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Jennifer Donau, Dirk Lindemann, Richard Funk, Monika Valtink; Comparison of Two Viral Vector Systems for Transient Gene-Delivery Into Primary Human Retinal Pigment Epithelial (RPE) Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1179.
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© ARVO (1962-2015); The Authors (2016-present)
Current methods to achieve target gene overexpression are either membrane damaging or may lead to undesired permanent genomic changes. We compared transient transgene expression from episomes of integrase-deficient lentiviral vectors (IDLVs) with mRNA delivered by enzymatically inactive Prototype Foamy Virus (PFV) particles, using the example of proliferation stimulation with Human Papillomavirus type 16 E6/E7 (E6E7) or Simian Virus 40 T-antigens (SV40Ts) gene transfer in human RPE cells.
RPE cells were transduced with IDLVs or by PFV-mediated mRNA-transfer to express either E6E7 or SV40Ts (n=3 donors, 3 replicates each). Uninfected cells, or cells transduced with transgene-free vector particles or integration-competent lentiviral vectors for permanent transgene expression served as controls. Vector cytotoxicity was modified by pseudotyping with alternative viral envelope proteins. Transgene-induced proliferation was determined by calculating cumulative population doublings. Metabolic activity was measured by resazurin conversion for determining cytotoxic effects. Replicative senescence was identified by senescence-associated β-galactosidase (SA-b-Gal) activity. Episome integration (IDLVs) was determined by recording EGFP marker gene fluorescence, and mRNA transduction (PFV) was verified by qPCR.
Transient transduction with IDLVs or PFV-mediated mRNA-transfer was proven by marker gene detection. Vector cytotoxicity could be reduced by pseudotyping (PFV). IDLVs encoding for either transgene induced permanent proliferation and reduced SA-b-Gal expression compared to negative controls. E6E7 mRNA-transfer could only marginally stimulate proliferation compared to uninfected cells, but could overcome cytotoxic side effects of PFV particle transduction. SV40Ts mRNA-transfer failed to stimulate proliferation and could also not counteract cytotoxic effects.
IDLVs show an integrase-independent integration capacity leading to permanent transgene expression, but PFV-mediated mRNA-transfer is suitable for transient transgene expression without manipulating the host cell genome. Though primary cell proliferation could not be achieved, PFV-mediated mRNA-transfer might be useful for transiently modifying other cellular targets, e.g. genes involved in phagocytosis, ion flux, replicative senescence or apoptosis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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