Somatic CAG expansion in Huntington's disease is dependent on the MLH3 endonuclease domain, which can be excluded via splice redirection

Abstract

Somatic expansion of the CAG repeat tract that causes Huntington's disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect Mlh3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.

Di-valent siRNA Mediated Silencing of MSH3 Blocks Somatic Repeat Expansion in Mouse Models of Huntington’s Disease

“…The heterozygous Hdh Q111 (C57BL/6J background) mouse model is a validated knock-in model of HD, in which human mutant HTT exon 1 is inserted in the context of the mouse Htt locus ( 36, 37 ). This model possesses a 109-111 CAG repeat tract that undergoes somatic repeat expansion within two months in the striatum ( 37-39 ).…”

“…While the MSH3 is an obvious top target to explore for modulation of somatic repeat expansion, other genes involved in the MMR pathway might be of interest. For example, genetic knockout of MLH1 and MLH3 have been shown to impact somatic repeat expansion in HD mouse models ( 13, 36, 60, 61 ). The inherent sequence specificity of siRNAs and their duration of effect provide a powerful therapeutic paradigm for treating neurodegenerative disorders.…”

Mismatch repair is a double-edged sword in the battle against microsatellite instability

“…Although MutL γ is the least abundant of all the MutL complexes, its nuclease activity is required for repeat expansion (Refs 103 – 105 ). Thus, expansion either involves a substrate that is bound preferentially by MutS β /MutL γ or MutL γ cleavage is uniquely able to generate an intermediate that can be processed to generate an expansion.…”

Msh2-Msh3 interferes with DNA metabolismin vivo

“…This suggests a model in which Msh2-msh3 mis-directs one or more MLH complexes when bound to 5' ssDNA flaps to interfere with Rad27-mediated DNA metabolic pathways. Notably, all three Mlh complexes play a role in Msh2-Msh3-mediated TNR expansion (118)(119)(120)(121)(122)(123). Based on our data we suggest a model in which the endonuclease activity of Mlh complexes is misdirected by an altered nucleotide cycling of Msh2-Msh3 bound to a 5' ssDNA flap intermediate, similar to what has been proposed in TNR expansion studies.…”