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Gene Therapy for Diabetic Corneal Stem Cells
Delayed wound healing, erosions, and keratitis, the most serious complications of diabetes in the cornea, are treated only symptomatically. We have described a number of markers altered in diabetic corneas. Using adenoviral gene therapy, we were able to correct aberrant wound healing and several marker protein expression abnormalities in human organ-cultured diabetic corneas. We have also uncovered epithelial stem cell changes in diabetic corneas and are working on gene therapy corrections to alleviate these changes. The studies are geared to translating gene therapy for corneal diabetes to clinical applications. This research is funded by NIH R01 grant.
C-met gene therapy of organ-cultured human diabetic corneas normalizes staining of putative epithelial stem cell markers keratin 15 (K15), keratin 17 (K17) and ΔNp63∝. Limbal staining is shown. e, epithelium; s, stroma. Reproduced with permission from Mol Vis. 2011;17:2177-90.
Limbal Stem Cell Deficiency
Limbal stem cell deficiency is a consequence of genetic diseases, chronic inflammation, and eye burns. It results in partial to complete vision loss due to conjunctival ingrowth with neovascularization and is hard to cure even with corneal transplantation. We have generated induced pluripotent stem cells (iPSC) from corneal epithelial cultures. Currently, optimized protocol for their differentiation into corneal cells is being developed in collaboration with several labs in the RMI. This would enable the creation of a bankable source of corneal epithelial cells for future transplantation to patients. This research is funded by a recent NIH R01 grant.
Cornea-derived iPSC on top of denuded organ-cultured human cornea (two weeks). They express differentiated corneal marker, keratin 3 (K3). DAPI, nuclear stain. S, stroma.
MicroRNA Therapy for Corneal Diabetes
miRNA are potent regulators of gene expression. Using gene arrays, several miRNA have been identified with altered expression in diabetic corneas, which correlates with their effects on corneal cell wound healing. Most recently, deep sequencing analysis of a large group of normal and diabetic corneas has been performed resulting in the identification of promising miRNA candidates for further analysis. If successful, these studies may complement our gene therapy efforts to restore normal functions to diabetic corneal stem cells. Dr. Saghizadeh Ghiam is leading this research area supported by NIH R21 grant.
Corneal epithelial cells heal scratch wounds significantly slower in the presence of diabetes-upregulated miRNA miR-146a compared to control miR-Cy3 (24 hours healing).
Our lab was the first to show that the ubiquitous protein kinase CK2 plays an important role in pathologic retinal neovascularization, which could be greatly reduced by CK2 inhibitors. Moreover, specific CK2 inhibition suppresses engraftment of endothelial progenitor cells into newly formed retinal capillaries in the mouse retinopathy model (collaboration with Dr. Maria Grant, University of Florida). This may be a new mechanism of action of antiangiogenic drugs. Recent data by Dr. Kramerov suggest that CK2 inhibition may act through cell rounding caused by alterations of the actin cytoskeleton.
Retinas of neonatal mice treated with vehicle or CK2 inhibitor emodin. Intravitreal neovascularization is seen in vehicle (arrowheads) but is absent in emodin (below inner limiting membrane, ILM). Reproduced with permission from Mol Cell Biochem. 2008;316:177-86.