In The journal of gene medicine
BACKGROUND : Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult due to large length of genes. Here, we used engineered human induced pluripotent stem cells (hiPSCs) and artificial intelligence (AI) to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene.
METHODS : We explored whether AI detected differentiation of human OTX2, PAX6, and MITF (hOPM) cells, in which OTX2, PAX6, and MITF expression were induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6, was constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death, and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay, and Western blotting.
RESULTS : AI-based evaluation of RPE differentiation was successful. RPE cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity, and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM.
CONCLUSIONS : CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress.
Iwagawa Toshiro, Masumoto Hiroki, Tabuchi Hitoshi, Tani Kenzaburo, Conklin Bruce R, Watanabe Sumiko
2022-Nov-22
CHM, CRISPR/Cas9, RPE, choroideremia, exon-skipping, hiPSC
