Abstract
Purpose :
Usher Syndrome type II (USH2A) is a retinal degenerative disease leading to hearing and vision loss that is most commonly caused by recessive mutations in exon 13. USH2A protein is localized in the connecting cilium between inner and outer segments, supporting inner/outer segment formation in photoreceptors. A human iPSC-derived retinal organoid model was developed to test the hypothesis that deleting or inverting USH2A exon 13 (redundant protein domain) could restore deficits caused by USH2A mutation.
Methods :
Human iPSC lines were generated from a USH2A patient containing homozygous c.2299delG mutation in exon 13 and healthy control individuals. In patient iPSC clones, exon 13 was deleted (or inverted) by high fidelity CRISPR gene editing. iPSCs from control (n=2), patient (n=5), exon 13-deleted (n=3) and exon 13-inverted (n=3) clones were differentiated at least for 250 days to form high-quality retinal organoids. These organoids were characterized at various time points during organogenesis by gene expression (RT-qPCR), immunohistochemistry (IHC), and morphology.
Results :
Control and exon 13-deleted and inverted iPSC clones showed similar gene and protein expression patterns and overall retinal organoid morphologies that are consistent with normal development and published results. In contrast, USH2A mutant iPSC-derived organoids showed irregularity in photoreceptor morphology and deficits in cilia and inner/outer segment organization. Moreover, USH2A, Whirlin and Vlgr1 proteins were stably expressed in WT but not in mutant retinal organoids. These USH2A mutant abnormalities were rescued by exon 13 deletion (and inversion) using precision gene editing.
Conclusions :
USH2A-c.2299delG homozygous mutation caused deficits in photoreceptor morphology, cilia and inner/outer segment formation and destabilization of Usher protein complex (Whirlin/Vlgr1/USH2A) during retinal organogenesis. These abnormalities were rescued by precision CRISPR-mediated deletion (and inversion) of exon 13, providing strong support for our exon 13-deletion therapeutic strategy for the treatment of USH2A-associated retinal degenerative diseases.
This is a 2021 ARVO Annual Meeting abstract.