Head-to-head comparison of relevant cell sources of small extracellular vesicles for cardiac repair: Superiority of embryonic stem cells.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

Creating Designer Engineered Extracellular Vesicles for Diverse Ligand Display, Target Recognition, and Controlled Protein Loading and Delivery.

Efficient and targeted delivery of therapeutic agents remains a bottleneck in modern medicine. Here, biochemical engineering approaches to advance the repurposing of extracellular vesicles (EVs) as drug delivery vehicles are explored. Targeting ligands such as the sugar GalNAc are displayed on the surface of EVs using a HaloTag-fused to a protein anchor that is enriched on engineered EVs. These EVs are successfully targeted to human primary hepatocytes. In addition, the authors are able to decorate EVs with an antibody that recognizes a GLP1 cell surface receptor by using an Fc and Fab region binding moiety fused to an anchor protein, and they show that this improves EV targeting to cells that overexpress the receptor. The authors also use two different protein-engineering approaches to improve the loading of Cre recombinase into the EV lumen and demonstrate that functional Cre protein is delivered into cells in the presence of chloroquine, an endosomal escape enhancer. Lastly, engineered EVs are well tolerated upon intravenous injection into mice without detectable signs of liver toxicity. Collectively, the data show that EVs can be engineered to improve cargo loading and specific cell targeting, which will aid their transformation into tailored drug delivery vehicles.

Oncostatin M-Enriched Small Extracellular Vesicles Derived from Mesenchymal Stem Cells Prevent Isoproterenol-Induced Fibrosis and Enhance Angiogenesis.

Myocardial fibrosis is a pathological hallmark of cardiac dysfunction. Oncostatin M (OSM) is a pleiotropic cytokine that can promote fibrosis in different organs after sustained exposure. However, OSM released by macrophages during cardiac fibrosis suppresses cardiac fibroblast activation by modulating transforming growth factor beta 1 (TGF-ß1) expression and extracellular matrix deposition. Small extracellular vesicles (SEVs) from mesenchymal stromal cells (MSCs) are being investigated to treat myocardial infarction, using different strategies to bolster their therapeutic ability. Here, we generated TERT-immortalized human MSC cell lines (MSC-T) engineered to overexpress two forms of cleavage-resistant OSM fused to CD81TM (OSM-SEVs), which allows the display of the cytokine at the surface of secreted SEVs. The therapeutic potential of OSM-SEVs was assessed in vitro using human cardiac ventricular fibroblasts (HCF-Vs) activated by TGF-ß1. Compared with control SEVs, OSM-loaded SEVs reduced proliferation in HCF-V and blunted telo-collagen expression. When injected intraperitoneally into mice treated with isoproterenol, OSM-loaded SEVs reduced fibrosis, prevented cardiac hypertrophy, and increased angiogenesis. Overall, we demonstrate that the enrichment of functional OSM on the surface of MSC-T-SEVs increases their potency in terms of anti-fibrotic and pro-angiogenic properties, which opens new perspectives for this novel biological product in cell-free-based therapies.

Exosomes, microvesicles, and other extracellular vesicles-a Keystone Symposia report.

Extracellular vesicles (EVs) are small, lipid-bilayer-bound particles released by cells that can contain important bioactive molecules, including lipids, RNAs, and proteins. Once released in the extracellular environment, EVs can act as messengers locally as well as to distant tissues to coordinate tissue homeostasis and systemic responses. There is a growing interest in not only understanding the physiology of EVs as signaling particles but also leveraging them as minimally invasive diagnostic and prognostic biomarkers (e.g., they can be found in biofluids) and drug-delivery vehicles. On October 30-November 2, 2022, researchers in the EV field convened for the Keystone symposium "Exosomes, Microvesicles, and Other Extracellular Vesicles" to discuss developing standardized language and methodology, new data on the basic biology of EVs and potential clinical utility, as well as novel technologies to isolate and characterize EVs.

TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery.

Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles' (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

Subcutaneous delivery of FGF21 mRNA therapy reverses obesity, insulin resistance, and hepatic steatosis in diet-induced obese mice.

Fibroblast growth factor 21 (FGF21) is a promising therapeutic agent for treatment of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). We show that therapeutic levels of FGF21 were achieved following subcutaneous (s.c.) administration of mRNA encoding human FGF21 proteins. The efficacy of mRNA was assessed following 2-weeks repeated s.c. dosing in diet-induced obese (DIO), mice which resulted in marked decreases in body weight, plasma insulin levels, and hepatic steatosis. Pharmacokinetic/pharmacodynamic (PK/PD) modelling of several studies in both lean and DIO mice showed that mRNA encoding human proteins provided improved therapeutic coverage over recombinant dosed proteins in vivo. This study is the first example of s.c. mRNA therapy showing pre-clinical efficacy in a disease-relevant model, thus, showing the potential for this modality in the treatment of chronic diseases, including T2D and NASH.

Engineered Cas9 extracellular vesicles as a novel gene editing tool.

Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

Chemogenetics defines a short-chain fatty acid receptor gut-brain axis.

Volatile small molecules, including the short-chain fatty acids (SCFAs), acetate and propionate, released by the gut microbiota from the catabolism of nondigestible starches, can act in a hormone-like fashion via specific G-protein-coupled receptors (GPCRs). The primary GPCR targets for these SCFAs are FFA2 and FFA3. Using transgenic mice in which FFA2 was replaced by an altered form called a Designer Receptor Exclusively Activated by Designer Drugs (FFA2-DREADD), but in which FFA3 is unaltered, and a newly identified FFA2-DREADD agonist 4-methoxy-3-methyl-benzoic acid (MOMBA), we demonstrate how specific functions of FFA2 and FFA3 define a SCFA-gut-brain axis. Activation of both FFA2/3 in the lumen of the gut stimulates spinal cord activity and activation of gut FFA3 directly regulates sensory afferent neuronal firing. Moreover, we demonstrate that FFA2 and FFA3 are both functionally expressed in dorsal root- and nodose ganglia where they signal through different G proteins and mechanisms to regulate cellular calcium levels. We conclude that FFA2 and FFA3, acting at distinct levels, provide an axis by which SCFAs originating from the gut microbiota can regulate central activity.

Pharmacological treatment with FGF21 strongly improves plasma cholesterol metabolism to reduce atherosclerosis.

AIMS: Fibroblast growth factor (FGF) 21, a key regulator of energy metabolism, is currently evaluated in humans for treatment of type 2 diabetes and non-alcoholic steatohepatitis. However, the effects of FGF21 on cardiovascular benefit, particularly on lipoprotein metabolism in relation to atherogenesis, remain elusive. METHODS AND RESULTS: Here, the role of FGF21 in lipoprotein metabolism in relation to atherosclerosis development was investigated by pharmacological administration of a half-life extended recombinant FGF21 protein to hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model mimicking atherosclerosis initiation and development in humans. FGF21 reduced plasma total cholesterol, explained by a reduction in non-HDL-cholesterol. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into browned WAT, consequently accelerating the clearance of the cholesterol-enriched remnants by the liver. In addition, FGF21 reduced body fat, ameliorated glucose tolerance and markedly reduced hepatic steatosis, related to up-regulated hepatic expression of genes involved in fatty acid oxidation and increased hepatic VLDL-triglyceride secretion. Ultimately, FGF21 largely decreased atherosclerotic lesion area, which was mainly explained by the reduction in non-HDL-cholesterol as shown by linear regression analysis, decreased lesion severity, and increased atherosclerotic plaque stability index. CONCLUSION: FGF21 improves hypercholesterolaemia by accelerating triglyceride-rich lipoprotein turnover as a result of activating BAT and browning of WAT, thereby reducing atherosclerotic lesion severity and increasing atherosclerotic lesion stability index. We have thus provided additional support for the clinical use of FGF21 in the treatment of atherosclerotic cardiovascular disease.

Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.

Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRß transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50-170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications.

Extracellular vesicles for tissue repair and regeneration: Evidence, challenges and opportunities.

Extracellular vesicles (EVs) are biological nanoparticles naturally secreted by cells, acting as delivery vehicles for molecular messages. During the last decade, EVs have been assigned multiple functions that have established their potential as therapeutic mediators for a variety of diseases and conditions. In this review paper, we report on the potential of EVs in tissue repair and regeneration. The regenerative properties that have been associated with EVs are explored, detailing the molecular cargo they carry that is capable of mediating such effects, the signaling cascades triggered in target cells and the functional outcome achieved. EV interactions and biodistribution in vivo that influence their regenerative effects are also described, particularly upon administration in combination with biomaterials. Finally, we review the progress that has been made for the successful implementation of EV regenerative therapies in a clinical setting.

Selection of Fluorescent, Bioluminescent, and Radioactive Tracers to Accurately Reflect Extracellular Vesicle Biodistribution in Vivo.

The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics.

Protease-activated receptor-2 ligands reveal orthosteric and allosteric mechanisms of receptor inhibition.

Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2.

Endosomal escape enhancing compounds facilitate functional delivery of extracellular vesicle cargo.

Aim: Extracellular vesicles (EVs) are desirable delivery vehicles for therapeutic cargoes. We aimed to load EVs with Cre recombinase protein and determine whether functional delivery to cells could be improved by using endosomal escape enhancing compounds. Materials & methods: Overexpressed CreFRB protein was actively loaded into EVs by rapalog-induced dimerization to CD81FKBP, or passively loaded by overexpression in the absence of rapalog. Functional delivery of CreFRB was analysed using a HEK293 Cre reporter cell line in the absence and presence of endosomal escape enhancing compounds. Results: The EVs loaded with CreFRB by both active and passive mechanisms were able to deliver functional CreFRB to recipient cells only in the presence of endosomal escape enhancing compounds chloroquine and UNC10217938A. Conclusion: The use of endosomal escape enhancing compounds in conjunction with EVs loaded with therapeutic cargoes may improve efficacy of future EV based therapeutics.

CETSA beyond Soluble Targets: a Broad Application to Multipass Transmembrane Proteins.

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.

Molecular tuning of farnesoid X receptor partial agonism.

The bile acid-sensing transcription factor farnesoid X receptor (FXR) regulates multiple metabolic processes. Modulation of FXR is desired to overcome several metabolic pathologies but pharmacological administration of full FXR agonists has been plagued by mechanism-based side effects. We have developed a modulator that partially activates FXR in vitro and in mice. Here we report the elucidation of the molecular mechanism that drives partial FXR activation by crystallography- and NMR-based structural biology. Natural and synthetic FXR agonists stabilize formation of an extended helix α11 and the α11-α12 loop upon binding. This strengthens a network of hydrogen bonds, repositions helix α12 and enables co-activator recruitment. Partial agonism in contrast is conferred by a kink in helix α11 that destabilizes the α11-α12 loop, a critical determinant for helix α12 orientation. Thereby, the synthetic partial agonist induces conformational states, capable of recruiting both co-repressors and co-activators leading to an equilibrium of co-activator and co-repressor binding.

Extracellular vesicles induce minimal hepatotoxicity and immunogenicity.

Extracellular vesicles (EVs) mediate cellular communication through the transfer of active biomolecules, raising interest in using them as biological delivery vehicles for therapeutic drugs. For drug delivery applications, it is important to understand the intrinsic safety and toxicity liabilities of EVs. Nanoparticles, including EVs, typically demonstrate significant accumulation in the liver after systemic administration in vivo. We confirmed uptake of EVs derived from Expi293F cells into HepG2 cells and did not detect any signs of hepatotoxicity measured by cell viability, functional secretion of albumin, plasma membrane integrity, and mitochondrial and lysosomal activity even at high exposures of up to 5 × 1010 EVs per mL. Whole genome transcriptome analysis was used to measure potential effects on the gene expression in the recipient HepG2 cells at 24 h following exposure to EVs. Only 0.6% of all genes were found to be differentially expressed displaying less than 2-fold expression change, with genes related to inflammation or toxicity being unaffected. EVs did not trigger any proinflammatory cytokine response in HepG2 cells. However, minor changes were noted in human blood for interleukin (IL)-8, IL-6, and monocyte chemotactic protein 1 (MCP-1). Administration of 5 × 1010 Expi293F-derived EVs to BALB/c mice did not result in any histopathological changes or increases of liver transaminases or cytokine levels, apart from a modest increase in keratinocyte chemoattractant (KC). The absence of any significant toxicity associated with EVs in vitro and in vivo supports the prospective use of EVs for therapeutic applications and for drug delivery.

An Affinity-Based Probe for the Human Adenosine A2A Receptor.

Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A2A receptor (hA2AR) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA2AR ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA2AR. Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA2AR by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA2AR, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.

Rapid isolation and enrichment of extracellular vesicle preparations using anion exchange chromatography.

Extracellular vesicles (EVs) have important roles in physiology, pathology, and more recently have been identified as efficient carriers of therapeutic cargoes. For efficient study of EVs, a single-step, rapid and scalable isolation strategy is necessary. Chromatography techniques are widely used for isolation of biological material for clinical applications and as EVs have a net negative charge, anion exchange chromatography (AIEX) is a strong candidate for column based EV isolation. We isolated EVs by AIEX and compared them to EVs isolated by ultracentrifugation (UC) and tangential flow filtration (TFF). EVs isolated by AIEX had comparable yield, EV marker presence, size and morphology to those isolated by UC and had decreased protein and debris contamination as compared to TFF purified EVs. An improved AIEX protocol allowing for higher flow rates and step elution isolated 2.4*1011 EVs from 1 litre of cell culture supernatant within 3 hours and removed multiple contaminating proteins. Importantly AIEX isolated EVs from different cell lines including HEK293T, H1299, HCT116 and Expi293F cells. The AIEX protocol described here can be used to isolate and enrich intact EVs in a rapid and scalable manner and shows great promise for further use in the field for both research and clinical purposes.

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.