Mieux comprendre la cascade de signalisation des cellules bipolaires ON et développement de thérapies pour la cécité nocturne congénitale stationnaire

Transmission of the signal from photoreceptors to ON-bipolar cells (ON-BCs) in the retina is partially mediated by the ON-BCs signaling cascade. This cascade involves five major proteins: GRM6, TRPM1, Nyctalopin, GPR179 and LRIT3. The complete form of Congenital Stationary Night Blindness (cCSNB) is an incurable disease caused by mutations in genes coding for these proteines. Affected individuals present an absence of the b-wave on the electroretinogram (ERG) under scotopic conditions, an impairment of dim-light vision often associated with severe myopia, nystagmus and strabismus. This absence of b-wave is also found in patients displaying a melanoma-associated retinopathy (MAR). In this thesis we studied both disorders in order to better understand the signaling cascade of ON-BCs and develop gene therapy approaches for cCSNB due to mutations in LRIT3 or GRM6. We identified TRPM1 autoantibodies in the sera of three new cases of MAR, explaining the absence of b-wave on the ERG. In Grm6-/- mice, we restored GRM6 at the dendritic tips of ON-BCs along with other partners of the ON-BCs signaling cascade, however no functional rescue was obtained in these mice. This gives basis for a GRM6 therapy approach of which the efficacy needs to be improved. Furthermore, we demonstrated the feasibility of gene therapy in adult Lrit3-/- mice which presented a restoration at the protein level and at the functional level up to four months after treatment. This thesis highlights the key role of the ON-BCs signaling cascade in the transmission of the visual signal and the success, to different extents, of gene addition strategy to treat cCSNB.La transmission du signal entre les photorécepteurs et les cellules bipolaires ON (ON-BCs) dans la rétine est en partie médiée par la cascade de signalisation des ON-BCs. Cette cascade implique cinq protéines majeures : GRM6, TRPM1, Nyctalopin, GPR179 et LRIT3. La forme complète de la cécité nocturne congénitale stationnaire (CNCSc) est une maladie incurable causée par des mutations sur les gènes codants ces protéines. Les patients atteints de CNCSc n’ont plus d’onde b à l’électrorétinogramme (ERG) et ont des difficultés à voir en condition de faible lumière souvent associé à une forte myopie. L’absence d’onde b à l’ERG est aussi observée chez les patients atteints de rétinopathie associée au mélanome (MAR). Dans cette thèse nous avons étudié ces deux maladies afin de mieux comprendre la cascade de signalisation des ON-BCs et développer un traitement pour la CNCSc due à des mutations dans GRM6 ou LRIT3. Nous avons identifié des auto-anticorps dirigés contre TRPM1 expliquant le phénotype de trois nouveaux patients atteints de MAR. Nous avons également démontré la faisabilité d’une approche de thérapie génique pour les mutations dans LRIT2 dans un modèle de souris adulte Lrit3-/-. Une restauration au niveau protéique et fonctionnel a été enregistrée chez les souris traitées. Chez les souris Grm6-/- traitées, la localization de GRM6 a été restaurée ainsi que celle d’autres protéines de la cascade mais aucune restauration fonctionnelle n’a été observée. Cette thèse met en avant le rôle majeur de la cascade des ON-BCs dans la transmission du signal visuel dans la rétine et le succès, à différents niveaux, de la stratégie d’addition de gène pour traiter la CNCSc

Mieux comprendre la cascade de signalisation des cellules bipolaires ON et développement de thérapies pour la cécité nocturne congénitale stationnaire

Transmission of the signal from photoreceptors to ON-bipolar cells (ON-BCs) in the retina is partially mediated by the ON-BCs signaling cascade. This cascade involves five major proteins: GRM6, TRPM1, Nyctalopin, GPR179 and LRIT3. The complete form of Congenital Stationary Night Blindness (cCSNB) is an incurable disease caused by mutations in genes coding for these proteines. Affected individuals present an absence of the b-wave on the electroretinogram (ERG) under scotopic conditions, an impairment of dim-light vision often associated with severe myopia, nystagmus and strabismus. This absence of b-wave is also found in patients displaying a melanoma-associated retinopathy (MAR). In this thesis we studied both disorders in order to better understand the signaling cascade of ON-BCs and develop gene therapy approaches for cCSNB due to mutations in LRIT3 or GRM6. We identified TRPM1 autoantibodies in the sera of three new cases of MAR, explaining the absence of b-wave on the ERG. In Grm6-/- mice, we restored GRM6 at the dendritic tips of ON-BCs along with other partners of the ON-BCs signaling cascade, however no functional rescue was obtained in these mice. This gives basis for a GRM6 therapy approach of which the efficacy needs to be improved. Furthermore, we demonstrated the feasibility of gene therapy in adult Lrit3-/- mice which presented a restoration at the protein level and at the functional level up to four months after treatment. This thesis highlights the key role of the ON-BCs signaling cascade in the transmission of the visual signal and the success, to different extents, of gene addition strategy to treat cCSNB.La transmission du signal entre les photorécepteurs et les cellules bipolaires ON (ON-BCs) dans la rétine est en partie médiée par la cascade de signalisation des ON-BCs. Cette cascade implique cinq protéines majeures : GRM6, TRPM1, Nyctalopin, GPR179 et LRIT3. La forme complète de la cécité nocturne congénitale stationnaire (CNCSc) est une maladie incurable causée par des mutations sur les gènes codants ces protéines. Les patients atteints de CNCSc n’ont plus d’onde b à l’électrorétinogramme (ERG) et ont des difficultés à voir en condition de faible lumière souvent associé à une forte myopie. L’absence d’onde b à l’ERG est aussi observée chez les patients atteints de rétinopathie associée au mélanome (MAR). Dans cette thèse nous avons étudié ces deux maladies afin de mieux comprendre la cascade de signalisation des ON-BCs et développer un traitement pour la CNCSc due à des mutations dans GRM6 ou LRIT3. Nous avons identifié des auto-anticorps dirigés contre TRPM1 expliquant le phénotype de trois nouveaux patients atteints de MAR. Nous avons également démontré la faisabilité d’une approche de thérapie génique pour les mutations dans LRIT2 dans un modèle de souris adulte Lrit3-/-. Une restauration au niveau protéique et fonctionnel a été enregistrée chez les souris traitées. Chez les souris Grm6-/- traitées, la localization de GRM6 a été restaurée ainsi que celle d’autres protéines de la cascade mais aucune restauration fonctionnelle n’a été observée. Cette thèse met en avant le rôle majeur de la cascade des ON-BCs dans la transmission du signal visuel dans la rétine et le succès, à différents niveaux, de la stratégie d’addition de gène pour traiter la CNCSc

Risk Mitigation of Immunogenicity: A Key to Personalized Retinal Gene Therapy

Gene therapy (GT) for ocular disorders has advanced the most among adeno-associated virus (AAV)-mediated therapies, with one product already approved in the market. The bank of retinal gene mutations carefully compiled over 30 years, the small retinal surface that does not require high clinical vector stocks, and the relatively immune-privileged environment of the eye explain such success. However, adverse effects due to AAV-delivery, though rare in the retina have led to the interruption of clinical trials. Risk mitigation, as the key to safe and efficient GT, has become the focus of ‘bedside-back-to-bench’ studies. Herein, we overview the inflammatory adverse events described in retinal GT trials and analyze which components of the retinal immunological environment might be the most involved in these immune responses, with a focus on the innate immune system composed of microglial surveillance. We consider the factors that can influence inflammation in the retina after GT such as viral sensors in the retinal tissue and CpG content in promoters or transgene sequences. Finally, we consider options to reduce the immunological risk, including dose, modified capsids or exclusion criteria for clinical trials. A better understanding and mitigation of immune risk factors inducing host immunity in AAV-mediated retinal GT is the key to achieving safe and efficient GT

Risk Mitigation of Immunogenicity: A Key to Personalized Retinal Gene Therapy

Gene therapy (GT) for ocular disorders has advanced the most among adeno-associated virus (AAV)-mediated therapies, with one product already approved in the market. The bank of retinal gene mutations carefully compiled over 30 years, the small retinal surface that does not require high clinical vector stocks, and the relatively immune-privileged environment of the eye explain such success. However, adverse effects due to AAV-delivery, though rare in the retina have led to the interruption of clinical trials. Risk mitigation, as the key to safe and efficient GT, has become the focus of ‘bedside-back-to-bench’ studies. Herein, we overview the inflammatory adverse events described in retinal GT trials and analyze which components of the retinal immunological environment might be the most involved in these immune responses, with a focus on the innate immune system composed of microglial surveillance. We consider the factors that can influence inflammation in the retina after GT such as viral sensors in the retinal tissue and CpG content in promoters or transgene sequences. Finally, we consider options to reduce the immunological risk, including dose, modified capsids or exclusion criteria for clinical trials. A better understanding and mitigation of immune risk factors inducing host immunity in AAV-mediated retinal GT is the key to achieving safe and efficient GT

Impact of the COVID-19 lockdown on basic science research in ophthalmology: the experience of a highly specialized research facility in France

International audienceno abstrac

Identification and characterization of novel TRPM1 autoantibodies from serum of patients with melanoma-associated retinopathy

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1+/+ but not on Trpm1-/- mouse retina as shown by co-immunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry). Therefore, we propose that a combination of different methods should be used to test for the presence of anti-TRPM1 autoantibodies in the sera of MAR patients

Prevalence of ABCA4 Deep-Intronic Variants and Related Phenotype in An Unsolved “One-Hit” Cohort with Stargardt Disease

We investigated the prevalence of reported deep-intronic variants in a French cohort of 70 patients with Stargardt disease harboring a monoallelic pathogenic variant on the exonic regions of ABCA4. Direct Sanger sequencing of selected intronic regions of ABCA4 was conducted. Complete phenotypic analysis and correlation with the genotype was performed in case a known intronic pathogenic variant was identified. All other variants found on the analyzed sequences were queried for minor allele frequency and possible pathogenicity by in silico predictions. The second mutated allele was found in 14 (20%) subjects. The three known deep-intronic variants found were c.5196+1137G>A in intron 36 (6 subjects), c.4539+2064C>T in intron 30 (4 subjects) and c.4253+43G>A in intron 28 (4 subjects). Even though the phenotype depends on the compound effect of the biallelic variants, a genotype-phenotype correlation suggests that the c.5196+1137G>A was mostly associated with a mild phenotype and the c.4539+2064C>T with a more severe one. A variable effect was instead associated with the variant c.4253+43G>A. In addition, two novel variants, c.768+508A>G and c.859-245_859-243delinsTGA never associated with Stargardt disease before, were identified and a possible splice defect was predicted in silico. Our study calls for a larger cohort analysis including targeted locus sequencing and 3D protein modeling to better understand phenotype-genotype correlations associated with deep-intronic changes and patients’ selection for clinical trials

Restoration of mGluR6 Localization Following AAV-Mediated Delivery in a Mouse Model of Congenital Stationary Night Blindness

International audiencePurpose: Complete congenital stationary night blindness (cCSNB) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. GRM6 mutations are the third most prevalent cause of cCSNB. The Grm6−/− mouse model mimics the human phenotype, showing no b-wave in the electroretinogram (ERG) and a loss of mGluR6 and other proteins of the same cascade at the outer plexiform layer (OPL). Our aim was to restore protein localization and function in Grm6−/− adult mice targeting specifically ON-BCs or the whole retina.Methods: Adeno-associated virus-encoding Grm6 under two different promoters (GRM6-Grm6 and CAG-Grm6) were injected intravitreally in P15 Grm6−/− mice. ERG recordings at 2 and 4 months were performed in Grm6+/+, untreated and treated Grm6−/− mice. Similarly, immunolocalization studies were performed on retinal slices before or after treatment using antibodies against mGluR6, TRPM1, GPR179, RGS7, RGS11, Gβ5, and dystrophin.Results: Following treatment, mGluR6 was localized to the dendritic tips of ON-BCs when expressed with either promoter. The relocalization efficiency in mGluR6-transduced retinas at the OPL was 2.5% versus 11% when the GRM6-Grm6 and CAG-Grm6 were used, respectively. Albeit no functional rescue was seen in ERGs, relocalization of TRPM1, GPR179, and Gβ5 was also noted using both constructs. The restoration of the localization of RGS7, RGS11, and dystrophin was more obvious in retinas treated with GRM6-Grm6 than in retinas treated with CAG-Grm6.Conclusions: Our findings show the potential of treating cCSNB with GRM6 mutations; however, it appears that the transduction rate must be improved to restore visual function

Near-infrared fundus autofluorescence alterations correlate with swept-source optical coherence tomography angiography findings in patients with retinitis pigmentosa

International audienceThirty-eight patients from 37 families with retinitis pigmentosa (RP) underwent macular 6 × 6-mm swept-source optical coherence tomography angiography (SS-OCTA) and 30° near-infrared fundus autofluorescence (NIR-FAF) acquisitions in one eye. Superficial vascular complex (SVC), deep capillary complex (DCC) and choriocapillaris (CC) angiograms were registered with NIR-FAF acquisitions to comparatively assess subjects with and without central area of preserved NIR-FAF (APA). On the subset of patients showing an APA, the vessel densities for SVC and DCC and flow deficits for CC were assessed in three directions (superior, inferior and temporal) from the fovea and compared to healthy 1:1 age-matched controls. Nine patients with no APA had evidence of severe central OCTA alterations at all levels, especially in the DCC. In the other 29 subjects presenting APA, all OCTA parameters were similar to healthy eyes within the APA, where the retina preserves its structural integrity. Outside the APA, both the DCC and CC were significantly reduced in all directions. These alterations are probably related to the outer retinal atrophy outside the APA. Comparing OCTA to other imaging modalities is helpful to determine the potential interest of OCTA findings as an outcome measure for disease status and progression

Substantial restoration of night vision in adult mice with congenital stationary night blindness

International audienceComplete congenital stationary night blindness (cCSNB) due to mutations in TRPM1, GRM6, GPR179, NYX, or leucinerich repeat immunoglobulin-like transmembrane domain 3 (LRIT3) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. Since the disease is nondegenerative and stable, treatment could theoretically be administrated at any time in life, making it a promising target for gene therapy. Until now, adeno-associated virus (AAV)mediated therapies lead to significant functional improvements only in newborn cCSNB mice. Here we aimed to restore protein localization and function in adult Lrit3 À/À mice. LRIT3 localizes in the outer plexiform layer and is crucial for TRPM1 localization at the dendritic tips of ON-BCs and the electroretinogram (ERG)-b-wave. AAV2-7m8-Lrit3 intravitreal injections were performed targeting either ON-BCs, photoreceptors (PRs), or both. Protein localization of LRIT3 and TRPM1 at the rod-to-rod BC synapse, functional rescue of scotopic responses, and ON-responses detection at the ganglion cell level were achieved in a few mice when ON-BCs alone or both PRs and ON-BCs, were targeted. More importantly, a significant number of treated adult Lrit3 À/À mice revealed an ERG b-wave recovery under scotopic conditions, improved optomotor responses, and on-time ON-responses at the ganglion cell level when PRs were targeted. Functional rescue was maintained for at least 4 months after treatment