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Toru Matsunaga, Koji Kitazawa, Kenta Yamasaki, Takao Sato, Yasuo Watanabe, Toshinari Funaki, Akira Matsuda, Nobuyuki Ebihara, Satoshi Kawasaki, Akira Murakami; Transfer of TACSTD2 Gene into Corneal Epithelial Cells of Gelatinous Drop-Like Corneal Dystrophy and Its Functional Expression. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2734.
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© ARVO (1962-2015); The Authors (2016-present)
Gelatinous drop-like corneal dystrophy (GDLD) is a refractory disease that deposits amyloid into the subepithelium through increased intercellular permeability in the corneal epithelium. As a novel treatment for the disease, we have been investigating the transfer of TACSTD2 gene that is responsible for the disease, into a contact lens (CL) to use as the device. For the purpose of this study, we have used corneal epithelial cells that were obtained from a GDLD patient and investigated the transfer and functional expression of TACSTD2 gene.
Corneal epithelial cells that had been obtained from a GDLD patient were immortalized with the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcription (hTERT) genes. CL that had phosphate groups as its side chains was synthesized as the gene transfer device to allow it to form a complex, mediated by calcium ions, with plasmid DNA obtained through implanting pLenti6.3_V5-TOPO vector with the TACSTD2 gene (TACSTD2-V5). Using this DNA-CL, gene transfer was performed into the immortalized corneal epithelial cell line from the GDLD patient (imHCE-T_GDLD) cells, followed by observation for expression of TACSTD2 as well as claudin (CLDN).
The imHCE-T_GDLD cells processed with DNA-CL observed fluorescence in the cellular cytoplasm in the immunostaining with an anti-TACSTD2 antibody. Localization of CLDN1 and CLDN7 were also observed. Western blotting recognized protein expression as well.
Transfer into imHCE-T_GDLD cells and functional expression of TACSTD gene recognized through the use of DNA-CL has indicated that gene transfer can be achieved by non-invasive treatments.
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