Corneal Transduction by Intra-Stromal Injection of AAV Vectors In Vivo in the Mouse and Ex Vivo in Human Explants

The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea

The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models

Etudes de transfert de gène pour une maladie de surcharge lysosomale, la cystinose

La cystinose appartient au groupe des maladies de surcharge lysosomale (MSL) et est caractérisée par un défaut d'efflux de cystine hors du lysosome. Le gène en cause, CTNS, code la cystinosine, le transporteur lysosomal de la cystine. L'accumulation de cystine va perturber la fonction de la majorité des organes à différents niveaux. La cystéamine, seul traitement actuellement disponible présente des contraintes et des inconvénients majeurs, ce qui nous a conduit à explorer une nouvelle alternative de traitement. La majeure partie de mes travaux de thèse a consisté à apporter la preuve que le transfert de gène est applicable à une MSL due à un transporteur déficient. Nos études réalisées in vivo ciblant le foie de souris Ctns-/- avec un vecteur adénoviral humain (hAd) montrent que la thérapie génique pour la cystinose semble plus préventive que curative des atteintes déjà présentes. Au lieu d'appliquer une thérapie multi-systémique nous avons choisi une approche tissu-spécifique. Pour cela, nous nous sommes orientés vers la caractérisation et le ciblage des anomalies oculaires et neurologiques des souris Ctns-/-. Nous avons montré que les atteintes de ces souris sont similaires à celles des patients. En parallèle, nous avons établi un guide spati-temporel de leurs apparitions, outil indispensable pour nos études de transfert de gène. Ces travaux nous ont permis de démarrer nos études de transfert de gène ciblées dans lesquelles nous évaluons l'efficacité de deux vecteurs plus pertinent au niveau clinique que le vecteur hAd: le vecteur adénoviral canin dépendant d'un virus helper et le vecteur associé à l'adénovirusCystinosis is a lysosomal storage disorder characterised by a defective lysosomal cystine efflux. The causative gene, CTNS, encodes the lysosomal cystine transporter, cystinosin. Storage of cystine, which forms crystals at high levels, disrupts the function of different organs at different rates. Cysteamine, the only treatment currently available to reduce lysosomal cystine levels, has dramatically improved the life of patients. However, due to its administration constraints and side effects, we explored the feasibility of viral-mediated CTNS gene therapy to reduce cystine levels in cystinosin-deficient (Ctns-/-) mice. The main part of my PhD was concentrated on in vivo gene transfer studies to the liver using CTNS-expressing human adenovirus vectors (hAd). This work provided for the first time the proof-of-concept that viral-mediated CTNS gene transfer can correct a lysosomal transport defect, but suggests that, in the case of cystinosis, it could be preventive but not curative in some tissues. Rather than attempting a mulisystemic gene therapy to cystinosis, we specifically concentrated on treating the ocular and CNS anomalies, which are incapacitating and have the potential to be life-threatening. Thus, along this line, we characterised these anomalies in Ctns-/- mice : we showed that they mimic those of patients and, in parallel, generated a temporospatial guide to their appearance, an essential tool for testing novel ocular and CNS cystine-depleting therapies. The next step of this work was to begin gene transfer studies in these structures using more clinically relevant vectors than hAd vector: an helper dependant canin adenovirus and an adeno-associated virusMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Structure locale dans un ferroélectrique relaxeur : BaTi(1-x)Zr(x)O3

Les ferroélectriques relaxeurs se caractérisent par un large pic de permittivité en fonction de la température, dépendant de la fréquence du champ de mesure. Ce comportement est généralement attribué à la présence de régions polaires de taille nanométrique. L un des enjeux expérimentaux est la détermination de la nature structurale de ces régions, nécessitant entre autres l utilisation de sondes de la structure locale. L objet de ce travail est l étude de la structure locale dans les pérovskites relaxeurs BaTi1-xZrxO3 (0.25 <= x <= 0.50), présentant une substitution isovalente Ti4+/Zr4+. Les techniques expérimentales utilisées sont l absorption des rayons X (EXAFS et XANES) et la détermination de la fonction de distribution de paires par diffusion totale des neutrons. Les déplacements des cations Ti4+ et Zr4+ dans leur cage d oxygènes ont pu être déterminés. Le principal résultat est que les cations Ti4+ jouent un rôle majeur dans la polarisation locale des relaxeurs BaTi1-xZrxO3. Par ailleurs, il est montré que la déformation des octaèdres ZrO6 dépend directement de la répartition locale des Ti et des Zr dans la solution solide.Relaxor ferroelectrics are characterized by a large, frequency-dependent peak of the dielectric permittivity as a function of temperature. This behaviour is generally attributed to the presence of nanometre-sized polar regions. One of the experimental challenges is the determination of the structural nature of such regions, which requires the use of local structural probes. This manuscript presents the study of the local structure in the perovskite-type relaxor ferroelectrics BaTi1-xZrxO3 (0.25 <= x <= 0.50), by means of X-ray absorption (EXAFS and XANES) and pair distribution function determination from neutron total scattering. We could determine the Ti4+ and Zr4+ cation displacements with respect to the centre of their oxygen cage. The main result is that the Ti4+ cations play a major role in the local polarisation in BaTi1-xZrxO3 relaxors. Moreover, it is shown that the deformation of the ZrO6 octahedra directly depends on the local distribution of the Ti and Zr atoms in the solid solution.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Local structure in BaTi_1−<i>x</i>Zr_<i>x</i>O₃ relaxors from neutron pair distribution function analysis

International audienceThe pair distribution functions (PDF) of BaTi1−xZrxO3 (BTZ) relaxors (x=0.25,0.32,0.35), as well as those of the end members BaTiO3 and BaZrO3, were determined at 300 K from neutron powder scattering data. In the relaxors, the PDF provides direct evidence that the Ti and Zr atoms do not occupy the equivalent octahedral sites expected from the crystallographic cubic perovskite structure. It is shown that the TiO6 and ZrO6 octahedra in BTZ relaxors are instead similar to those observed in BaTiO3 and BaZrO3, respectively. In BaZrO3, the Zr atoms lie at the center of regular oxygen octahedra, forming nonpolar ZrO6 units. In the tetragonal ferroelectric phase of BaTiO3, the distribution of Ti-O distances within TiO6 octahedra is found compatible with a displacement of the Ti atoms in the [111]p direction of the pseudocubic perovskite cell. We conclude that the local polarization in BTZ relaxors is mainly due to the displacements of the Ti atoms and that moreover the Ti displacements are very similar in BTZ relaxors and in the classical ferroelectric BaTiO3

Photoreceptor replacement therapy:challenges presented by the diseased recipient retinal environment

AbstractVision loss caused by the death of photoreceptors is the leading cause of irreversible blindness in the developed world. Rapid advances in stem cell biology and techniques in cell transplantation have made photoreceptor replacement by transplantation a very plausible therapeutic strategy. These advances include the demonstration of restoration of vision following photoreceptor transplantation and the generation of transplantable populations of donor cells from stem cells. In this review, we present a brief overview of the recent progress in photoreceptor transplantation. We then consider in more detail some of the challenges presented by the degenerating retinal environment that must play host to these transplanted cells, how these may influence transplanted photoreceptor cell integration and survival, and some of the progress in developing strategies to circumnavigate these issues.</jats:p

Random local strain effects in the relaxor ferroelectric BaTi_1−<i>x</i>Zr_<i>x</i>O₃: experimental and theoretical investigation

We report an investigation of the local structure in homovalent-substituted BaTi1−x Zr x O3 relaxors by a combination of experimental and theoretical methods, namely neutron total scattering, X-ray absorption spectroscopy, and supercell ab-initio calculations. It is shown that unlike Zr atoms, Ti atoms are largely displaced in their octahedra, and are thus associated with strong local dipole moments. Besides, we give evidence that the difference in the size of Ti4+ and Zr4+ cations leads to a significant size mismatch of the Ti-O6 and Zr-O6 octahedra. When they link to form the perovskite structure of BaTi1−x Zr x O3, the O6 octahedra undergo slight distortions in order to accommodate their different sizes. It is shown that they are compressed in the direction of Zr neighbors, and expanded in the direction of Ti neighbors. The polar Ti displacements, which are sensitive to the octahedral distortions, then become constrained in their orientation according to the local Zr/Ti distribution. Such constraints impede a perfect alignment of all the Ti displacements as existing in the classic ferroelectric BaTiO3. Our results shed light on the structural mechanisms that lead to disordered Ti displacements in BaTi1−x Zr x O3 relaxors, and probably in other BaTiO3-based relaxors with homovalent substitution

Improvement of Phase‐Change Memory Performance by Means of GeTe/Sb 2 Te 3 Superlattices

International audienc

Transduction efficiency of AAV vectors in the mouse cornea.

<p>EGFP expression (indicated by arrows) in the mouse cornea detected by <i>in vivo</i> epifluorescence microscopy 1-wk post-injection of AAV2/1 (<b>a</b>), AAV2/2 (<b>e</b>), AAV2/5 (<b>i</b>) and AAV2/8 (<b>m</b>) vectors. (<b>b</b>, <b>f</b>, <b>j</b>, <b>n</b>) Higher magnification of <b>a</b>, <b>e</b>, <b>i</b> and <b>m</b>, respectively. (<b>c</b>, <b>g</b>, <b>k</b>, <b>o</b>) EGFP expression in the same corneas detected 4-wk post-injection. (<b>d</b>, <b>h</b>, <b>l</b>, <b>p</b>) Higher magnification of <b>c</b>, <b>g</b>, <b>k</b> and <b>o</b>, respectively. Magnifications <b>a</b>, <b>e</b>, <b>g</b>, <b>i</b>, <b>k</b>, <b>m</b>, <b>o</b>: 20×; <b>b</b>: 43×; <b>c</b>: 25×, <b>d</b>: 53×; <b>f</b>, <b>j</b>: 35×; <b>h</b>: 44×, <b>l</b>: 33×; <b>n</b>: 40×; <b>p</b>: 45×. For reference, the diameter of an adult mouse eye is ∼3.5 mm. The large green spot in centre of photos is the pupil of the mouse eye. The asterisk in panels <b>i</b> to <b>l</b> indicates an opaque lesion on the mouse eye that was present from the beginning of the experiments.</p