Diabetic eye disease is a leading cause of blindness in working-age adults. More than 70% of diabetic patients suffer from corneal problems, mainly from neuropathy and epithelial keratopathy (impaired barrier function and wound healing, recurrent erosions, epithelial keratitis, etc.), which can reduce vision, and cause discomfort and pain. Diabetic keratopathy is underdiagnosed, and therapy is limited to symptom control. We have discovered downregulation of putative limbal epithelial stem cells (LESC) markers in diabetic corneas and cultured diabetic LESC; normalized marker expression and epithelial wound healing in organ-cultured human diabetic corneas by adenoviral gene therapy of LESC-harboring limbus; designed a new fast method of denuding human amniotic membrane (HAM) for cell growth; showed that diabetic cells on HAM could be transplanted to the debrided corneas. Overall, we uncovered alterations in diabetic LESCs, normalized their marker expression, corrected impaired wound healing by gene therapy, and were able to transplant them on HAM to denuded corneas. We designed diabetic cornea-tailored non-toxic gene therapy vehicles, natural-derived polymalic acid-based nanobioconjugates (NBC). They bind to targeted cells by attached antibodies and modulate specific markers with antisense oligonucleotides (AON). We propose to use these new NBCs to safely and efficiently normalize diabetic LESC. Diabetes is associated with stable epigenetic changes, e.g., altered DNA methylation. A novel way of normalizing diabetic LESC would be to remove methylation changes. We propose to achieve this by generating induced pluripotent stem cells (iPSC) from diabetic LESC and differentiating iPSCs to limbal cells. Our main hypothesis is that normalization of diabetic limbal epithelial stem cells and wound healing could be achieved by using new non-toxic nanobioconjugate gene therapy and/or by transplantable functionally normal limbal epithelial cells obtained from diabetic LESC-derived iPSCs.
Specific Aim 1. To develop a safe gene therapy for diabetic LESC using novel nanobioconjugates modulating diabetic markers by antisense inhibition. The NBCs will be designed to downregulate diabetes- increased cathepsin F and MMP-10, and upregulate diabetes-decreased c-met.
Specific Aim 2. Specific Aim 2. To develop a novel way of normalizing cultured diabetic LESC by generating iPSC from LESC and redifferentiating the iPSCs back to limbal epithelial cells. We predict that iPSC would shed some diabetic epigenetic methylation, allowing to convert them to normal limbal cells.
Specific Aim 3. Specific Aim 3. To achieve transplantation of nanodrug-treated and of diabetic iPSC- derived limbal cells on HAM on the diabetic corneas and compare restoration of normal stem cell marker expression and wound healing. iPSC and nano gene therapy will be compared for efficacy.
Our aims fit the priorities of NEI Vision Research: improving transplantation of cultured corneal epithelial cells, understanding epigenetics of wound healing, and developing methods to enhance wound healing.

Public Health Relevance

of the proposed research project to public health The application addresses PA-13-302 goal as a Research Project Grant on new ways of treating stem cell and wound healing defects in diabetic corneal disease. We will focus on normalizing human diabetic corneal epithelial stem cells, which are the source of corneal surface lining, through generation of transplantable cells made from autologous cornea-derived induced pluripotent stem cells (iPSC), as well as through gene therapy with innovative non-toxic and biodegradable nano polymer conjugates. If successful, the proposal may have a high impact for future treatment of frequent wound healing-related corneal problems in diabetic patients.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Mckie, George Ann
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Cedars-Sinai Medical Center
Los Angeles
United States
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Leszczynska, Aleksandra; Kulkarni, Mangesh; Ljubimov, Alexander V et al. (2018) Exosomes from normal and diabetic human corneolimbal keratocytes differentially regulate migration, proliferation and marker expression of limbal epithelial cells. Sci Rep 8:15173
Ljubimov, Alexander V (2017) Diabetic complications in the cornea. Vision Res 139:138-152
Daliri, Karim; Ljubimov, Alexander V; Hekmatimoghaddam, Seyedhossein (2017) Glaucoma, Stem Cells, and Gene Therapy: Where Are We Now? Int J Stem Cells 10:119-128
Kulkarni, Mangesh; Leszczynska, Aleksandra; Wei, Gabbie et al. (2017) Genome-wide analysis suggests a differential microRNA signature associated with normal and diabetic human corneal limbus. Sci Rep 7:3448
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Ljubimov, Alexander V (2017) Cell Therapy for Age-Related Macular Degeneration: A New Vision for the Bone Marrow? Mol Ther 25:832-833
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
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
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