Abnormal cellular RS1 expression and retinal organoid Development in RS1(E72K) Mutation of X-Linked Juvenile Retinoschisis

Abstract

Purpose : X-linked juvenile retinoschisis (XLRS) is a common early-onset in males characterized by retinal splitting and visual loss inherited disease, caused by an RS1 gene mutation. We differentiated human induced pluripotent stem cells (hiPSCs) into three-dimensional retinal organoids (ROs) to study the pathogenesis of XLRS in vitro.

Methods : Peripheral blood mononuclear cells (PBMC) from two XLRS patients and two control donors were reprogrammed into human induced pluripotent stem cells (hiPSCs) using the Sendai virus. Additionally, CRISPR/Cas9 gene editing introduced the RS1 (E72K) mutation into the hiPSCs line. ROs derived from hiPSC were assayed by immunofluorescence (IF) and qPCR. Wild type (WT) and RS1-E72K were obtained from transfected HEK293 cells. Whereafter, western blotting (WB) under reducing and non-reducing conditions was conducted to measure the homo-octameric complex formation of E72K.

Results : All hiPSCs lines expressed human PSC pluripotency markers OCT4, SSEA4, SOX2, and Nanog. All hiPSCs lines had the capacity of multipotent differentiation potential, which was confirmed by the ectodermal marker (PAX6), mesodermal marker (SMA), and endodermal marker (AFP). The E72K mutation and synonymous point mutation were introduced into the RS1 gene of hiPSC by CRISPR/Cas9 gene editing technique (Figure 1A-1B). There were no off-target mutations at the top 10 most likely gRNA off-target sites. WT HEK293 cells and E72K mutation HEK293 cells robustly expressed RS1 monomer in the cellular part. However, E72K mutation cells failed to secrete RS1 monomer into the culture medium and couldn’t produce mature RS1 octamers compared to WT cells (Figure 2A-2C). The control ROs mimicked the human retinal neural layer and expressed retinal relative markers. Compared to the control, the E72K mutation ROs showed overt schisis phenotype. E72K mutation affected the development of photoreceptor and RS1 protein expression on ROs.

Conclusions : This study demonstrates that E72K ROs can be used as an in vitro model of XLRS and may unleash ROs for drug development.

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

 

Figure 1. Sanger sequencing of RS1 gene: The RS1 genome sequence of WT (A) and E72K mutation (B).

 

Figure 2. Overexpression of RS1 in HEK293 cells: WB of WT and E72K in HEK293 cells under reducing conditions (A) and non-reducing conditions(B). WB of WT and E72K in the medium under reducing conditions (C).