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Corneal transparency restoration by gene therapy
Author(s) -
Mohan Rajiv R.
Publication year - 2019
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2019.8248
Subject(s) - myod , myofibroblast , gene silencing , rna interference , microbiology and biotechnology , stromal cell , myod protein , biology , cancer research , pathology , medicine , fibrosis , gene , genetics , myogenesis , rna , myocyte
Loss of corneal transparency after trauma, injury or infection results in blindness. Compromised wound healing because of excessive production of myofibroblasts has been identified a major mechanism for the loss of corneal transparency. We tested a hypothesis that de‐differentiation of corneal myofibroblast into precursor corneal stromal fibroblast by silencing of MyoD (basic helix‐loop‐helix) gene will restore corneal transparency by inhibiting alpha smooth muscle actin (αSMA) transcriptional machinery responsible for myofibroblast production. To test the hypothesis, donor human corneas, human corneal stromal fibroblasts (CSFs) and myofibroblasts (CMFs), MyoD/scrambled‐RNAi plasmids, nanoparticles, immunofluorescence, western blotting, qPCR, 2D/3D confocal microscopy, and slot blots were used. The fibrotic human corneas showed significantly greater MyoD expression compared to the normal human cornea (p < 0.05). Likewise, CMFs showed markedly increased MyoD compared to the CSFs in vitro (p < 0.01). The MyoD‐RNAi or scrambled‐RNAi delivery into CSFs and CMFs did not alter their appearance, viability, or proliferation in normal culture conditions. MyoD‐RNAi delivery into CMFs led to phenotype similar to CSFs in a time‐dependent manner. Furthermore, MyoD‐RNAi silencing in CMFs demonstrated significantly deceased αSMA mRNA and protein levels in a time‐dependent manner (p < 0.001). MyoD gene silencing is a novel approach for the restoration of corneal transparency by promoting de‐differentiation of opaque corneal myofibroblasts to transparent corneal keratocytes.