Delayed differentiation of retinal organoids derived from a patient with CRB1-associated retinitis pigmentosa.

Abstract

Purpose : Over 190 mutations in the human crumbs homologue 1 (CRB1) gene causes various forms of inherited retinal diseases, however little is known about the mechanisms by which these mutations lead to retinal pathology. To investigate the pathophysiological mechanisms of CRB1-associated retinal disease we examined retinal organoids derived from a patient with adult-onset retinitis pigmentosa caused by compound heterozygous mutations in CRB1.

Methods : Dermal fibroblasts were obtained from the proband with early-onset retinal degeneration due to compound heterozygous mutation in CRB1 (OMIM#604210, NM_201253.2; c.1892A>G; p. (Tyr631Cys) and c.2548G>A; p. (Gly850Ser)) affecting the laminin A/G-like domain. Targeted sequencing confirmed same mutations were present in proband’s sister who has the same retinal phenotype. Induced pluripotent stem cells (iPSC) were generated from fibroblasts using episomal reprogramming and differentiated into neural retinal (NR) organoids. Development of photoreceptor connecting cilia was examined by transmission electron microscopy. NR protein and RNA expression were analyzed by western blot and quantitative RT-PCR.

Results : During retinal organoid differentiation, expression of PAX6 and N-cadherin protein was induced in control NR from day 2 and day 6 respectively, while expression of these proteins was delayed until day 12 in patient NR. In control NR connecting cilia formed on photoreceptor inner segments after 35 days of differentiation but were not detectable in patient NR until day 70. Expression of CRB1 was significantly reduced in day 35 patient NR compared with control NR (p<0.0001), while expression of RAX, CRX and RHO was similar. Interestingly, CRB2 expression was increased in patient NR compared with controls (p<0.05).

Conclusions : Retinal tissues derived from a patient carrying the c.1892A>G and c.2548G>A CRB1 variants displayed reduced CRB1 mRNA expression as well as delayed onset of retinal differentiation and maturation of photoreceptors. Patient -derived retinal organoids are a useful system for understanding the mechanisms of ocular dysgenesis in CRB1-associated retinopathy.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.