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Curtis R Brandt, Hongyu Rao, B'Ann T Gabelt, Zeynep Aktas, Sarah Slauson, Paul L Kaufman; Genome Deficient Lentiviral Particles Improve FIV Transduction Efficiency of Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3280.
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© ARVO (1962-2015); The Authors (2016-present)
Cells contain innate systems, such as TRIM 5α, that signal destruction of incoming capsids blocking infection. The goal of the study was to determine if genome-deficient (gd)FIV particles, acting as decoys for TRIM 5α, would improve lentiviral trabecular meshwork transduction.
FIV.copGFP and gd FIV were packaged in HEK293TN cells. For genome-deficient particles, the vector plasmid was omitted from the packaging reaction. Biological titers were determined in CrFK cells by immunofluorescence. The titers of gd particles were estimated by quantitative Western blot standardized using serial dilutions of FIV.copGFP stocks with known biological titer.<br /> <br /> TM-1 cells (5x104 cells/well) in 12-well plates were pre-treated with 1, 2.5, 5, 10 and 20 transducing units (TU) of gd FIV, for two hours at 37oC. The cells were then transduced with FIV.copGFP vector (MOI 20). Controls included TM-1 cells only and TM-1 cells treated with FIV.copGFP vector. Four days later, harvested and used for copGFP Western blotting, qRT-PCR, and percentage of GFP+ cells was determined. For monkey-organ-cultured anterior segments (MOCAS) one rhesus eye (OD) was pre-treated with 2.5 x 107 TU of genome-deficient FIV for two hours then transduced with 2x107 TU FIV.copGFP. The other eye (OS) was only transduced with 2x107 TU FIV.copGFP. Three days later the eyes were harvested for GFP imaging, histology, and Western blotting. Five pairs of monkey eyes were used.
In TM-1 cells, GFP positive cells were increased by 15.4%, 19.5%, 50.5%, and 120.1% with gdFIV pretreatment of 1, 2.5, 5, 10 and 20 TU, respectively. qRT-PCR analysis showed significantly improved copGFP expression level in FIV.copGFP samples with gdFIV pretreatment. In MOCAS, more intense GFP fluorescence and immunofluorescence were observed in trabecular meshwork of FIV.copGFP-transduced monkey eyes with gdFIV pretreatment compared to no gdFIV pretreatment.
The results indicate that gdFIV pretreatment increased FIV.copGFP transduction efficiency both in TM-1 cells and in trabecular meshwork of organ cultured monkey eyes which could potentially improve FIV vector-mediated glaucoma gene therapy.
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