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H. Yang, H. E. Grossniklaus; Pigment Epithelium-Derived Factor (PEDF) Gene Therapy Inhibits Hepatic Microetastasis in Murine Uveal Melanoma Model. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4753.
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© ARVO (1962-2015); The Authors (2016-present)
Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of angiogenesis. We have previously demonstrated the expression of PEDF in mouse melanoma cell lines and human uveal melanoma cells and showed that low expression of PEDF is associated with increased murine uveal melanoma hepatic micrometastasis. The purpose of this study is to investigate whether the PEDF gene is a potent tumor suppressor and a potential candidate for cancer gene therapy.
Mouse PEDF gene expressive sequence was inserted into an entry vector pENTRTM/D-TOPO. The entry vector containing the mouse PEDF gene expressive sequence was recombined with lentivirual vector pLenti6.2/N-LumioTM/V5-DEST to generate the expression construct. The expression construct was sequenced to confirm the sequence and position of the mouse PEDF gene. PEDF expression was verified by transiently transfecting the expression construct into the HEK 293 cell line with LipofectamineTM 2000 and Western blot analysis. Lentiviral stocks were produced and titered in the 293FT cell line. pLenti6.2/N-LumioTM/V5-DEST/mPEDF was transduced into mouse melanoma B16LS9 cells, transduced cell clones were selected by 4 µg/ml Blasticidin for 2weeks, and cells were expanded in complete culture medium with Blasticidin without antibiotics.PEDF expression was detected by Western blot and LumioTM-tagged PEDF protein in live B16LS9 cells. Stably PEDF overexpressing B16LS9 and control B16LS9 cells were inoculated into C57BL/6 mice, the eye was enucleated at 7 days after inoculation, hepatic tissue was collected at 4 weeks after inoculation and the number of hepatic micrometastasis was counted.
Sequencing confirmed that the inserted sequence in lentivirual vector pLenti6.2/N-LumioTM/V5-DEST matched with the PEDF expressive sequence (Gene bank accession number NM_011340.2). Western blot showed overexpression of PEDF in HEK 293 cells transiently transfected with pLenti6.2/N-LumioTM/V5-DEST/mPEDF, and in B16LS9 cells transduced by the lentiviral vector. The mouse uveal melanoma model demonstrated fewer hepatic micrometastases in stably overexpressing PEDF B16LS9 cells versus the B16LS9 control group.
Lentivirus-mediated gene transfer of PEDF decreased the number of hepatic micrometastasis in a mouse uveal melanoma model. Dual antitumor/anti-angiogenic activities of PEDF suggest that PEDF gene therapy may be considered as a new approach for treatment of uveal melanoma.
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