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Anitha Krishnan, Fei Fei, Alexander Jones, Bruce R Ksander, Ann Marshak Rothstein, Meredith S Gregory-Ksander; Gene therapy treatment with AAV-soluble Fas ligand protects retinal ganglion cells during development of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2594.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma and vision loss is ultimately due to the loss of retinal ganglion cells (RGCs). We demonstrated previously that apoptosis of RGCs is mediated by Fas ligand (FasL), which exists in two forms, either pro-apoptotic membrane-bound (mFasL), or anti-apoptotic soluble (sFasL). RGC death was accelerated in mice that only expressed mFasL. Since sFasL is anti-apoptotic, we hypothesize that AAV2.sFasL treatment will prevent glaucoma, even in the presence of elevated intraocular pressure (IOP).
We used two mouse models of elevated IOP: (i) DBA/2J (D2) mice that develop age-related elevated IOP and loss of RGCs and (ii) a microbead induced model of elevated IOP in C57BL/6 (B6) mice. D2 mice received an intravitreal injection of AAV2.CB6.sFasL, or AAV2.CB6.eGFP (neg. control) at 3 months. D2 untreated mice were positive controls and D2-Gpnmb+ untreated mice (no elevated IOP or glaucoma) were negative controls. B6 mice received AAV2 treatment 3 wks prior to microbead injection. Rebound tonometry monitored IOP. Groups of D2 and B6 mice were analyzed at 9 mons and 28 days post microbead injection, respectively for: (i) RGC counts (β-III tubulin), (ii) axon counts (PPD), (iii) western blotting for FasL, and (iv) mRNA expression of apoptotic and inflammatory mediators..
Treatment with either AAV2.eGFP, or AAV2.sFasL did not alter IOP elevation in either model. Treatment with AAV2.sFasL increased significantly retinal sFasL levels as compared to controls. At 9 months of age D2 untreated and AAV2.eGFP treated mice displayed a significant loss of RGCs and axons as compared to control D2-Gpnmb+ mice. By contrast, D2.AAV2.sFasL treated mice were completely protected, displaying no significant loss of RGCs or axons. At 28 days after microbead injection, B6 mice and AAV2.eGFP treated B6 mice displayed a significant loss of RGCs and axons as compared to B6 mice without microbeads. However, AAV2.sFasL treated B6 microbead mice were completely protected, displaying no significant loss of RGC’s or axons. Protection correlated with increased cFLIP mRNA and decreased mRNA for: FADD, Fas, IL-1β, TNFα, and CD11b.
In two mouse models of IOP-induced glaucoma, sFasL was neuroprotective and prevented loss of RGCs and axons, even in the presence of elevated IOP. Moreover, AAV delivered long-term expression of sFasL, which is critical in a chronic disease such as glaucoma.
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