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Dive into the research topics where Mélissa Desrosiers is active.

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Featured researches published by Mélissa Desrosiers.


Molecular Therapy | 2015

Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice.

Emilie Macé; Romain Caplette; Olivier Marre; Abhishek Sengupta; Antoine Chaffiol; Peggy Barbe; Mélissa Desrosiers; Ernst Bamberg; José-Alain Sahel; Serge Picaud; Jens Duebel; Deniz Dalkara

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eyes easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.


Embo Molecular Medicine | 2016

Red‐shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina

Abhishek Sengupta; Antoine Chaffiol; Emilie Macé; Romain Caplette; Mélissa Desrosiers; Maruša Lampič; Valérie Forster; Olivier Marre; John Y. Lin; José-Alain Sahel; Serge Picaud; Deniz Dalkara; Jens Duebel

Targeting the photosensitive ion channel channelrhodopsin‐2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin‐2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red‐shifted light is vastly lower than that of blue light. Here, we show that a red‐shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV‐ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV‐ and lentivirus‐mediated optogenetic spike responses in ganglion cells of the postmortem human retina.


Molecular Therapy | 2015

Repair of Rhodopsin mRNA by Spliceosome-Mediated RNA Trans-Splicing: A New Approach for Autosomal Dominant Retinitis Pigmentosa

Adeline Berger; Stéphanie Lorain; Charlène Joséphine; Mélissa Desrosiers; Cécile Peccate; Thomas Voit; Luis F. García; José-Alain Sahel; Alexis-Pierre Bemelmans

The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.


PLOS ONE | 2013

A Single Intravenous AAV9 Injection Mediates Bilateral Gene Transfer to the Adult Mouse Retina

Alexis-Pierre Bemelmans; Sandra Duque; Christel Rivière; Stéphanie Astord; Mélissa Desrosiers; Thibault Marais; José-Alain Sahel; Thomas Voit; Martine Barkats

Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.


Biotechnology and Bioengineering | 2016

Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8

Hanen Khabou; Mélissa Desrosiers; Céline Winckler; Stéphane Fouquet; Gwenaelle Auregan; Alexis-Pierre Bemelmans; José-Alain Sahel; Deniz Dalkara

Recently, we described a modified AAV2 vector—AAV2‐7m8—having a capsid‐displayed peptide insertion of 10 amino acids with enhanced retinal transduction properties. The insertion of the peptide referred to as 7m8 is responsible for high‐level gene delivery into deep layers of the retina when virus is delivered into the eyes vitreous. Here, we further characterize AAV2‐7m8 mediated gene delivery to neural tissue and investigate the mechanisms by which the inserted peptide provides better transduction away from the injection site. First, in order to understand if the peptide exerts its effect on its own or in conjunction with the neighboring amino acids, we inserted the 7m8 peptide at equivalent positions on three other AAV capsids, AAV5, AAV8, and AAV9, and evaluated its effect on their infectivity. Intravitreal delivery of these peptide insertion vectors revealed that only AAV9 benefited from 7m8 insertion in the context of the retina. We then investigated AAV2‐7m8 and AAV9‐7m8 properties in the brain, to better evaluate the spread and efficacy of viral transduction in view of the peptide insertion. While 7m8 insertion led to higher intensity gene expression, the spread of gene expression remained unchanged compared to the parental serotypes. Our results indicate that the 7m8 peptide insertion acts by increasing efficacy of cellular entry, with little effect on the spread of viral particles in neural tissue. The effects of peptide insertion are capsid and tissue dependent, highlighting the importance of the microenvironment in gene delivery using AAV. Biotechnol. Bioeng. 2016;113: 2712–2724.


JCI insight | 2018

Noninvasive gene delivery to foveal cones for vision restoration

Hanen Khabou; Marcela Garita-Hernandez; Antoine Chaffiol; Sacha Reichman; Céline Jaillard; Elena Brazhnikova; Stéphane Bertin; Valérie Forster; Mélissa Desrosiers; Céline Winckler; Olivier Goureau; Serge Picaud; Jens Duebel; José-Alain Sahel; Deniz Dalkara

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.


Archive | 2018

Neutralizing Antibodies Against Adeno-Associated Virus (AAV): Measurement and Influence on Retinal Gene Delivery

Mélissa Desrosiers; Deniz Dalkara

Adeno-associated viral vectors have become widely used in the clinic for retinal gene therapy. Thanks to AAVs impeccable safety profile and positive functional outcomes in its clinical application, interest in retinal gene therapy has increased exponentially over the past decade. Although early clinical trials have shown there is little influence of neutralizing antibodies on the performance of AAV when vector is administered into the subretinal space, recent findings suggest neutralizing antibodies may play a role when AAV is delivered via the intravitreal route. These findings highlight the importance of microenvironment on gene delivery and stress the need for a versatile assay to screen subjects for the presence of AAV-neutralizing antibodies. Measuring NAb titers against AAV prior and after gene therapy will help us better understand the impact of preexisting immunity on gene transfer, especially when the vector is administered intravitreally.


Human Molecular Genetics | 2018

Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype

Peter M Quinn; Aat A. Mulder; C Henrique Alves; Mélissa Desrosiers; Sharon I. de Vries; Jan Klooster; Deniz Dalkara; Abraham J. Koster; Carolina R. Jost; Jan Wijnholds

Abstract Variations in the human Crumbs homolog‐1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a‐wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non‐human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1‐associated RP‐like phenotype towards an LCA‐like phenotype.


Molecular Therapy | 2015

495. In Vivo Evidence of trans-Splicing in a Humanized Mouse Model of Autosomal Dominant Retinitis Pigmentosa Induced By Mutation of the Rhodopsin Gene

Adeline Berger; Stéphanie Lorain; Charlène Joséphine; Mélissa Desrosiers; Cécile Peccate; Thomas Voit; Luis F. García; José-Alain Sahel; Alexis-Pierre Bemelmans

The most frequent cause of retinitis pigmentosa (RP), a group of hereditary retinal dystrophies leading to blindness, is the occurrence of point mutations in the Rhodopsin (RHO) gene. Most of these mutations lead to gain of functions or dominant negative effects deleterious for photoreceptors, thus resulting in a dominant mode of transmission. Moreover, it has been shown that variations in RHO expression level are also deleterious for the retina. Gene transfer strategies modifying RHO mutations should therefore lead both to suppression of mutant protein expression and restoration of the normal one at a physiological level. Spliceosome-mediated RNA trans-splicing should in theory respect these constraints by repairing mutations at the pre-mRNA level. Using this technology, the expression level of the repaired mRNA will indeed depend solely on that of the gene endogenous promoter. To achieve this goal, this approach consists of introducing, by gene transfer, an exogenous RNA – called PTM, for Pre-Trans-splicing Molecule – able to bind the pre-mRNA and promote splicing in trans, leading to replacement of the mutant part of the RHO pre-mRNA.We engineered fourteen different RHO-PTMs able to repair any mutation in RHO exons 2, 3, 4 and 5, which differ only on their binding sequence to RHO pre-mRNA. To determine the efficiency of each PTM, we transiently co-transfected HEK293T cells with a plasmid encoding a PTM and another encoding the wild-type or mutant RHO. The maximum trans-splicing efficiency observed at the mRNA level was about 25%. We improved this efficiency to 40% by refinement of the PTM sequence, through reintroduction of an endogenous RHO intron in the cDNA part of the PTM. We then tested PTM at the protein level in a cellular model expressing RHO stably. While wild-type RHO was localized to the plasma membrane, mutant RHO was sequestrated in the cytoplasmic compartment. Using a flow-imaging methodology (ImageStreamX), we were able to precisely quantify this phenotype and showed that trans-splicing partially restored a correct localization of RHO in this cellular model. Thus, we constructed an AAV vector containing our best PTM to assess its activity in vivo in a humanized mouse model of rhodopsin mutation following subretinal injection. Using co-injection with an AAV-GFP and micro-dissection of the transduced part of the retina, we demonstrated that trans-splicing reached 30% at the RNA level, while it was not detectable in the untransduced part, or in animals that received a control PTM. This study demonstrates that trans-splicing may provide a therapeutic benefit in case of dominant mutations of the rhodopsin gene.


Molecular Therapy | 2017

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

Antoine Chaffiol; Romaine Caplette; Céline Jaillard; Elena Brazhnikova; Mélissa Desrosiers; Elisabeth Dubus; Laëtitia Duhamel; Emilie Macé; Olivier Marre; Patrick Benoit; Philippe Hantraye; Alexis-Pierre Bemelmans; Ernst Bamberg; Jens Duebel; José-Alain Sahel; Serge Picaud; Deniz Dalkara

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Deniz Dalkara

Helen Wills Neuroscience Institute

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Alexis-Pierre Bemelmans

Centre national de la recherche scientifique

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Anne M. Douar

Centre national de la recherche scientifique

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Thomas Voit

University College London

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