Diabetic eye disease is the leading cause of blindness worldwide, affecting all eye tissues including the cornea. Over 70% of diabetics suffer from corneal problems that can reduce vision and cause pain, such as neuropathy and various epithelial alterations referred to as diabetic keratopathy. Keratopathy is more pronounced in severe diabetes but is under-diagnosed;its therapy remains symptomatic. Our gene therapy in organ-cultured human corneas using sufficiently safe replication-deficient adenovirus (AV) showed that: (1) Sildenafil enhanced AV epithelial delivery;(2) In diabetic corneas, c-met proto-oncogene upregulation normalized expression of specific proteins and epithelial wound healing via p38 activation;(3) In normal corneas, upregulation of cathepsin F and matrix metalloproteinase (MMP)-10 that are increased in diabetic corneas delayed wound healing and changed marker expression towards diabetic;this occurred via inhibiting Akt activity;(4) When proteinase genes were silenced using AV-shRNA, diabetic corneas healed faster. AV gene therapy was able to correct key epithelial abnormalities associated with diabetic keratopathy. We also show that several presumed stem cell markers had a decreased expression in the diabetic limbal epithelium making it an important target of gene therapy. Our main hypothesis is that deficiencies in the limbal epithelium of diabetic patients ultimately lead to corneal epithelial abnormalities. These defects may be corrected by targeting limbal epithelial cells with gene therapy as a promising approach to the treatment of diabetic keratopathy. Based on this hypothesis, we propose the following Specific Aims:
Specific Aim 1. To examine whether AV-driven gene therapy on limbal epithelial cells reverses the diabetic marker protein expression pattern and facilitates epithelial wound healing of organ-cultured diabetic corneas. We predict that our gene therapy will restore presumed stem cell marker expression and epithelial wound healing in diabetic corneas towards normal.
Specific Aim 2. To determine if overexpression of c-met and downregulation of MMP-10 and cathepsin F normalizes marker expression pattern and wound healing of diabetic limbal epithelial cells cultured on amniotic membrane. We predict that gene therapy will normalize cultured diabetic limbal cells for their use as substitutes for pathologically altered diabetic limbal epithelium in Aim 3.
Specific Aim 3. To achieve transplantation of genetically modified cultured limbal cells to the limbal zone of diabetic corneas in order to restore normal stem cell marker expression and wound healing. We predict that gene therapy will normalize diabetic limbal epithelial cells, so that transplanting them into the limbal region of a diabetic cornea will restore its normal wound healing and marker protein expression. Health relevance: our novel strategy of targeting corneal epithelial progenitor cell niche by gene therapy may lead to a new treatment for diabetic keratopathy, a serious clinical problem affecting the majority of diabetics.
The application addresses PA-10-067 goal as a Research Project Grant on a new mechanism and treatment approach of diabetic corneal disease. We will focus on corneal epithelial progenitor cells, which are the source of corneal surface lining, as a new target of specific gene therapy aiming at normalizing their functions in order to restore normal protein expression and wound healing in diabetic corneas. If successful, the application may have a high impact for future treatment of frequent corneal problems in diabetic patients.
|Saghizadeh, Mehrnoosh; Dib, Christian M; Brunken, William J et al. (2014) Normalization of wound healing and stem cell marker patterns in organ-cultured human diabetic corneas by gene therapy of limbal cells. Exp Eye Res 129:66-73|
|Kramerov, A A; Ahmed, K; Ljubimov, A V (2012) Cell rounding in cultured human astrocytes and vascular endothelial cells upon inhibition of CK2 is mediated by actomyosin cytoskeleton alterations. J Cell Biochem 113:2948-56|
|Inoue, Satoshi; Ding, Hui; Portilla-Arias, Jose et al. (2011) Polymalic acid-based nanobiopolymer provides efficient systemic breast cancer treatment by inhibiting both HER2/neu receptor synthesis and activity. Cancer Res 71:1454-64|
|Koronyo-Hamaoui, Maya; Koronyo, Yosef; Ljubimov, Alexander V et al. (2011) Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model. Neuroimage 54 Suppl 1:S204-17|
|Saghizadeh, Mehrnoosh; Soleymani, Siavash; Harounian, Angel et al. (2011) Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy. Mol Vis 17:2177-90|
|Gao, Nan; Yin, Jia; Yoon, Gi Sang et al. (2011) Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair. Am J Pathol 179:2243-53|
|Kramerov, A A; Golub, A G; Bdzhola, V G et al. (2011) Treatment of cultured human astrocytes and vascular endothelial cells with protein kinase CK2 inhibitors induces early changes in cell shape and cytoskeleton. Mol Cell Biochem 349:125-37|
|Mankus, Courtney; Rich, Celeste; Minns, Martin et al. (2011) Corneal epithelium expresses a variant of P2X(7) receptor in health and disease. PLoS One 6:e28541|
|Ding, Hui; Inoue, Satoshi; Ljubimov, Alexander V et al. (2010) Inhibition of brain tumor growth by intravenous poly (?-L-malic acid) nanobioconjugate with pH-dependent drug release [corrected]. Proc Natl Acad Sci U S A 107:18143-8|
|Allen, Sariah J; Mott, Kevin R; Ljubimov, Alexander V et al. (2010) Exacerbation of corneal scarring in HSV-1 gK-immunized mice correlates with elevation of CD8+CD25+ T cells in corneas of ocularly infected mice. Virology 399:11-22|
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