An engineered influenza virus to deliver antigens for lung cancer vaccination.

The development of cancer neoantigen vaccines that prime the anti-tumor immune responses has been hindered in part by challenges in delivery of neoantigens to the tumor. Here, using the model antigen ovalbumin (OVA) in a melanoma model, we demonstrate a chimeric antigenic peptide influenza virus (CAP-Flu) system for delivery of antigenic peptides bound to influenza A virus (IAV) to the lung. We conjugated attenuated IAVs with the innate immunostimulatory agent CpG and, after intranasal administration to the mouse lung, observed increased immune cell infiltration to the tumor. OVA was then covalently displayed on IAV-CPG using click chemistry. Vaccination with this construct yielded robust antigen uptake by dendritic cells, a specific immune cell response and a significant increase in tumor-infiltrating lymphocytes compared to peptides alone. Lastly, we engineered the IAV to express anti-PD1-L1 nanobodies that further enhanced regression of lung metastases and prolonged mouse survival after rechallenge. Engineered IAVs can be equipped with any tumor neoantigen of interest to generate lung cancer vaccines.

Proximity-enabled covalent binding of IL-2 to IL-2Rα selectively activates regulatory T cells and suppresses autoimmunity.

Interleukin-2 (IL-2) is a pleiotropic cytokine that orchestrates bidirectional immune responses via regulatory T cells (Tregs) and effector cells, leading to paradoxical consequences. Here, we report a strategy that exploited genetic code expansion-guided incorporation of the latent bioreactive artificial amino acid fluorosulfate-L-tyrosine (FSY) into IL-2 for proximity-enabled covalent binding to IL-2Rα to selectively promote Treg activation. We found that FSY-bearing IL-2 variants, such as L72-FSY, covalently bound to IL-2Rα via sulfur-fluoride exchange when in proximity, resulting in persistent recycling of IL-2 and selectively promoting the expansion of Tregs but not effector cells. Further assessment of L72-FSY-expanded Tregs demonstrated that L72-FSY maintained Tregs in a central memory phenotype without driving terminal differentiation, as demonstrated by simultaneously attenuated expression of lymphocyte activation gene-3 (LAG-3) and enhanced expression of programmed cell death protein-1 (PD-1). Subcutaneous administration of L72-FSY in murine models of pristane-induced lupus and graft-versus-host disease (GvHD) resulted in enhanced and sustained therapeutic efficacy compared with wild-type IL-2 treatment. The efficacy of L72-FSY was further improved by N-terminal PEGylation, which increased its circulatory retention for preferential and sustained effects. This proximity-enabled covalent binding strategy may accelerate the development of pleiotropic cytokines as a new class of immunomodulatory therapies.

Synthesis of Novel Pentacyclic Triterpenoid Derivatives that Induce Apoptosis in Cancer Cells through a ROS-dependent, Mitochondrial-Mediated Pathway.

Betulinic acid (BA) and oleanolic acid (OA) are plant-derived conjugates found in various medicinal plants that have emerged as potential antitumor agents. Herein, a series of novel BA and OA derivatives were synthesized by conjugation with per-O-methylated-ß-cyclodextrin (PM-ß-CD), and their anticancer properties against a panel of three human cancer cell lines were evaluated. Two OA-PM-ß-CD conjugates (48 and 50) were observed to be the most potent conjugates against the three cell lines (MCF-7, BGC-823, and HL-60), with a 15- to 20-fold decrease in the IC50 values (IC50: 6.06-8.47 µM) compared with their parental conjugate (OA). Annexin V-FITC/propidium iodide staining and Western blot analysis revealed that both conjugates induced apoptosis in HL-60 cells. Additionally, in the representative conjugate 48-treated HL-60 cells, a decrease in mitochondrial membrane potential and subsequent release of cytochrome c into the cytosol were observed, indicating the activation of the intrinsic apoptosis pathway. Furthermore, 48 dramatically induced the generation of reactive oxygen species (ROS) in HL-60 cells, and the corresponding effect could be reversed using the ROS scavenger N-acetylcysteine. Collectively, these results suggest that the novel pentacyclic triterpenoid derivatives trigger the intrinsic apoptotic pathways via the ROS-mediated activation of caspase-3 signaling, inducing cell death in human cancer cells.

Kill two birds with one stone: Engineered exosome-mediated delivery of cholesterol modified YY1-siRNA enhances chemoradiotherapy sensitivity of glioblastoma.

Glioblastoma (GBM) is the most malignant tumor of the central nervous system in adults. Irradiation (IR) and temozolomide (TMZ) play an extremely important role in the treatment of GBM. However, major impediments to effective treatment are postoperative tumor recurrence and acquired resistance to chemoradiotherapy. Our previous studies confirm that Yin Yang 1 (YY1) is highly expressed in GBM, whereby it is associated with cell dedifferentiation, survival, and therapeutic resistance. Targeted delivery of small interfering RNA (siRNA) without blood-brain barrier (BBB) restriction for eradication of GBM represents a promising approach for therapeutic interventions. In this study, we utilize the engineering technology to generate T7 peptide-decorated exosome (T7-exo). T7 is a peptide specifically binding to the transferrin receptor. T7-exo shows excellent packaging and protection of cholesterol-modified Cy3-siYY1 while quickly releasing payloads in a cytoplasmic reductive environment. The engineered exosomes T7-siYY1-exo could deliver more effciently to GBM cells both in vitro and in vivo. Notably, in vitro experiments demonstrate that T7-siYY1-exo can enhance chemoradiotherapy sensitivity and reverse therapeutic resistance. Moreover, T7-siYY1-exo and TMZ/IR exert synergistic anti-GBM effect and significantly improves the survival time of GBM bearing mice. Our findings indicate that T7-siYY1-exo may be a potential approach to reverse the chemoradiotherapy resistance in GBM.

Discovery of Pentacyclic Triterpenoid PROTACs as a Class of Effective Hemagglutinin Protein Degraders.

Influenza hemagglutinin that drives viral entry into cells via the membrane fusion process is an up-and-coming antiviral drug target. Herein, we described for the first time the design, synthesis, and biological characteristics of a new class of pentacyclic triterpenoid-based proteolysis targeting chimeras (PROTACs) to enhance the degradation of hemagglutinin target. Among these PROTACs, V3 showed the best degradation effect on the hemagglutinin with a median degradation concentration of 1.44 µM in a ubiquitin and proteasome-dependent manner and broad-spectrum anti-influenza A virus activity but not affected the entry of influenza virus. Moreover, intravenous injection of V3 protected mice against influenza A virus-induced toxic effects. Further diazirine-containing photo-crosslinking mass spectrometric analysis of hemagglutinin complexes indicated crosslinking to Asn15, Thr31, and Asn27, a novel target of hemagglutinin. Taken together, our data revealed that oleanolic acid-based PROTACs could degrade hemagglutinin protein, providing a new direction toward the discovery of potential anti-influenza drugs.

Intelligent Nanodelivery System-Generated 1 O2 Mediates Tumor Vessel Normalization by Activating Endothelial TRPV4-eNOS Signaling.

Tumor microenvironment (TME)-responsive intelligent photodynamic therapy (PDT) systems have attracted increasing interest in anticancer therapy, due to their potential to address significant and unsatisfactory therapeutic issues, such as limited tissue penetration, inevitable normal tissue damage, and excessive impaired vessels. Here, an H2 O2 -triggered intelligent LCL/ZnO PDT nanodelivery system is elaborately designed. LCL/ZnO can selectively regulate tumor-derived endothelial cells (TECs) and specifically kill tumor cells, by responding to different H2 O2 gradients in TECs and tumor cells. The LCL/ZnO is able to normalize tumor vessels, thereby resulting in decreased metastases, and ameliorating the immunosuppressive TME. Further analysis demonstrates that singlet oxygen (1 O2 )-activated transient receptor potential vanilloid-4-endothelial nitric oxide synthase signals generated in TECs by LCL/ZnO induce tumor vascular normalization, which is identified as a novel mechanism contributing to the increased ability of PDT to promote cancer therapy. In conclusion, designing an intelligent PDT nanodelivery system response to the TME, that includes both selective TECs regulation and specific tumor-killing, will facilitate the development of effective interventions for future clinical applications.

LncRNA FTX promotes the malignant progression of colorectal cancer by regulating the miR-214-5p-JAG1 axis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have recently been found to be vital regulators of various cancers, including colorectal cancer (CRC). It has been previously reported that the dysregulated expression of lncRNA Five prime to Xist (FTX) is involved in carcinogenesis. However, the role of lncRNA FTX in the progression of CRC is still unclear. METHODS: Fluorescence in situ hybridization (FISH) was used to detect the expression of lncRNA FTX and miR-214-5p in CRC tissues. Cell Counting Kit-8 assay, transwell assay, wound-healing assay, and proliferation assay were used to explore the function of lncRNA FTX in CRC cells. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and luciferase reporter assay were used to confirm the relationship between lncRNA FTX and miR-214-5p-jagged canonical Notch ligand 1 (JAG1). We further explored the role of lncRNA FTX in vivo using xenograft tumor assay. RESULTS: lncRNA FTX was found to be upregulated in CRC tissues by FISH. The downregulation of endogenous lncRNA FTX expression inhibited CRC cell proliferation, migration, and invasion. Mechanistically, lncRNA FTX sequestered miR-214-5p and thus released its repression on JAG1, driving the malignant progression of CRC. CONCLUSIONS: These findings give rise to a new perspective, the lncRNA FTX-miR-214-5p-JAG1 regulatory axis, in exploring the cancer-promoting mechanism of lncRNA FTX in CRC.

Influenza Virus Precision Diagnosis and Continuous Purification Enabled by Neuraminidase-Resistant Glycopolymer-Coated Microbeads.

Rapid diagnosis and vaccine development are critical to prevent the threat posed by viruses. However, rapid tests, such as colloidal gold assays, yield false-negative results due to the low quantities of viruses; moreover, conventional virus purification, including ultracentrifugation and nanofiltration, is multistep and time-consuming, which limits laboratory research and commercial development of viral vaccines. A rapid virus enrichment and purification technique will improve clinical diagnosis sensitivity and simplify vaccine production. Hence, we developed the surface-glycosylated microbeads (glycobeads) featuring chemically synthetic glycoclusters and reversible linkers to selectively capture the influenza virus. The surface plasmon resonance (SPR) evaluation indicated broad spectrum affinity of S-linked glycosides to various influenza viruses. The magnetic glycobeads were integrated into clinical rapid diagnosis, leading to a 30-fold lower limit of detection. Additionally, the captured viruses can be released under physiological conditions, delivering purified viruses with >50% recovery and without decreasing their native infectivity. Notably, this glycobead platform will facilitate the sensitive detection and continuous one-step purification of the target virus that contributes to future vaccine production.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation*.

There is an urgent need to develop antiviral drugs and alleviate the current COVID-19 pandemic. Herein we report the design and construction of chimeric oligonucleotides comprising a 2'-OMe-modified antisense oligonucleotide and a 5'-phosphorylated 2'-5' poly(A)4 (4A2-5 ) to degrade envelope and spike RNAs of SARS-CoV-2. The oligonucleotide was used for searching and recognizing target viral RNA sequence, and the conjugated 4A2-5 was used for guided RNase L activation to sequence-specifically degrade viral RNAs. Since RNase L can potently cleave single-stranded RNA during innate antiviral response, degradation efficiencies with these chimeras were twice as much as those with only antisense oligonucleotides for both SARS-CoV-2 RNA targets. In pseudovirus infection models, chimera-S4 achieved potent and broad-spectrum inhibition of SARS-CoV-2 and its N501Y and/or ΔH69/ΔV70 mutants, indicating a promising antiviral agent based on the nucleic acid-hydrolysis targeting chimera (NATAC) strategy.