Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing

Abstract

Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced ( TGFBI ) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI -targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy.

Mini-Review: Clinical Features and Management of Granular Corneal Dystrophy Type 2

“…Genome editing technique such as CRISPR/Cas9 may be used to target genes site specifically. However, one of the problems of using gene therapy technique is the unwanted off-target effects, which may affect or silence the opposite normal allele or other genes [ 31 , 76 ]. Developing safe delivery methods free from unwanted off-target effects would be essential for specifically targeted gene therapy for GCD2.…”

In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study

“…Gene therapies may provide an alternative and potentially permanent treatment to correct the defective or missing transcript. Previous studies using clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR-Cas9)-mediated genome editing technology have shown promising results in targeting the pathogenic TGFBI mutations in isolated corneal keratocytes and epithelial stem cells 6 , 7 . However, to our knowledge, in situ genetic modification of these cells has not been demonstrated.…”

“…The global shortage of donor corneas and diminishing returns of repeated surgery further highlights the pressing need for an alternative management modality.Gene therapies may provide an alternative and potentially permanent treatment to correct the defective or missing transcript. Previous studies using clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR-Cas9)-mediated genome editing technology have shown promising results in targeting the pathogenic TGFBI mutations in isolated corneal keratocytes and epithelial stem cells 6,7 . However, to our knowledge, in situ genetic modification of these cells has not been demonstrated.In recent years, adeno-associated viruses (AAVs) have emerged as vectors of choice for ocular gene therapy thanks to their established clinical efficacy, biosafety profile, and low immunogenicity [8][9][10] .…”