A cell-based screen for small molecule binders of mutant huntingtin messenger RNA

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DESCRIPTION (provided by applicant): Huntington's Disease (HD) is a member of a family of dominantly inherited neurodegenerative diseases caused by expansion in a glutamine-encoding CAG tract. Mutant huntingtin (Htt) protein containing an expanded polyglutamine (polyQ) tract acquires novel properties that are believed to directly cause cellular dysfunction and disease. Proteins containing expanded polyQ tracts have been shown to be toxic when expressed in a wide range of experimental transgenic systems including yeast, cultured mammalian cells, C. elegans, Drosophila and mouse. The pathologic phenotype in HD is therefore thought to be driven primarily by a toxic gain of function mechanism. The overwhelming majority of HD patients are heterozygous for the mutation and therefore have one expanded and one wild-type copy of the HD gene. Studies in HD knock- out mice have shown that HD is an essential gene, but heterozygous null animals have no apparent phenotype. These observations taken together indicate that specifically inhibiting expression of the expanded HD gene would be of therapeutic benefit in a heterozygous HD patient. Secondary-structure prediction algorithms indicate that the 5' regions of the mutant and wild-type HD messenger RNAs have different folds. This suggests that the CAG-expanded message may contain a mutant-specific RNA epitope that could exploited as a drug target. We are developing a human cell based assay to screen for small molecules that bind to the mutant HD mRNA in such a way as to slow or prevent its translation into protein. This cell-based assay uses a translational reporter gene consisting of the 5' region of a mutant HD gene (with 48 CAG codons) fused to the luciferase gene. Since the HD part of the fusion contains no start codon, it serves as a 5' untranslated region (UTR) for luciferase, and the ribosome must read through the HD message to produce luciferase protein. The reporter construct also contains a GFP reporter to monitor for non-specific inhibitors of gene-expression. This assay will be used to screen small molecules libraries to identify compounds that down-regulate expression of the HD-UTR-luciferase fusion without affecting GFP expression. Hits will be counter screened against control reporter genes including non expanded HD fused to luciferase as a UTR. The ultimate goal of this project is to identify compounds that bind specifically to the mutant HD messenger RNA so as to inhibit its translation into mutant HD protein.