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Project description:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.
| S-EPMC8216720 | biostudies-literature
Project description:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs’ endothelial corneal dystrophy (FECD), which can cause blindness through progressive loss of corneal endothelial cells. We established a novel procedure for achieving structural and functional rescue of the post-mitotic corneal endothelium without surgery, using CRISPR/Cas9-based postnatal gene editing in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) at a titer below the cytotoxic threshold, efficiently knocked down COL8A2 protein expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. Here, to determine the indel rate in mouse corneal endothelium, we performed deep sequencing of PCR products (including the target site) amplified from genomic DNA of corneal endothelium. We found that the indel rate was 23.7 ± 4.5% in mouse corneal endothelium. Most insertions were 1bp insertions (19.8 ± 4.0% in total reads), while 2bp deletions were the most frequent (1.0 ± 0.3% in total reads). We moreover found that A or T insertion was predominant, with the proportion of A:T:G:C being 48.7 : 44.6 : 1.8 : 4.9.
2020-09-01 | GSE146998 | GEO
Project description:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs’ endothelial corneal dystrophy (FECD), which can cause blindness through progressive loss of corneal endothelial cells. We established a novel procedure for achieving structural and functional rescue of the post-mitotic corneal endothelium without surgery, using CRISPR/Cas9-based postnatal gene editing in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) at a titer below the cytotoxic threshold, efficiently knocked down COL8A2 protein expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. Here, we evaluated the off-target activity of humanized gRNA targeting COL8A2 gene by a modified digenome analysis (Nat Methods 12, 237-243). Briefly, in vitro SpCas9 digested genomic DNA with/without gRNA are compared by deep sequencing. The target sequence is CGTCCACGGACGCCATGCT. In this study, we identified 13 candidate sites with homology to the gRNA and 8 candidate sites without homology to the gRNA.
2020-09-01 | GSE146997 | GEO
Project description:Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy
| PRJNA612701 | ENA
Project description:Indel rate analysis:Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy
| PRJNA612702 | ENA
Project description:Digenome for off-target analysis: Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy
| PRJNA612703 | ENA
Project description:PURPOSE:To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS:We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n?=?11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS:CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (?50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ?91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION:CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.
| S-EPMC6752322 | biostudies-literature
Project description:Fuchs endothelial corneal dystrophy (FECD) is the most common primary corneal endothelial dystrophy and the leading indication for corneal transplantation worldwide. FECD is characterized by the progressive decline of corneal endothelial cells (CECs) and the formation of extracellular matrix (ECM) excrescences in Descemet's membrane (DM), called guttae, that lead to corneal edema and loss of vision. FECD typically manifests in the fifth decades of life and has a greater incidence in women. FECD is a complex and heterogeneous genetic disease where interaction between genetic and environmental factors results in cellular apoptosis and aberrant ECM deposition. In this review, we will discuss a complex interplay of genetic, epigenetic, and exogenous factors in inciting oxidative stress, auto(mito)phagy, unfolded protein response, and mitochondrial dysfunction during CEC degeneration. Specifically, we explore the factors that influence cellular fate to undergo apoptosis, senescence, and endothelial-to-mesenchymal transition. These findings will highlight the importance of abnormal CEC-DM interactions in triggering the vicious cycle of FECD pathogenesis. We will also review clinical characteristics, diagnostic tools, and current medical and surgical management options for FECD patients. These new paradigms in FECD pathogenesis present an opportunity to develop novel therapeutics for the treatment of FECD.
| S-EPMC7648733 | biostudies-literature
Project description:Fuchs endothelial corneal dystrophy (FECD) is amongst one of the most common indications for endothelial keratoplasty worldwide. Despite being originally described among Caucasians, it is now known to be prevalent among a large number of populations, including Asians. While the FECD phenotype is classically described as that of central guttate and pigment deposits associated with corneal endothelial dysfunction, there are subtle yet important differences in how FECD and its phenocopies may present in Caucasians vs Asians. Such differences are paralled by genotypic variations and disease management preferences which appear to be geographically and ethnically delineated. This article provides a succinct review of such differences, with a focus on diagnostic and management issues which may be encountered by ophthalmologists practicing in the different geographic regions, when evaluating a patient with FECD.
| S-EPMC7042366 | biostudies-literature
Project description:PurposeRNA toxicity from CTG trinucleotide repeat (TNR) expansion within noncoding DNA of the transcription factor 4 (TCF4) and DM1 protein kinase (DMPK) genes has been described in Fuchs' endothelial corneal dystrophy (FECD) and myotonic dystrophy, type 1 (DM1), respectively. We prospectively evaluated DM1 patients and their families for phenotypic FECD and report the analysis of CTG expansion in the TCF4 gene and DMPK expression in corneal endothelium.MethodsFECD grade was evaluated by slit lamp biomicroscopy in 26 participants from 14 families with DM1. CTG TNR length in TCF4 and DMPK was determined by a combination of Gene Scan and Southern blotting of peripheral blood leukocyte DNA.ResultsFECD grade was 2 or higher in 5 (36%) of 14 probands, significantly greater than the general population (5%) (P < 0.001). FECD segregated with DM1; six of eight members of the largest family had both FECD and DM1, while the other two family members had neither disease. All DNA samples from 24 subjects, including four FECD-affected probands, were bi-allelic for nonexpanded TNR length in TCF4 (<40 repeats). Considering a 75% prevalence of TCF4 TNR expansion in FECD, the probability of four FECD probands lacking TNR expansion was 0.4%. Neither severity of DM1 nor DMPK TNR length predicted the presence of FECD in DM1 patients.ConclusionsFECD was common in DM1 families, and the diseases cosegregated. TCF4 TNR expansion was lacking in DM1 families. These findings support a hypothesis that DMPK TNR expansion contributes to clinical FECD.
| S-EPMC6005624 | biostudies-literature