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Jingbo Liu, Victor Guaiquil, Aihong Liu, Mark Rosenblatt; Ex vivo gene transfer of anti-angiogenic soluble Flt-1 to corneal epithelial cells sheets suitable for ocular surface reconstruction. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2717.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate ex vivo gene transfer of anti-angiogenic soluble Flt-1 (sFlt1) to corneal epithelial cell sheets transplanted in a rabbit model of total limbal stem cell deficiency (TLSCD).
Mouse sFlt1 was cloned into pLenti6.3/V5-DEST vector using the Gateway systems. The vector was cotransfected with viral packaging plasmids into 293FT cells and the produced viruses were harvested and titrated. Primary rabbit corneal epithelial cells (RCEC) were transduced with the lentivirus and the sFlt1 mRNA expression and protein levels were analyzed by qRT-PCR, immunofluorescence staining and ELISA, respectively. Functional assays using aortic ring were performed to evaluate the inhibitory effect of sFlt1 on VEGF164 induced endothelial cell proliferation. RCEC transduced with sFlt1 were grown as sheet on decellularized amniotic membranes and transplanted on the rabbit corneas with TLSCD. The biocompatibility of the cell sheet was monitored by slit lamp biomicroscopy.
Lentiviruses containing msFlt1 gene were successfully produced. qRT-PCR analysis showed high level of expression of msFlt1 mRNA in the transduced RCEC. The sFlt1 protein was detected by immunofluorescence staining in the cytoplasm and up to one week by ELISA in the culture medium. The aortic ring assays showed that sFlt1 inhibits the VEGF164 induced proliferative effect of aortic endothelial cells (p<0.01). Transduced corneal epithelial cells sheets were well tolerated on the limbal stem cell depleted rabbit corneas.
We have successfully created and transplanted cells sheets expressing heterologous anti-angiogenic molecules. Genetically modified cultivated epithelial stem cell transplants provide a basis for ex vivo corneal gene therapy and could be used to treat corneal disease.
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