Triplex-directed and other oligonucleotide-based strategies will be tested for feasibility as gene-specific therapies for autosomal dominant retinitis pigmentosa (ADRP) caused by defects in the rhodopsin gene. In the course of this work the investigator will develop technologies that can be used for testing a variety of therapeutic approaches to ADRP and other autosomal dominant genetic disorders. As any therapy for ADRP must ultimately be tested and optimized in animals, the investigator has chosen mice because of the ease with which their genomes can be modified. The investigator proposes to create a mouse ES cell line selectable segment of DNA that can be efficiently targeted by site-specific recombination. These modified ES cells will permit efficient, selectable 'knock-in' of any form of rhodopsin, cDNA or genomic, wild-type or mutant, from any species, for a broad range of physiological studies. The investigator will """"""""knock-in"""""""" modified human rhodopsin genes, designed to serve as sensitive cellular detectors of the effects of oligonucleotide treatments on rhodopsin gene expression, correction and knockout. For whole mouse studies, the investigator will use fusions of rhodopsin with green fluorescent protein (GFP). Oligonucleotide effects on transcription will be measured as a decrease in fluorescence intensity; oligonucleotide effects on gene correction and mutation will be measured by the appearance of GFP fluorescence starting with rhodopsin-GFP fusion constructs that are not expressed (GFP-). Cellular studies will precede whole animal experiments in order to test various treatment parameters. Although oligonucleotide-mediated effects on transcription can be measured readily, detection of recombination-based correction and mutational knockout requires specialized constructs, which the investigator will initially test at the selectable adenosine phosphoribosyl transferase (APRT) gene in CHO cells. A sensitive detection system will allow us to detect weak signals that can then be improved and optimized. Using CHO oligonucleotides (TFOs), RNA/DNA chimeric oligonucleotides and oligonucleotide analogues such as peptide nucleic acids (PNAs), for their ability to inhibit transcription, correct gene defects, and introduce mutations. These results will be used as the basis for developing treatment protocols in mice.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Special Emphasis Panel (ZRG1 (01))
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Dudley, Peter A
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Baylor College of Medicine
Schools of Medicine
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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
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
Roman-Sanchez, Ramon; Wensel, Theodore G; Wilson, John H (2016) Nonsense mutations in the rhodopsin gene that give rise to mild phenotypes trigger mRNA degradation in human cells by nonsense-mediated decay. Exp Eye Res 145:444-449
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
Eberle, Dominic; Kurth, Thomas; Santos-Ferreira, Tiago et al. (2012) Outer segment formation of transplanted photoreceptor precursor cells. PLoS One 7:e46305
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
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

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