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CRISPR-Cas9 Genome Editing of Limbal Stem Cells Generates Faithful Model of Panocular Condition

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Review of “Modeling of Aniridia‐Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein” from STEM CELLS by Stuart P. Atkinson

Mutations in the PAX6 gene lead to nonsense‐mediated mRNA decay and haploinsufficiency [1, 2] and the development of aniridia, a panocular condition characterized by conjunctival cell ingrowth, corneal neovascularization, corneal opacity, and eventual visual loss [3], aniridia‐related keratopathy (ARK). While studies have speculated that PAX6 loss promotes dysfunction of the corneal limbal niche [4], the condition suffers from a lack of faithful models required to describe disease progression and develop therapeutic approaches.

To remedy this situation, researchers led by Daniel Aberdam (INSERM U976, Hôpital Saint‐Louis, Paris, France) have developed and characterized a cell model of PAX6 haploinsufficiency using CRISPR-Cas9 genome editing of limbal stem cells (LSCs) [5]. Reporting in STEM CELLS, Roux et al. have employed this model system to demonstrate the potential utility of recombinant PAX6 protein as a therapeutic tool for aniridia patients.

Employing CRISPR-Cas9-mediated genome editing of intron 7 of the PAX6 gene in telomerase-immortalized LSCs, the authors obtained nine clones that carried a p.E109X nonsense mutation on one allele (with no noted off-target effects). The resultant heterozygous mutant LSCs expressed reduced levels of PAX6, which prompted a significant reduction in proliferation and migration and enhanced culture substrate adhesion. However, culture medium supplementation with a recombinant PAX6 protein fused to a cell penetrating peptide rescued the phenotypic defects of mutant LSCs, suggesting this approach as a promising therapy for aniridia/ARK patients. 

The authors suggest that this successful first example of CRISPR-Cas9 genome editing in LSCs to create a faithful model of ocular disease will prove highly useful in the search for new and effective therapeutic approaches. Furthermore, they suggest that the identification of recombinant PAX6 as a potentially effective treatment option may facilitate the treatment of PAX6 dysfunction in other eye tissues (lens, retina) and other PAX6-expressing tissues such as the pancreas.

For more on CRISPR-Cas9 genome editing in limbal stem cells, new stem cell therapies for other ocular pathologies, and the generation of faithful disease models stay tuned to the Stem Cells Portal!

References

  1. Robinson DO, Howarth RJ, Williamson KA, et al., Genetic analysis of chromosome 11p13 and the PAX6 gene in a series of 125 cases referred with aniridia. Am J Med Genet A 2008;146A:558-69.
  2. Axton R, Hanson I, Danes S, et al., The incidence of PAX6 mutation in patients with simple aniridia: an evaluation of mutation detection in 12 cases. J Med Genet 1997;34:279-86.
  3. Secker GA and Daniels JT, Corneal epithelial stem cells: deficiency and regulation. Stem Cell Rev 2008;4:159-68.
  4. Ramaesh T, Ramaesh K, Martin Collinson J, et al., Developmental and cellular factors underlying corneal epithelial dysgenesis in the Pax6+/- mouse model of aniridia. Exp Eye Res 2005;81:224-35.
  5. Roux LN, Petit I, Domart R, et al., Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein. STEM CELLS 2018;36:1421-1429.