Abstract
Purpose :
Mutations in the PRPF6 gene cause retinitis pigmentosa (RP) characterized by progressive degeneration of retinal pigment epithelium and photoreceptors. This study aimed to investigate the therapeutic effect of PRPF6 gene correction on RP patient-derived retinal organoids (ROs) by CRISPR/Cas9 gene editing technology.
Methods :
Induced pluripotent stem cells (iPSCs) of a patient with RP carrying the PRPF6 heterozygous mutation (c.2699G>A) were induced into ROs (RP-iROs). CRISPR/Cas9 system was used to repair the PRPF6 point mutation in patient-derived iPSCs via electroporation transfection and was identified by Sanger sequencing. Mutant ROs were compared with those derived from normal control iPSCs (NC-iROs) and gene-corrected iPSCs (GC-iROs). The expression levels of photoreceptor-specific proteins of rhodopsin and opsin were examined by immunofluorescence staining analysis. The ultrastructural phenotype of ROs outer layer was evaluated by transmission electron microscopy (TEM). P < 0.05 was considered to be statistically significant.
Results :
Sanger sequencing analysis showed that the PRPF6 c.2699 G > A (p.R900H) mutation in the patient-derived iPSCs had been corrected by CRISPR/Cas9 gene editing technology, using a single-stranded donor template to replace the hemizygous point mutation “A” with a wild-type nucleotide “G” (Figure 1A). 3D ROs from the NC-iPSCs, RP-iPSCs, and GC-iPSCs groups exhibited neuroretinal structure during days 3 - 242 (Figure 1B). At day 242 of differentiation, the photoreceptor inner segment/outer segment layers around ROs were arranged in a neat brush pattern, and the mature photoreceptor markers rhodopsin and opsin S/M/L were positively expressed. However, the number of photoreceptor cells in the RP-iRO group was significantly reduced compared with the NC-iRO group. In contrast, less photoreceptor loss was observed in the GC-iRO group (Figure 1C-D). TEM images also revealed abnormal photoreceptor changes in the RP-iRO group, which were reversed by gene correction in the GC-iRO group.
Conclusions :
Our study suggests that CRISPR/Cas9-mediated PRPF6 correction in RP patient-derived iPSCs rescues photoreceptor loss in mutant ROs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.