C006 Insights into the DNA mismatch repair system exploiting a frameshift mutator cell-based assay to identify new targets and potential treatments for Huntington’s disease

<h3></h3> DNA mismatch repair (MMR) has been linked to Huntington’s disease (HD) through GWA studies. Mouse models have highlighted the crucial role of MMR in promoting somatic CAG instability, which is related to early HD onset and worsens its progression. The development of cell-based assays enabling investigation of the molecular mechanisms underlying the MMR pathway could provide insight into the pathogenesis of HD and aid in identifying potential targets for therapeutic intervention. Therefore, we have engineered HeLa and TK6 single cell clones with a NanoLuc reporter downstream of a tetranucleotide repeat sequence, which is known to generate frameshift mutations sensitive to MutSβ activity. The frameshift mutator assay (FSMA) was developed and validated by CRISPR/Cas9 to KO MSH3 and/or MLH1, key components of the MMR system. Rescue experiments were conducted for structure/function investigations and specific mutations in MSH3 and MLH1 were found to be important for MMR functionality. Furthermore, CRISPR/Cas9 technology was used in the FSMA to explore the role of the GWAS hits in the instability of tetranucleotide repeats. The effect of their depletion was also evaluated on the expression of the other MMR proteins, revealing interdependencies among some of them. Finally, the FSMA has been optimized in TK6 for medium-throughput screening studies and was employed to assess the impact of specific MMR inhibitors. In conclusion, we have developed and validated a highly versatile tool that can be utilized for a wide range of purposes within the funnel of MMR modulator discovery, including target validation, compound screening and structure-activity relationship studies.