63 results on '"Mäger, I."'
Search Results
2. The Aachen MiniHLM - A Miniaturized Heart Lung Machine for Neonates with Congenital Heart Defect
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Arens, J., Schnöring, H., Pfennig, M., Mager, I., Vázquez-Jiménez, J., Schmitz-Rode, T., Steinseifer, U., Magjarevic, Ratko, editor, Dössel, Olaf, editor, and Schlegel, Wolfgang C., editor
- Published
- 2009
- Full Text
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3. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper
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Mateescu, B., Kowal, E., van Balkom, B., Bartel, S., Bhattacharyya, S., Buzá, E., Buck, A., de Candia, P., Chow, F., Das, S., Driedonks, T., Fernández-Messina, L., Haderk, F., Hil, L, A., Jones, J., Van Keuren-Jensen, K., Lai, C., Lässer, C., Liegro, D., I, Lunavat, T., Lorenowicz, M., Maas, S., Mäger, I., Mittelbrunn, M., Momma, S., Mukherjee, K., Nawaz, M., Pegtel, D., Pfaffl, M., Schiffelers, R., Tahara, H., Théry, C., Tosar, J., Wauben, M., Witwer, K., Nolte-'t Hoen, E., Mateescu, B, Kowal, EJ, van Balkom, BW, Bartel, S, Bhattacharyya, SN, Buzá, EI, Buck, AH, de Candia, P, Chow, FW, Das, S, Driedonks, TA, Fernández-Messina, L, Haderk, F, Hil,l AF, Jones, JC, Van Keuren-Jensen, KR, Lai, CP, Lässer, C, Di Liegro, I, Lunavat, TR, Lorenowicz, MJ, Maas, S, Mäger,I, Mittelbrunn, M, Momma, S, Mukherjee, K, Nawaz, M, Pegtel, DM, Pfaffl, MW, Schiffelers, RM, Tahara, H, Théry, C, Tosar, JP, Wauben, MH, Witwer, KW, Nolte-'t Hoen, EN., Mateescu, B., Kowal, E. J. K., van Balkom, B. W. M., Bartel, S., Bhattacharyya, S. N., Buzas, E. I., Buck, A. H., de Candia, P., Chow, F. W. N., Das, S., Driedonks, T. A. P., Fernandez-Messina, L., Haderk, F., Hill, A. F., Jones, J. C., Van Keuren-Jensen, K. R., Lai, C. P., Lasser, C., di Liegro, I., Lunavat, T. R., Lorenowicz, M. J., Maas, S. L. N., Mager, I., Mittelbrunn, M., Momma, S., Mukherjee, K., Nawaz, M., Pegtel, D. M., Pfaffl, M. W., Schiffelers, R. M., Tahara, H., Thery, C., Tosar, J. P., Wauben, M. H. M., Witwer, K. W., Nolte-'t Hoen, E. N. M., dB&C I&I, LS Celbiologie-Algemeen, CCA - Imaging and biomarkers, Amsterdam Neuroscience - Cellular & Molecular Mechanisms, and Pathology
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0301 basic medicine ,Histology ,media_common.quotation_subject ,mRNA ,non-coding RNA ,exosomes ,Biology ,Exosomes ,Article ,03 medical and health sciences ,Quantification ,Settore BIO/10 - Biochimica ,Journal Article ,MRNA ,exosome ,Original Research Article ,ddc:610 ,Function ,lcsh:QH573-671 ,Function (engineering) ,Non-coding RNA ,media_common ,function ,lcsh:Cytology ,Sorting ,RNA ,Extracellular vesicle ,Cell Biology ,Extracellular vesicles ,Data science ,RNA binding protein ,quantification ,RNA binding proteins ,sorting ,Cell biology ,Variety (cybernetics) ,030104 developmental biology ,Signalling ,Position paper ,Functional analysis (psychology) - Abstract
The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data., Journal of Extracellular Vesicles, 6 (1), ISSN:2001-3078
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- 2017
4. Cells release subpopulations of exosomes with distinct molecular and biological properties
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Willms, E, Johansson, H, Mäger, I, Lee, Y, Blomberg, K, Sadik, M, Alaarg, A, Smith, C, Lehtiö, J, El Andaloussi, S, Wood, M, Vader, P, Faculty of Science and Technology, Biomaterials Science and Technology, Sub General Pharmaceutics, and Pharmaceutics
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Mice ,Cell Line, Tumor ,Research Support, Non-U.S. Gov't ,Melanoma, Experimental ,Journal Article ,Animals ,Exosomes ,Article - Abstract
Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics.
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- 2016
5. C9ORF72 and RAB7L1 regulate vesicle traffi cking in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia
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Manzano, R., primary, Aoki, Y., additional, Lee, Y., additional, Dafinca, R., additional, Aoki, M., additional, Douglas, A.G.L., additional, Varela, M.A., additional, Sathyaprakash, C., additional, Scaber, J., additional, Barbagallo, P., additional, Vader, P., additional, Mäger, I., additional, Ezzat, K., additional, Turner, M.R., additional, Ito, N., additional, Gasco, S., additional, Ohbayashi, N., additional, El Andaloussi, S., additional, Taked, S., additional, Fukuda, M., additional, Talbot, K., additional, and Wood, M.J.A., additional
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- 2017
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6. Prediction of Cell-Penetrating Peptides using Artificial Neural Networks
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Dobchev, D. A., Mäger, I., Tulp, I., Karelson, G., Tamm, T., Tamm, K., Jänes, J., Langel, Ülo, Karelson, M., Dobchev, D. A., Mäger, I., Tulp, I., Karelson, G., Tamm, T., Tamm, K., Jänes, J., Langel, Ülo, and Karelson, M.
- Abstract
An investigation of cell-penetrating peptides (CPPs) by using combination of Artificial Neural Networks (ANN) and Principle Component Analysis (PCA) revealed that the penetration capability (penetrating/non-penetrating) of 101 examined peptides can be predicted with accuracy of 80%-100%. The inputs of the ANN are the main characteristics classifying the penetration. These molecular characteristics (descriptors) were calculated for each peptide and they provide bio-chemical insights for the criteria of penetration. Deeper analysis of the PCA results also showed clear clusterization of the peptides according to their molecular features.
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- 2010
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7. Finger beat-to-beat blood pressure responses to successive hand elevations
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Raamat, R., primary, Jagomägi, K., additional, Talts, J., additional, and Mäger, I., additional
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- 2009
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8. Understanding extracellular vesicle biology for the development of bioinspired nanotechnology platforms
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Bonner, SE, Dustin, M, Ferarri, E, Wood, M, Mäger, I, and Carter, D
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Molecular biology ,Biological products--Therapeutic use ,Nanoparticles ,Extracellular vesicles ,Biotechnology - Abstract
Extracellular vesicles (EVs) are membranous nanoparticles released by cells that are important mediators of intercellular communication via transfer of their cargoes to recipient cells. For this reason, EVs have been harnessed as a novel targeted therapeutic delivery platform. However, EV research faces numerous challenges that need to be addressed if their potential as therapeutic devices is to be fully realised. EV heterogeneity is arguably the most crucial challenge to be addressed. EVs differ in size, biogenesis, and composition, and these characteristic differences have been linked to functional differences which may impact EV therapeutic activity. Here, functionally heterogeneous EV populations were identified among size-based and subclonal EV populations. EVs were characterised revealing differences in marker and functional protein expression, including integrins, cell adhesion proteins and EV marker proteins. These differences may explain functional differences between EV subpopulations and could aid selective purification of therapeutically advantageous EVs. Furthermore, the subclonal nature of the analysed EV populations also indicated that EV heterogeneity might be linked to cellular heterogeneity. Separately, changes in EV protein expression were documented among EVs harvested at different time points, highlighting another critical consideration for EV therapeutic development. Advancement of in vivo EV biodistribution and purification techniques was also sought to improve EV therapeutic development and characterisation. While requiring further experimentation, EV DNA barcoding, a technique that labels EVs with synthetic DNA identifiers, showed potential as a novel EV biodistribution and therapeutic siRNA delivery tool. Additionally, Capto Core binding chromatography showed significant improvement over size exclusion chromatography for purification of high concentrations of EVs from high cell density cultures. Overall, this research offers insights into EV heterogeneity and novel techniques to aid therapeutics EV production. It also highlights the importance of understanding EV biology and overcoming the challenges associated with EV research to improve EV therapeutic development.
- Published
- 2023
9. Stress-induced extracellular vesicle and cytoophidium biology in breast cancer
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Kassim, H, Goberdhan, D, Liu, J, and Mäger, I
- Abstract
Cells respond to metabolic stresses by reprogramming gene expression, degrading unneeded proteins and altering post-translational modifications of other proteins. Herein, I reported that breast cancer (BC) cells can adapt to cellular stress by modulating the protein composition and function of extracellular vesicles (EVs) and reorganizing metabolic enzymes into filamentous structures, termed cytoophidia. Glutamine is a non-essential amino acid that activates the mechanistic target of rapamycin complex 1 (mTORC1) pathway. I confirmed that luminal and basal lineages of BC cells differ in their glutamine dependencies, and that EVs may mediate intercellular glutamine symbiosis between the two cell lineages (Chapter 3). Despite the difference in sensitivity towards INK128 (an mTOR kinase inhibitor) between lung and brain metastatic clones of basal BC cells, I demonstrated that, blocking mTOR led to secretion of EVs containing proteins associated with cell migration and activation of epithelial-mesenchymal transition (EMT) (Chapter 4). I further demonstrated that the glutamine dependency was not only variable across BC subtypes but also between lung and brain metastatic clones of basal BC cells. Furthermore, my data shows that inhibition of glutamine consumption by 6-Diazo-5-oxo-L-norleucine (DON) significantly promoted higher abundance and length of glutamine-dependent cytidine triphosphate synthase 1 (CTPS1) and inosine-5’-monophosphate dehydrogenase 1 (IMPDH1) cytoophidia in brain metastatic than lung metastatic clone of basal BC cells, highlighting the variation in glutamine dependency across different metastatic clones of basal BC (Chapter 5). The assemblies of cytoophidia of CTPS and many other metabolic enzymes is a stress adaptation strategy to maintain metabolic homeostasis during nutrient starvation, as I demonstrated by conducting a genetic screen in budding yeast (Chapter 5). In summary, my work suggests that stress-induced alteration of protein composition and function of EVs and reorganization of metabolic enzymes into cytoophidia are strategies for BC cell survival in hostile microenvironment.
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- 2019
10. RNA-based therapeutics for Alzheimer's disease
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Woffindale, C, Wood, M, and Mäger, I
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Neuroscience - Abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterised pathologically by the accumulation of amyloid-β plaques and neurofibrillary tangles within the brain. Currently, there are no disease-modifying therapeutic interventions available. This thesis describes the development and delivery of novel RNA-based therapeutics for AD. Recently, significant advances have occurred in the field of RNA interference (RNAi)-based therapeutics, allowing targeted silencing of disease-relevant genes through the use of short interfering RNAs (siRNAs) or microRNAs (miRNAs). However, development has been impeded by poor penetrance of the blood-brain-barrier (BBB) in vivo. Exosomes, as secreted extracellular vesicles (EVs), may overcome this problem as they naturally deliver RNA between cells and cross the BBB. Furthermore, recent evidence suggests that they may be loaded with exogenous RNA and preferentially targeted to neuronal tissue. Thus, this study investigated EV-mediated delivery of RNAi-based therapeutics shown to regulate AD-relevant genes. A comparison of EV-loading methods revealed that electroporation was confounded by the formation of large siRNA aggregates and indicated that endogenous loading strategies may be preferable. Endogenous loading of RNAi molecules was shown to be enhanced by co-expression with RNA-binding proteins, particularly following inclusion of an acylation domain. These findings highlight the potential for EV-mediated delivery of RNAi-based therapeutics. A combinatorial therapeutic approach is increasingly recognised to hold the most promise for the future of AD. Thus, this study also focused upon the development of novel multi-gene targeting locked nucleic acid-modified antisense oligonucleotides (LNA-ASOs) as a combinatorial approach towards AD therapy. Two multi-gene LNA-ASOs each produced potent silencing of two separate target genes simultaneously, resulting in a significant reduction in amyloid-β production in vitro. A murine-specific multi-gene LNA-ASO was also identified, paving the way for future in vivo studies. These findings suggest that multi-gene targeting LNA-ASOs may represent a promising novel therapeutic strategy for AD. In summary, this thesis has investigated novel strategies for both RNA delivery and multi-gene targeting, demonstrating the potential of RNA-based therapeutics for the treatment of AD.
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- 2015
11. MN11 - C9ORF72 and RAB7L1 regulate vesicle traffi cking in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.
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Manzano, R., Aoki, Y., Lee, Y., Dafinca, R., Aoki, M., Douglas, A.G.L., Varela, M.A., Sathyaprakash, C., Scaber, J., Barbagallo, P., Vader, P., Mäger, I., Ezzat, K., Turner, M.R., Ito, N., Gasco, S., Ohbayashi, N., El Andaloussi, S., Taked, S., and Fukuda, M.
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- *
VESICLES (Cytology) , *AMYOTROPHIC lateral sclerosis , *FRONTOTEMPORAL dementia , *NEUROSCIENCES , *NEUROBLASTOMA - Published
- 2017
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12. Antibody-displaying extracellular vesicles for targeted cancer therapy.
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Wiklander OPB, Mamand DR, Mohammad DK, Zheng W, Jawad Wiklander R, Sych T, Zickler AM, Liang X, Sharma H, Lavado A, Bost J, Roudi S, Corso G, Lennaárd AJ, Abedi-Valugerdi M, Mäger I, Alici E, Sezgin E, Nordin JZ, Gupta D, Görgens A, and El Andaloussi S
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Extracellular vesicles (EVs) function as natural delivery vectors and mediators of biological signals across tissues. Here, by leveraging these functionalities, we show that EVs decorated with an antibody-binding moiety specific for the fragment crystallizable (Fc) domain can be used as a modular delivery system for targeted cancer therapy. The Fc-EVs can be decorated with different types of immunoglobulin G antibody and thus be targeted to virtually any tissue of interest. Following optimization of the engineered EVs by screening Fc-binding and EV-sorting moieties, we show the targeting of EVs to cancer cells displaying the human epidermal receptor 2 or the programmed-death ligand 1, as well as lower tumour burden and extended survival of mice with subcutaneous melanoma tumours when systemically injected with EVs displaying an antibody for the programmed-death ligand 1 and loaded with the chemotherapeutic doxorubicin. EVs with Fc-binding domains may be adapted to display other Fc-fused proteins, bispecific antibodies and antibody-drug conjugates., (© 2024. The Author(s).)
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- 2024
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13. EV-mediated promotion of myogenic differentiation is dependent on dose, collection medium, and isolation method.
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Hanson B, Vorobieva I, Zheng W, Conceição M, Lomonosova Y, Mäger I, Puri PL, El Andaloussi S, Wood MJA, and Roberts TC
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Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. C2C12 murine myoblast differentiation was reduced following treatment with GW4869 or heparin (to inhibit exosome biogenesis and EV uptake, respectively). Conversely, treatment with C2C12 myotube-conditioned medium enhanced myogenic differentiation. Ultrafiltration-size exclusion liquid chromatography (UF-SEC) was used to isolate EVs and non-EV extracellular protein in parallel from C2C12 myoblast- and myotube-conditioned medium. UF-SEC-purified EVs promoted myogenic differentiation at low doses (≤2 × 10
8 particles/mL) and were inhibitory at the highest dose tested (2 × 1011 particles/mL). Conversely, extracellular protein fractions had no effect on myogenic differentiation. While the transfer of muscle-enriched miRNAs (myomiRs) has been proposed to mediate the pro-myogenic effects of EVs, we observed that they are scarce in EVs (e.g., 1 copy of miR-133a-3p per 195 EVs). Furthermore, we observed pro-myogenic effects with undifferentiated myoblast-derived EVs, in which myomiR concentrations are even lower, suggestive of a myomiR-independent mechanism underlying the observed pro-myogenic effects. During these investigations we identified technical factors with profound confounding effects on myogenic differentiation. Specifically, co-purification of insulin (a component of Opti-MEM) in non-EV LC fractions and polymer precipitated EV preparations. These findings provide further evidence that polymer-based precipitation techniques should be avoided in EV research., Competing Interests: M.J.A.W. and S.ELA. are founders, shareholders, and consultants for Evox Therapeutics, a biotech company that aims to commercialize extracellular vesicles., (© 2023 The Authors.)- Published
- 2023
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14. Choroid plexus-derived extracellular vesicles exhibit brain targeting characteristics.
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Pauwels MJ, Xie J, Ceroi A, Balusu S, Castelein J, Van Wonterghem E, Van Imschoot G, Ward A, Menheniott TR, Gustafsson O, Combes F, El Andaloussi S, Sanders NN, Mäger I, Van Hoecke L, and Vandenbroucke RE
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- Brain, Blood-Brain Barrier physiology, Central Nervous System, Choroid Plexus, Extracellular Vesicles
- Abstract
The brain is protected against invading organisms and other unwanted substances by tightly regulated barriers. However, these central nervous system (CNS) barriers impede the delivery of drugs into the brain via the blood circulation and are therefore considered major hurdles in the treatment of neurological disorders. Consequently, there is a high need for efficient delivery systems that are able to cross these strict barriers. While most research focuses on the blood-brain barrier (BBB), the design of drug delivery platforms that are able to cross the blood-cerebrospinal fluid (CSF) barrier, formed by a single layer of choroid plexus epithelial cells, remains a largely unexplored domain. The discovery that extracellular vesicles (EVs) make up a natural mechanism for information transfer between cells and across cell layers, has stimulated interest in their potential use as drug delivery platform. Here, we report that choroid plexus epithelial cell-derived EVs exhibit the capacity to home to the brain after peripheral administration. Moreover, these vesicles are able to functionally deliver cargo into the brain. Our findings underline the therapeutic potential of choroid plexus-derived EVs as a brain drug delivery vehicle via targeting of the blood-CSF interface., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)
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- 2022
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15. Author Correction: GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.
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Dar GH, Mendes CC, Kuan WL, Speciale AA, Conceição M, Görgens A, Uliyakina I, Lobo MJ, Lim WF, El Andaloussi S, Mäger I, Roberts TC, Barker RA, Goberdhan DCI, Wilson C, and Wood MJA
- Published
- 2021
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16. GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.
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Dar GH, Mendes CC, Kuan WL, Speciale AA, Conceição M, Görgens A, Uliyakina I, Lobo MJ, Lim WF, El Andaloussi S, Mäger I, Roberts TC, Barker RA, Goberdhan DCI, Wilson C, and Wood MJA
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- Animals, Cell Line, Tumor, Disease Models, Animal, Drug Delivery Systems methods, Extracellular Vesicles ultrastructure, Glyceraldehyde-3-Phosphate Dehydrogenases genetics, HEK293 Cells, HeLa Cells, Humans, Huntingtin Protein genetics, Huntingtin Protein metabolism, Huntington Disease genetics, Huntington Disease metabolism, Mesenchymal Stem Cells cytology, Mice, Inbred C57BL, Phosphatidylserines metabolism, Protein Binding, RNA, Small Interfering administration & dosage, RNA, Small Interfering genetics, Mice, Brain metabolism, Extracellular Vesicles metabolism, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Mesenchymal Stem Cells metabolism, RNA, Small Interfering metabolism
- Abstract
Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington's disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain., (© 2021. The Author(s).)
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- 2021
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17. Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation.
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Roig-Carles D, Willms E, Fontijn RD, Martinez-Pacheco S, Mäger I, de Vries HE, Hirst M, Sharrack B, Male DK, Hawkes CA, and Romero IA
- Abstract
Blood-brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders.
- Published
- 2021
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18. Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.
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Gupta D, Wiklander OPB, Görgens A, Conceição M, Corso G, Liang X, Seow Y, Balusu S, Feldin U, Bostancioglu B, Jawad R, Mamand DR, Lee YXF, Hean J, Mäger I, Roberts TC, Gustafsson M, Mohammad DK, Sork H, Backlund A, Lundin P, de Fougerolles A, Smith CIE, Wood MJA, Vandenbroucke RE, Nordin JZ, and El-Andaloussi S
- Subjects
- Animals, Cytokines, Inflammation, Mice, Tumor Necrosis Factor-alpha, Extracellular Vesicles, Neuroinflammatory Diseases
- Abstract
Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2021
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19. Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion.
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Sork H, Conceicao M, Corso G, Nordin J, Lee YXF, Krjutskov K, Orzechowski Westholm J, Vader P, Pauwels M, Vandenbroucke RE, Wood MJ, El Andaloussi S, and Mäger I
- Subjects
- Cell Line, HEK293 Cells, Humans, MicroRNAs genetics, Sequence Analysis, RNA methods, Extracellular Vesicles genetics, Extracellular Vesicles physiology, RNA, Small Interfering genetics
- Abstract
The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R
2 = 0.69-0.87), quantitatively an outstanding 96.2-99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.- Published
- 2021
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20. Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle.
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Conceição M, Forcina L, Wiklander OPB, Gupta D, Nordin JZ, Vrellaku B, McClorey G, Mäger I, Gӧrgens A, Lundin P, Musarò A, Wood MJA, Andaloussi SE, and Roberts TC
- Subjects
- Animals, Interleukin-6, Mice, Muscle Fibers, Skeletal, Signal Transduction, Extracellular Vesicles, Muscular Dystrophy, Duchenne
- Abstract
The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 trans-signalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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21. An ALS-linked mutation in TDP-43 disrupts normal protein interactions in the motor neuron response to oxidative stress.
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Feneberg E, Gordon D, Thompson AG, Finelli MJ, Dafinca R, Candalija A, Charles PD, Mäger I, Wood MJ, Fischer R, Kessler BM, Gray E, Turner MR, and Talbot K
- Subjects
- Amyotrophic Lateral Sclerosis genetics, Animals, Cells, Cultured, Embryonic Stem Cells, Endoplasmic Reticulum Chaperone BiP, Humans, Mice, Mice, Transgenic, Mutation, Protein Biosynthesis genetics, RNA, Messenger metabolism, RNA-Binding Proteins metabolism, Transcription, Genetic genetics, Amyotrophic Lateral Sclerosis metabolism, DNA-Binding Proteins genetics, Gene Regulatory Networks, Motor Neurons metabolism, Oxidative Stress, Protein Interaction Maps
- Abstract
TDP-43 pathology is a key feature of amyotrophic lateral sclerosis (ALS), but the mechanisms linking TDP-43 to altered cellular function and neurodegeneration remain unclear. We have recently described a mouse model in which human wild-type or mutant TDP-43 are expressed at low levels and where altered stress granule formation is a robust phenotype of TDP-43
M337V/- expressing cells. In the present study we use this model to investigate the functional connectivity of human TDP-43 in primary motor neurons under resting conditions and in response to oxidative stress. The interactome of human TDP-43WT or TDP-43M337V was compared by mass spectrometry, and gene ontology enrichment analysis identified pathways dysregulated by the M337V mutation. We found that under normal conditions the interactome of human TDP-43WT was enriched for proteins involved in transcription, translation and poly(A)-RNA binding. In response to oxidative stress, TDP-43WT recruits proteins of the endoplasmic reticulum and endosomal-extracellular transport pathways, interactions which are reduced in the presence of the M337V mutation. Specifically, TDP-43M337V impaired protein-protein interactions involved in stress granule formation including reduced binding to the translation initiation factors Poly(A)-binding protein and Eif4a1 and the endoplasmic reticulum chaperone Grp78. The M337V mutation also affected interactions involved in endosomal-extracellular transport and this this was associated with reduced extracellular vesicle secretion in primary motor neurons from TDP-43M337V/- mice and in human iPSCs-derived motor neurons. Taken together, our analysis highlights a TDP-43 interaction network in motor neurons and demonstrates that an ALS associated mutation may alter the interactome to drive aberrant pathways involved in the pathogenesis of ALS., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020. Published by Elsevier Inc.)- Published
- 2020
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22. CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis.
- Author
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Thompson AG, Gray E, Mäger I, Thézénas ML, Charles PD, Talbot K, Fischer R, Kessler BM, Wood M, and Turner MR
- Abstract
Background: Extracellular vesicles (EVs) released by neurons and glia reach the cerebrospinal fluid (CSF). Studying the proteome of CSF-derived EVs offers a novel perspective on the key intracellular processes associated with the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and a potential source from which to develop biomarkers., Methods: CSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatography-tandem mass spectrometry proteomic analysis performed., Results: CSF EV concentration and size distribution did not differ between ALS and control groups, nor between a sub-group of ALS patients with or without an associated hexanucleotide repeat expansion (HRE) in C9orf72 . Univariate proteomic analysis identified downregulation of the pentameric proteasome-like protein Bleomycin hydrolase in ALS patients, whilst Gene Ontology enrichment analysis demonstrated downregulation of proteasome core complex proteins (8/8 proteins, normalized enrichment ratio -1.77, FDR-adjusted p = 0.057) in the ALS group. The sub-group of ALS patients associated with the C9orf72 HRE showed upregulation in Ubiquitin-like modifying-activating protein 1 (UBA1) compared to non- C9orf72 cases., Conclusions: Proteomic analysis of CSF EVs in ALS detects intracellular alterations in protein homeostatic mechanisms, previously only identified in pathological tissues. This supports the wider use of CSF EVs as a source of novel biomarkers reflecting key and potentially druggable pathological intracellular pathway alterations in ALS., Competing Interests: Competing interestsMW has filed patent applications in relation to EVs and is a founder and shareholder in Evox Therapeutics., (© The Author(s) 2020.)
- Published
- 2020
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23. Publisher Correction: A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.
- Author
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de Jong OG, Murphy DE, Mäger I, Willms E, Garcia-Guerra A, Gitz-Francois JJ, Lefferts J, Gupta D, Steenbeek SC, van Rheenen J, El Andaloussi S, Schiffelers RM, Wood MJA, and Vader P
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2020
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24. A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.
- Author
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de Jong OG, Murphy DE, Mäger I, Willms E, Garcia-Guerra A, Gitz-Francois JJ, Lefferts J, Gupta D, Steenbeek SC, van Rheenen J, El Andaloussi S, Schiffelers RM, Wood MJA, and Vader P
- Subjects
- Biological Transport, Cell Communication, Cell Line, Endocytosis genetics, Extracellular Vesicles genetics, Fluorescence, Genes, Reporter genetics, HEK293 Cells, Humans, RNA, Guide, CRISPR-Cas Systems genetics, RNA, Guide, CRISPR-Cas Systems metabolism, RNA, Small Untranslated genetics, CRISPR-Cas Systems, Extracellular Vesicles metabolism, RNA, Small Untranslated metabolism
- Abstract
Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.
- Published
- 2020
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25. MiR-219a-5p Enriched Extracellular Vesicles Induce OPC Differentiation and EAE Improvement More Efficiently Than Liposomes and Polymeric Nanoparticles.
- Author
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Osorio-Querejeta I, Carregal-Romero S, Ayerdi-Izquierdo A, Mäger I, A NL, Wood M, Egimendia A, Betanzos M, Alberro A, Iparraguirre L, Moles L, Llarena I, Möller M, Goñi-de-Cerio F, Bijelic G, Ramos-Cabrer P, Muñoz-Culla M, and Otaegui D
- Abstract
Remyelination is a key aspect in multiple sclerosis pathology and a special effort is being made to promote it. However, there is still no available treatment to regenerate myelin and several strategies are being scrutinized. Myelination is naturally performed by oligodendrocytes and microRNAs have been postulated as a promising tool to induce oligodendrocyte precursor cell differentiation and therefore remyelination. Herein, DSPC liposomes and PLGA nanoparticles were studied for miR-219a-5p encapsulation, release and remyelination promotion. In parallel, they were compared with biologically engineered extracellular vesicles overexpressing miR-219a-5p. Interestingly, extracellular vesicles showed the highest oligodendrocyte precursor cell differentiation levels and were more effective than liposomes and polymeric nanoparticles crossing the blood-brain barrier. Finally, extracellular vesicles were able to improve EAE animal model clinical evolution. Our results indicate that the use of extracellular vesicles as miR-219a-5p delivery system can be a feasible and promising strategy to induce remyelination in multiple sclerosis patients., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results
- Published
- 2020
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26. Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics.
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Coenen-Stass AML, Pauwels MJ, Hanson B, Martin Perez C, Conceição M, Wood MJA, Mäger I, and Roberts TC
- Subjects
- Animals, Cell Line, Culture Media, Conditioned chemistry, Female, Humans, Mice, Muscle Fibers, Skeletal chemistry, Muscle Fibers, Skeletal cytology, Preservation, Biological, RNA Stability, Serum chemistry, Temperature, Extracellular Vesicles genetics, MicroRNAs chemistry, MicroRNAs genetics
- Abstract
Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4ºC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37ºC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from ~1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells.
- Published
- 2019
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27. Therapeutic Potential of Extracellular Vesicles for Demyelinating Diseases; Challenges and Opportunities.
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Osorio-Querejeta I, Alberro A, Muñoz-Culla M, Mäger I, and Otaegui D
- Abstract
Multiple Sclerosis is a demyelinating disease of the central nervous system for which no remyelination therapy is available and alternative strategies are being tested. Extracellular vesicles (EVs) have emerged as players in physiological and pathological processes and are being proposed as therapeutic targets and mediators. More concretely, EVs have shown to be involved in myelination related processes such as axon-oligodendrocyte communication or oligodendrocyte precursor cell migration. In addition, EVs have been shown to carry genetic material and small compounds, and to be able to cross the Blood Brain Barrier. This scenario led scientists to test the ability of EVs as myelin regeneration promoters in demyelinating diseases. In this review we will address the use of EVs as remyelination promoters and the challenges and opportunities of this therapy will be discussed.
- Published
- 2018
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28. Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome.
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Sork H, Corso G, Krjutskov K, Johansson HJ, Nordin JZ, Wiklander OPB, Lee YXF, Westholm JO, Lehtiö J, Wood MJA, Mäger I, and El Andaloussi S
- Subjects
- Cell Line, Chromatography, High Pressure Liquid, HEK293 Cells, Humans, RNA, Small Interfering chemistry, RNA, Small Interfering metabolism, RNA, Small Nuclear chemistry, Sequence Analysis, RNA, Tandem Mass Spectrometry, Extracellular Vesicles metabolism, Proteome analysis, RNA, Small Nuclear metabolism, Transcriptome
- Abstract
Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.
- Published
- 2018
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29. Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression.
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Willms E, Cabañas C, Mäger I, Wood MJA, and Vader P
- Subjects
- Animals, Disease Progression, Humans, Extracellular Vesicles physiology, Neoplasms pathology
- Abstract
Cells release membrane enclosed nano-sized vesicles termed extracellular vesicles (EVs) that function as mediators of intercellular communication by transferring biological information between cells. Tumor-derived EVs have emerged as important mediators in cancer development and progression, mainly through transfer of their bioactive content which can include oncoproteins, oncogenes, chemokine receptors, as well as soluble factors, transcripts of proteins and miRNAs involved in angiogenesis or inflammation. This transfer has been shown to influence the metastatic behavior of primary tumors. Moreover, tumor-derived EVs have been shown to influence distant cellular niches, establishing favorable microenvironments that support growth of disseminated cancer cells upon their arrival at these pre-metastatic niches. It is generally accepted that cells release a number of major EV populations with distinct biophysical properties and biological functions. Exosomes, microvesicles, and apoptotic bodies are EV populations most widely studied and characterized. They are discriminated based primarily on their intracellular origin. However, increasing evidence suggests that even within these EV populations various subpopulations may exist. This heterogeneity introduces an extra level of complexity in the study of EV biology and function. For example, EV subpopulations could have unique roles in the intricate biological processes underlying cancer biology. Here, we discuss current knowledge regarding the role of subpopulations of EVs in cancer development and progression and highlight the relevance of EV heterogeneity. The position of tetraspanins and integrins therein will be highlighted. Since addressing EV heterogeneity has become essential for the EV field, current and novel techniques for isolating EV subpopulations will also be discussed. Further dissection of EV heterogeneity will advance our understanding of the critical roles of EVs in health and disease.
- Published
- 2018
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30. Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography.
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Corso G, Mäger I, Lee Y, Görgens A, Bultema J, Giebel B, Wood MJA, Nordin JZ, and Andaloussi SE
- Subjects
- Animals, Cell Line, Humans, Mice, Proteins metabolism, RNA metabolism, Chemical Fractionation methods, Chromatography, Affinity, Chromatography, Gel methods, Extracellular Vesicles metabolism, Extracellular Vesicles ultrastructure
- Abstract
Extracellular vesicles (EVs) play a pivotal role in cell-to-cell communication and have been shown to take part in several physiological and pathological processes. EVs have traditionally been purified by ultracentrifugation (UC), however UC has limitations, including resulting in, operator-dependant yields, EV aggregation and altered EV morphology, and moreover is time consuming. Here we show that commercially available bind-elute size exclusion chromatography (BE-SEC) columns purify EVs with high yield (recovery ~ 80%) in a time-efficient manner compared to current methodologies. This technique is reproducible and scalable, and surface marker analysis by bead-based flow cytometry revealed highly similar expression signatures compared with UC-purified samples. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. Hence, the BE-SEC based EV purification method represents an important methodological advance likely to facilitate robust and reproducible studies of EV biology and therapeutic application.
- Published
- 2017
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31. Functional Delivery of Lipid-Conjugated siRNA by Extracellular Vesicles.
- Author
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O'Loughlin AJ, Mäger I, de Jong OG, Varela MA, Schiffelers RM, El Andaloussi S, Wood MJA, and Vader P
- Subjects
- Animals, Base Sequence, Biological Transport, Cell Line, Cell Line, Tumor, Cholesterol metabolism, ELAV-Like Protein 1 genetics, ELAV-Like Protein 1 metabolism, Extracellular Vesicles chemistry, Fibroblasts cytology, Fibroblasts metabolism, Gene Expression, HEK293 Cells, Humans, Mice, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Neurons metabolism, Neurons pathology, Permeability, RNA, Small Interfering genetics, Temperature, Cholesterol chemistry, Drug Delivery Systems, ELAV-Like Protein 1 antagonists & inhibitors, Extracellular Vesicles metabolism, Neoplasm Proteins antagonists & inhibitors, RNA, Small Interfering metabolism
- Abstract
Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules, including proteins and RNA, between cells. As a result of their suggested natural capability to functionally deliver RNA, EVs may be harnessed as therapeutic RNA carriers. One major limitation for their translation to therapeutic use is the lack of an efficient, robust, and scalable method to load EVs with RNA molecules of interest. Here, we evaluated and optimized methods to load EVs with cholesterol-conjugated small interfering RNAs (cc-siRNAs) by systematic evaluation of the influence of key parameters, including incubation time, volume, temperature, and EV:cc-siRNA ratio. EV loading under conditions that resulted in the highest siRNA retention percentage, incubating 15 molecules of cc-siRNA per EV at 37°C for 1 hr in 100 μL, facilitated concentration-dependent silencing of human antigen R (HuR), a therapeutic target in cancer, in EV-treated cells. These results may accelerate the development of EV-based therapeutics., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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32. Targeting blood-brain-barrier transcytosis - perspectives for drug delivery.
- Author
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Mäger I, Meyer AH, Li J, Lenter M, Hildebrandt T, Leparc G, and Wood MJA
- Subjects
- Animals, Humans, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Proteomics, Transcytosis drug effects, Biological Transport physiology, Blood-Brain Barrier physiology, Drug Delivery Systems, Transcytosis physiology
- Abstract
Efficient transcytosis across the blood-brain-barrier (BBB) is an important strategy for accessing drug targets within the central nervous system (CNS). Despite extensive research the number of studies reporting successful delivery of macromolecules or macromolecular complexes to the CNS has remained very low. In order to expand current research it is important to know which receptors are selective and abundant on the BBB so that novel CNS-targeting antibodies or other ligands could be developed, targeting those receptors for transcytosis. To do that, we have set up a proteomics- and transcriptomics-based workflow within the COMPACT project (Collaboration on the Optimization of Macromolecular Pharmaceutical Access to Cellular Targets) of the Innovative Medicines Initiative (IMI) of the EU. Here we summarise our overall strategy in endothelial transcytosis research, describe in detail the related challenges, and discuss future perspectives of these studies. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders"., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2017
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33. Methodological Guidelines to Study Extracellular Vesicles.
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Coumans FAW, Brisson AR, Buzas EI, Dignat-George F, Drees EEE, El-Andaloussi S, Emanueli C, Gasecka A, Hendrix A, Hill AF, Lacroix R, Lee Y, van Leeuwen TG, Mackman N, Mäger I, Nolan JP, van der Pol E, Pegtel DM, Sahoo S, Siljander PRM, Sturk G, de Wever O, and Nieuwland R
- Subjects
- Biomarkers blood, Cardiovascular Diseases blood, Cardiovascular Diseases diagnosis, Exosomes metabolism, Flow Cytometry methods, Humans, Blood Specimen Collection methods, Blood Specimen Collection standards, Extracellular Vesicles metabolism
- Abstract
Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research., (© 2017 American Heart Association, Inc.)
- Published
- 2017
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34. C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.
- Author
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Aoki Y, Manzano R, Lee Y, Dafinca R, Aoki M, Douglas AGL, Varela MA, Sathyaprakash C, Scaber J, Barbagallo P, Vader P, Mäger I, Ezzat K, Turner MR, Ito N, Gasco S, Ohbayashi N, El Andaloussi S, Takeda S, Fukuda M, Talbot K, and Wood MJA
- Subjects
- Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis pathology, Animals, Biological Transport, C9orf72 Protein, COS Cells, Cell Line, Chlorocebus aethiops, DNA Repeat Expansion, Fibroblasts drug effects, Fibroblasts pathology, Frontotemporal Dementia genetics, Frontotemporal Dementia pathology, Humans, Introns, Motor Neurons drug effects, Motor Neurons pathology, Neurons drug effects, Neurons pathology, Oligonucleotides, Antisense pharmacology, Pedigree, Pluripotent Stem Cells drug effects, Pluripotent Stem Cells pathology, Proteins genetics, rab GTP-Binding Proteins, rab1 GTP-Binding Proteins genetics, Amyotrophic Lateral Sclerosis metabolism, Frontotemporal Dementia metabolism, Proteins metabolism, rab1 GTP-Binding Proteins metabolism
- Abstract
A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2017
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35. Preparation and Isolation of siRNA-Loaded Extracellular Vesicles.
- Author
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Vader P, Mäger I, Lee Y, Nordin JZ, Andaloussi SE, and Wood MJ
- Subjects
- Cell Line, Humans, Polymerase Chain Reaction, RNA, Small Interfering genetics, RNA, Small Interfering isolation & purification, Cell Fractionation methods, Extracellular Vesicles metabolism, RNA, Small Interfering metabolism
- Abstract
RNA interference (RNAi) has tremendous potential for specific silencing of disease-causing genes. Its clinical usage however critically depends on the development of carrier systems that can transport the RNAi-mediating small interfering RNA (siRNA) molecules to the cytosol of target cells. Recent reports have suggested that extracellular vesicles (EVs) form a natural transport system through which biomolecules, including RNA, is exchanged between cells. Therefore, EVs are increasingly being considered as potential therapeutic siRNA delivery systems.In this chapter we describe a method for preparing siRNA-loaded EVs, including a robust, scalable method to isolate them from cell culture supernatants.
- Published
- 2017
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36. Proteostasis and Diseases of the Motor Unit.
- Author
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Rinaldi C, Mäger I, and Wood MJ
- Abstract
The accumulation in neurons of aberrant protein species, the pathological hallmark of many neurodegenerative diseases, results from a global impairment of key cellular processes governing protein synthesis/degradation and repair mechanisms, also known as the proteostasis network (PN). The growing number of connections between dysfunction of this intricate network of pathways and diseases of the motor unit, where both motor neurons and muscle are primarily affected, has provided momentum to investigate the muscle- and motor neuron-specific response to physiological and pathological stressors and to explore the therapeutic opportunities that manipulation of this process may offer. Furthermore, these diseases offer an unparalleled opportunity to deepen our understanding of the molecular mechanisms behind the intertissue communication and transfer of signals of proteostasis. The most compelling aspect of these investigations is their immediate potential for therapeutic impact: targeting muscle to stem degeneration of the motor unit would represent a dramatic paradigm therapeutic shift for treating these devastating diseases. Here we will review the current state of the art of the research on the alterations of the PN in diseases of the motor unit and its potential to result in effective treatments for these devastating neuromuscular disorders.
- Published
- 2016
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37. Selective release of muscle-specific, extracellular microRNAs during myogenic differentiation.
- Author
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Coenen-Stass AM, Betts CA, Lee YF, Mäger I, Turunen MP, El Andaloussi S, Morgan JE, Wood MJ, and Roberts TC
- Subjects
- Animals, Cell Differentiation genetics, Cell Proliferation genetics, Extracellular Space genetics, Humans, Mice, MicroRNAs blood, Muscle, Skeletal growth & development, Muscle, Skeletal pathology, Muscular Dystrophy, Duchenne blood, Muscular Dystrophy, Duchenne pathology, Myoblasts metabolism, Myoblasts pathology, Organ Specificity, Primary Cell Culture, MicroRNAs genetics, Muscle Development genetics, Muscle, Skeletal metabolism, Muscular Dystrophy, Duchenne genetics
- Abstract
MyomiRs are muscle-specific microRNAs (miRNAs) that regulate myoblast proliferation and differentiation. Extracellular myomiRs (ex-myomiRs) are highly enriched in the serum of Duchenne Muscular Dystrophy (DMD) patients and dystrophic mouse models and consequently have potential as disease biomarkers. The biological significance of miRNAs present in the extracellular space is not currently well understood. Here we demonstrate that ex-myomiR levels are elevated in perinatal muscle development, during the regenerative phase that follows exercise-induced myoinjury, and concomitant with myoblast differentiation in culture. Whereas ex-myomiRs are progressively and specifically released by differentiating human primary myoblasts and C2C12 cultures, chemical induction of apoptosis in C2C12 cells results in indiscriminate miRNA release. The selective release of myomiRs as a consequence of cellular differentiation argues against the idea that they are solely waste products of muscle breakdown, and suggests they may serve a biological function in specific physiological contexts. Ex-myomiRs in culture supernatant and serum are predominantly non-vesicular, and their release is independent of ceramide-mediated vesicle secretion. Furthermore, ex-myomiRs levels are reduced in aged dystrophic mice, likely as a consequence of chronic muscle wasting. In conclusion, we show that myomiR release accompanies periods of myogenic differentiation in cell culture and in vivo. Serum myomiR abundance is therefore a function of the regenerative/degenerative status of the muscle, overall muscle mass, and tissue expression levels. These findings have implications for the use of ex-myomiRs as biomarkers for DMD disease progression and monitoring response to therapy., (© The Author 2016. Published by Oxford University Press.)
- Published
- 2016
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38. Extracellular vesicles in neurodegenerative disease - pathogenesis to biomarkers.
- Author
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Thompson AG, Gray E, Heman-Ackah SM, Mäger I, Talbot K, Andaloussi SE, Wood MJ, and Turner MR
- Subjects
- Humans, Neurodegenerative Diseases blood, Neurodegenerative Diseases cerebrospinal fluid, Biomarkers, Extracellular Vesicles, Neurodegenerative Diseases diagnosis
- Abstract
To develop effective disease-modifying therapies for neurodegenerative diseases, reliable markers of diagnosis, disease activity and progression are a research priority. The fact that neurodegenerative pathology is primarily associated with distinct subsets of cells in discrete areas of the CNS makes the identification of relevant biomarker molecules a challenge. The trafficking of macromolecules from the CNS to the cerebrospinal fluid and blood, mediated by extracellular vesicles (EVs), presents a promising source of CNS-specific biomarkers. EVs are released by almost all cell types and carry a cargo of protein and nucleic acid that varies according to the cell of origin. EV output changes with cell status and reflects intracellular events, so surface marker expression can be used to identify the cell type from which EVs originate. EVs could, therefore, provide an enriched pool of information about core neuropathogenic, cell-specific processes. This Review examines the current knowledge of the biology and function of EVs, discusses the evidence for their involvement in the pathogenesis of neurodegenerative diseases, and considers their potential as biomarkers of disease.
- Published
- 2016
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39. Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.
- Author
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Baranyai T, Herczeg K, Onódi Z, Voszka I, Módos K, Marton N, Nagy G, Mäger I, Wood MJ, El Andaloussi S, Pálinkás Z, Kumar V, Nagy P, Kittel Á, Buzás EI, Ferdinandy P, and Giricz Z
- Subjects
- Animals, Male, Plasma chemistry, Rats, Wistar, Chromatography, Gel methods, Exosomes chemistry, Plasma cytology, Ultracentrifugation methods
- Abstract
Background: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described., Aim: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC)., Methods and Results: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin., Conclusion: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.
- Published
- 2015
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40. Therapeutic Potential of Multipotent Mesenchymal Stromal Cells and Their Extracellular Vesicles.
- Author
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Heldring N, Mäger I, Wood MJ, Le Blanc K, and Andaloussi SE
- Subjects
- Animals, Cell Differentiation, Humans, Immunotherapy, Mesenchymal Stem Cells physiology, MicroRNAs genetics, Paracrine Communication, Extracellular Vesicles physiology, Mesenchymal Stem Cell Transplantation
- Abstract
The therapeutic potential of mesenchymal stromal cells (MSCs) is evident by the number of new and ongoing trials targeting an impressive variety of conditions. In bone and cartilage repair, MSCs are expected to replace the damaged tissue, while in other therapies they modulate a therapeutic response by the secretion of bioactive molecules. MSCs possess a phenotypic plasticity and harbor an arsenal of bioactive molecules that can be released upon sensing signals in the local milieu either directly or packaged in extracellular vesicles (EVs). The reported paracrine effects comprise many of the important functions of MSCs, including supporting hematopoietic stem cells in the bone marrow, promoting angiogenesis, and modulating the immune system. The major drawback in MSC therapy is the incomplete understanding of cell fate following systemic administration as well as the mechanisms by which these cells correct disease. In this review we discuss what is known about MSC engraftment, hemocompatibility, and immunomodulation, as well as the potential of bringing the MSC-EV field toward a clinical translation.
- Published
- 2015
- Full Text
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41. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles.
- Author
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Li J, Lee Y, Johansson HJ, Mäger I, Vader P, Nordin JZ, Wiklander OP, Lehtiö J, Wood MJ, and Andaloussi SE
- Abstract
Extracellular vesicles (EVs) play a significant role in cell-cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.
- Published
- 2015
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42. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.
- Author
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Nordin JZ, Lee Y, Vader P, Mäger I, Johansson HJ, Heusermann W, Wiklander OP, Hällbrink M, Seow Y, Bultema JJ, Gilthorpe J, Davies T, Fairchild PJ, Gabrielsson S, Meisner-Kober NC, Lehtiö J, Smith CI, Wood MJ, and El Andaloussi S
- Subjects
- Chromatography, Gel, HEK293 Cells, Humans, Ultrafiltration, Cell-Derived Microparticles chemistry, Cell-Derived Microparticles ultrastructure
- Abstract
Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs., From the Clinical Editor: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
43. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting.
- Author
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Wiklander OP, Nordin JZ, O'Loughlin A, Gustafsson Y, Corso G, Mäger I, Vader P, Lee Y, Sork H, Seow Y, Heldring N, Alvarez-Erviti L, Smith CI, Le Blanc K, Macchiarini P, Jungebluth P, Wood MJ, and Andaloussi SE
- Abstract
Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.
- Published
- 2015
- Full Text
- View/download PDF
44. Extracellular microRNAs in Membrane Vesicles and Non-vesicular Carriers.
- Author
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Coenen-Stass AML, Mäger I, and Wood MJA
- Abstract
Great excitement has surrounded the finding that small RNAs are stable in various biofluids and carry specific signatures reflecting physiological and pathological states. In this chapter, we briefly describe the impact of this revolutionary discovery and introduce different subclasses of circulating microRNAs based on their mode of transport. Subsequently, we review the current state-of-the art knowledge on microRNA selection for export, secretion and possible uptake mechanisms and their potential function in circulation. Furthermore, we give an overview on the possible use of cell-free microRNAs as biomarkers and as therapeutic targets. Overall, we aim to highlight open questions and address some of the pitfalls of current extracellular RNA research.
- Published
- 2015
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45. From gut to brain: bioencapsulated therapeutic protein reduces amyloid load upon oral delivery.
- Author
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Mäger I, Roberts TC, Wood MJ, and El Andaloussi S
- Subjects
- Animals, Female, Humans, Male, Tripeptidyl-Peptidase 1, Alzheimer Disease drug therapy, Aminopeptidases metabolism, Apolipoproteins E metabolism, Blood-Brain Barrier metabolism, Blood-Retinal Barrier metabolism, Chloroplasts metabolism, Cholera Toxin metabolism, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases metabolism, Myelin Basic Protein metabolism, Neuronal Ceroid-Lipofuscinoses drug therapy, Neuronal Ceroid-Lipofuscinoses physiopathology, Peptides administration & dosage, Plaque, Amyloid drug therapy, Serine Proteases metabolism
- Published
- 2014
- Full Text
- View/download PDF
46. Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples.
- Author
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Krõlov K, Frolova J, Tudoran O, Suhorutsenko J, Lehto T, Sibul H, Mäger I, Laanpere M, Tulp I, and Langel Ü
- Subjects
- Chlamydia Infections genetics, Chlamydia trachomatis enzymology, DNA, Bacterial analysis, DNA-Directed DNA Polymerase chemistry, Diacylglycerol Cholinephosphotransferase genetics, Female, Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) genetics, Humans, Molecular Diagnostic Techniques methods, Nucleic Acid Amplification Techniques, Polymerase Chain Reaction, Recombinases chemistry, Sensitivity and Specificity, Sexually Transmitted Diseases, Bacterial genetics, Chlamydia Infections diagnosis, Chlamydia Infections urine, Chlamydia trachomatis genetics, Sexually Transmitted Diseases, Bacterial diagnosis, Sexually Transmitted Diseases, Bacterial urine
- Abstract
Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%-100%) and a sensitivity of 83% (95% CI, 51%-97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings., (Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
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47. Extracellular vesicles: biology and emerging therapeutic opportunities.
- Author
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EL Andaloussi S, Mäger I, Breakefield XO, and Wood MJ
- Subjects
- Cell-Derived Microparticles, Drug Delivery Systems, Exosomes, Extracellular Space drug effects, Humans, Translational Research, Biomedical, Cell Communication drug effects, Cell Communication physiology, Drug Therapy, Extracellular Space physiology
- Abstract
Within the past decade, extracellular vesicles have emerged as important mediators of intercellular communication, being involved in the transmission of biological signals between cells in both prokaryotes and higher eukaryotes to regulate a diverse range of biological processes. In addition, pathophysiological roles for extracellular vesicles are beginning to be recognized in diseases including cancer, infectious diseases and neurodegenerative disorders, highlighting potential novel targets for therapeutic intervention. Moreover, both unmodified and engineered extracellular vesicles are likely to have applications in macromolecular drug delivery. Here, we review recent progress in understanding extracellular vesicle biology and the role of extracellular vesicles in disease, discuss emerging therapeutic opportunities and consider the associated challenges.
- Published
- 2013
- Full Text
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48. Exosomes for targeted siRNA delivery across biological barriers.
- Author
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El Andaloussi S, Lakhal S, Mäger I, and Wood MJ
- Subjects
- Animals, Biological Transport, Blood-Brain Barrier metabolism, Cell Membrane metabolism, Drug Delivery Systems, Drug Design, Gene Expression Regulation, Gene Transfer Techniques, Humans, Mast Cells metabolism, Exosomes metabolism, Oligonucleotides administration & dosage, RNA, Small Interfering administration & dosage
- Abstract
Using oligonucleotide-based drugs to modulate gene expression has opened a new avenue for drug discovery. In particular small interfering RNAs (siRNAs) are being rapidly recognized as promising therapeutic tools, but their poor bioavailability limits the full realization of their clinical potential. In recent years, cumulating evidence has emerged for the role of membrane vesicles, secreted by most cells and found in all body fluids, as key mediators of information transmission between cells. Importantly, a sub-group of these termed exosomes, have recently been shown to contain various RNA species and to mediate their horizontal transfer to neighbouring- or distant recipient cells. Here, we provide a brief overview on membrane vesicles and their role in exchange of genetic information. We also describe how these natural carriers of genetic material can be harnessed to overcome the obstacle of poor delivery and allow efficient systemic delivery of exogenous siRNA across biological barriers such as the blood-brain barrier., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
49. PepFect14 peptide vector for efficient gene delivery in cell cultures.
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Veiman KL, Mäger I, Ezzat K, Margus H, Lehto T, Langel K, Kurrikoff K, Arukuusk P, Suhorutšenko J, Padari K, Pooga M, Lehto T, and Langel Ü
- Subjects
- Animals, CHO Cells, Cell Culture Techniques, Cell-Penetrating Peptides metabolism, Cricetinae, DNA genetics, Endocytosis genetics, Genetic Vectors metabolism, HEK293 Cells, Humans, Lipopeptides metabolism, Nanoparticles administration & dosage, Oligonucleotides administration & dosage, Oligonucleotides genetics, Oligonucleotides metabolism, Particle Size, Plasmids genetics, Plasmids metabolism, Transfection methods, Cell-Penetrating Peptides administration & dosage, Cell-Penetrating Peptides genetics, Gene Transfer Techniques, Genetic Vectors administration & dosage, Genetic Vectors genetics, Lipopeptides administration & dosage, Lipopeptides genetics
- Abstract
The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.
- Published
- 2013
- Full Text
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50. Use of cell-penetrating-peptides in oligonucleotide splice switching therapy.
- Author
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El Andaloussi SA, Hammond SM, Mäger I, and Wood MJ
- Subjects
- Cell Membrane metabolism, Gene Expression Regulation, Humans, MicroRNAs genetics, MicroRNAs metabolism, Molecular Targeted Therapy, Nanoparticles chemistry, Nanoparticles therapeutic use, RNA Precursors genetics, RNA Precursors metabolism, RNA, Double-Stranded genetics, RNA, Double-Stranded therapeutic use, Alternative Splicing genetics, Cell-Penetrating Peptides genetics, Cell-Penetrating Peptides metabolism, Cell-Penetrating Peptides therapeutic use, Muscular Atrophy, Spinal genetics, Muscular Atrophy, Spinal therapy, Muscular Dystrophy, Duchenne genetics, Muscular Dystrophy, Duchenne therapy, Oligonucleotides genetics, Oligonucleotides metabolism, Oligonucleotides therapeutic use
- Abstract
The hydrophobic plasma membrane constitutes an indispensable barrier for cells, allowing influx of essential molecules while preventing access to other macromolecules. Although pivotal for the maintenance of cells, the inability to cross the plasma membrane is one of the major obstacles toward current drug development. Oligonucleotides (ONs) are a group of substances that display great therapeutic potential to interfere with gene expression. Several classes of ONs have emerged either based on double stranded RNAs, such as short interfering RNAs that are utilized to confer gene silencing, or single stranded ONs of various chemistries for antisense targeting of small regulatory micro RNAs or mRNAs. In particular the use of splice switching oligonucleotides (SSOs) to manipulate alternative splicing, by targeting pre-mRNA, has proven to be a highly promising therapeutic strategy to treat various genetic disorders, including Duchenne muscular dystrophy and spinal muscular atrophy. Despite being efficient compounds to alter splicing patterns, their hydrophilic macromolecular nature prohibits efficient cellular internalization.Various chemical drug delivery vehicles have been developed aiming at improving the bioavailability of nucleic acid-based drugs. In the context of SSOs, one group of peptidebased delivery vectors, i.e. cell-penetrating peptides (CPPs), display extremely high potency. CPPs have a remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This review provides insight into the application of CPPs and ONs in gene regulation with particular focus on CPP-assisted delivery of therapeutic SSOs.
- Published
- 2012
- Full Text
- View/download PDF
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