AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model

Thilo M. Buck,C. Henrique Alves,Rogier M. Vos,Jan Wijnholds

doi:10.1016/j.omtm.2020.12.012

Abstract

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1KOCrb2LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in Crb1KOCrb2LowMGC retinas but not in wild-type or Crb1-deficient retinas. Crb1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Müller cells of Crb1KOCrb2LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.

Highlights

Mutations in the Crumbs homolog 1 (CRB1) gene are associated with retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and cone-rod dystrophies and are sporadically found in foveal retinoschisis and macular dystrophy.[1,2,3] The human and nonhuman primate retina express and localize CRB1 and CRB2 proteins in Müller glial cells (MGCs) and photoreceptor cells (PRCs) at the outer limiting membrane (OLM).[4,5,6] The mouse retina expresses and localizes the CRB2 protein at the OLM in MGCs and PRCs
We show that (2) reduction of CRB2 protein levels worsens the retinal phenotype in the inferior quadrants as found in Crb1KO mice, (3) Crb1KOCrb2LowMGC RP mice are more susceptible to stress on MGCs than are Crb1KOCrb2Flox/WT and Crb2Flox/Flox mice, and (4) Recombinant adeno-associated viral (rAAV)-human CRB2 (hCRB2) therapy protects Crb1KOCrb2LowMGC retinas against loss of vision due to exposure to DL-a-aminoadipate acid (DL-AAA)
We assessed whether Cre expression in Crb1KO MGCs (Crb1À/ÀPdgfraCreTg/+) has an impact on retinal morphology, retinal transmission (ERG responses), or vision-guided optokinetic head-tracking thresholds

Summary

Introduction

Mutations in the Crumbs homolog 1 (CRB1) gene are associated with retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and cone-rod dystrophies and are sporadically found in foveal retinoschisis and macular dystrophy.[1,2,3] The human and nonhuman primate retina express and localize CRB1 and CRB2 proteins in Müller glial cells (MGCs) and photoreceptor cells (PRCs) at the outer limiting membrane (OLM).[4,5,6] The mouse retina expresses and localizes the CRB2 protein at the OLM in MGCs and PRCs. Recombinant adeno-associated viral (rAAV) vector-mediated gene supplementation may provide a lasting therapy to CRB1 RP patients. We previously showed that CRB2 can rescue retinas lacking CRB1 or CRB2 proteins from retinal degeneration in two fastprogression RP mouse models by increasing the levels of CRB2 into both MGCs and PRCs.[13] rescue at mid-stage retinal disease could not be achieved with CRB1 cDNA supplementation, or by supplementation of CRB2 cDNA only in PRCs or only in MGCs. In the present study, we developed a sensitive RP MGC-specific mouse model to test for protection at early stage retinal disease by rAAV-human CRB (hCRB) gene therapy vectors

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Conclusion