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D. Mittelman, J. Wilson; Zinc Finger Mediated Repair of Disease Causing Alleles in Rhodopsin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5095.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Retinitis pigmentosa is a class of hereditary disorders of the photoreceptor that degrade vision and cause blindness in nearly 1.5 million worldwide. The most common form of the disorder is autosomal dominant RP. While several genetic factors have been related to ADRP, the majority of cases correlate to SNPs in the rhodopsin gene. We seek to develop molecular therapeutics that can be targeted to repair genetic mutations in the rhodopsin gene, promote the expression of normal rhodopsin, and potentially reverse or reduce the rate of photoreceptor degeneration in ADRP patients. Methods:Engineered synthetic molecules that recognize and bind specific DNA targets are the foundation for a new class of technologies that promise quantum gains in the development of novel therapeutics and molecular research tools. The Cys2His2 zinc finger, the most common DNA binding domain in the genome, is a 30 amino acid beta–beta–alpha DNA domain that confers binding specificity to a triplet DNA sequence. These zinc–finger proteins can be attached in tandem to other protein domains to direct various functions in the genome of cells and whole organisms. We are exploiting one application of this technology, chimeric restriction enzymes. These molecules use zinc finger domains to direct the activity of a nonspecific DNA cleavage domain to induce double–strand cleavage and promote gene correction via homologous recombination. When coupled with a molecular delivery system, such as adeno–associated virus, the technology could eventually be used as the basis of gene therapy to repair disease–causing alleles in whole organisms. Results:The specific aims involve developing computational tools to design chimeric restriction enzymes, determining their cleavage specificity in vitro, and demonstrating their utility in gene therapy by repairing inactivating mutations in the rhodopsin gene. Transgenic mice with a defective rhodopsin gene fused to the GFP will be utilized, so that upon repair, GFP will be expressed on the retina of the mice, providing a visible measure of repair efficiency. Currently, we are applying a novel weighting scheme for scoring putative zinc finger proteins by predicted binding specificity. A catalog of all potential chimeric zinc finger nucleases for almost 100 known point mutations in Rhodopsin has being compiled. We are experimentally validating the specificity of candidate zinc fingers with mobility shift assays. Conclusions: Ultimately, we hope to develop chimeric restriction enzymes that can be used to repair disease–causing alleles in order to halt or even reverse the effects of ADRP.
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