Abstract
Purpose :
Mutations in the Eyes Shut Drosophila homolog (EYS) gene are a major cause of autosomal recessive retinitis pigmentosa (RP) reaching up to 25% of Asian RP cohorts. Despite the high prevalence of EYS-associated retinal degeneration, little progress has been achieved toward the development of genetic therapies for this form of RP. Challenges arise from the gene's considerable size and the absence of suitable mammalian models, given that EYS is not expressed in rodents. In this study we employ a recently developed zebrafish disease model to assess the therapeutic effectiveness of human EYS midigenes.
Methods :
We created a CRISPR-generated knock-out eys zebrafish model, utilizing a previously published gRNA sequence (GTGCAGGAAAACTCCCCTG). Retinal degeneration assessment involved histology, immunostaining, RT-qPCR, and visual motor response (VMR) assay. To explore therapeutic potential, three human EYS codon-optimized midigene constructs were generated, each featuring distinct truncated segments (N-terminal, C-terminal, and middle) while maintaining the in-frame structure. mRNA was synthesized via in vitro transcription and injected into the eys mutant zebrafish embryos at the 1-4 cell stage. EYS co-immunostaining with photoreceptor and ciliary markers, along with functional rescue evaluation using VMR assay characterized the outcomes.
Results :
CRISPR editing of zebrafish embryos resulted in a founder fish carrying the c.3482_c.3509del mutation, causing a frameshift (p.Thr1161fs). Immunostaining confirmed the absence of the eys protein in the mutant fish. The eys knockout zebrafish exhibited a reduced response to light, as measured by VMR at 5 dpf, and significant changes in retinal organization at 60 dpf. Injection of eys knockout embryos with wild-type full-length human EYS mRNA revealed a similar protein localization to the endogenous eys. Moreover, these injections improved VMR response at 5 dpf. Testing different truncated EYS constructs identified a potentially therapeutic midigene.
Conclusions :
We developed a novel eys knockout zebrafish model, providing a valuable platform for exploring the therapeutic potential of human EYS midigenes. Our model facilitated the examination of the significance of specific EYS domains, leading to the identification of a potentially therapeutic midigene featuring a truncated hEYS protein.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.