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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY013431-13
Application #
8657044
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
2001-08-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
13
Fiscal Year
2014
Total Cost
$616,499
Indirect Cost
$247,338
Name
Cedars-Sinai Medical Center
Department
Type
DUNS #
075307785
City
Los Angeles
State
CA
Country
United States
Zip Code
90048
Kramerov, Andrei A; Ljubimov, Alexander V (2016) Stem cell therapies in the treatment of diabetic retinopathy and keratopathy. Exp Biol Med (Maywood) 241:559-68
Kramerov, Andrei A; Saghizadeh, Mehrnoosh; Ljubimov, Alexander V (2016) Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells. J Vis Exp :e54058
Ljubimov, Alexander V; Saghizadeh, Mehrnoosh (2015) Progress in corneal wound healing. Prog Retin Eye Res 49:17-45
Patil, Rameshwar; Ljubimov, Alexander V; Gangalum, Pallavi R et al. (2015) MRI virtual biopsy and treatment of brain metastatic tumors with targeted nanobioconjugates: nanoclinic in the brain. ACS Nano 9:5594-608
Kramerov, Andrei A; Saghizadeh, Mehrnoosh; Maguen, Ezra et al. (2015) Persistence of reduced expression of putative stem cell markers and slow wound healing in cultured diabetic limbal epithelial cells. Mol Vis 21:1357-67
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
Sareen, Dhruv; Saghizadeh, Mehrnoosh; Ornelas, Loren et al. (2014) Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium. Stem Cells Transl Med 3:1002-12
Winkler, Michael A; Dib, Christian; Ljubimov, Alexander V et al. (2014) Targeting miR-146a to treat delayed wound healing in human diabetic organ-cultured corneas. PLoS One 9:e114692
Saghizadeh, Mehrnoosh; Epifantseva, Irina; Hemmati, David M et al. (2013) Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing. Invest Ophthalmol Vis Sci 54:8172-80
Saghizadeh, Mehrnoosh; Winkler, Michael A; Kramerov, Andrei A et al. (2013) A simple alkaline method for decellularizing human amniotic membrane for cell culture. PLoS One 8:e79632

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