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Andrei A Kramerov, Ruchi Shah, Hui Ding, Mehrnoosh Saghizadeh, Julia Ljubimova, Alexander V Ljubimov; Validation of nanoconjugates targeting various diabetes-associated protein markers for gene therapy of diabetic keratopathy. Invest. Ophthalmol. Vis. Sci. 2021;62(8):757.
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The purposes were to 1) develop nanoconjugates for effective delivery of antisense oligonucleotides (AON) to human stem cell-enriched limbal epithelial cells (LEC) for gene therapy of diabetic keratopathy, and 2) to validate the effect of a novel nanoconjugate with AON to miR-203a suppressing Wnt5a (decreased in diabetes) and to compare it to nanoconjugate with AON to cathepsin F (CF) (increased in diabetes), and miR-409 targeting c-met proto-oncogene (decreased in diabetes), in human diabetic LEC and organ-cultured corneas.
Stem cell-enriched LEC cultures and corneal organ cultures were obtained from postmortem human donor eyes. Nanoconjugates based on polymalic acid scaffold (1) were synthesized and contained a cell-targeting antibody to LEC transferrin receptor, and morpholino AON to CF and to miR-409 that targets c-met, or to miR-203a (all AON at 10 or 20 μM). Control nanoconjugate had a scrambled sequence AON (Gene Tools). Healing of scratch-induced (LEC) and heptanol-induced corneal epithelial wounds was monitored microscopically.
Treatment with nanoconjugate bearing AON to miR409+CF caused an increase of its target c-met and a decrease of CF protein levels in diabetic LEC and in organ-cultured diabetic corneas. This treatment also upregulated corneal epithelial stem cell markers and accelerated wound healing of cultured LEC and organ-cultured diabetic corneas, indicating that non-toxic nanoconjugates provide a new tool for gene therapy for normalizing diabetic limbal epithelial cells and organ-cultured corneas. Recently, we found a significantly decreased expression of Wnt5a protein and upregulation of its suppressing miR-203a in diabetic LEC and corneal epithelium. Also, miR-203a antagomir inhibitor increased Wnt5a expression and promoted wound healing in diabetic LEC. We made a nanoconjugate with AON to miR-203a to restore (upregulate) Wnt5a expression and validated its ability to normalize wound healing by diabetic corneal epithelial cells. The data will be presented to support the validity of this approach and possible use for combined treatment with nanoconjugates carrying AON miR409+CF.
Cell-targeting nanoconjugates may be used as a new tool for gene therapy in normalizing diabetic limbal epithelial cells and diabetic corneal wound healing.1. Proc Natl Acad Sci USA, 2010;107:18143-18148.
This is a 2021 ARVO Annual Meeting abstract.
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