Expanded trinucleotide repeats cause many human diseases, including Huntington’s disease (HD). Recent studies indicate that somatic instability of these repeats contributes to pathogenesis in several expansion disorders. We find that lowering huntingtin protein (HTT) levels reduces somatic instability of both the Htt and Atxn2 CAG tracts in knockin mouse models, and the HTT CAG tract in human iPSC-derived neurons, revealing an unexpected role for HTT in regulating somatic instability.

Huntington's Disease (HD) is a severe neurodegenerative disorder caused by expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS Homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify fully chemically modified short interfering RNA (siRNA) that robustly silence MSH3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of MSH3 effectively blocked CAG repeat expansion in striatum of two HD mouse models without impacting tumor-associated microsatellite instability. Our findings establish a novel paradigm for treating patients with HD and other repeat expansion diseases.

Repeat expansion diseases (REDs) are a large group of human genetic disorders caused by expansion of a specific short tandem repeat sequence. Expansion in somatic cells affects age at onset and disease severity in some of these disorders. However, alleles in blood, a commonly used source of DNA, usually show much less expansion than disease-relevant cells in the central nervous system (CNS) in both humans and mouse models. Here we examined the extent of expansion in different DNA sources from mouse models of the Fragile X related disorders (FXDs), Huntington's disease (HD), Spinocerebellar ataxia type 1 (SCA1) and Spinocerebellar ataxia type 2 (SCA2). We found that stool is a much better indicator of somatic expansion than blood. Since stool is a sensitive and non-invasive source of DNA, it may be useful for studies of factors affecting expansion risk or the monitoring of treatments aimed at reducing expansion in preclinical trials since it would allow expansions to be examined longitudinally in the same animal and allow significant effects to be ascertained much earlier than is possible with other DNA sources.

Huntington's disease arises from a toxic gain of function in the huntingtin (HTT) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological and plasma metabolite level. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic β-catenin function.