Abstract
Purpose :
To investigate the effects of mutant O-mannose 1,2-N-acetylglucosaminyltransferase (POMGnT1) protein and its molecular mechanism in a mouse model of nonsyndromic retinitis pigmentosa
Methods :
Knock-in (KI) mouse model was generated by CRISPR/Cas9 based technology. The KI pathogenic variant (p.L120R) was created in exon 3 of POMGnT1 using the guide RNA. POMGnT1 knockout (KO) immortalized human retinal pigment epithelial cells (RPE) cell line was generated using CRISPR/Cas9 gene editing system to explore the cellular function and efficacy of gene therapy. Adeno-associated virus (AAV) carrying human POMGnT1 gene, AAV8-hPOMGnT1, was transfected in KO RPE cells or delivered to KI mice via subretinal injection at postnatal day 90. The trans-epithelial electric resistance (TEER) was used to confirm the cell barrier integrity and permeability of KO RPE. Evaluation methods include co-immunoprecipitation (CO-IP), TUNEL assay, immunohistochemical studies, and western blotting analysis for exploring the molecular mechanism. The visual function was evaluated via the electroretinogram (ERG) approach.
Results :
The Pomgnt1L120R/L120R (KI) mice showed the significantly decrease of ERG value compared with WT mice. A significantly GFAP-positive cells accumulated in the KI retina indicated the inflammatory response. Furthermore, a RIP3 and LC3B-positive cells significantly expressed in the KI retina. Western blot of CO-IP assay from retina demonstrated mutant POMGnT1 protein cannot bind to enolase1 and s-arrestin complex, which induced inflammation in the retina. Immunoblot results demonstrated the increase of necroptotic markers in the KI retina compared with control retina. In vitro study, the phosphorylation of necroptotic markers were significantly increased in the KO RPE. The TEER level of KO RPE was lower than WT cell line. KO RPE transfected with AAV8-hPOMGnT1 showed recovery of the TEER value and suppression of the necroptotic markers. In the AAV8-hPOMGnT1-treated mice, the ERG results showed the b-wave amplitudes were statistically similar to the age-mated WT mice for at least 6 months after subretinal injection.
Conclusions :
Our results demonstrated that mutant Pomgnt1 gene induced the inflammatory response and the necroptotic death of neuron cells in the KI mouse retina. AAV8-hPOMGnT1 shows promise for improving retinal phenotype in the Pomgnt1L120R/L120R-KI mouse model.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.