Safety and efficacy of bempedoic acid: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND AND AIMS: Bempedoic Acid (BA) is a novel Lipid-Lowering Therapy (LLT). We performed a systematic review and meta-analysis to assess the efficacy and safety of BA in patients with hypercholesterolemia. METHODS: PubMed, Scopus, and Cochrane library databases were searched for randomised controlled trials evaluating the efficacy and/or safety of BA compared with placebo. Trials investigating dosages other than 180 mg/die were excluded. Major adverse cardiovascular events (MACE) were the primary efficacy endpoint. LDL-cholesterol reduction was the primary laboratory endpoint. Pre-specified safety endpoints included muscle-related adverse events, new-onset diabetes, and gout. The protocol was registered on PROSPERO (temporary ID:399,867). RESULTS: Study search identified 275 deduplicated results. 11 studies, encompassing 18,315 patients (9854 on BA vs 8461 on placebo/no treatment) were included. BA was associated with a reduced risk of MACE (OR 0.86, 95% CI 0.79-0.95), myocardial infarction (OR 0.76, 95% CI 0.64-0.88) and unstable angina (OR 0.69, 95% CI 0.54-0.88) compared to control, over a median follow up of 87 (15-162) weeks. BA was associated with a reduction of LDL-Cholesterol (mean difference [MD]-22.42,95% CI - 24.02% to - 20.82%), total cholesterol (- 16.50%,95% - 19.21% to - 13.79%), Apo-B lipoprotein (- 19.55%, - 22.68% to - 16.42%) and high-sensitivity CRP (- 27.83%, - 31.71% to - 23.96%) at 12 weeks. BA was associated with a higher risk of gout (OR 1.55, 95% CI 1.27-1.90) as compared with placebo. Efficacy on laboratory endpoints was confirmed, with a variable extent, across patients on statin or ezetimibe background therapy. CONCLUSIONS: The improved cholesterol control achieved with BA translates into a reduced risk of MACE, including myocardial infarction and coronary revascularisation. The drug has a satisfactory safety profile except for an increased risk of gout.

Extracellular Vesicles: Emergent and Multiple Sources in Wound Healing Treatment.

Non-healing wound- and tissue-injury are commonly experienced worldwide by the aging population. The persistence of disease commonly leads to tissue infection, resulting in severe clinical complications. In the last decade, extracellular vesicles (EVs) have been considered promising and emergent therapeutic tools to improve the healing processes. Therefore, efforts have been directed to develop a cell-free therapeutic platform based on EV administration to orchestrate tissue repair. EVs derived from different cell types, including fibroblast, epithelial, and immune cells are recruited to the injured sites and in turn take part in scar formation. EVs are nano-sized particles containing a heterogeneous cargo consisting of lipids, proteins, and nucleic acids protected from degradation by their lipid bilayer. Noteworthy, since EVs have natural biocompatibility and low immunogenicity, they represent the ideal therapeutic candidates for regenerative purposes. Indeed, EVs are released by several cell types, and even if they possess unique biological properties, their functional capability can be further improved by engineering their content and functionalizing their surface, allowing a specific cell cargo delivery. Herein, we provide an overview of preclinical data supporting the contribution of EVs in the repair and regenerative processes, focusing on different naïve EV sources, as well as on their engineering, to offer a scalable and low-cost therapeutic option for tissue repair.

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells.

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1ß, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1ß secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities.

Circulating extracellular vesicles derived from tumor endothelial cells hijack the local and systemic anti-tumor immune response: Role of mTOR/G-CSF pathway.

Circulating tumour-derived extracellular vesicles are supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumour-endothelial cells (TEVs) in the expansion of the metastatic bulk. We focus on the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model. 4T1 cells were intravenously injected and exposed to circulating TEVs from day 7. The lung, spleen, and bone marrow (BM) were recovered and analysed. We demonstrated that circulating TEVs boost lung metastasis and angiogenesis. FACS and immunohistochemically analyses revealed a significant enrichment of Ly6G+/F4/80+/CD11b+ cells and Ly6G+/F4/80-/CD11b+ in the lung and in the spleen, while Ly6G+/F4/80-/CD11b+ in the BM, indicating the occurrence of a systemic and local immune suppression. TEV immune suppressive properties were further supported by the increased expression of PD-L1, PD-1, and iNOS in the tumour mass. In addition, in vitro experiments demonstrated an increase of CD11+ cells, PD-L1+ myeloid and cancer cells, upregulation of LAG3, CTLA4 and PD-1 in T-cells, release of ROS and NOS, and impaired T-cell-mediated cytotoxic effect in co-culture of TEVs-preconditioned PBMCs and cancer cells. Granulocyte-colony stimulating factor (G-CSF) level was increased in vivo, and was involved in reshaping the immune response. Mechanistically, we also found that mTOR enriched TEVs support G-CSF release and trigger the phosphorylation of the S6 (Ser235/236) mTOR downstream target. Overall, we provided evidence that circulating TEVs enriched in mTOR supported G-CSF release thereby granting tumour immune suppression and metastasis outgrowth.

Insight on Infections in Diabetic Setting.

The correlation between diabetes mellitus and infectious diseases is widely recognized. DM patients are characterized by the impaired function of the immune system. This translates into the occurrence of a variety of infections, including urinary tract, skin and surgical site infections, pneumonia, tuberculosis, and, more recently, SARS-CoV-2. Hyperglycemia has been identified as a relevant factor contributing to unfavorable outcomes in hospitalized patients including SARS-CoV-2 patients. Several studies have been performed proving that to maintain the proper and stringent monitoring of glycemia, a balanced diet and physical activity is mandatory to reduce the risk of infections and their associated complications. This review is focused on the mechanisms accounting for the increased susceptibility of DM patients to infections, with particular attention to the impact of newly introduced hypoglycemic drugs in sepsis management.

First pilot case-control interventional study using autologous extracellular vesicles to treat chronic venous ulcers unresponsive to conventional treatments.

Current therapeutic approaches for chronic venous ulcers (CVUs) still require evidence of effectiveness. Diverse sources of extracellular vesicles (EVs) have been proposed for tissue regeneration, however the lack of potency tests, to predict in-vivo effectiveness, and a reliable scalability have delayed their clinical application. This study aimed to investigate whether autologous serum-derived EVs (s-EVs), recovered from patients with CVUs, may be a proper therapeutic approach to improve the healing process. A pilot case-control interventional study (CS2/1095/0090491) has been designed and s-EVs recovered from patients. Patient eligibility included two or more distinct chronic lesions in the same limb with 11 months as median persistence of active ulcer before enrollment. Patients were treated three times a week, for 2 weeks. Qualitative CVU analysis demonstrated that s-EVs-treated lesions displayed a higher percentage of granulation tissue compared to the control group (Sham) (s-EVs 3 out of 5: 75-100 % vs Sham: none), further confirmed at day 30. s-EVs-treated lesions also displayed higher sloughy tissue reduction at the end of treatment even increased at day 30. Additionally, s-EV treatment led to a median surface reduction of 151 mm2 compared to 84 mm2 in the Sham group, difference even more evident at day 30 (s-EVs 385 mm2vs Sham 106 mm2p = 0.004). Consistent with the enrichment of transforming growth factor-ß1 in s-EVs, histological analyses showed a regenerative tissue with an increase in microvascular proliferation areas. This study first demonstrates the clinical effectiveness of autologous s-EVs in promoting the healing process of CVUs unresponsive to conventional treatments.

Upregulation of miR145 and miR126 in EVs from Renal Cells Undergoing EMT and Urine of Diabetic Nephropathy Patients.

Diabetic nephropathy (DN) is a severe kidney-related complication of type 1 and type 2 diabetes and the most frequent cause of end-stage kidney disease. Extracellular vesicles (EVs) present in the urine mainly derive from the cells of the nephron, thus representing an interesting tool mirroring the kidney's physiological state. In search of the biomarkers of disease progression, we here assessed a panel of urinary EV miRNAs previously related to DN in type 2 diabetic patients stratified based on proteinuria levels. We found that during DN progression, miR145 and miR126 specifically increased in urinary EVs from diabetic patients together with albuminuria. In vitro, miRNA modulation was assessed in a model of TGF-ß1-induced glomerular damage within a three-dimensional perfusion system, as well as in a model of tubular damage induced by albumin and glucose overload. Both renal tubular cells and podocytes undergoing epithelial to mesenchymal transition released EVs containing increased miR145 and miR126 levels. At the same time, miR126 levels were reduced in EVs released by glomerular endothelial cells. This work highlights a modulation of miR126 and miR145 during the progression of kidney damage in diabetes as biomarkers of epithelial to mesenchymal transition.

Extracellular vesicles in cardiac repair and regeneration: Beyond stem-cell-based approaches.

The adult human heart poorly regenerate after injury due to the low self-renewal capability retained by adult cardiomyocytes. In the last two decades, several clinical studies have reported the ability of stem cells to induce cardiac regeneration. However, low cell integration and survival into the tissue has limited stem-cell-based clinical approaches. More recently, the release of paracrine mediators including extracellular vesicles (EV) has been recognized as the most relevant mechanism driving benefits upon cell-based therapy. In particular, EV have emerged as key mediators of cardiac repair after damage, in terms of reduction of apoptosis, resolution of inflammation and new blood vessel formation. Herein, mechanisms involved in cardiac damage and regeneration, and current applications of EV and their small non-coding RNAs (miRNAs) in regenerative medicine are discussed.

Interleukin-3-Receptor-α in Triple-Negative Breast Cancer (TNBC): An Additional Novel Biomarker of TNBC Aggressiveness and a Therapeutic Target.

Tumour molecular annotation is mandatory for biomarker discovery and personalised approaches, particularly in triple-negative breast cancer (TNBC) lacking effective treatment options. In this study, the interleukin-3 receptor α (IL-3Rα) was investigated as a prognostic biomarker and therapeutic target in TNBC. IL-3Rα expression and patients' clinical and pathological features were retrospectively analysed in 421 TNBC patients. IL-3Rα was expressed in 69% human TNBC samples, and its expression was associated with nodal metastases (p = 0.026) and poor overall survival (hazard ratio = 1.50; 95% CI = 1.01-2.2; p = 0.04). The bioinformatics analysis on the Breast Invasive Carcinoma dataset of The Cancer Genome Atlas (TCGA) proved that IL-3Rα was highly expressed in TNBC compared with luminal breast cancers (p = 0.017, padj = 0.026). Functional studies demonstrated that IL-3Rα activation induced epithelial-to-endothelial and epithelial-to-mesenchymal transition, promoted large blood lacunae and lung metastasis formation, and increased programmed-cell death ligand-1 (PD-L1) in primary tumours and metastases. Based on the TCGA data, IL-3Rα, PD-L1, and EMT coding genes were proposed to discriminate against TNBC aggressiveness (AUC = 0.86 95% CI = 0.82-0.89). Overall, this study identified IL-3Rα as an additional novel biomarker of TNBC aggressiveness and provided the rationale to further investigate its relevance as a therapeutic target.

Tumour Derived Extracellular Vesicles: Challenging Target to Blunt Tumour Immune Evasion.

Control of the immune response is crucial for tumour onset and progression. Tumour cells handle the immune reaction by means of secreted factors and extracellular vesicles (EV). Tumour-derived extracellular vesicles (TEV) play key roles in immune reprogramming by delivering their cargo to different immune cells. Tumour-surrounding tissues also contribute to tumour immune editing and evasion, tumour progression, and drug resistance via locally released TEV. Moreover, the increase in circulating TEV has suggested their underpinning role in tumour dissemination. This review brings together data referring to TEV-driven immune regulation and antitumour immune suppression. Attention was also dedicated to TEV-mediated drug resistance.

IL-3 signalling in the tumour microenvironment shapes the immune response via tumour endothelial cell-derived extracellular vesicles.

Antibody-based anti-cancer therapy is considered a successful approach to impair tumour progression. This study aimed to investigate the clinical impact of targeting the IL-3 signalling in the microenvironment of solid tumours. We intended to investigate whether the IL-3Rα blockade on tumour-derived endothelial cells (TEC) can modulate PD-L1 expression in tumour cells and peripheral blood mononuclear cells (PBMC) to reshape the anti-tumour immune response. Extracellular vesicles released by TEC after IL-3Rα blockade (aTEV) were used as the ultimate effectors of the antibody-based approach, while naive TEC-derived extracellular vesicles (nTEV) served as control. Firstly, we demonstrated that, either directly or indirectly via nTEV, IL-3 controls the expression of its receptor on TEC and PBMC respectively. Moreover, we found that nTEV, moulded by the autocrine secretion of IL-3, increased PD-L1 expression in myeloid cells both in vitro and in vivo. In addition, we found that nTEV-primed PBMC favour tumour cell growth (TEC and MDA-MB-231 cells), whereas PBMC-primed with aTEV still retain their anti-tumour properties. Isolated T-cells pre-conditioned with nTEV or aTEV and co-cultured with TEC or MDA-MB-231 cells have no effects, thereby sustaining the key role of myeloid cells in tumour immune editing. In vivo nTEV, but not aTEV, increased the expression of PD-L1 in primary tumours, lung and liver metastases. Finally, we demonstrated that the enrichment of miR-214 in aTEV impacts on PD-L1 expression in vivo. Overall, these data indicate that an approach based on IL-3Rα blockade in TEC rearranges EV cargo and may reshape the anti-tumour immune response.

Percutaneous Coronary Intervention (PCI) Reprograms Circulating Extracellular Vesicles from ACS Patients Impairing Their Cardio-Protective Properties.

Extracellular vesicles (EVs) are promising therapeutic tools in the treatment of cardiovascular disorders. We have recently shown that EVs from patients with Acute Coronary Syndrome (ACS) undergoing sham pre-conditioning, before percutaneous coronary intervention (PCI) were cardio-protective, while EVs from patients experiencing remote ischemic pre-conditioning (RIPC) failed to induce protection against ischemia/reperfusion Injury (IRI). No data on EVs from ACS patients recovered after PCI are currently available. Therefore, we herein investigated the cardio-protective properties of EVs, collected after PCI from the same patients. EVs recovered from 30 patients randomly assigned (1:1) to RIPC (EV-RIPC) or sham procedures (EV-naive) (NCT02195726) were characterized by TEM, FACS and Western blot analysis and evaluated for their mRNA content. The impact of EVs on hypoxia/reoxygenation damage and IRI, as well as the cardio-protective signaling pathways, were investigated in vitro (HMEC-1 + H9c2 co-culture) and ex vivo (isolated rat heart). Both EV-naive and EV-RIPC failed to drive cardio-protection both in vitro and ex vivo. Consistently, EV treatment failed to activate the canonical cardio-protective pathways. Specifically, PCI reduced the EV-naive Dusp6 mRNA content, found to be crucial for their cardio-protective action, and upregulated some stress- and cell-cycle-related genes in EV-RIPC. We provide the first evidence that in ACS patients, PCI reprograms the EV cargo, impairing EV-naive cardio-protective properties without improving EV-RIPC functional capability.

Human Liver Stem Cell Derived Extracellular Vesicles Alleviate Kidney Fibrosis by Interfering with the ß-Catenin Pathway through miR29b.

Human liver stem-cell-derived extracellular vesicles (HLSC-EVs) exhibit therapeutic properties in various pre-clinical models of kidney injury. We previously reported an overall improvement in kidney function following treatment with HLSC-EVs in a model of aristolochic acid nephropathy (AAN). Here, we provide evidence that HLSC-EVs exert anti-fibrotic effects by interfering with ß-catenin signalling. A mouse model of AAN and an in vitro pro-fibrotic model were used. The ß-catenin mRNA and protein expression, together with the pro-fibrotic markers α-SMA and collagen 1, were evaluated in vivo and in vitro following treatment with HLSC-EVs. Expression and functional analysis of miR29b was performed in vitro following HLSC-EV treatments through loss-of-function experiments. Results showed that expression of ß-catenin was amplified both in vivo and in vitro, and ß-catenin gene silencing in fibroblasts prevented AA-induced up-regulation of pro-fibrotic genes, revealing that ß-catenin is an important factor in fibroblast activation. Treatment with HLSC-EVs caused increased expression of miR29b, which was significantly inhibited in the presence of α-amanitin. The suppression of the miR29b function with a selective inhibitor abolished the anti-fibrotic effects of HLSC-EVs, resulting in the up-regulation of ß-catenin and pro-fibrotic α-Sma and collagen type 1 genes. Together, these data suggest a novel HLSC-EV-dependent regulatory mechanism in which ß-catenin is down regulated by HLSC-EVs-induced miR29b expression.

Extracellular Vesicles: New Tools for Early Diagnosis of Breast and Genitourinary Cancers.

Breast cancers and cancers of the genitourinary tract are the most common malignancies among men and women and are still characterized by high mortality rates. In order to improve the outcomes, early diagnosis is crucial, ideally by applying non-invasive and specific biomarkers. A key role in this field is played by extracellular vesicles (EVs), lipid bilayer-delimited structures shed from the surface of almost all cell types, including cancer cells. Subcellular structures contained in EVs such as nucleic acids, proteins, and lipids can be isolated and exploited as biomarkers, since they directly stem from parental cells. Furthermore, it is becoming even more evident that different body fluids can also serve as sources of EVs for diagnostic purposes. In this review, EV isolation and characterization methods are described. Moreover, the potential contribution of EV cargo for diagnostic discovery purposes is described for each tumor.

Extracellular Vesicles as a Novel Liquid Biopsy-Based Diagnosis for the Central Nervous System, Head and Neck, Lung, and Gastrointestinal Cancers: Current and Future Perspectives.

Early diagnosis, along with innovative treatment options, are crucial to increase the overall survival of cancer patients. In the last decade, extracellular vesicles (EVs) have gained great interest in biomarker discovery. EVs are bilayer lipid membrane limited structures, released by almost all cell types, including cancer cells. The EV cargo, which consists of RNAs, proteins, DNA, and lipids, directly mirrors the cells of origin. EVs can be recovered from several body fluids, including blood, cerebral spinal fluid (CSF), saliva, and Broncho-Alveolar Lavage Fluid (BALF), by non-invasive or minimally invasive approaches, and are therefore proposed as feasible cancer diagnostic tools. In this review, methodologies for EV isolation and characterization and their impact as diagnostics for the central nervous system, head and neck, lung, and gastrointestinal cancers are outlined. For each of these tumours, recent data on the potential clinical applications of the EV's unique cargo, alone or in combination with currently available tumour biomarkers, have been deeply discussed.

Extracellular vesicles from patients with Acute Coronary Syndrome impact on ischemia-reperfusion injury.

The relevance of extracellular vesicles (EV) as mediators of cardiac damage or recovery upon Ischemia Reperfusion Injury (IRI) and Remote Ischemic PreConditioning (RIPC) is controversial. This study aimed to investigate whether serum-derived EV, recovered from patients with Acute Coronary Syndrome (ACS) and subjected to the RIPC or sham procedures, may be a suitable therapeutic approach to prevent IRI during Percutaneous-Coronary-Intervention (PCI). A double-blind, randomized, sham-controlled study (NCT02195726) has been extended, and EV were recovered from 30 patients who were randomly assigned (1:1) to undergo the RIPC- (EV-RIPC) or sham-procedures (EV-naive) before PCI. Patient-derived EV were analyzed by TEM, FACS and western blot. We found that troponin (TnT) was enriched in EV, compared to healthy subjects, regardless of diagnosis. EV-naive induced protection against IRI, both in-vitro and in the rat heart, unlike EV-RIPC. We noticed that EV-naive led to STAT-3 phosphorylation, while EV-RIPC to Erk-1/2 activation in the rat heart. Pre-treatment of the rat heart with specific STAT-3 and Erk-1/2 inhibitors led us to demonstrate that STAT-3 is crucial for EV-naive-mediated protection. In the same model, Erk-1/2 inhibition rescued STAT-3 activation and protection upon EV-RIPC treatment. 84 Human Cardiovascular Disease mRNAs were screened and DUSP6 mRNA was found enriched in patient-derived EV-naive. Indeed, DUSP6 silencing in EV-naive prevented STAT-3 phosphorylation and cardio-protection in the rat heart. This analysis of ACS-patients' EV proved: (i) EV-naive cardio-protective activity and mechanism of action; (ii) the lack of EV-RIPC-mediated cardio-protection; (iii) the properness of the in-vitro assay to predict EV effectiveness in-vivo.

Type 2 diabetes mellitus and sepsis: state of the art, certainties and missing evidence.

Diabetes and sepsis are important causes of morbidity and mortality worldwide, and diabetic patients represent the largest population experiencing post-sepsis complications and rising mortality. Dysregulated immune pathways commonly found in both sepsis and diabetes contribute to worsen the host response in diabetic patients with sepsis. The impact of diabetes on mortality from sepsis is still controversial. Whereas a substantial proportion of severe infections can be attributed to poor glycemic control, treatment with insulin, metformin and thiazolidinediones may be associated with lower incidence and mortality for sepsis. It has been suggested that chronic exposure to high glucose might enhance immune adaptation, leading to reduced mortality rate in septic diabetic patients. On the other hand, higher risk of acute kidney injury has been extensively documented and a suggested lower risk of acute respiratory distress syndrome has been recently questioned. Additional investigations are ongoing to confirm the protective role of some anti-diabetic treatments, the occurrence of acute organ dysfunction, and the risk/benefit of less stringent glycemic control in diabetic patients experiencing sepsis. Based on a MEDLINE/PubMed search from inception to December 31, 2020, the aim of this review is therefore to summarize the strengths and weaknesses of current knowledge on the interplay between diabetes and sepsis.

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.