AM AAV 2 Mediated Expression of Intraceptor Flt 23 k Shows No Endoplasmic Reticulum Stress Response

Program Number: 3321 Poster Board Number: D0124 Presentation Time: 8:30 AM–10:15 AM AAV2 Mediated Expression of Intraceptor Flt23k Shows No Endoplasmic Reticulum Stress Response Austin Bohner, Xiaohui Zhang, Hironori Uehara, Bonnie Archer, Balamurali Ambati. John Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT. Purpose: Current therapy of neovascular age-related macular degeneration is a multiple intravitreal injection of an anti-VEGF-A drug. Previously we demonstrated that single subretinal injections of a AAV2 mediated expression of Flt23k was able to inhibit vascular endothelial growth factor (VEGF) in mice and effectively inhibit laser-induced murine choroidal neovascularization (CNV) for up to six months. Because our novel treatment method acts by maintaining VEGF in the endoplasmic reticulum (ER) and thus blocks extracellular release of VEGF, further study has now been conducted to determine the effect of sequestered VEGF on ER stress. Methods: An overexpression of protein in the ER has been shown to activate signalling pathways that lead to an increase in the production of molecular chaperones GRP78/BiP and result in increased splicing of XBP-1 mRNA. We examined whether Flt23k overexpression upregulated GRP78/BiP and XBP-1 mRNA splicing as markers for ER stress in Hela cells via western blot and RT-PCR compared with soluble Flt-1 overexpression. To investigate the effects on ER stress of AAV2.Flt23k in a murine model, mice were given a single subretinal injection of 1 μl (5x108 vector genomes) of AAV2.Flt23k. Control groups received a single injection of an equivalent amount of AAV2.AcGFP, PBS or no injection. Six months post subretinal injection all groups had RPE/ choroid tissues harvested, sectioned, and analyzed. ER stress and apoptosis was assessed in RPE cells using Anti-GRP78/BiP staining and TUNEL staining method. Results: AAV2.Flt23k overexpression in Hela cells did not induce an upregulated response of ER stress markers at mRNA or protein level compared to positive control cells. Mice treated with a subretinal injection AAV2.Flt23k also did not exhibit increased GRP78 BiP expression or apoptosis in RPE of treated mice compared to positive control mice. Conclusions: The absence of ER stress increases confidence in the safety of AAV2.Flt23k as a novel treatment approach to neovascular AMD. Potentially, AAV2.Flt23k may induce efficient degradation of VEGF-A. Future study will focus on the cellular methods of the degradation of sequestered VEGF which prevent the cell from experiencing ER stress. Commercial Relationships: Austin Bohner, None; Xiaohui Zhang, None; Hironori Uehara, None; Bonnie Archer, None; Balamurali Ambati, None Support: RPB Unrestricted Award and NEI 5R01EY017182