Molecular insights and treatment of IFT140-mediated retinal dystrophy in iPSCs-derived retinal organoids and RPE

Abstract

Purpose : Biallelic pathogenic variants in IFT140 can cause non-syndromic retinal dystrophy or syndromic ciliopathies, for which therapeutic approaches are lacking. Here, we investigated the molecular changes associated with IFT140-mediated retinal dystrophy in isogenic CRISPR-developed knock-out (IFT140-KO) and patient-derived retinal organoids and RPE. We also explored the potential of small molecule treatments as potential therapies.

Methods : Fibroblasts from three IFT140 patients with either non-syndromic or syndromic retinal dystrophy were characterized for ciliary defects. Induced pluripotent stem cells (iPSC) were generated from three patient lines carrying IFT140 missense variants. An isogenic IFT140-KO iPSC line was generated using CRISPR. These iPSC were differentiated to retinal organoids and RPE cells and their morphology characterized by immunocytochemistry. The transcriptome of isogenic IFT140-KO organoids was studied by bulk RNA sequencing. iPSC-RPE were treated for 24 hours with different concentrations of each small molecule, and cilium length was analysed using CiliaQ. Mature retinal organoids (over day 200) were treated twice a week for 30 days.

Results : In the patient fibroblasts, deficiency of IFT140 resulted in accumulation of proteins at the ciliary tip but no other major ciliary defects. IFT140-KO RPE had stubby cilia and irregular cell-cell junctions compared to isogenic controls, whereas patient-derived iPSC-RPE showed slightly shorter cilia than controls, with accumulation of proteins at the ciliary tip, but normal cellular junctions. Molecular characterization of IFT140-deficient retinal organoids showed retention of rhodopsin in the photoreceptor inner segments and outer nuclear layer. In addition, cilia analyses showed an extension of IFT88 and LCA5 immunoreactivity along the photoreceptor cilium. Transcriptomic analyses of the organoids revealed an increased expression of stress response and cell adhesion genes. The flavonoid eupatilin was found to rescue cilium length in iPSC-RPE after treatment, as well as rhodopsin and IFT transport in retinal organoids.

Conclusions : IFT140-deficient retinal organoids and RPE show characteristic ciliary defects that can be ameliorated by treatment with the flavonoid eupatilin. This work supports eupatilin as a drug candidate for the treatment of IFT140-mediated retinal dystrophy and associated ciliopathies.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.