Generation of an in vitro stem cell based model for researching gyrate atrophy of the choroid and retina

Abstract

Purpose : Gyrate atrophy of the choroid and retina (GACR) is an rare autosomal recessive metabolic eye disorder. GACR patients suffer from progressive vision loss, starting with near sightedness and night blindness in the first decade of life which progresses to complete blindness around the age of 50. Mutations in the gene encoding for ornithine amino transferase (OAT) impair the function of the encoded enzyme and thereby the ability of the cell to convert ornithine to 1-Pyrroline-5-carboxylic acid (P5C). It is currently unknown by which mechanism OAT deficiency results in deterioration in vision. In this study we developed GACR in vitro models which can be used to gain a better understanding of the disease mechanism responsible for inducing retinal pigment epithelial (RPE) cell death.

Methods : Skin biopts and blood was collected from 2 clinically well characterized GACR patients, 1 of which has a start codon mutation. From these materials, we generated 2 induced pluripotent stem cell lines (iPSCs). We performed RNA analysis, western blot and immunostainings to determine the presence of OAT in all cell lines. Both patient and healthy control iPSCs were exposed to a range of ornithine and arginine concentrations and subsequently assayed for cell viability.

Results : DNA mutation analysis showed that the patient iPSCs carry the c.1 A>G and c.1058 G>A OAT mutations. Using RT-PCR, western blot and immunostainings we have been able to determine the presence of OAT in our healthy control cell lines. Interestingly, 1 of the patient cell lines showed almost no loss of OAT activity. In contrast, the patient cell line with a start codon mutation showed only residual expression of OAT protein. The cell viability assay demonstrated an increased sensitivity towards ornithine in our patient iPSCs compared to the healthy controls. Similar increased sensitivity was also observed in the patient iPSCs when exposing them to arginine.

Conclusions : We have demonstrated that our patient derived iPSC model displays the key characteristic of GACR, which is toxicity as a result of hyperornithinemia. Our first functional results are promising and encourages further use of the iPSC model to further identify the disease mechanism responsible for ornithine induced toxicity. We are currently differentiating our iPSCs to iRPE cells yielding more specifically relevant information about the effect of OAT deficiency in the eye.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.