AsPC-1 and H1299 demonstrated innate resistance to PARP inhibitor olaparib compared to BRCA1-mutant, p53-null MDA-MB-436 cells. We quantified the interaction between olaparib and a selective Chk1 inhibitor MK-8776, which produced synergistic effects under sub-IC50 concentrations in p53-depleted AsPC-1 and H1299 cells. Olaparib in combination with MK-8776 showed enhanced antitumor effects through prohibiting proliferation and secondarily inducing apoptosis in two cell lines. Of note, we observed that MK-8776 significantly sensitized cells to olaparib by broad DNA and chromosomal breaks. Mechanistically, MK-8776 abrogated olaparib-induced BRCA1 intranuclear foci formation, MCM7-mediated replication machineries, and ultimately triggered an accumulation of γH2AX, a well-recognized marker of DNA double-strand breaks. Additionally, we established ectopic expression of hotspot mutant p53 in H1299 cells. Introduction of p53R175 H promoted olaparib resistance as single-agent treatment, but the synergy between olaparib and MK-8776 was still achievable and the region of synergy was produced by lower combination concentrations. These data provide insight into how Chk1 inhibition could be effectively targeted and confer sensitivity to olaparib toward p53-deficient and HR-proficient cancers.

Betulinic acid, a major therapeutic triterpene of Celastrus orbiculatus Thunb., acts as a chemosensitizer of gemcitabine by promoting Chk1 degradation.

ETHNOPHARMACOLOGICAL RELEVANCE: Celastrus orbiculatus Thunb., also called as oriental bittersweet vine or climbing spindle berry, a traditional Chinese herbal medicine has been used to treat a spectrum of painful and inflammatory diseases for centuries. Explored for their unique medicinal properties, C.orbiculatus offers additional therapeutic effects on cancerous diseases. The effect of single-agent gemcitabine on survival has not long been encouraging, combination therapies provide patients multiple chances of benefit for improved clinical response. AIMS OF THIS STUDY: This study aims at expounding the chemopotentiating effects and underlying mechanisms of betulinic acid, a primary therapeutic triterpene of C. orbiculatus in combination with gemcitabine chemotherapy. MATERIALS AND METHODS: The preparation of betulinic acid was optimized using ultrasonic-assisted extraction method. Gemcitabine-resistant cell model was established by induction of the cytidine deaminase. MTT, colony formation, EdU incorporation and Annexin V/PI staining assays were used to evaluate cytotoxicity, cell proliferation and apoptosis in BxPC-3 pancreatic cancer cell line and H1299 non-small cell lung carcinoma cell line. Comet assay, metaphase chromosome spread and γH2AX immunostaining were applied for DNA damage assessment. Western blot and co-immunoprecipitation was used to detect the phosphorylation and ubiquitination of Chk1. Mode of action of gemcitabine in combination with betulinic acid was further captured in BxPC-3-derived mouse xenograft model. RESULTS: We noticed that the extraction method had an impact on the thermal stability of C. orbiculatus. Ultrasound-assisted extraction at room temperature in shorter processing time could maximize the overall yields and biological activities of C. orbiculatus. The major constituent was identified as betulinic acid, and the pentacyclic triterpene represented the prominent anticancer activity of C. orbiculatus. Forced expression of cytidine deaminase conferred acquired resistance to gemcitabine, while betulinic acid displayed equivalent cytotoxicity toward gemcitabine-resistant and sensitive cells. A combination therapy of gemcitabine with betulinic acid produced synergistic pharmacologic interaction on cell viability, apoptosis and DNA double-strand breaks. Moreover, betulinic acid abrogated gemcitabine-triggered Chk1 activation by destabilizing Chk1 loading via proteasomal degradation. The combination of gemcitabine and betulinic acid significantly retarded BxPC-3 tumor growth in vivo compared to single-agent gemcitabine treatment alone, accompanied with reduced Chk1 expression. CONCLUSIONS: These data provide evidence that betulinic acid is a potential candidate for chemosensitization as a naturally occurring Chk1 inhibitor and warrants further preclinical evaluation.

Current Progress in the Development of Zika Virus Vaccines.

Zika virus (ZIKV) is an arbovirus first discovered in the Americas. ZIKV infection is insidious based on its mild clinical symptoms observed after infection. In Brazil, after 2015, ZIKV infection broke out on a large scale, and many infected pregnant women gave birth to babies with microcephaly. The teratogenic effects of the virus on the fetus and its effects on nerves and the immune system have attracted great attention. Currently, no specific prophylactics or therapeutics are clinically available to treat ZIKV infection. Development of a safe and effective vaccine is essential to prevent the rise of any potential pandemic. In this review, we summarize the latest research on Zika vaccine development based on different strategies, including DNA vaccines, subunit vaccines, live-attenuated vaccines, virus-vector-based vaccines, inactivated vaccines, virus-like particles (VLPs), mRNA-based vaccines, and others. We anticipate that this review will facilitate further progress toward the development of effective and safe vaccines against ZIKV infection.

An overview of Middle East respiratory syndrome coronavirus vaccines in preclinical studies.

INTRODUCTION: Middle East respiratory syndrome coronavirus (MERS-CoV) causes high mortality in humans. No vaccines are approved for use in humans; therefore, a consistent effort to develop safe and effective MERS vaccines is needed. AREAS COVERED: This review describes the structure of MERS-CoV and the function of its proteins, summarizes MERS vaccine candidates under preclinical study (based on spike and non-spike structural proteins, inactivated virus, and live-attenuated virus), and highlights potential problems that could prevent these vaccines entering clinical trials. It provides guidance for the development of safe and effective MERS-CoV vaccines. EXPERT OPINION: Although many MERS-CoV vaccines have been developed, most remain at the preclinical stage. Some vaccines demonstrate immunogenicity and efficacy in animal models, while others have potential adverse effects or low efficacy against high-dose or divergent virus strains. Novel strategies are needed to design safe and effective MERS vaccines to induce broad-spectrum immune responses and improve protective efficacy against multiple strains of MERS-CoV and MERS-like coronaviruses with pandemic potential. More funds should be invested to move vaccine candidates into human clinical trials.