Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein

Roux, L.; Petit, Isabelle; Domart, Romain; Concordet, Jean‐Paul; Qu, Jing; Zhou, Huiqing; Joliot, Alain; Ferrigno, Olivier; Aberdam, Daniel

doi:10.1002/stem.2858

Cited by 46 publications

(21 citation statements)

References 32 publications

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“…PAX6 transcription factor is known to be the master gene of eye formation (for review, Shaham et al, 2012). Previous studies reported that in the context of Pax6 mutation, corneal cells were unable to differentiate properly, and adopt the corneal epithelial cell identity (Roux et al, 2018). After Pax6a expression was validated by qPCR in control and wounded corneas (Fig.…”

Section: Resultsmentioning

confidence: 99%

Unilateral corneal insult in Zebrafish results in a bilateral cell shape and identity modification, supporting wound closure

Ikkala

Stratoulias

Michon

2021

Preprint

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Most of terrestrial and aquatic vertebrates are equipped with camera-type eyes, offering a focused and clear sight. This apparatus is rendered inefficient if its most superficial and transparent element, the cornea, is opaque. This structure, prone to environmental aggressions, bears excellent wound healing capabilities to preserve vision. Up to date, most of the corneal wound healing studies are made on mammals. Here, for the first time, zebrafish is used as model to study wound closure of corneal epithelium after abrasion. Our study demonstrates a swift wound closure after corneal insult. Interestingly, a unilateral wound induces a bilateral response. While cell proliferation is increased during wound closure, this parameter is not crucial, and cell rearrangements seems to be the driving force. Furthermore, we discovered a profound change in epithelial cell transcriptomic signature after abrasion, reflecting a modulation of cell identity and increase of phenotypic plasticity. The latter seems to unlock terminally differentiated cell capacities for wound healing, which could be the key for a speed up organ regeneration. Our results prove that zebrafish cornea is a powerful model to investigate, not only corneal wound healing, but ectodermal organ pathophysiology.

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Section: Resultsmentioning

confidence: 99%

Unilateral corneal insult in Zebrafish results in a bilateral cell shape and identity modification, supporting wound closure

Ikkala

Stratoulias

Michon

2021

Preprint

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“…LSC deficiency is the underlying mechanism responsible for cornea opacification and ultimately blindness in aniridia. [57][58][59][60] Hence, in future studies, we aim to deliver our CRISPR strategy to LSCs of post-weaned Fey mice to model gene therapy in recently diagnosed children with aniridia.…”

Section: Discussionmentioning

confidence: 99%

Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia

Mohanna

Hickmott

Lam

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the Sey allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy in vitro then tested it in vivo in the germline of our new mouse to validate the causality of the Sey mutation. The genomic manipulations were analyzed by PCR, as well as by Sanger and next-generation sequencing. The mice were studied by slit lamp imaging, immunohistochemistry, and western blot analyses. We successfully achieved both in vitro and in vivo germline correction of the Sey mutation, with the former resulting in an average 34.8% ± 4.6% SD correction, and the latter in restoration of 3xFLAG-tagged PAX6 expression and normal eyes. Hence, in this study we have created a novel mouse model for aniridia, demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia.

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“…This consequence might due to metabolic abnormalities of the ocular surface caused by haploinsufficiency of PAX 6 . 17 Studies demonstrated the individuals harbouring PAX 6 mutation displayed various clinical symptoms, including aniridia, keratitis, cataract, lens dislocation, ciliary body hypoplasia, foveal hypoplasia and brain structure abnormalities. In this study, nystagmus, ptosis, glaucoma, corneal pannus and other abnormalities were observed, and OCT showed that all affected individual had foveal hypoplasia.…”

Section: Discussionmentioning

confidence: 99%

Recurrent PAX 6 mutation in a Chinese family with congenital aniridia, progressive cataracts and mental retardation

Chen

Yang

Zhu

2018

European Journal of Ophthalmology

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Background: One prominent pathological feature of congenital aniridia is hypoplasia of the iris, often accompanied by other eye abnormalities. The objective of this study is to identify gene mutations responsible for autosomal dominance in a Chinese family with congenital aniridia, progressive cataracts and mental retardation. Methods: A total of 11 family members, including 6 affected and 5 unaffected individuals were recruited. Whole exome sequencing was performed on the proband and Sanger sequencing was applied to identify the causal mutation in the other family members and control samples. Results: A heterozygous mutation, c. 112delC (p. Arg38fs) in PAX 6, was identified in the family that was associated with congenital aniridia, progressive cataracts and mental retardation. The mutation was exclusively observed in all affected individuals but not in unaffected family members or unrelated healthy controls without aniridia recruited from Beijing Tongren Hospital. Bioinformatics analysis indicated that the mutation c. 112delC (p. Arg38fs) in PAX 6 affected sugar phosphate backbone construction, leading to half reduction of the full-length protein. Other symptoms such as lens opacity, keratitis, lens dislocation, ciliary body hypoplasia, foveal hypoplasia and mental development retardation were also observed in this family. Conclusion: These results provided a new insight into the effects of PAX 6 as a mutational hotspot, with a symptom complex that includes congenital aniridia, progressive cataracts and mental retardation. These findings suggested the cognitive treatment of PAX 6-mutated individuals could be considered earlier clinically, combined with medication or surgery of congenital aniridia and progressive cataracts.

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Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein

Cited by 46 publications

References 32 publications

Unilateral corneal insult in Zebrafish results in a bilateral cell shape and identity modification, supporting wound closure

Unilateral corneal insult in Zebrafish results in a bilateral cell shape and identity modification, supporting wound closure

Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia

Recurrent PAX 6 mutation in a Chinese family with congenital aniridia, progressive cataracts and mental retardation

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