AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier permeability restoration

Abstract

 

Purpose

 

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells, its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (Sene, 2009). Dp71-null mouse is thus an excellent model to approach the study of retinal pathologies showing BRB permeability. The Adeno-associated virus (AAV) variant, ShH10, engineered to target Müller cells specifically, has been characterized to transduce more efficiently Müller cells through intravitreal injection in the Dp71-null mouse (Vacca, 2014). Here, we use ShH10 to restore Dp71 expression in Müller cells to study molecular and functional effects of this restoration in an adult mouse.

 

Methods

 

The hGFP-2A-Dp71 sequence was cloned in the pTR-SB-smCBA to produce by triple transfection method, the recombinant AAV, ShH10-hGFP-2A-Dp71. 1.8x10E10 particles of AAV was injected into the vitreous of 8-weeks-old Dp71-null mice. GFP expression was followed by fundus imaging and retinal thickness by OCT. Mice were sacrificed two months after injection. RNA and proteins were extracted to evaluate Dp71 and related proteins expression level. The localisation of these proteins was highlighted by immunocytochemistry on isolated Müller cells. The BRB permeability was evaluated by the Evans blue method.

 

Results

 

Fundus imaging revealed strong, pan-retinal expression of GFP fluorescent reporter, indicating robust transgene expression. The OCT revealed no significant thickness variation between Dp71-null and treated mouse. Dp71 mRNA and protein were overexpressed (n=7) in the treated Dp71-null mouse as well as several Dp71 related proteins (AQP4, β-dystroglycan, laminins). Moreover, the immunochemical analysis showed that exogenous Dp71 reaches a similar location than in WT mice. Surprisingly, the BRB permeability of Dp71-null mouse was completely restored two months after ShH10-hGFP-2A-Dp71 injection (see figure below).

 

Conclusions

 

Here we clearly demonstrate that in Dp71-null mouse with compromised BRB we can restore a normal BRB permeability by restoring Dp71 expression via ShH10. This study is an important step forwards the development of new treatments of diseases with a BRB breakdown (AMD and diabetic retinopathy).

 

 

Keywords: 538 gene transfer/gene therapy • 540 glia