Abstract
Purpose :
Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinitis pigmentosa (RP) or Leber congenital amaurosis (LCA). The brown Norway rat strain from Janvier has an inherited retinal degenerative phenotype due to a mutation in exon6 of Crb1. These rats exhibit disruptions at the outer limiting membrane and loss of retinal lamination. The ultrastructural localization of CRB1 and CRB2 proteins is unknown in healthy and Crb1 rat retinas. In addition, little is known about the tropism of different AAV serotypes upon subretinal (SR) or intravitreal (IV) injection in prenatal retina. Here, we describe the ultrastructural localization of CRB1 and CRB2, and the tropism of different AAV capsids in healthy and Crb1 mutant rat retinas.
Methods :
Postnatal day (P)17 and 3-months-old (3M) eyes from Crb1 mutant and control rats were collected and prepared for immuno-electron microscopy. Electroretinography (ERG) was performed on 1M Crb1 mutant and control rats. To determine tropism, different AAV.CMV.GFP vectors (AAV5, AAV9, or AAV6 variant ShH10Y445F) were SR or IV injected in P5 or P8 control and Crb1 mutant rat retinas. At 1M of age the retinas were collected for histology and confocal microscopy.
Results :
In the Crb1 mutant rat the ERG was severely decreased at 1M, suggesting early loss of retinal activity under photopic and scotopic conditions. In control retinas the CRB1 protein was located in Müller glial cells (MG) but not in PRC, whereas CRB2 was located in MG and PRC. In the Crb1 rat, the variant CRB1 protein was mostly lost whereas the CRB2 protein resided in MG and PRC. In addition, SR injection of AAV5 and AAV9.CMV.GFP at P5 resulted in mainly transduction of PRC and retinal pigment epithelium (RPE). However, IV injection at P5 and P8 with ShH10Y445F.CMV.GFP transduces MG in both rats.
Conclusions :
We show that the CRB1 protein in control rat retina is located in MG but not PRC, while CRB2 is located in MG and PRC. Preliminary data from control rat retinas suggest lower levels of CRB2 protein at P10 compared to P5. Localization of the CRB1 variant protein in the Crb1 mutant rat is predominantly lost, whereas levels of CRB2 appear relatively low. SR injection of AAV5 and AAV9.CMV.GFP results in transduction of PRC and RPE, while IV injection of ShH10Y445F.CMV.GFP results in the transduction of MG. These data suggest that ShH10Y445F serotype could be a tool for testing CRB gene therapy in the Crb1 mutant rat.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.