September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Gene Therapy for CSNB associated with GRM6 gene defects
Author Affiliations & Notes
  • Miguel Miranda de Sousa Dias
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Thomas Pugliese
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
    CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC, 28 rue de Charenton, 75012 Paris, France
  • Marion Neuille
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Elise Orhan
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Christelle Michiels
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Melissa Desrosiers
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Jose Sahel
    CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC, 28 rue de Charenton, 75012 Paris, France
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Deniz Dalkara
    Institute of Ophthalmology, University College of London, London, United Kingdom
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Isabelle S Audo
    CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC, 28 rue de Charenton, 75012 Paris, France
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
  • Christina Zeitz
    Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, 75012 Paris, France
    CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS CIC, 28 rue de Charenton, 75012 Paris, France
  • Footnotes
    Commercial Relationships   Miguel Miranda de Sousa Dias, None; Thomas Pugliese, None; Marion Neuille, None; Elise Orhan, None; Christelle Michiels, None; Melissa Desrosiers, None; Jose Sahel, None; Deniz Dalkara, None; Isabelle Audo, None; Christina Zeitz, None
  • Footnotes
    Support  Retina France, Fondation Voir et Entendre, Prix Dalloz for “la recherche en ophtalmologie”, Agence Nationale de la Recherche (ANR-12-BSVS1-001201_GPR179), the Fondation pour la Recherche Médicale (FRM DVS20131228918) in partnership with the Fondation Roland Bailly, Union Nationale des Aveugles et Déficients Visuels (UNADEV), alliance nationale pour les sciences de la vie et de la santé (aviesan), AFM-Téléthon (C.Z.), Fédération des Aveugles et Handicapés visuels de France (TP, MN), Ville de Paris and Région 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)
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1182. doi:
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    • Get Citation

      Miguel Miranda de Sousa Dias, Thomas Pugliese, Marion Neuille, Elise Orhan, Christelle Michiels, Melissa Desrosiers, Jose Sahel, Deniz Dalkara, Isabelle S Audo, Christina Zeitz; Gene Therapy for CSNB associated with GRM6 gene defects. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1182.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Congenital Stationary Night Blindness (CSNB) is characterized by a signalling dysfunction from the photoreceptors to the adjacent ON-bipolar cells (BCs). The purpose of this study was to target BCs in wild-type (WT) mice and mice lacking Grm6 (Grm6tm1Nak/tm1Nak) using adeno-associated virus (AAV) and to document the phenotype before/after treatment by electroretinography (ERG) and immunolocalization studies.

Methods : Two distinct constructs were produced from an AAV2 based capsid mutant (AAV2-7m8). The first construct contained GFP under the control of 200-bp enhancer sequence of Grm6 gene, fused to the SV40 eukaryotic promoter (AAV2-7m8-GFP), and the second construct encompassed Grm6 cDNA driven by the same promoter (AAV2-7m8-Grm6). WT mice were injected intravitreally with AAV2-7m8-GFP at P60; fundus pictures were documented and co-immunostaining of GFP was performed with GRM6 or PKC-α at P100. AAV2-7m8-Grm6 was intravitreally injected into Grm6tm1Nak/tm1Nak mice eyes at P60. The rescue of the rod ON-BCs function was evaluated through ERGs, under scotopic conditions, at P60 before treatment and at P90 and P120 after treatment. ERG responses from treated eyes were compared with untreated contralateral eyes. Subsequently, mice were sacrificed and expression and immunolocalization of GRM6 studied by quantitative real-time experiments (RT-qPCR) and immunohistochemistry.

Results : Fundus fluorescence photographs taken after AAV2-7m8-GFP delivery to WT mice revealed a widespread expression of GFP. Most GFP staining was observed in the cell-body and tips of BCs dendrites in close vicinity to GRM6. ERGs from eyes of Grm6tm1Nak/tm1Nak mice treated with AAV2-7m8-Grm6 presented an overall less negative polarity, but no measurable improvement in the ERG b-wave. RT-qPCR showed 91% expression of Grm6 on the retina of the treated eyes in comparison with WT. Immunolocalization confirmed the presence of GRM6 on the outer-plexiform layer of the treated eyes, but the staining pattern was slightly different compared to WT mice.

Conclusions : This study presents the first gene replacement therapy attempt for CSNB associated with the Grm6 gene defects. AAV2-7m8 efficiency in combination with the Grm6 promoter to deliver the GFP/Grm6 cDNA to the BCs was demonstrated. A helper peptide insertion and early viral delivery on post-natal eyes will most likely improve correct GRM6 localization and phenotype recovery.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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