Nous remercions les Drs. R. Jude Samulski et Xiao Xiao pour leurs dons en nature de recombinaison AAV3 et AAV-EGFP plasmides, respectivement, et le Dr Gail Tomlinson pour avoir généreusement fourni les cellules Hep293TT. Cette recherche a été financée en partie par Service de santé publique accorde bourse R01 HL-076901, R01 HL-097088, et P01 DK-058327 (Projet 1) de la National Institutes of Health (AS).

1. Emballage de virus recombinants adéno-associé de sérotype 3 (rAAV3) Vecteurs <ol><li> Allez sur le site: &quot; <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> &quot;et de calculer la quantité d&#39;ADN. </li><li> Pré-mélange plasmides pHelper (aide adénovirus), pACG2c3 (AAV helper) et pdsAAV-CB-EGFP (rAAV vecteur) dans un milieu sans SVF et des antibiotiques. Le volume total devrait être 8750 uL de dix à 15 cm 2 plaques. </li><li> Ajouter directement 1250 uL de PEI (0,1% m / v, Polysciences, Cat # 23966, pH 4,5) dans la solution d&#39;ADN à l&#39;étape 1.2. Vortex la solution d&#39;ADN-Prince-Édouard pendant quelques secondes. </li><li> Incuber l&#39;ADN-Prince-Édouard solution pour former un complexe de 5 min à température ambiante (RT). </li><li> Ajouter 1 mL de l&#39;ADN-Prince-Édouard solution par 15 cm 2 plaques. </li><li> Retirer moyenne après 4 heures, en remplaçant avec des produits frais 25 ml de milieu complet 10% de FBS (C-DMEM). </li><li> Soixante-douze heures après transfection, les cellules gratter et verser dans un tube de 250 ml à centrifuger conique. </li><li> Centrifuger à 3000 rpm à 4 ° C pendant 10 min. Verser le liquide surnageant. </li><li> Re-suspendre le culot cellulaire dans 5 ml de RB TMS tampon (50 mM Tris-HCl, NaCl 150 mM, pH 8,0). Transfert à un nouveau 15 ml tube conique. </li><li> Congeler dans la glace sèche-éthanol de bain pendant 10 minutes et le dégel à 37 ° C pendant 10 min. Répétez trois fois. </li><li> Ajouter 1 ul de 4,8 M MgCl 2 et 2 pi de Benzonase (25 U / uL, Novagen, Cat # 70664-3). Vortex et laisser incuber à 37 ° C pendant 40 min. </li><li> Isoler à 4000 rpm à 4 ° C pendant 40 minutes et recueillir le surnageant (lysat clarifié). </li><li> Dans un tube de 13 ml à centrifuger Quick-Seal (Beckman, Cat # 342413), la charge iodixanol gradient (OptiPrep, Cat # 1114542) de haut en bas: 2 mL de 15%, 2 ml de 25%, 1,5 mL de 40% et 1,5 ml de iodixanol 60%. Chargez le surnageant (étape 1.12) sur le haut de la pente iodixanol. Remplir les tubes avec RB TMS Tampon. </li><li> Centrifuger à 75000 rpm pendant 1 heure à 16 ° C en utilisant Beckman 90TI rotor. </li><li> Recueillir la fraction iodixanol 40% en insérant une seringue à la limite de 40-60%. </li><li> Transfert de la fraction iodixanol 40% en un tube conique 50 ml et porter à 40 ml avec le tampon A. (Tris 20 mM, NaCl 15 mM, pH 8,5) </li><li> Assembler l&#39;appareil de filtration à l&#39;aide HiTrap Q HP colonne (GE Healthcare, Cat # 17-1154-01). Laver la colonne avec le tampon suivant: 25 ml de tampon A (Tris 20 mM, NaCl 15 mM, pH 8,5);; 25 ml de tampon B (Tris 10 mM, NaCl 500 mM, pH 8,5), 50 ml de tampon A 40 ml d&#39;échantillon de virus dans le tampon A, 50 ml de tampon A (Tris 20 mM, NaCl 15 mM, pH 8,5);; 20 ml de tampon C (20 mM Tris, 175 mM NaCl, pH 8,5) </li><li> Recueillir C dans un tampon concentrateurs 20 mL d&#39;Apollo (Orbital Biosciences). Centrifuger à 3000 rpm à 4 ° C pendant 10 min. </li><li> Jeter l&#39;effluent à travers et ajouter 20 ml de PBS et centrifuger refroidi à 3.000 tpm, à 4 ° C pendant 10 min. </li><li> Jeter l&#39;effluent à travers et ajouter 500 ul de PBS refroidi. Lavez les vecteurs hors de la membrane et le transfert d&#39;traité à la silicone et de stocker des tubes Eppendorf congelés dans de petites aliquotes à -80 ° C. </li></ol> 2. Détermination de rAAV3 Les titres Vecteur <ol><li> Dégeler 10 uL de virus purifié dans un tube Eppendorf sur la glace. Ajouter 40 uL de DDH 2 O. </li><li> Ajouter 0,2 ul de Benzonase (25 U / uL, Novagen, Cat # 70664) et incuber à 37 ° C pendant 1 heure. </li><li> Prenez 50 uL de standard plasmidique (0,2 ng / uL) dans un autre tube Eppendorf. </li><li> Ajouter 50 ul de 100 mM NaOH dans chaque tube et incuber à 65 ° C pendant 30 min. </li><li> Réfrigérer la glace immédiatement pendant au moins 5 min. </li><li> Couper la membrane de transfert (Millipore, Cat # INYC00010) selon Bio-Dot Papier filtre (Bio-Rad, Cat # 1620161). Rincer la membrane en 10x SSC avec trois papiers filtres pendant 20 min. </li><li> Set slot blot SF Appareillage (Bio-Dot, Cat # 170-6542). Connectez-vous à une fiole à vide et ajouter 100 ul de 10x SSC aux appareils slot blot. </li><li> Ajouter 100 ul de 20x SSC et 1 pl de colorant de chargement 6x à chacun des échantillons à l&#39;étape 2.5. </li><li> Charge 100 ul de chaque échantillon sur la membrane de l&#39;appareil slot blot. </li><li> Ajouter un autre uL 100 de 10x SSC dans chacun des échantillons. </li><li> Répétez les étapes 2.9 et 2.10 jusqu&#39;à ce que tous les échantillons va à travers la membrane. </li><li> Démonter l&#39;appareil et le slot blot retirer la membrane. Mettez-le en Linker inspecteur SL-1000 Crosslinker UV. Tourner sur &quot;l&#39;énergie&quot;, &quot;Optimal Crosslink&quot; (spectacle 1200) et pousser &quot;Démarrer&quot;. </li><li> Faire bouillir 1 ml de sperme de saumon (SS) ADN (Fisher, Cat # NC9753983) pendant 5 min. Réfrigérer la glace pendant au moins 5 min. </li><li> Préparer la solution de pré-hybridation en mélangeant 27,5 ml de DDH 2 O, 15 ml de SSC 20x, 5 ml de solution 50x Denhardt, 1,25 mL de SDS 20%, 0,25 mL de Polya et 1 ml de SS ADN de l&#39;étape 2.13. </li><li> Suspendre la membrane dans 25 ml de solution de pré-hybridation dans un cylindre de verre bouché et incuber à 65 ° C dans une chambre d&#39;hybridation pendant la nuit. </li><li> Diluer 50 modèles ng d&#39;ADN dans 10 ul de DDH 2 O. Faire bouillir à 100 ° C pendant 5 min et laisser refroidir sur la glace immédiatement pendant au moins 5 min. </li><li> Centrifuger à 3000 rpm à température ambiante pendant 1 min. Ajouter 2 ul de 10 mM dNTP 3 (manque dCTP), 2 pl de Mix hexanucléotide (Roche, Cat # 11277081001), 1 pl de Klenow (Roche, Cat # 11008404001) et 5 pi de α -32 P-dCTP. Incuber à 37 ° C pendant 10 min. </li><li> Centrifugeuse G-50 colonnes (GE Healthcare, Cat # 27-5330-01) à 3000 rpm pendant 2 min. la charge de la sonde à l&#39;étape 2.17 dans la colonne. Centrifuger à 3000 rpm pendant 2 min et recueillir les actions accréditives. </li><li> Comptez radioactivité. Faire bouillir la sonde à 100 ° C pendant 5 min et laisser refroidir sur la glace immédiatement pendant au moins 5 min. </li><li> Ajouter la sonde (6x10 5 cpm / ml) dans la solution de pré-hybridation avec la membrane. </li><li> Incuber une nuit à 65 ° C dans une chambre d&#39;hybridation. </li><li> Jeter la solution d&#39;hybridation et rincer la membrane dans 2x SSC 0,1% SDS à température ambiante pendant 15 min. </li><li> Jeter la solution et rincer la membrane de 0,1 x SSC + 0,1% SDS à 65 ° C pendant 30 min. </li><li> Jeter la solution et rincer la membrane de 0,1 x SSC à brève RT. </li><li> Exposer le film à -80 ° C pendant 6 heures et de développer le film. Un résultat typique est montré dans la figure 1. </li></ol> 3. rAAV3 Vecteur médiée Transduction et l&#39;expression du transgène dans les cellules humaines de cancer du foie <ol><li> Graine 1x10 4 cellules (soit Huh7 ou Hep293TT) dans chaque puits de plaque de 96 puits. Incuber dans un incubateur humidifié CO 2 pendant au moins 18 heures. </li><li> Retirer de la plaque de support de 96 puits. </li><li> Laver les cellules deux fois avec 50 ul de milieu sans SVF et des antibiotiques et à enlever. </li><li> Ajouter 50 uL de milieu sans SVF et des antibiotiques et des vecteurs rAAV à 5000 vgs / cellule. </li><li> Incuber les plaques dans l&#39;incubateur CO 2 pendant 4 heures. </li><li> Jeter la moyenne. Laver les cellules deux fois avec 50 ul de milieu complet et à enlever. </li><li> Ajouter 150 pi de DMEM-C dans chaque puits et incuber la plaque dans incubateur à CO 2 pendant 72 heures, et de visualiser l&#39;expression de l&#39;EGFP en utilisant un microscope à fluorescence (DMI 4000B; Leica Microsystems). </li><li> Images à partir de trois puits de cellules infectées sont analysées quantitativement par ImageJ logiciels d&#39;analyse (NIH, Bethesda, MD, USA). </li><li> L&#39;expression du transgène est évalué comme surface totale de fluorescence verte (2 pixels) par le champ visuel. Un résultat typique est montré dans la figure 2. </li><li> L&#39;expression du transgène peut également être déterminée par cytométrie en flux. </li></ol> 4. Les résultats représentatifs: En suivant le protocole décrit ci-dessus, on peut générer des vecteurs AAV de sérotype efficacement. Le rendement typique est d&#39;environ 500 uL contenant ~ 10 11 vgs / uL d&#39;purifiée stock de vecteur. La pureté du stock de vecteur est déterminé en comparant les signaux d&#39;hybridation sur taches quantitatives emplacement de l&#39;ADN, avec et sans la digestion Benzonase. Purifié rAAV3 vecteurs transduire un cancer du foie humain - hépatoblastome (HB) et le carcinome hépatocellulaire (CHC) - lignées cellulaires de façon efficace. <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="Figure 1" /> Figure 1. Quantitative ADN slot-blot pour déterminer les titres des rAAV3 vecteurs. Deux dilutions des stocks viraux purifiés, digérés avec Benzonase (rangée du haut) ont été analysés sur quantitatives taches slot d&#39;ADN avec 32 P-étiquetés sonde d&#39;ADN spécifique EGFP. Le titre du AAV3 vecteur a été déterminée par comparaison avec 1 ng (rangée du milieu) ou 10 ng (rangée du bas) de l&#39;AAV-EGFP normes plasmide chargé sur la membrane. Les numéros correspondent aux copies d&#39;ADN. <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="Figure 2" /> Figure 2. Vecteur AAV recombinant médiée par l&#39;expression du transgène dans les cellules humaines de cancer du foie. Cellules Hep293TT, une création récente lignée humaine de cellules d&#39;hépatoblastome 4, ont été soit (à gauche) maquette infectés, ou transduites avec 5000 vgs / cellule soit scAAV2-EGFP ou scAAV3-EGFP vecteurs à 37 ° C pendant 2 heures. L&#39;expression du transgène a été visualisée 72 heures après la transduction utilisant un microscope à fluorescence.

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Des vecteurs recombinants basés sur un parvovirus non pathogène pour l&#39;homme, le virus adéno-associé (AAV) ont été développés et sont actuellement en usage dans un certain nombre d&#39;essais cliniques de thérapie génique 5. Auparavant, les 10 sérotypes les plus couramment utilisés AAV, l&#39;efficacité de la transduction des vecteurs AAV3 en général a été signalé comme étant particulièrement faible, tant in vitro et in vivo 1. Cependant, notre observation récente que AAV3 vecteurs transduire des lignées cellulaires de cancer du foie, excessivement bien, tel que déterminé quantitativement par deux cytométrie en flux, et que AAV3 utilise le récepteur hépatocytaire humaine du facteur de croissance (hHGFR) comme un co-récepteur cellulaire d&#39;entrée obligatoire et dans ces cellules 3, suggèrent fortement une approche sélective des tissus tropisme du AAV3 des cellules hépatiques humaines en général, et des cellules humaines de cancer du foie en particulier. Toutefois, depuis AAV3 vecteurs aussi transduire efficacement les hépatocytes humains normaux 2, leur utilisation potentielle dans le traitement du cancer de demande de gène in vivo seraient affectées négativement. Une stratégie possible pour contourner ce problème potentiel transcriptionnel implique le ciblage des cellules cancéreuses en identifiant un produit génique qui est sélectivement produites par la tumeur, mais pas par les hépatocytes normaux. Par exemple, des études antérieures ont montré que les concentrations sériques de α-foetoprotéine (AFP) sont utilisés comme un marqueur spécifique pour le suivi de la présence, la progression et / ou à la réapparition de certains types de cancers du foie, puisque les hépatocytes normaux produisent généralement de très petites quantités cette protéine. En effet, nous avons utilisé AAV3 vecteurs contenant le promoteur AFP pour cibler l&#39;expression du transgène dans les cellules de cancer du foie, mais pas dans les hépatocytes normaux 2, et des études sont actuellement en cours pour tester l&#39;efficacité de cette approche dans un modèle murin de xénogreffe de cancer du foie. Dans nos études supplémentaires, nous avons observé que l&#39;efficacité de transduction des différents vecteurs AAV de sérotype peut être considérablement augmentée par mutagenèse dirigée de la surface exposée résidus tyrosines dans les capsides virales 6-14. Depuis 6 sur 7 exposées à la surface tyrosines sont également conservées dans AAV3, nous avons effectué une mutagenèse dirigée de ces résidus ainsi observé que l&#39;efficacité de transduction de la tyrosine-mutant AAV3 vecteurs est significativement accrue dans les cellules humaines de cancer du foie (données non publiées). Des études sont également en cours pour évaluer l&#39;innocuité et l&#39;efficacité de la tyrosine-AAV3 mutant vecteurs dans des modèles de xénogreffe de souris pour hépatoblastome humain et le carcinome hépatocellulaire, et en cas de succès, la tyrosine-optimale mutant vecteurs sérotype AAV3 peuvent se révéler utiles pour le ciblage du foie humain cancers de la thérapie génique potentielle.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-0170CM</td>
<td>&nbsp;</td>
<tr>
<td>PEI</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>23966</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70664-3</td>
<td>&nbsp;</td>
<tr>
<td>HiTrap Q HP column</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>17-1154-01</td>
<td>&nbsp;</td>
<tr>
<td>Salmon sperm DNA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9753983</td>
<td>&nbsp;</td>
<tr>
<td>Iodixanol gradient</td>
<td>&nbsp;</td>
<td>OptiPrep</td>
<td>1114542</td>
<td>&nbsp;</td>
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<td>G-50 Columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>27-5330-01</td>
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high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

Des vecteurs recombinants basés sur un parvovirus non-pathogènes humains, le virus adéno-associé 2 (AAV2) ont été développés et sont actuellement en usage dans un certain nombre d&#39;essais cliniques de thérapie génique. Plus récemment, un certain nombre de sérotypes AAV supplémentaires ont également été isolées, qui ont été montrés à l&#39;exposition sélective des tissus tropisme dans divers modèles animaux petits et grands 1. Parmi les 10 sérotypes les plus couramment utilisés AAV, AAV3 est de loin la moins efficace dans la transduction de cellules et de tissus in vitro ainsi que in vivo. Cependant, dans nos études publiées récemment, nous avons documenté que AAV3 vecteurs transduire un cancer du foie humain - hépatoblastome (HB) et le carcinome hépatocellulaire (CHC) - lignées cellulaires de manière extrêmement efficace, car AAV3 utilise des récepteurs humains facteur de croissance des hépatocytes comme un co-récepteur cellulaire pour la liaison et l&#39;entrée dans ces cellules 2,3. Dans cet article, nous décrivons les étapes nécessaires pour atteindre de haute efficacité de transduction des cellules humaines de cancer du foie par recombinaison AAV3 vecteurs portant un gène rapporteur. L&#39;utilisation de recombinants AAV3 vecteurs portant un gène thérapeutique peut éventuellement conduire à la thérapie génique potentiel de cancers du foie chez les humains.

Watch this Scientific Journal Video about High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors at JoVE.com

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Dans cet article, nous décrivons l&#39;identification du virus adéno-associé de sérotype 3 (AAV3) comme le vecteur le plus efficace pour cibler les cellules humaines de cancer du foie.

Haute-efficacité de la transduction des cellules cancéreuses du foie par recombinants sérotype virus adéno-associé 3 Vecteurs

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

high-efficiency-transduction-liver-cancer-cells-recombinant-adeno

Research

JoVE Journal

Medicine

13.1K vues.  University of Florida. Dans cet article, nous décrivons l'identification du virus adéno-associé de sérotype 3 (AAV3) comme le vecteur le plus efficace pour cibler les cellules humaines de cancer du foie.

Vidéo: High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modélisation et Imagerie en 3 dimensions l&#39;invasion des cellules collectives

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Recherche

Médecine

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Une plateforme de criblage à base de luciférase multiplexé pour interroger associée au cancer transduction du signal dans des cellules cultivées

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Profil moléculaire du microenvironnement de la tumeur invasive dans un modèle 3-dimensionnelle des cellules du cancer colorectal et<em&gt; Ex vivo</em&gt; fibroblastes

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Utilisant le virus adéno-associé comme un outil pour étudier les obstacles dans la maladie de la rétine

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Élaboration d&#39;un modèle de rat à base alpha-synucléine pour la maladie de Parkinson par injection stéréotaxique d&#39;un vecteur viral adéno-associé recombinant

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Génération rapide et efficace des Recombinant pluripotentes humaines cellules souches par recombinase médiée Cassette Exchange dans le<em&gt; AAVS1</em&gt; Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Adéno-associés de livraison induite par le Virus de CRISPR gène cardiaque édition chez la souris

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Administration sous-conjonctivale de vecteurs de virus adéno-associés dans des modèles de petits animaux

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

Isolation AAV par centrifugation à double gradient de densité iodixanol

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Pleins feux sur l’auteur : Isolement efficace des virus adéno-associés pour les applications précliniques

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

Culture en suspension, production et purification de virus adéno-associés par centrifugation par gradient de densité d’iodixanol pour des applications in vivo