18 research outputs found
Physical and interdisciplinary approaches of the extracellular vesicle field : new tools and techniques toward clinical translation in regenerative medicine and drug delivery
Les vĂ©sicules extracellulaires sont des nano-vĂ©sicules (100nm) comprenant les exosomes, microvesicules et corps apoptotiques, sĂ©crĂ©tĂ©es par toutes les cellules de lâorganisme. Elles ont des rĂŽles physiologiques et physiopathologiques dans lâhĂ©mostase, lâinflammation, la transmission dâinformation et de molĂ©cules biologiques, les mĂ©tastases, ou la rĂ©gĂ©nĂ©ration tissulaire. Par exemple, des vĂ©sicules issues de cellules souches mĂ©senchymateuses ont le mĂȘme effet que leur cellule mĂšre dans la rĂ©gĂ©nĂ©ration du myocarde infarci, mais ont beaucoup dâavantages par rapport Ă ces derniĂšres : possibilitĂ© de les conserver au congĂ©lateur, peu immunogĂšnes, ne provoquant pas dâembolies, pas de risque de diffĂ©rentiation anarchique, etc. Toutefois, lâutilisation en clinique de ces vĂ©sicules reste difficile pour des raisons pratiques : trĂšs faible production par les cellules, difficultĂ© Ă les caractĂ©riser, mĂ©thodes de chargement avec des agents thĂ©rapeutique peu reproductible, mĂ©thodes dâimagerie ou de dâingĂ©nierie peu efficientes, etc. Nous avons dĂ©veloppĂ© des mĂ©thodes pour rĂ©pondre Ă ces dĂ©fis, croisant la biologie, la pharmacie et la physique, et avons pu dĂ©couvrir que certaines de ces techniques pouvaient ĂȘtre utilisĂ©es dans dâautres domaines et pour dâautres indications. En pratique, pour rĂ©pondre au problĂšme de la caractĂ©risation des vĂ©sicules, nous avons proposĂ© une nouvelle mĂ©thode dâimagerie utilisant le microscope Ă©lectronique en cellule liquide « in situ » pour observer des vĂ©sicules dans leur milieu liquide en temps rĂ©el, et peut ĂȘtre utilisĂ© pour observer dâautres matĂ©riaux et phĂ©nomĂšnes comme les liposomes ou des processus biologiques. Nous avons proposĂ© une nouvelle mĂ©thode de chargement des vĂ©sicules avec des molĂ©cules thĂ©rapeutiques en les fusionnant avec des liposomes, permettant la dĂ©livrance dâagents de chimiothĂ©rapie plus efficacement que des liposomes. La mĂ©thode de production des vĂ©sicules Ă grand rendement a nĂ©cessitĂ© 3 dâitĂ©rations successives, toutes basĂ©es sur le mĂȘme concept de vĂ©siculation induite par une contrainte mĂ©canique, et a abouti Ă une mĂ©thode efficace, scalable, et conformes aux standards de production pharmaceutique. La protĂ©omique de ces vĂ©sicules montre des expressions de protĂ©ines plus proche dâun sous type de vĂ©sicule issues de la membrane plasmique appelĂ©es microvĂ©sicules. Les vĂ©sicules issues de cette mĂ©thode ont Ă©tĂ© testĂ©es in vitro avec succĂšs, induisent un phĂ©notype rĂ©gĂ©nĂ©ratif dans des modĂšles cicatrisation de fistule cutanĂ©o-digestives et des modĂšles murins dâinsuffisance cardiaque chronique. Dâautres Ă©tudes sur les vĂ©sicules produites par cette mĂ©thode sont en cours sur la rĂ©gĂ©nĂ©ration osseuse, articulaire et cĂ©rĂ©brale, ou la dĂ©livrance de mĂ©dicaments et sur lâinhibition du phĂ©nomĂšne mĂ©tastatique en cancĂ©rologie. Nous commençons aujourdâhui Ă dĂ©fricher le transfert de ces vĂ©sicules en clinique par le biais de productions en conditions pharmaceutiques dites et de la mise en place dâune start-up.Extracellular Vesicles, encompassing exosomes, microvesicles, apoptotic bodies are nanosized vesicles secreted by most cells of the organism, that demonstrated physiologic and physio-pathologic roles in various processes like hemostasis, metastasis, information transfer through biological macromolecules or more recently in inflammation resolution in regenerative medicine. Therapeutic use of these EVs, in particular as drug delivery systems or as a regeneration triggering agent is of a major interest, for example the use Mesenchymal Stem Cells derived EVs after myocardial infarction or stroke. EV recapitulate their parental cell effect and benefit from unique opportunities like off the shelf availability, low immunogenicity and no anarchic differentiation or pulmonary embolism. However, major obstacles are still to be faced in the field, like the EV drug loading, engineering, targeting, characterization, delivery method and GMP high yield production toward clinical translation. We developed new methods to respond to these needs at the crossroad of biology, physics, pharmacy and medicine, and discovered meanwhile that some of these techniques can be used in other fields and indications. As an example, a new liquid cell transmission electron microscopy labeling method was used to investigate live in situ at the nanoscale level EVs behavior, and can be used for other âsoftâ materials like liposomes or biology processes. The PEG induced liposome/EV fusion method was designed to produce biological/synthetic hybrids with engineered membrane properties and drug loading. A first response to the production problem was made designing a microfluidic chip allowing shear stress application to trigger EV production. The concept of shear stress triggered EV release was also used in the design of 2nd generation system for high yield, scalable and compliant with Good Manufacturing Practice, EV production method that uses a controlled shear stress to induce EV secretion. These EVs were tested in regenerative medicine models of fistula healing and chronic heart insufficiency confirming the interest of a new local delivery method using thermosensitive gels and their potency compared to parental cells. Our team is now exploring the scale-up, immunogenicity, and stability of these EVs and benchmarking their cost/efficiency in various models to pave the way toward the democratization of EV-based regenerative medicine through a company/platform creation
Thinking Quantitatively of RNA-Based Information Transfer via Extracellular Vesicles: Lessons to Learn for the Design of RNA-Loaded EVs
Extracellular vesicles (EVs) are 50â1000 nm vesicles secreted by virtually any cell type in the body. They are expected to transfer information from one cell or tissue to another in a short- or long-distance way. RNA-based transfer of information via EVs at long distances is an interesting well-worn hypothesis which is ~15 years old. We review from a quantitative point of view the different facets of this hypothesis, ranging from natural RNA loading in EVs, EV pharmacokinetic modeling, EV targeting, endosomal escape and RNA delivery efficiency. Despite the unique intracellular delivery properties endowed by EVs, we show that the transfer of RNA naturally present in EVs might be limited in a physiological context and discuss the lessons we can learn from this example to design efficient RNA-loaded engineered EVs for biotherapies. We also discuss other potential EV mediated information transfer mechanisms, among which are ligandâreceptor mechanisms
Combining independent protein and cellular SELEX with bioinformatic analysis may allow high affinity aptamer hit discovery
Autophagy as a therapeutic target in pancreatic cancer
International audiencePancreatic ductal adenocarcinoma (PDAC) is characterised by early metastasis and resistance to anti-cancer therapy, leading to an overall poor prognosis. Despite continued research efforts, no targeted therapy has yet shown meaningful efficacy in PDAC; mutations in the oncogene KRAS and the tumour suppressor TP53, which are the most common genomic alterations in PDAC, have so far shown poor clinical actionability. Autophagy, a conserved process allowing cells to recycle altered or unused organelles and cellular components, has been shown to be upregulated in PDAC and is implicated in resistance to both cytotoxic chemotherapy and targeted therapy. Autophagy is thus regarded as a potential therapeutic target in PDAC and other cancers. Although the molecular mechanisms of autophagy activation in PDAC are only beginning to emerge, several groups have reported interesting results when combining inhibitors of the extracellular-signal-regulated kinase/mitogen-activated protein kinase pathway and inhibitors of autophagy in models of PDAC and other KRAS-driven cancers. In this article, we review the existing preclinical data regarding the role of autophagy in PDAC, as well as results of relevant clinical trials with agents that modulate autophagy in this cancer
Potential of onâchip analysis and engineering techniques for extracellular vesicle bioproduction for therapeutics
International audienceThe clinical interest around extracellular vesicles (EVs) started during the year 2000s, now leading to new clinical diagnostic and therapeutic strategies. Due to their outstanding properties as biogenic drug delivery systems and as alternatives to cell therapy, the need to produce EVs with sufficiently high yield and quality for clinical use expedited the development of analytical techniques and dedicated bioproduction methods. Though preclinical studies revealed the potential of EVs to become next generation subcellular therapies that could lead to major breakthroughs in current therapeutics possibilities, they remain complex objects on both a physicochemical and biological level. Here, we review the capacity of microfluidic technologies to match EV-based therapeutics need for clinical translation via standardized and intensified bioproduction methods. Indeed, some of the current routine tools used in bioproduction are already achieved on chips such as micromixers or particle sorting and analysis using field flow fraction or nanoparticle tracking analyzer. Also, microfluidics communities have developed a wide set of new techniques to isolate and quantify EVs, but the few that are adopted in a bioproduction workflow are well-established since the 1990s. We first review the different EV generation and loading methods embedded on chip. We focus on EVs preparation methods, from purification to in-line separative techniques. We finally describe the on-chip analytical tools to analyze physicochemistry and phenotypes of EVs
Les doubles cursus médecine-sciences en France
Les doubles cursus mĂ©decine-sciences (DC/MS) permettent lâacquisition dâune formation Ă la recherche et dâun doctorat de sciences au cours des Ă©tudes mĂ©dicales. En France, avant les annĂ©es 2000, la formation Ă la recherche Ă©tait rĂ©alisĂ©e durant, voire aprĂšs, le troisiĂšme cycle des Ă©tudes mĂ©dicales (internat). Des DC/MS intĂ©grĂ©s, dits « prĂ©coces », ont Ă©tĂ© dĂ©veloppĂ©s depuis 2003 Ă lâinitiative du cursus national de lâĂcole de lâInserm Liliane Bettencourt, suivie par la crĂ©ation de DC/MS par diverses universitĂ©s. Quel que soit le mode de rĂ©alisation du double cursus, les Ă©tudiants engagĂ©s dans ces voies dâexcellence se heurtent Ă des difficultĂ©s qui rĂ©sultent essentiellement du manque dâarticulation entre les formations mĂ©dicale et scientifique. Les objectifs de ce texte sont de prĂ©senter les filiĂšres DC/MS de France, de recenser les principales difficultĂ©s rencontrĂ©es par les Ă©tudiants, ainsi que de formaliser un ensemble de propositions dâamĂ©nagements pour faciliter et consolider la formation des mĂ©decins/chercheurs
Modification of Extracellular Vesicles by Fusion with Liposomes for the Design of Personalized Biogenic Drug Delivery Systems
International audienceExtracellular vesicles (EVs) are recognized as nature's own carriers to transport macromolecules throughout the body. Hijacking this endogenous communication system represents an attractive strategy for advanced drug delivery. However, efficient and reproducible loading of EVs with therapeutic or imaging agents still represents a bottleneck for their use as a drug delivery system. Here, we developed a method for modifying cell-derived EVs through their fusion with liposomes containing both membrane and soluble cargoes. The fusion of EVs with functionalized liposomes was triggered by polyethylene glycol (PEG) to create smart biosynthetic hybrid vectors. This versatile method proved to be efficient to enrich EVs with exogenous lipophilic or hydrophilic compounds , while preserving their intrinsic content and biological properties. Hybrid EVs improved cellular delivery efficiency of a chemotherapeutic compound by a factor of 3â4, as compared to the free drug or the drug-loaded liposome precursor. On one side, this method allows the biocamouflage of liposomes by enriching their lipid bilayer and inner compartment with biogenic molecules. On the other side, the proposed fusion strategy enables efficient EV loading, and the pharmaceutical development of EVs with adaptable activity and drug delivery property
Thinking Quantitatively of RNA-Based Information Transfer via Extracellular Vesicles: Lessons to Learn for the Design of RNA-Loaded EVs
Extracellular vesicles (EVs) are 50â1000 nm vesicles secreted by virtually any cell type in the body. They are expected to transfer information from one cell or tissue to another in a short- or long-distance way. RNA-based transfer of information via EVs at long distances is an interesting well-worn hypothesis which is ~15 years old. We review from a quantitative point of view the different facets of this hypothesis, ranging from natural RNA loading in EVs, EV pharmacokinetic modeling, EV targeting, endosomal escape and RNA delivery efficiency. Despite the unique intracellular delivery properties endowed by EVs, we show that the transfer of RNA naturally present in EVs might be limited in a physiological context and discuss the lessons we can learn from this example to design efficient RNA-loaded engineered EVs for biotherapies. We also discuss other potential EV mediated information transfer mechanisms, among which are ligandâreceptor mechanisms
Applications thérapeutiques des vésicules extracellulaires
Les vĂ©sicules extracellulaires, sĂ©crĂ©tĂ©es spontanĂ©ment ou en rĂ©ponse Ă un stress par tous les types cellulaires, sont proposĂ©s comme des biothĂ©rapies alternatives aux thĂ©rapies cellulaires et aux nanomĂ©dicaments synthĂ©tiques. Leurs atouts logistiques (stockage, stabilitĂ©, disponibilitĂ©, tolĂ©rance), leur capacitĂ© Ă franchir les barriĂšres biologiques, Ă dĂ©livrer leurs contenus (protĂ©ines, lipides et acides nuclĂ©iques) pour modifier leurs cellules cibles, ainsi que leurs activitĂ©s immunomodulatrice et rĂ©gĂ©nĂ©rative, suscitent un intĂ©rĂȘt grandissant pour un trĂšs large spectre de maladies. Cette synthĂšse prĂ©sente les dĂ©fis qui restent Ă relever pour appliquer ces biothĂ©rapies en clinique. Quelques applications prometteuses dans les domaines du cancer et de la mĂ©decine rĂ©gĂ©nĂ©rative seront proposĂ©es