Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

Covid-19: Is the placenta a safe space

Problem: Several large studies have demonstrated that COVID-19 pregnant individuals are at a significant risk for severe disease and adverse pregnancy outcomes. The mechanisms underlying these phenomena remain to be elucidated and are the focus of our project. Although fetal and placental infection is rare, placental abnormalities and adverse pregnancy outcomes associated with placental dysfunction in COVID-19 cases have been widely reported. In particular, placental thrombosis and lesions consistent with maternal vascular malperfusion (MVM) of the placenta are common in individuals with COVID-19. Since thrombotic complications have been associated with COVID-19, it is not surprising that pregnant individuals with COVID- 19 are at risk for placental thrombosis. Method of Study: Placentas were evaluated histologically. Extracellular vesicles were isolated by serial centrifugation. Result(s): Adverse pregnancy outcomes associated with these placental lesions, including hypertensive disorders of pregnancy (gestational hypertension and preeclampsia), small for gestational age (SGA, birthweight < 10th percentile for gestational age), and preterm birth (PTB, < 37 weeks) are significantly increased among pregnant individuals with COVID-19. Placental infection with SARSCoV- 2 is uncommon, but multiple inflammatory and metabolic factors are likely to affect the placenta, including circulating extracellular vesicles (EVs) derived from various organs that have been associated with COVID-19 pathology and disease severity.We have analyzed over 500 placentas from COVID-19 pregnancies and found marked changes in placental morphology, characterized by abnormal maternal and fetal vessels, intervillous thrombi, and fibrin deposition, even in the face of mild or asymptomatic disease. We detected increased levels of small EVs in maternal serum from COVID-19 cases compared to controls and increased levels of mitochondrial DNA in EVs from COVID-19 cases. In in vitro experiments, we found increased oxidative stress in uterine endothelial cells and primary trophoblasts. Syncytialization of trophoblast cells following exposure to EVs from pregnant COVID-19 patients was markedly reduced. RNAseq of trophoblast cells exposed to EVs from pregnant COVID-19 patients revealed disruption of multiple pathways related to mitochondria function, oxidative stress, coagulation defects, and inflammation. Timing of infection during pregnancy (first, second, and third trimester) altered EV size distribution, cargo content, and functional consequences of trophoblast EV exposure. Conclusion(s): Our studies show that COVID-19 infection during pregnancy has profound effects on placenta morphology and function. It remains to be determined what the long-term consequences are on the offspring.

Nanomedicine "New Food for an Old Mouth": Novel Approaches for the Treatment of COVID-19

Coronavirus disease (COVID-19) is an infectious disease caused by coronavirus. Devel-oping specific drugs for inhibiting replication and viral entry is crucial. Several clinical trial studies are underway to evaluate the efficacy of anti-viral drugs for COVID-19 patients. Nanomedicine formulations can present a novel strategy for targeting the virus life cycle. Nano-drug delivery systems can modify the pharmacodynamics and pharmacokinetics properties of anti-viral drugs and reduce their adverse effects. Moreover, nanocarriers can directly exhibit anti-viral effects. A number of nanocarriers have been studied for this purpose, including liposomes, dendrimers, exosomes and decoy nanoparticles (NPs). Among them, decoy NPs have been considered more as nanodecoys can efficiently protect host cells from the infection of SARS-CoV-2. The aim of this review article is to highlight the probable nanomedicine therapeutic strategies to develop anti-viral drug delivery systems for the treatment of COVID-19.Copyright © 2023 Bentham Science Publishers.

Exosomes/EVs: BIOXOME: A NATURAL LIPOSOME MIMETIC AS DRUG DELIVERY SYSTEM

Background & Aim: Liposomes are spherical-shaped vesicles composed of one or more lipid bilayers. The ability of liposomes to encapsulate hydro- or lipophilic drugs allowed these vesicles to become a useful drug delivery system. Natural cell membranes, such as Bioxome, have newly emerged as new source of materials for molecular delivery systems. Bioxome are biocompatible and GMP-compliant liposome-like membrane that can be produced from more than 200 cell types. Bioxome self-assemble, with in-process self-loading capacity and can be loaded with a variety of therapeutic compounds. Once close to the target tissue, Bioxome naturally fuse with the cell membrane and release the inner compound. Orgenesis is interested in evaluating the potential of Bioxome as new drug delivery system for treatment of several diseases, including skin repair, local tumour or COVID19. Methods, Results & Conclusion(s): Bioxome were obtained from adipose- derived Mesenchymal Stem Cells, with a process of organic- solvent lipid extraction, followed by lyophilization and sonication assemblage. During the sonication process, Bioxome were charged or not with several cargos. Size distribution of empty Bioxome was detected by Particle Size Analyzer (NanoSight). Electron Microscopy (EM) was performed to assess Bioxome morphology. Lipid content was evaluated by electrospray ionization system. Dose response in vitro test on human lung fibroblasts treated or not with Bioxome encapsulating a specific cargo (API) against COVID19 were performed. NanoSight analysis showed that nanoparticle size in Bioxome samples ranged between 170+/-50 nm, with a concentration ranging between 109-1010+/-106 particles/mL. EM clearly showed the double phospholipid layers that composes the Bioxome. Stability study demonstrated that Bioxome are stable in size and concentration up to 90 days at +4Cdegree or even at RT. No change in size between encapsulated Bioxome with small size (~340 Da) cargo vs empty Bioxome was observed up to 30 days storage. Lipidomic analysis approach revealed that the yield of lipids and their composition are satisfactory for a therapeutic product using Bioxome. Lastly, in the in vitro model of COVID19, Bioxome encapsulating API effectively saved cells from death (20x vs untreated cells) and at lower doses of API than these of non-encapsulated cargo (0.005 microM vs 0.1 microM). Bioxome seems to be an excellent candidate for liposome mimetic tool as drug delivery system for targeting specific organs and diseases treatment.Copyright © 2023 International Society for Cell & Gene Therapy

Exosomes/EVs: IMMUNOMODULATORY EFFECTS OF AMNITOIC FLUID EXTRACELLULAR VESICLES IN AN IN-VITRO MODEL OF IMMUNE ACTIVATION

Background & Aim: Amniotic fluid (AF)-derived EVs are currently under investigation for use as anti-inflammatory therapeutics in COVID-19 and COVID-19 long haulers. The dysregulation of the immune response induced by SARS-COV-2 is a key driver of both acute COVID-19 induced lung injury and long term COVID-19 sequela. There is a clear need to identify therapeutics that suppress excessive inflammation and reduce immune cell exhaustion to improve patient short term and long-term outcomes. Amniotic fluid (AF)- derived extracellular vesicles (EVs) have previously been shown to deliver anti-inflammatory and immune-modulatory signals to diverse cellular targets. We aimed to test if AF-EVs carry immune-suppressive molecules and can suppress T-cell immune activation and exhaustion in vitro. Methods, Results & Conclusion(s): The AF-EV biologic tested was derived from AF collected from consenting donors during planned, fullterm cesarean sections. AF was centrifuged and filtered to remove cellular debris and create a product containing AF-EVs and soluble extracellular components. Fluorescent EXODOT analysis was performed to demonstrate the presence of EV markers CD9, CD81, ALIX, and immune suppressive molecule PD-L1. T-cell activation/exhaustion was induced in vitro by treating human peripheral blood mononuclear cells with activation agent PHA for 3 days with the addition of AF-EVs or saline control. Immune activation/exhaustion was measured by flow cytometry to determine the expression of PD-1 on CD3+ T-cells. The AF-EV biologic was characterized to contain EVs with positive expression of CD9, CD81, ALIX, and PL-L1. T-cell activation/exhaustion was upregulated in response to PHA and was significantly reduced by 8% in AF-EV treated T-cells compared to saline control (77.7% vs 85.7%, respectively P<0.05). These findings demonstrate that AF-EVs do express PD-L1, a surface marker that has previously been demonstrated to contribute to exosome-mediated immunosuppression. Furthermore, we confirmed in vitro that AF-EVs suppress T-cell activation/ exhaustion in the presence of a T-cell activation agent. COVID-19 long haulers have been described to have upregulated and pro-longed immune activation and T-cell exhaustion, marked by an increase in PD1+ T-cells. Therefore, this finding serves as a starting point for the development of a potential mechanism of action that may describe AF-EV's therapeutic effect in COVID-19 long hauler patients.Copyright © 2023 International Society for Cell & Gene Therapy

Viruses and the risk of lung cancer: prevention and treatment

Lung cancer is the most common cancer in males and the second most common among females both in Europe and worldwide. Moreover, lung cancer is the leading cause of death due to cancer in males. The European region accounts for 23% of total cancer cases and 20% of cancer-related deaths. Relationships have been described between a number of infectious agents and cancers, but our knowledge of the role of viruses, both respiratory and systemic, in the pathogenesis of lung cancer is still rudimentary and has been poorly disseminated. In this chapter, we review the available evidence on the involvement of HPV, Epstein-Barr virus, HIV, cytomegalovirus and measles virus in the epidemiology and pathogenesis of lung cancer.Copyright © ERS 2021.

Treatment of diseases through stem cell bioengineering techniques

The development of stem cell transplantation technology has opened up the possibility of curing many diseases that were difficult to treat in the past. There are ethical issues in the field of widespread clinical use of human embryonic stem cells, and tissue rejection may also occurs after transplantation. One way to solve these problems is to generate specific pluripotent stem cells directly from patient cells to study specific treatments. Induced stem cells refer to a type of cell produced by the reprogramming of human somatic cells into exogenous transcription factors, which are very similar to embryonic stem cells. Both types of cells express human pluripotent factors and embryonic stem cell surface markers, and have the potential to differentiate into 3 germ layers.The induced pluripotent stem cells can be induced to differentiate into different cells under different conditions. At present, stem cell therapy has entered the clinical trial stage in many fields, and this paper discusses the stem cell regenerative medicine in the field of cardiovascular disease, eye disease, and COVID-19. This paper is a review of the current status of stem cell treatment and the challenges it is facing. © 2023 SPIE.

2022 CAS Virtual Annual Meeting

The proceedings contain 63 papers. The topics discussed include: a retrospective study to optimize post-anesthetic recovery time after ambulatory lower limb orthopedic procedures at a tertiary care hospital in Canada;a virtual airway evaluation as good as the real thing?;airway management during in hospital cardiac arrest by a consultant led airway management team during the COVID-19 pandemic: a prospective and retrospective quality assurance project;prevention of cautery induced airway fire using saline filled endotracheal tube cuffs: a study in a trachea airway fire model;smart phone assisted retrograde illumination versus conventional laryngoscope illumination for orotracheal intubation: a prospective comparative trial;time to single lung isolation in massive pulmonary hemorrhage simulation using a novel bronchial blocker and traditional techniques;cannabinoid type 2 receptor activation ameliorates acute lung injury induced systemic inflammation;bleeding in patients with end-stage liver disease undergoing liver transplantation and fibrinogen level: a cohort study;endovascular Vena Cavae occlusion in right anterior mini-thoracoscopic approach for tricuspid valve in patients with previous cardiac surgery;and mesenchymal stem cell extracellular vesicles as a novel, regenerative nanotherapeutic for myocardial infarction: a preclinical systematic review.

Development and Utilization of an at Home Urine Exosome Biomarker Collection Test Kit to Support Urologic Telehealth

INTRODUCTION AND OBJECTIVE: COVID-19 led to paradigm shifts in telemedicine due to patient's fear of office visits and travel avoidance. With widespread cancellation of office visits and reduction of diagnostic biopsy procedures in men with elevated PSAs, the need for a non-invasive/non-DRE At Home Collection Kit for assessing risk of aggressive prostate cancer and to prioritize biopsy procedures became apparent. We adapted the existing ExoDx Prostate (EPI) office liquid biomarker kit into an At Home Collection Kit physician/ patient shared decisions for prostate biopsy. METHOD(S): A 2-stage program for an At Home Collection Testing Kit program for the ExoDx Test was initiated in April 2020 at the onset of the COVID-19 pandemic. The Phase 1 Pilot study (100 patients, 6 sites) was completed in June 2020. Based on the findings in the pilot, the program was streamlined based on feedback from physicians, patients, and office mangers before making it available in Phase 2 to all urologists in the US. The utilization of the At Home Collection Kits have been measured. RESULT(S): Extensive feedback from the pilot program led to improvements and streamlining before the Phase 2 rollout. As of October 31st, 2022, >30% of all the ExoDx Prostate Tests are At Home Collection Kits. EPI Score distributions are identical (mean 28.4 and 29.7), (median 23.0 and 24.7) in home or clinic sample collection respectively (Figure 1). CONCLUSION(S): The COVID-19 pandemic accelerated major shifts to telehealth and increased use of At Home Testing. The ExoDx Prostate (EPI) At Home Collection Kit was successfully developed and employed to help men (>50 years old) with elevated PSAs (2-10 ng/ml) considering initial or repeat diagnostic biopsy but with pandemic-related fears of visiting offices/hospitals or wanting to avoid long distance travel from rural areas. As COVID becomes manageable and clinical practices have opened, some pandemicadopted approaches remain relevant: the ExoDxTM Prostate, (EPI) At Home Collection Kit is one such approach.

Extracellular vesicles as delivery vectors to enhance killing of bacteria and viruses by airway-recruited human neutrophils

Background: Polymorphonuclear neutrophils (PMNs) recruited to the airway lumen in cystic fibrosis (CF) undergo a rapid transcriptional program, resulting in exocytosis of granules and inhibition of bacterial killing. As a result, chronic infection, feed-forward inflammation, and structural tissue damage occur. Because CF airway PMNs are also highly pinocytic, we hypothesized that we could deliver protein- and ribonucleic acid (RNA)-based therapies to modulate their function to benefit patients. We elected to use extracellular vesicles (EVs) as a delivery vector because they are highly customizable, and airway PMNs have previously been shown by our group to process and use their cargo efficiently [1]. Furthermore, our prior work on CF airway PMNs [2] led to identification of the long noncoding RNA MALAT1, the transcription factor Ehf, and the histone deacetylase/long-chain fatty deacylase HDAC11 as potential targets to modulate CF airway PMN dysfunction. Method(s): H441 human club epithelial cells were chosen for EV production because they efficiently communicate with lung-recruited primary human PMNs [1]. Relevant constructs were cloned into an expression plasmid downstream of a constitutive cytomegalovirus or U6 promoter with an additional puromycin selection cassette. EVs were generated in serumdepleted media and purified by differential centrifugation. Quality and concentration of EVs was determined by electron microscopy and nanoparticle tracking analysis and cargo content by western blot (protein) or qualitative reverse transcription polymerase chain reaction (RNA). Enhanced green fluorescent protein and messenger ribonucleic acid (mRNA) were used as controls. To test delivery to primary human PMNs, generated EVs were applied in the apical fluid of an airway transmigration model [2]. PMN activation was assessed by flow cytometry, and bacterial (PA01 and Staphylococcus aureus 8325-4) killing and viral (influenza Avirus [IAV] H1N1/PR/8/34;SARS-CoV-2/Washington) clearance assays were conducted. Result(s): To package protein, we used EV-loading motifs such as the tetraspanin CD63, Basp1 amino acids 1-9, and the palmitoylation signal of Lyn kinase. To load mRNA, a C'D box motif recognized by the RNA-binding protein L7Ae was included in the 3' untranslated region of the expressed RNA, and CD63-L7Ae was co-expressed. Airway-recruited PMNs treated with EVs containing small interfering RNAs against MALAT1 or HDAC11 showed greater ability to clear bacteria. Conversely, PMNs treated with constructs encasing MALAT1 or HDAC11 efficiently cleared IAV and SARSCoV- 2. PMNs expressing Ehf showed greater clearance of bacteria and viruses. Conclusion(s): Our findings suggest mutually exclusive roles of MALAT-1 and HDAC11 in regulating bacterial and viral clearance by airway-recruited PMNs. Expression of Ehf in airway PMNs may be a pathogen-agnostic approach to enhancing clearance by airway-recruited PMNs. Overall, our study brings proof-of-concept data for therapeutic RNA/protein transfer to airway-recruited PMNs in CF and other lung diseases and for use of EVs as a promising method for cargo delivery to these cells. It is our expectation that, by treating the immune compartment of CF airway disease, pathogentherapies, such as antibiotics will be more effective, and epithelial-targeted therapies, such as CFTR modulators, will have greater penetrance into the cell types of interest.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Exosome-Based COVID-19 Vaccine.

Extracellular vesicles (EVs) enable cell-to-cell communication and, by delivering antigens, can stimulate the immune response strongly. Approved in use SARS-CoV-2 vaccine, candidates immunize with the viral spike protein delivered via viral vectors, translated by injected mRNAs, or as a pure protein. Here, we outline a novel methodological approach for generating SARS-CoV-2 vaccine using exosome that delivers antigens from the SARS-CoV-2 structural proteins. Engineered EVs can be loaded with viral antigens, thus acting as antigens presenting EVs, eliciting strong and targeted CD8(+) T cell and B cell, offering a unique approach to vaccine development. Engineered EVs thus portray a safe, adaptable, and effective approach for a virus-free vaccine development.

Opportunities and challenges of COVID-19 therapy using mesenchymal stem cells and their exosomes.

BACKGROUND: Corona Virus Disease 2019 (COVID-19) is a highly contagious, rapidly variable, and dangerous infectious disease. However, no specific and effective treatment for COVID-19 is available until now. The safety and efficacy of mesenchymal stem cells and their exosomes have been well verified in numerous clinical trials. Their immunomodulatory and tissue regeneration capabilities may support them as a prospective therapy for COVID-19 application in the clinic. OBJECTIVE: To focus on the development, pathogenesis and the current treatment status of COVID-19, efficacy and possible immunomodulatory mechanisms of mesenchymal stem cells and their exosomes for COVID-19 so as to provide new insights into the clinical treatment for the disease in the future. METHODS: Articles were searched on PubMed and CNKI with the key words of "SARS-CoV-2, COVID-19, cytokine storm, acute respiratory distress syndrome, mesenchymal stem cells, exosomes, immune regulation, tissue repair” in Chinese and English. Finally, 64 articles were collected for this review. RESULTS AND CONCLUSION: Acute respiratory distress syndrome and acute lung injury caused by cytokine storm are the primary precipitating factors of death in individuals with COVID-19. Mesenchymal stem cells and their exosomes can effectively treat the symptoms of acute respiratory distress syndrome and repair the damaged lung tissue in COVID-19 patients by reducing the cytokine storm and promoting the regeneration of alveolar epithelial cells through the interaction with immune cells and their paracrine effects. All of these investigations confirmed that mesenchymal stem cells and their exosomes can fight the COVID-19 infection, and this might be a promising, safe and effective strategy. However, more preclinical studies and randomized, controlled clinical trials are needed to conduct the biodistribution, metabolic fate, and the potential treatment risks of mesenchymal stem cells and their derived exosomes in vivo to fully exploit their clinical efficacy. (English) [ FROM AUTHOR] 背景：2019 冠状病毒病 (Corona Virus Disease 2019，COVID-19) 的传播性强、变异速度快、且危害较大，目前没有针对 COVID-19 的特异治疗 策略。间充质干细胞及其外泌体的安全性和有效性已在众多临床试验中得到证实，其具有的免疫调节和组织修复能力，可作为COVID-19 前 瞻性疗法的主要应用依据，具有巨大的治疗潜力。 目的：重点阐述 COVID-19 的发生发展、致病机制、治疗现状，以及间充质干细胞与其衍生外泌体治疗 COVID-19 患者的有效性和可能的免疫 调控机制，为该疾病的临床治疗提供更多的理论参考。 方法：通过检索PubMed、中国知网数据库中收录的相关文献，英文搜索词为："SARS-CoV-2，COVID-19，cytokine storm，acute respiratory distress syndrome，mesenchymal stem cells，exosomes，immune regulation，tissue repair”，中文搜索词为："新型冠状病毒，2019 冠状病 毒病，细胞因子风暴，急性呼吸窘迫综合征，间充质干细胞，外泌体，免疫调节，组织修复”，最终对64篇文献进行归纳总结。 结果与结论：由细胞因子风暴所引起的急性呼吸窘迫综合征和急性肺损伤是导致 COVID-19 重症患者出现死亡的主要原因。间充质干细胞及 其外泌体通过与免疫细胞之间的相互作用及其旁分泌效应，降低 COVID-19 患者体内细胞因子风暴同时促进其肺泡上皮细胞再生，可有效治 疗急性呼吸窘迫综合征且能够修复其损伤肺组织，证明是一种能够对抗 COVID-19 感染且安全、有效的治疗策略。不过仍然需要更多的临床 前和随机对照临床试验对间充质干细胞及其外泌体移植后的生物分布、体内代谢命运、潜在风险进行更多的研究，以便于更充分发挥其临 床疗效。 (Chinese) [ FROM AUTHOR] Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo zu zhi gong cheng yan jiu is the property of Chinese Journal of Tissue Engineering Research and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Investigational Use of Mesenchymal Stem/Stromal Cells and Their Secretome as Add-On Therapy in Severe Respiratory Virus Infections: Challenges and Perspectives.

Serious manifestations of respiratory virus infections such as influenza and coronavirus disease 2019 (COVID-19) are associated with a dysregulated immune response and systemic inflammation. Treating the immunological/inflammatory dysfunction with glucocorticoids, Janus kinase inhibitors, and monoclonal antibodies against the interleukin-6 receptor has significantly reduced the risk of respiratory failure and death in hospitalized patients with severe COVID-19, but the proportion of those requiring invasive mechanical ventilation (IMV) and dying because of respiratory failure remains elevated. Treatment of severe influenza-associated pneumonia and acute respiratory distress syndrome (ARDS) with available immunomodulators and anti-inflammatory compounds is still not recommended. New therapies are therefore needed to reduce the use of IMV and the risk of death in hospitalized patients with rapidly increasing oxygen demand and systemic inflammation who do not respond to the current standard of care. This paper provides a critical assessment of the published clinical trials that have tested the investigational use of intravenously administered allogeneic mesenchymal stem/stromal cells (MSCs) and MSC-derived secretome with putative immunomodulatory/antiinflammatory/regenerative properties as add-on therapy to improve the outcome of these patients. Increased survival rates are reported in 5 of 12 placebo-controlled or open-label comparative trials involving patients with severe and critical COVID-19 and in the only study concerning patients with influenza-associated ARDS. Results are encouraging but inconclusive for the following reasons: small number of patients tested in each trial; differences in concomitant treatments and respiratory support; imbalances between study arms; differences in MSC source, MSC-derived product, dosing and starting time of the investigational therapy; insufficient/inappropriate reporting of clinical data. Solutions are proposed for improving the clinical development plan, with the aim of facilitating regulatory approval of the MSC-based investigational therapy for life-threatening respiratory virus infections in the future. Major issues are the absence of a biomarker predicting responsiveness to MSCs and MSC-derived secretome and the lack of pharmacoeconomic evaluations.

Extracellular Vesicles and Their Membranes: Exosomes vs. Virus-Related Particles.

Cells produce nanosized lipid membrane-enclosed vesicles which play important roles in intercellular communication. Interestingly, a certain type of extracellular vesicle, termed exosomes, share physical, chemical, and biological properties with enveloped virus particles. To date, most similarities have been discovered with lentiviral particles, however, other virus species also frequently interact with exosomes. In this review, we will take a closer look at the similarities and differences between exosomes and enveloped viral particles, with a focus on events taking place at the vesicle or virus membrane. Since these structures present an area with an opportunity for interaction with target cells, this is relevant for basic biology as well as any potential research or medical applications.

Exosome-Based Multivalent Vaccine: Achieving Potent Immunization, Broadened Reactivity, and Strong T-Cell Responses with Nanograms of Proteins.

Currently approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have focused solely on the spike protein to provide immunity. The first vaccines were developed rapidly using spike mRNA delivered by lipid nanoparticles but required ultralow-temperature storage and have had limited immunity against variations in spike. Subsequently, protein-based vaccines were developed, which offer broader immunity but require significant time for development and the use of an adjuvant to boost the immune response. Here, exosomes were used to deliver a bivalent protein-based vaccine in which two independent viral proteins were used. Exosomes were engineered to express either SARS-CoV-2 delta spike (Stealth X-Spike [STX-S]) or the more conserved nucleocapsid (Stealth X-Nucleocapsid [STX-N]) protein on the surface. When administered as a single product (STX-S or STX-N) or in combination (STX-S+N), both STX-S and STX-N induced strong immunization with the production of potent humoral and cellular immune responses. Interestingly, these results were obtained with the administration of only nanograms of protein and without an adjuvant. In two independent animal models (mouse and rabbit), the administration of nanograms of the STX-S+N vaccine resulted in increased antibody production, potent neutralizing antibodies with cross-reactivity to other variants of spike, and strong T-cell responses. Importantly, no competition of immune responses was observed, allowing the delivery of nucleocapsid with spike to offer improved SARS-CoV-2 immunity. These data show that the StealthX exosome platform has the enormous potential to revolutionize vaccinology by combining the advantages of mRNA and recombinant protein vaccines into a superior, rapidly generated, low-dose vaccine resulting in potent, broader immunity. IMPORTANCE The pandemic emergency has brought to light the need for a new generation of rapidly developed vaccines that induce longer-lasting, potent, and broader immune responses. While the mRNA vaccines played a critical role during the emergency in reducing SARS-CoV-2 hospitalization rates and deaths, more efficient approaches are needed. A multivalent, protein-based vaccine delivered by exosomes could meet this urgent need due to the high speed of development, manufacturability, and the ability to produce a strong antibody response, with neutralizing antibodies and a strong T-cell response able to broadly combat viral infection with a minimum number of injections.

Exosome and virus infection.

Exosomes are messengers of intercellular communication in monolayer vesicles derived from cells. It affects the pathophysiological process of the body in various diseases, such as tumors, inflammation, and infection. It has been confirmed that exosomes are similar to viruses in biogenesis, and exosome cargo is widely involved in many viruses' replication, transmission, and infection. Simultaneously, virus-associated exosomes can promote immune escape and activate the antiviral immune response of the body, which bidirectionally modulates the immune response. This review focuses on the role of exosomes in HIV, HBV, HCV, and SARS-CoV-2 infection and explores the prospects of exosome development. These insights may be translated into therapeutic measures for viral infections and reduce the disease burden.