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Andrei A Kramerov, Pallavi Gangalum, Hui Ding, Julia Y Ljubimova, Alexander V Ljubimov; Novel Nanoconjugates for Gene Therapy Normalization of Cultured Human Diabetic Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Our purpose was to normalize cultured human diabetic limbal epithelial cells (LEC) that have reduced expression of epithelial stem cell markers and slow wound healing using novel polymalic acid-based nanoconjugates inhibiting MMP-10 and cathepsin F (increased in diabetic corneas) and upregulating c-met (decreased in diabetic corneas).
LEC cultures enriched in stem cells were obtained from postmortem donor eyes. Nanoconjugates on natural-derived polymalic acid scaffold were synthesized as published (1) and contained a targeting antibody to LEC-expressed transferrin receptor (TfR), morpholino antisense oligos (AON) to cathepsin F and to miR-409-3p that targets c-met proto-oncogene, and trileucine endosome escape unit. A separate nanoconjugate contained an AON to MMP-10 and a tracking dye Alexa Fluor 488. Efficiency of target inhibition (proteinases) or upregulation (c-met) was tested with free AON and then with whole nanoconjugates (at 5 mM AON). Healing of scratch wounds was monitored microscopically.
Diabetic LEC from several donors were TfR-positive on western blots justifying the use of respective antibody for cell targeting. LEC internalized nanoconjugates mainly through receptor-mediated endocytosis as revealed by competition with free TfR antibody. By western blot, they were able to reduce the expression of MMP-10 and cathepsin F, and increase the expression of c-met (through inhibition of miR-409). Treatment with nanoconjugates accelerated healing of scratch wounds in LEC cultures with no toxicity observed.
Non-toxic nanoconjugates appear to be a new viable alternative to viral-based gene therapy in normalizing the diabetic limbal epithelial cells and wound healing. 1. Proc Natl Acad Sci USA, 2010;107:18143-18148.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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