High-throughput small molecule screen to reduce endogenous level of Msh3 for disease-modifying HD therapy - PROJECT SUMMARY Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG-repeat expansion encoding mutant Huntingtin (mHTT). Recent genome-wide association studies (GWAS) identified 10 genomic loci significantly modifying the age-of-onset or progression of HD. These loci are significantly enriched with DNA repair genes, including four DNA mismatch repair genes (Msh3, Mlh1, Pms1 and Pms2). Our mouse genetic study demonstrated that Msh3 deficiency significantly reduced mHtt aggregates and rescued mHtt-induced transcriptionopathy in Q140 knockin mouse model of HD. Our research initiative focuses on developing novel therapeutic strategies for HD by targeting the Msh3 protein. Leveraging a multi-faceted approach, we have established a robust screening pipeline utilizing a newly developed knockin mouse model expressing a luciferase-based reporter of endogenous Msh3, enabling high-throughput screening of small molecules that modulate Msh3 levels in cellular models. In the R61 phase, we aim to demonstrate the scalability, precision, and reproducibility of our primary mouse embryonic fibroblast (MEF)-based Msh3 reporter assay (Aim 1) and subsequently develop secondary assays to further interrogate and eliminate less desirable chemical hits (Aim 2). These assays include directly measuring endogenous MSH3 RNA and protein levels in human a human cell line, examining Msh3 lowering efficacy with a therapeutically relevant cell type (primary striatal neuron), and assessing the functional consequence of lowering MSH3 in cells. In the R33 phase, we will utilize the MEF- based Msh3 reporter assay optimized in Aim 1 to conduct a large-scale screening of 300,000 chemically diverse compounds to identify potential Msh3-lowering modulators. Subsequently, we will establish a testing funnel with the assays developed in Aim 1 and 2 to gradually eliminate and prioritize the hits and chemical scaffolds to identify potential leads. Throughout these endeavors, rigorous authentication of biological and chemical resources will be ensured, aligning with our commitment to scientific integrity. Through this comprehensive approach, we aim to identify lead compounds with therapeutic potential for HD and potentially other neurodegenerative diseases sharing similar MSH3-dependent, repeat-expansion related pathogenic mechanisms, ultimately advancing towards effective disease-modifying treatments.
