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Andrei A Kramerov, Mehrnoosh Saghizadeh Ghiam, Alexander V Ljubimov; Optimized gene therapy normalizes wound healing and stem cell marker expression in cultured diabetic limbal epithelial cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):149.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the expression of putative limbal epithelial stem cell (LESC) markers and wound healing rate in diabetic human limbal epithelial cells (LEC) after optimized adenoviral (AV) gene therapy with manipulation of the levels of previously identified diabetic markers.
Primary LEC from postmortem donor diabetic eyes were isolated with Dispase II and cultured in EpiLife medium on a mixture of human fibronectin, collagen type IV and laminin (1). Cells were transduced by AV with (AV-c-met) or without (AV-vector-GFP) c-met gene, in the presence of 1 µg/mL polylysine, for 24 hr in a small amount of medium (0.5 mL). Additional AV vectors harbored shRNA to genes of diabetes-increased markers MMP-10 or cathepsin F (CF). Transduced cells were incubated for 4 days. The expression of putative LESC markers DNp63α, keratins 15 and 17 was revealed by immunostaining. Scratch wound healing in cultures was quantified after 20 hrs on digitized photographs using ImageJ software.
Adenoviral transduction of diabetic LEC was optimized by lowering multiplicity of infection to 10-30 PFU/cell in the presence of transduction enhancing polylysine. This regimen yielded high transgene expression without toxicity. Transduction with AV-c-met as well as with AV-MMP-10-shRNA or AV-CF-shRNA significantly reduced scratch wound area vs. AV-vector control. By immunostaining, AV-c-met treated diabetic LEC cultures showed an increased expression vs. AV-vector of putative LESC markers keratins 15 and 17, and DNp63α that are decreased in diabetic LEC (1)
Optimized adenoviral gene therapy normalized wound healing rate and stem cells marker expression in primary diabetic LEC. These data may be used to develop gene therapy for diabetic corneal epithelial abnormalities by targeting limbal epithelial stem cells.1. Kramerov et al., Mol Vis 2015;21:1357-1367.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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