Defining Phenotype, Tropism, and Retinal Gene Therapy Using Adeno-Associated Viral Vectors (AAVs) in New-Born Brown Norway Rats with a Spontaneous Mutation in .

Mutations in the Crumbs homologue 1 () gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of . It has been reported that these mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult mutant rats in comparison with age-matched Brown Norway rats without a mutation in . A significantly decreased retinal function and visual acuity was observed in mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10 vectors in new-born control and mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10 at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10 to infect Müller glial cells, canonical h and h AAV-mediated gene therapy were explored in new-born mutant rats. Enhanced retinal function after gene therapy delivery in the rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in mutant rats.