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CRISPR-activation of SOX2 Promotes Corneal Endothelial Cell-mediated Wound Healing and Regeneration

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Review of “SOX2 Activation Using CRISPR/dCas9 Promotes Wound Healing in Corneal Endothelial Cells” from STEM CELLS by Stuart P. Atkinson

Other than corneal transplants [1], there currently exist no effective treatments for corneal endothelial dysfunction/disease [2]. While corneal endothelial cells (CECs) may seem to represent an obvious choice for the development of novel regenerative therapies, they do not proliferate in vivo even when in an injured state [3]. As wounded CECs express the pluripotency- and eye development-associated SOX2 transcription factor [4], researchers from the laboratory of Young Joo Shin (Hallym University, Seoul, Korea) sought to harness the power of CRISPR-activation technology [5, 6] to promote the overexpression of SOX2 in CECs in the hope of enhancing wound healing of the corneal endothelium.

In their new STEM CELLS study [7], Chang et al. first transfected SOX2 CRISPR/dCas9 activation plasmids into the cryo-injured corneal endothelium of Sprague-Dawley rats via electroporation. The authors confirmed the presence of dCas9, an increase in Sox2 expression, and an elevated number of proliferating CECs (Ki67-positive) by immunofluorescent staining. When compared to control, CRISPR-activation of Sox2 expression reduced both corneal opacity and central corneal thickness, with both measures suggesting an increased regenerative response. Furthermore, the study observed enhanced corneal endothelial wound healing and regeneration and reduced inflammatory cell infiltration and apoptosis upon Sox2 overexpression, leading to a higher density of CECs.

Correlative in vitro analysis of human CECs transfected with the SOX2 CRISPR/dCas9 activation plasmids discovered increased cell viability, proliferation rate, and number of cells in S‐phase. Furthermore, SOX2 activation also led to the overexpression of the Cyclin‐dependent kinase 1 (CDK1) and Cyclin D1 cell cycle progression regulators, the activation of the AKT pathway (which promotes proliferation and migration via G1/S‐phase transition), and an increase in mitochondrial oxidative stress and energy production (indicative of enhanced hCEC function).

In summary, the elevated expression of SOX2 expression in CECs prompted by CRISPR-activation, and the subsequent stimulation of corneal endothelium regeneration, represents a potentially exciting new method to treat CEC-related diseases in vivo.

For more on CRISPR-activation, applications of CRISPR in vivo, and more, stay tuned to the Stem Cells Portal!

References

  1. Wang F, Zhang T, Kang YW, et al., Endothelial keratoplasty versus repeat penetrating keratoplasty after failed penetrating keratoplasty: A systematic review and meta-analysis. PLoS One 2017;12:e0180468.
  2. Morishige N and Sonoda KH, Bullous keratopathy as a progressive disease: evidence from clinical and laboratory imaging studies. Cornea 2013;32 Suppl 1:S77-83.
  3. Joyce NC, Proliferative capacity of the corneal endothelium. Progress in Retinal and Eye Research 2003;22:359-89.
  4. McGowan SL, Edelhauser HF, Pfister RR, et al., Stem cell markers in the human posterior limbus and corneal endothelium of unwounded and wounded corneas. Molecular Vision 2007;13:1984-2000.
  5. Perez-Pinera P, Kocak DD, Vockley CM, et al., RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nature Methods 2013;10:973-6
  6. Park JJ, Dempewolf E, Zhang W, et al., RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. PLoS One 2017;12:e0179410.
  7. Chang YK, Hwang JS, Chung T-Y, et al., SOX2 Activation Using CRISPR/dCas9 Promotes Wound Healing in Corneal Endothelial Cells. STEM CELLS 2018;36:1851-1862.