Les vésicules extracellulaires comme vecteurs de macromolécules bioactives : modèle du transporteur ABCC7 (CFTR) et application à la biothérapie de la mucoviscidose

Cystic fibrosis is a genetic disease in which its prognosis depends on the lung damage. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), resulting in a dysfunctional CFTR protein normally located at the plasma membrane of epithelial cells. This thesis is a study of a novel therapeutic approach to use extracellular vesicles (EVs), microvesicles and exosomes, as transfer vectors for CFTR mRNA and protein to target cells. The proof of concept for the transfer of CFTR mRNA and protein was first done in the CHO hamster model. To validate this concept on human cells, we used human bronchial Calu-3 cells, which express the endogenous CFTR protein, and A549 lung epithelial cells transduced by the adenoviral vector Ad5-GFP-CFTR to overexpress the fusion exogenous protein GFP-CFTR. We show that EVs produced by these cells could transfer a new functionality to CF15 target cells carrying the CFTRdeltaF508 mutation and the transfer seems to be more efficient in a homologous cell system versus a heterologous system. Interestingly, the exosomes seem to be more efficient in CFTR transfer than the microvesicles. A study of the mechanism of EVs cellular uptake show that it is temperature dependent and that endocytosis and macropinocytosis are implicated. Collectively, this study demonstrates the potential application of EVs for CFTR transfer and functional correction of the genetic defect in human CF cellsLa mucoviscidose est une maladie génétique due à des mutations du gène CFTR (Cystic Fibrosis Transmembrane conductance Regulator), conduisant à un défaut d'adressage de la protéine CFTR à la membrane apicale des cellules épithéliales, ou à un déficit de sa fonction de canal à ions chlorure. Ce travail a consisté à étudier les vésicules extracellulaires (EV), microvésicules (MV) et exosomes (Exo), comme vecteurs de la protéine CFTR et de son ARN messager. La preuve de concept du transfert de matériel biologique d'intérêt par l'intermédiaire d'EV, d'abord apportée sur un modèle de cellules animales (CHO), a été validée en cellules humaines. Les EV ont été isolées à partir de surnageant de Calu-3, cellules exprimant la protéine CFTR de manière endogène, et de A549 transduites par le vecteur adenoviral Ad5-GFP-CFTR, surexprimant la protéine de fusion GFP-CFTR. Les cellules cibles choisies, A549 et CF15, étaient déficientes en CFTR. Le transfert s'est révélé plus efficace en système homologue (A549/A549) qu'en système hétérologue (A549/CF15). Par ailleurs, l'utilisation d'inhibiteurs métaboliques suggère que les EV ne suivent pas une voie d'internalisation cellulaire unique, mais que plusieurs mécanismes sont mis en jeu, dont l'endocytose clathrine dépendante et la macropinocytose. Les deux types d'EV sont capables de rétablir la fonction canal associée au CFTR dans les cellules CF15 de façon dose-dépendante, mais avec un effet de seuil minimum. L'activité CFTR reste stable pendant 3 jours, et à un niveau encore détectable après 5 jours. Notre travail démontre l'intérêt potentiel des MV et Exo comme vecteurs de biothérapie de pathologies génétique

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Extracellular vesicles as bioactive macromolecules vectors : model of the ABCC7 transporter (CFTR) and application to the biotherapy of cystic fibrosis

La mucoviscidose est une maladie génétique due à des mutations du gène CFTR (Cystic Fibrosis Transmembrane conductance Regulator), conduisant à un défaut d'adressage de la protéine CFTR à la membrane apicale des cellules épithéliales, ou à un déficit de sa fonction de canal à ions chlorure. Ce travail a consisté à étudier les vésicules extracellulaires (EV), microvésicules (MV) et exosomes (Exo), comme vecteurs de la protéine CFTR et de son ARN messager. La preuve de concept du transfert de matériel biologique d'intérêt par l'intermédiaire d'EV, d'abord apportée sur un modèle de cellules animales (CHO), a été validée en cellules humaines. Les EV ont été isolées à partir de surnageant de Calu-3, cellules exprimant la protéine CFTR de manière endogène, et de A549 transduites par le vecteur adenoviral Ad5-GFP-CFTR, surexprimant la protéine de fusion GFP-CFTR. Les cellules cibles choisies, A549 et CF15, étaient déficientes en CFTR. Le transfert s'est révélé plus efficace en système homologue (A549/A549) qu'en système hétérologue (A549/CF15). Par ailleurs, l'utilisation d'inhibiteurs métaboliques suggère que les EV ne suivent pas une voie d'internalisation cellulaire unique, mais que plusieurs mécanismes sont mis en jeu, dont l'endocytose clathrine dépendante et la macropinocytose. Les deux types d'EV sont capables de rétablir la fonction canal associée au CFTR dans les cellules CF15 de façon dose-dépendante, mais avec un effet de seuil minimum. L'activité CFTR reste stable pendant 3 jours, et à un niveau encore détectable après 5 jours. Notre travail démontre l'intérêt potentiel des MV et Exo comme vecteurs de biothérapie de pathologies génétiquesCystic fibrosis is a genetic disease in which its prognosis depends on the lung damage. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), resulting in a dysfunctional CFTR protein normally located at the plasma membrane of epithelial cells. This thesis is a study of a novel therapeutic approach to use extracellular vesicles (EVs), microvesicles and exosomes, as transfer vectors for CFTR mRNA and protein to target cells. The proof of concept for the transfer of CFTR mRNA and protein was first done in the CHO hamster model. To validate this concept on human cells, we used human bronchial Calu-3 cells, which express the endogenous CFTR protein, and A549 lung epithelial cells transduced by the adenoviral vector Ad5-GFP-CFTR to overexpress the fusion exogenous protein GFP-CFTR. We show that EVs produced by these cells could transfer a new functionality to CF15 target cells carrying the CFTRdeltaF508 mutation and the transfer seems to be more efficient in a homologous cell system versus a heterologous system. Interestingly, the exosomes seem to be more efficient in CFTR transfer than the microvesicles. A study of the mechanism of EVs cellular uptake show that it is temperature dependent and that endocytosis and macropinocytosis are implicated. Collectively, this study demonstrates the potential application of EVs for CFTR transfer and functional correction of the genetic defect in human CF cell

Obesity and cancer: role of adipose cells in preventing the transition from in situ to invasive breast cancer in a two-cell fluorescent spheroid model

International audienc

A novel platform for virus-like particle-display of flaviviral envelope domain III: induction of Dengue and West Nile virus neutralizing antibodies

International audienceCD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55Gag(HIV)) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV1) or West Nile virus Kunjin (WNVKun). The two resulting chimeric proteins, DIII-DENV1-RIgE and DIII-WNVKun-RIgE, were addressed to the plasma membrane, exposed at the surface of human and insect cells, and incorporated into extracellular VLPs when coexpressed with Pr55Gag(HIV) in insect cells. The DIII domains were accessible at the surface of retroviral VLPs, as shown by their reactivity with specific antibodies, and notably antibodies from patient sera. The DIII-RIgE proteins were found to be incorporated in VLPs made of SIV, MLV, or chimeric MLV-HIV Gag precursors, indicating that DIII-RIgE could pseudotype a wide variety of retroviral VLPs. VLP-displayed DIII were capable of inducing specific neutralizing antibodies against DENV and WNV in mice. Although the neutralization response was modest, our data confirmed the capability of DIII to induce a flavivirus neutralization response, and suggested that our VLP-displayed CD16-RIgE-based platform could be developed as a vaccine vector against different flaviviruses and other viral pathogens

CFTR channel activity

<p><b>in MP-transduced CHO recipient cells. (A), </b><b><i>Evolution of the DiSBAC₂(3) fluorescence signal</i></b>. The time-course analysis of DiSBAC₂(3) fluorescence changes was monitored in regions of the cell monolayer corresponding to 20–30 cells, taken at 72 h post-transfer, a time point corresponding to the maximal immunoreactivity of CFTR glycoprotein at the cell surface. Changes in the fluorescent signal were expressed as F_t/F₀ ratio values, in which F_t and F₀ were the fluorescence values at the times <i>t</i> and <i>t</i>₀, respectively, and <i>t</i>₀ the time when the cAMP-containing, CFTR-activating cocktail was added. The cocktail of CFTR activators was maintained throughout the experiment. The CFTR inhibitor GlyH-101 was added for 5 min (phase II, marked by two vertical arrows). Phase III shows reversibility of the CFTR block and recovery of the fluorescent signal. <b>(B), </b><b><i>Fluorescence microscopy</i></b>. Photographs of cell monolayers were taken at different time-points corresponding to the four successive phases, as indicated on the curve by the letters (a), (b), (c) and (d).</p

Electron microscopy (EM) of MPs

<p><b>isolated from CHO-CD46 cells.</b> (<b>A</b>), Negative staining and immuno-EM of MP₃₀CD46. (<b>B</b>), Negative staining (<b>a</b>), and immuno-EM (<b>b</b>) of MP₁₀₀CD46. In (A) and (B, b), immunogold labeling was performed using anti-C46 antibody and 10 nm-gold tagged complementary antibody. MP-associated gold grains are indicated by arrows. (<b>C</b>), Ultrathin sections of pelletable complexes of MP₃₀CD46-HAdV5F35, shown at low (a) and high (b) magnifications. Particles of HAdV5F35 vector (70–80 nm in diameter) in complex with MP₃₀CD46 are indicated by the letter V.</p

Time-course expression of GFP-CFTR glycoprotein at the surface of MP-transduced CHO cells.

<p>Cells harvested at different times after MP-cell transfer were analyzed by flow cytometry for the immunoreactivity of the first N-terminal loop of the CFTR ectodomain with anti-CFTR monoclonal antibody. (<b>A</b>), MP₃₀-transduced CHO cells. (<b>B</b>), MP₁₀₀-transduced CHO cells. Bars represent mean values ± SEM (<i>n</i> = 3).</p

MP₃₀-mediated transfer and functionality of (a) CAR, or (b) CD46 as adenoviral receptors in target cells.

<p>Aliquots of CHO cells (target cells) were incubated with MP₃₀CAR (a) or MP₃₀CD46 (b) at different MP doses per cell, as indicated in the <i>x</i>-axis. At 72 h after MP₃₀-cell interaction, cells were infected with HAdV5-GFP or HAdV5F35-GFP vector, at the same MOI (500 vp/cell). The degree of CHO permissiveness to HAdV5-GFP or HAdV5F35-GFP vector was evaluated by flow cytometry analysis of the intracellular GFP signal. In (<b>a</b>), HAdV5F35-GFP, which does not recognize CAR as cellular receptor, was used as the negative control. In (<b>b</b>), HAdV5-GFP, which does not recognize CD46 as cellular receptor, was used as the negative control. MP₃₀ from nontransduced CHO cells (Control MP) served as the negative controls in both panels.</p

Functionality of exogenous CD46 in MP₃₀CD46-transduced CHO cells. (A), CD46 as complement C3 regulator.

<p>CHO cells were harvested at 48 h posttransfer, and the CD46-induced protection against complement C3-mediated cell apoptosis was assayed by the percentage of Annexin V-positive cells determined by flow cytometry at increasing doses of complement C3. <b>(B), Kinetics of the gain of adenoviral receptor function by MP₃₀CD46-transduced CHO cells.</b> CHO cells were harvested at different times after MP₃₀CD46-transfer, and cell permissiveness to the HAdV5F35-GFP vector was assessed by infection with HAdV5F35-GFP at MOI 500. Cells were analyzed for GFP signal at 48 h postinfection. The degree of permissiveness to the vector was expressed as the percentage of GFP-positive cells (left <i>y</i>-axis), and the relative transduction efficiency (RTE; right <i>y</i>-axis). The RTE, in arbitrary units (AU), was given using the formula = (percentage of GFP-positive cells) x (MFI; mean fluorescence intensity).</p