Imaging Light Responses of Foveal Ganglion Cells in the Living Macaque Eye

The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses

Optogenetic Light Sensors in Human Retinal Organoids

Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules

A Novel Adeno-Associated Viral Variant for Efficient and Selective Intravitreal Transduction of Rat Müller Cells

BACKGROUND:The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection. METHODOLOGY/PRINCIPAL FINDINGS:We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6. CONCLUSIONS/SIGNIFICANCE:Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

The authors are grateful to Manuel Simonutti, Julie Dégardin, Jennifer Da Silva, Samantha Beck and Caroline Carvalho for their valuable help in phenotyping (platform of Institut de la Vision) and to Isabelle Renault, Léa Biedermann and André Tiffoche for animal care (platform of Institut de la Vision). The authors thank Stéphane Fouquet for his support in developing a custom-made Image J macro to measure thickness of retinal layers.This work was supported by Fondation Valentin Hauy (IA, EO), Retina France (IA, EO), e-rare RHORCOD (IA), Fondation de l’Oeil—Fondation de France (IA), Foundation Voir et Entendre (CZ), Foundation Fighting Blindness (FFB) (CD-CL-0808-0466-CHNO) (IA), and the FFB center grant (CD-CL-0808-0466-CHNO), Ville de Paris and Region Ile de France, Labex Lifesenses (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d’Avenir programme (ANR-11-IDEX-0004-0), the Regional Council of Ile de France (I09–1727/R) (EO), the National Institute of Health grants EY10609 (MIN), EY001919 (MML) and EY006842 (MML) and the Foundation Fighting Blindness (MIN and MML).Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.Yeshttp://www.plosone.org/static/editorial#pee

La conception de vecteurs adaptés à la thérapie génique oculaire

L’utilisation d’un gène comme médicament est l’un des concepts les plus passionnants de la médecine moderne. Les vecteurs viraux ont été largement utilisés à cette fin et ont montré une efficacité thérapeutique dans une variété de modèles animaux de dégénérescence rétinienne. L’extension de ce succès à une application clinique a été initialement lente, mais une expression à long terme de gènes thérapeutiques a récemment été obtenue chez des patients atteints de déficits immunitaires, d’hémophilie B ou de troubles héréditaires de la rétine. Ces résultats ont suscité des espoirs pour le traitement de nombreuses autres maladies, et ont ouvert la voie au développement de nouveaux outils de transfert de gènes. Comme nous le verrons ici, les perspectives et les défis de la thérapie génique sont dans une large mesure dépendants du tissu ciblé, de la maladie concernée et, surtout, de l’efficacité du transfert génique. L’acheminement du gène vers les cellules cibles dépend de vecteurs, qui doivent assurer une expression durable du gène sans engendrer ni toxicité ni réaction immunitaire de la part de l’hôte. La conception de tels vecteurs, proposée pour la première fois dans les années 1970, s’est avérée être plus compliquée que prévu, limitant pendant de nombreuses années le succès de la thérapie génique. Les vecteurs doivent donc être adaptés à chaque scénario ; nous discuterons ici la conception de tels vecteurs pour un transfert de gènes vers la rétine

Vectors and Gene Delivery to the Retina

International audienceOne of the great advantages of the retina as a target tissue for gene delivery is the wide array of genetic tools that have been developed in the past decade. This includes a variety of vectors for therapeutic gene delivery to most types of retinal neurons and glia, as well as cell type–specific promoters for restricted gene expression in distinct neuronal subtypes. Within the scope of neuroscience applications and for gene therapy, it is now routine to express reporter genes, replacement genes, neuronal activity indicators, and microbial opsins in specific neuronal types in the mouse retina. However, there are considerable anatomical, physiological, immunological, and behavioral differences between the mouse and the human that limit the usefulness of these tools in humans and nonhuman primates. Several advances are now being made toward the goal of applying viral targeting tools to understand the primate retina. Here, we describe these advances, consider their potential to advance our understanding of the primate retina, and describe what will be needed to move forward

Transfert intracellulaire de protéines

Les travaux réalisés au cours de cette thèse ont consisté à mettre au point des méthodes efficaces de transfert de protéines dans le cytoplasme des cellules mammifères. Pour cela, nous avons utilisé des formulations lipides cationiques protéines, puis étudié l inter-nalisation de ces complexes dans des cellules. La plupart des approches permettant l'expression spécifique d une protéine sont fondées sur la vectorisation du gène codant pour cette protéine dans le noyau des cellules. En considérant que le but du transfert de gène est la production de protéines et que les vecteurs synthétiques sont capables d effectuer efficacement un transfert de macromolécules dans le cytoplasme, nous avons étudié le transfert de protéines par ces mêmes vecteurs. Nous avons donc dans un premier temps étudié le transfert intracellulaire de protéines avec un lipide cationique, le dioctadécylamidoglycyl-spermine ou DOGS. Nous avons étudié l interaction du DOGS avec l albumine sérique, et ensuite déterminé les différents paramètres impliqués dans l obtention de complexes internalisables dans les cellules. Nous avons appliqué cette stratégie pour transférer d autres protéines avec des propriétés physico-chimiques différentes, et ainsi mieux analyser la contribution de chaque paramètre au processus de complexation et de vectorisation par le DOGS. Nous avons réussi à transférer avec grande efficacité une large variété de protéines anioniques comme des anticorps monoclonaux, une enzyme et une phycobiliprotéine. Au cours de cette thèse, nous avons donc développé une stratégie originale de transfert intracellulaire de protéines, qui devrait nous permettre de transférer in vitro, dans des cellules animales, une large variété de protéines à but thérapeutique ou fonctionnel.During this thesis we have developped an efficient method for intracytoplasmic protein delivery into mammalian cells. This method employs cationic lipids, previously used in gene delivery, to complex proteins and to transport them into the cytoplasm of cells in culture. In the last decade, the most commonly used approach for protein expression has been the transfection of its gene. Considering that the aim of transfection is to produce proteins and that synthetic vectors are capable of efficient gene delivery to mammalian cells; we studied direct intracellular protein delivery using these carriers. The direct delivery of functionally active proteins can be helpful in overcoming some bottlenecks of transfection mediated protein production. We thus initiated studying intracellular protein delivery using the cationic lipid DOGS. We studied the interaction of the cationic lipid with bovine serum albumin and determined the different parameters involved in creating complexes that are well internalised by cells. We applied the notions acquired during the intracellular delivery of this model protein to a variety of other proteins with different physico-chemical properties in order to apprehend the contribution of these properties to the processus of complexation and vectorisation by DOGS. We successfully delivered a variety of proteins such as monoclonal antibodies, an enzyme, and a phycobilliprotein into different mammalian cell lines. Furthermore, we demonstrated that the antibodies delivered using this method retain their ability to bind their antigens once they reach the cytoplasm. From this point of view, intracellular protein delivery with cationic lipids can be considered another way to interfere with cellular activities. During this thesis, we have thus developped an original strategy for the intracellular delivery of proteins which will allow us to deliver, in vivo, a large variety of proteins for therapeutic purposes or functional studies.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Sphingosine 1-Phosphate Receptor 1 Modulates CNTF-Induced Axonal Growth and Neuroprotection in the Mouse Visual System

The lack of axonal regeneration and neuronal cell death causes permanent neurological deficits in the injured CNS. Using the classical CNS injury model of optic nerve crush in mice, ciliary neurotrophic factor (CNTF) was found to stimulate retinal ganglion cell (RGC) survival and axonal growth, but in an incomplete fashion. The elucidation of molecular mechanisms impairing CNTF-induced axonal regeneration is paramount to promote visual recovery. In the present study, we sought to evaluate the contribution of sphingosine 1-phosphate receptor 1 (S1PR1) to the neuroprotective and regenerative effects of CNTF. The transduction of retinal cells with adeno-associated viruses (AAV) allowed to activate CNTF/signal transducer and activator of transcription 3 (Stat3) signaling and to modulate S1PR1 expression in RGCs. Our results showed that CNTF/Stat3 prevented injury-induced S1PR1 downregulation. Silencing S1PR1 in RGCs significantly enhanced CNTF-induced axonal growth in the injured optic nerve. In contrast, RGC survival was markedly decreased when S1PR1 was repressed with viral vectors. The level of phosphorylated Stat3 (P-Stat3), an intracellular mediator of CNTF, did not fluctuate after S1PR1 inhibition and CNTF stimulation. Collectively, these results suggest that S1PR1 acts as a major regulator of retinal neuron survival and restricts the RGC growth response induced by CNTF