Gene Therapy for CSNB associated with GRM6 gene defects

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 (Grm6^{tm1Nak/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 Grm6^{tm1Nak/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 Grm6^{tm1Nak/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.