Trinucleotide Repeat-Targeting dCas9 as a Therapeutic Strategy for Fuchs’ Endothelial Corneal Dystrophy

Abstract

Fuchs' endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation. Seventy percent of cases are caused by an intronic CTG triplet repeat expansion in the TCF4 gene that results in accumulation of pathogenic expanded CUG repeat RNA (CUG exp) as nuclear foci in corneal endothelium. A catalytically dead Cas9 (dCas9) can serve as an effective guide to target genomic DNA or RNA transcripts. Here, we examined the utility of the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 system to effectively target and reduce CUG exp. Methods: We delivered dCas9 and repeat-targeting single guide RNA (sgRNA) expression plasmids to patient-derived endothelial cells using lipofection or lentiviral transduction. We used fluorescence in situ hybridization (FISH) and RNA dot-blot hybridization to quantify CUG exp foci and repeat RNA levels, respectively. TCF4 expression levels were assessed using quantitative PCR (qPCR). Results: Using FISH, we found that expression of both dCas9 and a (CAG) n sgRNA complementary to CUG exp are necessary to reduce foci. We observed a reduction in percentage of cells with foci from 59% to 5.6% and number of foci per 100 cells from 73.4 to 7.45 (P < 0.001) in cells stably expressing dCas9-(CAG) n sgRNA but saw no decrease in cells expressing dCas9-(CUG) n sgRNA or nontargeting control sgRNA. In cells with dCas9-(CAG) n sgRNA, we detected a reduction in CUG exp RNA by dot-blot without any reduction in TCF4 mRNA levels using qPCR. Conclusions: Using CRISPR-dCas9 to target the trinucleotide repeat is a promising treatment for FECD contingent on effective in vivo delivery. Translational Relevance: This work advances a gene therapy for a common age-related degenerative disorder.

Loss of Corneal Nerves and Corneal Haze in Patients with Fuchs’ Endothelial Corneal Dystrophy with the Transcription Factor 4 Gene Trinucleotide Repeat Expansion

“…These observations open the possibility of preventing FECD disease onset or progression with molecular therapies early in the disease course before the onset of irreversible loss of endothelial cells, fibrosis, and corneal edema. We and others have proposed therapeutic strategies that target the TCF4 trinucleotide repeat expansion, including the use of gene editing, 12 antisense oligonucleotides, 13 , 14 , 15 duplex RNAs, 16 trinucleotide repeat-targeting catalytically dead Cas9, 17 and small molecules that bind with repeat RNA. 18 …”

RNA-Seq–Based Transcriptome Analysis of Corneal Endothelial Cells Derived from Patients with Fuchs Endothelial Corneal Dystrophy

“…The high prevalence of the CTG expansion in FECD has been con rmed in multiple ethnic cohorts, with the prevalence depending on ethnicity [27,[31][32][33][34][35][36][37]. Following those discoveries, several disease mechanisms induced by CTG repeat expansion have been proposed: 1) dysregulation of TCF4 transcripts [12,36,38,39]; 2) RNA-mediated toxicity [40][41][42][43]; 3) repeat-associated non-AUG dependent (RAN) translation [28,44]; and 4) somatic instability of CTG repeat expansion [45]. Although these hypothetical mechanisms have been actively investigated, inspired by the high prevalence of the CTG repeat expansion, the mechanism of FECD in cases that do not harbor the repeat expansion remains unclear.…”

From Bench to Bed: The Current Genome Editing Therapies for Glaucoma

“…Therefore, targeting the fibrous layer consisting of the cornea, sclera, and lamina cribrosa in patients with glaucoma is an alternative avenue for treatment. While genome editing therapy targeting the sclera and lamina cribrosa has not yet been performed, corneal gene modification using the CRISPR/Cas9 system has recently been successfully executed in several disease models, including for Meesmann’s epithelial corneal dystrophy ( Courtney et al, 2016 ) and Fuchs’ endothelial corneal dystrophy ( Rong et al, 2020 ), and has even entered clinical trials for viral keratitis (NCT04560790). The investigation of glaucoma-related cross-linking proteins, such as lysyl oxidase (LOX)/lysyl oxidase-like 1 (LOXL1), tissue trans-glutaminase (TG2), and advanced glycation end products, will allow corneal gene therapy to be further developed into a promising treatment of glaucoma.…”