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Ze Zhang, Harry Tseng, Robert N. Weinreb, Nils A. Loewen; Ablation Of Trabecular Meshwork Cells with a Conditionally Cytotoxic FIV Vector. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3253.
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© ARVO (1962-2015); The Authors (2016-present)
To use an inducible, trackable, cytotoxic viral vector for targeted removal of the cells that constitute the principal pathology in primary open angle glaucoma, trabecular meshwork cells.
Feline immunodeficiency viral (FIV) vectors were produced by transient transfection of 293T/C17 cells using a tripartite vector system. FIV vectors have a high transduction efficiency and allow targeted outflow tract modification in vivo. FHSVtkIG expressed herpes simples virus 1 thymidine kinase and eGFP via an internal ribosomal entry site (IRES) while the control vector expressed eGFP and IRES-mediated neomycin resistance.Filtered vector batches were used to transduce CrFK, GTM3 and TM5 cells. Vector titers were determined by fluorescence assisted flow cytometry. Successful transduction was confirmed by fluorescence at 48 hours followed by ablation with 30 mcg/mL ganciclovir (GCV). Cell death was assessed by visualization after 24 hours and 48 hours following ganciclovir treatment.
FIV.HSVtk/eGFP induced apoptosis within 48 hours of exposure to ganciclovir. Extent of ablation could be titrated both by vector dilution and concentration of GCV. A multiplicity of infection (MOI) of 5 transducing units (TU) per cell and treatment with 30 mcg/mL ganciclovir resulted in elimination of virtually all transduced cells. Cells transduced with the non-ablative control vector FGINSIN and untransduced cells had no response to ganciclovir and grew to full confluence after 48 hours.
We demonstrated successful ablation of trabecular meshwork cells using a conditionally cytotoxic FIV vector in vitro. These results allow proceeding to in vivo experimentation to target the outflow tract for ablation.
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