Strategies for correction and knockout of the rhodopsin gene in rod photoreceptor cells will be tested to determine what it takes to manipulate the structure and expression of deleterious genes in terminally differentiated neurons. We seek to understand the fundamental cellular processes that allow targeted gene repair and mutation in these cells, as well as those that can control expression of toxic proteins. In the process of gaining this understanding, we aim to develop effective methods for treating autosomal dominant retinitis pigmentosa (ADRP) caused by defects in the rhodopsin gene. Successful therapies for ADRP will provide a paradigm for treatment of dominant eye diseases and other neurodegenerative disorders. Mice are a natural choice for these studies because their eye physiology resembles that of humans;ADRP disease genes cause retinal degeneration in mice more quickly than in humans and large animals, allowing rapid testing of efficacy;and mouse genomes can be readily manipulated. Previously, we fused the complete human rhodopsin gene?the ultimate target for therapy?tothe GFP gene to generate a visible marker for rhodopsin expression that localizes properly to rod outer segments. Here, we propose to use our existing wild type rhodopsin-GTP mice and generate four new mouse lines carrying mutant human rhodopsin-GFP genes to provide targets for various kinds of genetic manipulation. These modified human rhodopsin-GFP genes will include three defined ADRP mutations?P23H, Q64ter, and Q344ter?and a gene that carries an internal duplication. Adeno-associated virus vectors will be used to deliver novel reagents for rhodopsin correction and knockout. For gene correction, we will test segments of rhodopsin DNA in the presence and absence of genes for zinc-finger nucleases (ZFNs) that have been rationally designedto cleave near the target mutations. For gene knockout, we will test exon-specific ZFNs to mutate the rhodopsin-GFP gene directly, and intron- specific ZFNs designed to stimulate incorporation of a 'killer'exon that will poison expression of the rhodopsin gene. Assays for rhodopsin gene correction and knockout will utilize the fluorescent properties of the humanrhodopsin-GFPtarget gene.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Neuhold, Lisa
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Baylor College of Medicine
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Wensel, Theodore G; Zhang, Zhixian; Anastassov, Ivan A et al. (2016) Structural and molecular bases of rod photoreceptor morphogenesis and disease. Prog Retin Eye Res 55:32-51
Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia et al. (2016) Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair. J Mol Biol 428:2978-80
Sandoval, Ivette M; Price, Brandee A; Gross, Alecia K et al. (2014) Abrupt onset of mutations in a developmentally regulated gene during terminal differentiation of post-mitotic photoreceptor neurons in mice. PLoS One 9:e108135
Santillan, Beatriz A; Moye, Christopher; Mittelman, David et al. (2014) GFP-based fluorescence assay for CAG repeat instability in cultured human cells. PLoS One 9:e113952
Price, Brandee A; Sandoval, Ivette M; Chan, Fung et al. (2012) Rhodopsin gene expression determines rod outer segment size and rod cell resistance to a dominant-negative neurodegeneration mutant. PLoS One 7:e49889
Eberle, Dominic; Kurth, Thomas; Santos-Ferreira, Tiago et al. (2012) Outer segment formation of transplanted photoreceptor precursor cells. PLoS One 7:e46305
Gilliam, Jared C; Chang, Juan T; Sandoval, Ivette M et al. (2012) Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration. Cell 151:1029-41
Price, Brandee A; Sandoval, Ivette M; Chan, Fung et al. (2011) Mislocalization and degradation of human P23H-rhodopsin-GFP in a knockin mouse model of retinitis pigmentosa. Invest Ophthalmol Vis Sci 52:9728-36
Chan, Fung; Hauswirth, William W; Wensel, Theodore G et al. (2011) Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice. Nucleic Acids Res 39:5955-66
Mojumder, Deb K; Concepcion, Francis A; Patel, Shil K et al. (2010) Evaluating retinal toxicity of intravitreal caspofungin in the mouse eye. Invest Ophthalmol Vis Sci 51:5796-803

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