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Anna-Lisa Doebley, Adam Crain, Maureen Neitz, Jay Neitz; Developing an AAV vector to target VEGF-Trap secretion from macular cones for the treatment of AMD via intravitreal injection. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4525.
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© ARVO (1962-2015); The Authors (2016-present)
Monthly injections of anti-vascular endothelial growth factor agents such as VEGF-Trap have proven successful in treating wet AMD, though, under-treatment is common. Gene therapy could provide long-term treatment with one injection, but, AMD gene therapy clinical trial results have been disappointing. Prior vectors have used ubiquitous promoters that don’t target cells in the macula, are poorly expressed, and use the SFLT signal sequence for secretion. We designed a VEGF-Trap gene with a signal sequence from the RS1 gene to direct secretion from cones and an AAV expression cassette to target high-level transgene expression in macular cones. We tested the expression cassette in nonhuman primates, and evaluated secretion of the modified VEGF-Trap gene.
We injected AAV carrying the gene for GFP in our new, cone specific expression cassette into the vitreous of nonhuman primate and monitored expression by retinal imaging. We analyzed secretion of VEGF-Trap in cell culture. HEK293T cells were transfected with 3 different plasmids. Two of the plasmids had the VEGF-Trap/citrine fusion within the optimized cone-targeting cassette and either the SFLT1 or RS1 signal sequence. The third plasmid had a non-secreted form of citrine. We compared cellular and extracellular fluorescence for untransfected cultures vs. cultures transfected with a plasmid. Media fluorescence was measured with a GloMax luminometer, cellular fluorescence was evaluated with fluorescence microscopy.
Fundus imaging demonstrated targeted expression in the macula. Intracellular fluorescence in HEK293T cells was high for cells transfected with the citrine plasmid, but low for cells transfected with VEGF-Trap/citrine fusion plasmids. Media fluorescence was similar to background for cells transfected with the citrine plasmid, but high for cells transfected with VEGF-Trap/citrine fusion plasmids. Media fluorescence from cells transfected with VEGF-Trap with the RS1 signal sequence was higher than for those with the SFLT1 signal sequence.
We developed an optimized AAV vector for delivering VEGF-Trap to cones in the macula and for secreting VEGF-Trap from cones. We verified that the vector targets macular cones in nonhuman primate, and that the RS1 signal sequences improves secretion over the SFLT1 signal sequence. Experiments to test the vector in mouse models of AMD are underway.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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