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D. Muramatsu, B. C. Oveson, T. Iwase, S. Lee, N. R. Berlon, J. Laganiere, Q. Yu, P. D. Gregory, S. H. Zhang, P. A. Campochiaro; Ocular Gene Transfer of an Engineered Zinc Finger Protein (ZFP) That Increases Expression of Glial Cell Line Derived Neurotrophic Factor (GDNF) Reduces Photoreceptor Cell Death in a Rat Model of Retinitis Pigmentosa (RP). Invest. Ophthalmol. Vis. Sci. 2010;51(13):4489.
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The purpose of this study was to test in a model of RP the effect of gene transfer of an engineered ZFP transcription factor (ZFP TF) designed to increase the expression of endogenous GDNF.
ZFP TFs were engineered to target the rat GDNF promoter. A lead ZFP activator (GdnfZFP) was identified using transient transfection studies. Six weeks after subretinal injection of AAV2.GFP in one eye and AAV2.GdnfZFP in the other eye, expression of GDNF was assessed. At P21, subretinal injections of AAV2.GFP or AAV2.GdnfZFP were done in RCS rats. After electroretinograms (ERGs) at P60, rod survival was assessed by measurements of outer nuclear layer (ONL) thickness and cone survival was measured by confocal microscopy of retinal flat mounts stained with rhodamine-labeled peanut agglutinin (PNA).
A ZFP activator that significantly increases expression of the endogenous GDNF gene was identified in cultured Rinm5f cells and C6 cells. In normal rats, the expression of Gdnf mRNA was 5 times greater in eyes injected subretinally with AAV2.GdnfZFP compared to those injected with AAV2.GFP. In P60 RCS rats, scotopic and photopic ERG b-wave amplitudes, ONL thickness, and cone density were significantly greater in eyes injected with AAV2.GdnfZFP compared to those injected with AAV2.GFP. With a stimulus intensity of 4cd-s/m2 , mean scotopic b-wave amplitude was 97.9±54.3µV in eyes injected with AAV2.GdnfZFP compared to 51.9±26.0µV in eyes injected with AAV2.GFP (p=0.0006). With a stimulus intensity of 25cd-s/m2, mean photopic b-wave amplitudes were 62.5±36.9 compared to 37.7±10.8µV (p=0.0058). Mean ONL thickness was 0.13±0.006mm in AAV2.GdnfZFP-treated eyes and 0.04±0.002mm in AAV2.GFP-treated eyes (p<0.0001).
Upregulation of the endogenous Gdnf gene by gene transfer of an engineered ZFP TF promotes survival and preservation of function of rod and cone photoreceptors in RCS rats.
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