Physical and interdisciplinary approaches of the extracellular vesicle field : new tools and techniques toward clinical translation in regenerative medicine and drug delivery

Les vésicules extracellulaires sont des nano-vésicules (100nm) comprenant les exosomes, microvesicules et corps apoptotiques, sécrétées par toutes les cellules de l’organisme. Elles ont des rôles physiologiques et physiopathologiques dans l’hémostase, l’inflammation, la transmission d’information et de molécules biologiques, les métastases, ou la régénération tissulaire. Par exemple, des vésicules issues de cellules souches mésenchymateuses ont le même effet que leur cellule mère dans la régénération du myocarde infarci, mais ont beaucoup d’avantages par rapport à ces dernières : possibilité de les conserver au congélateur, peu immunogènes, ne provoquant pas d’embolies, pas de risque de différentiation anarchique, etc. Toutefois, l’utilisation en clinique de ces vésicules reste difficile pour des raisons pratiques : très faible production par les cellules, difficulté à les caractériser, méthodes de chargement avec des agents thérapeutique peu reproductible, méthodes d’imagerie ou de d’ingénierie peu efficientes, etc. Nous avons développé des méthodes pour répondre à ces défis, croisant la biologie, la pharmacie et la physique, et avons pu découvrir que certaines de ces techniques pouvaient être utilisées dans d’autres domaines et pour d’autres indications. En pratique, pour répondre au problème de la caractérisation des vésicules, nous avons proposé une nouvelle méthode d’imagerie utilisant le microscope électronique en cellule liquide « in situ » pour observer des vésicules dans leur milieu liquide en temps réel, et peut être utilisé pour observer d’autres matériaux et phénomènes comme les liposomes ou des processus biologiques. Nous avons proposé une nouvelle méthode de chargement des vésicules avec des molécules thérapeutiques en les fusionnant avec des liposomes, permettant la délivrance d’agents de chimiothérapie plus efficacement que des liposomes. La méthode de production des vésicules à grand rendement a nécessité 3 d’itérations successives, toutes basées sur le même concept de vésiculation induite par une contrainte mécanique, et a abouti à une méthode efficace, scalable, et conformes aux standards de production pharmaceutique. La protéomique de ces vésicules montre des expressions de protéines plus proche d’un sous type de vésicule issues de la membrane plasmique appelées microvésicules. Les vésicules issues de cette méthode ont été testées in vitro avec succès, induisent un phénotype régénératif dans des modèles cicatrisation de fistule cutanéo-digestives et des modèles murins d’insuffisance cardiaque chronique. D’autres études sur les vésicules produites par cette méthode sont en cours sur la régénération osseuse, articulaire et cérébrale, ou la délivrance de médicaments et sur l’inhibition du phénomène métastatique en cancérologie. Nous commençons aujourd’hui à défricher le transfert de ces vésicules en clinique par le biais de productions en conditions pharmaceutiques dites et de la mise en place d’une 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 “soft” 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 50–1000 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 ligand–receptor mechanisms

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 on‐chip 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 médecine-sciences (DC/MS) permettent l’acquisition d’une formation à la recherche et d’un doctorat de sciences au cours des études médicales. En France, avant les années 2000, la formation à la recherche était réalisée durant, voire après, le troisième cycle des études médicales (internat). Des DC/MS intégrés, dits « précoces », ont été développés depuis 2003 à l’initiative du cursus national de l’École de l’Inserm Liliane Bettencourt, suivie par la création de DC/MS par diverses universités. Quel que soit le mode de réalisation du double cursus, les étudiants engagés dans ces voies d’excellence se heurtent à des difficultés qui résultent essentiellement du manque d’articulation entre les formations médicale et scientifique. Les objectifs de ce texte sont de présenter les filières DC/MS de France, de recenser les principales difficultés rencontrées par les étudiants, ainsi que de formaliser un ensemble de propositions d’aménagements pour faciliter et consolider la formation des médecins/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 3−4, 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 50–1000 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 ligand–receptor mechanisms

Applications thérapeutiques des vésicules extracellulaires

Les vésicules extracellulaires, sécrétées spontanément ou en réponse à un stress par tous les types cellulaires, sont proposés comme des biothérapies alternatives aux thérapies cellulaires et aux nanomédicaments synthétiques. Leurs atouts logistiques (stockage, stabilité, disponibilité, tolérance), leur capacité à franchir les barrières biologiques, à délivrer leurs contenus (protéines, lipides et acides nucléiques) pour modifier leurs cellules cibles, ainsi que leurs activités immunomodulatrice et régénérative, suscitent un intérêt grandissant pour un très large spectre de maladies. Cette synthèse présente les défis qui restent à relever pour appliquer ces biothérapies en clinique. Quelques applications prometteuses dans les domaines du cancer et de la médecine régénérative seront proposées