Biology subjects

Burch, Z.

Publications and source records attributed to Burch, Z..

3 recordsLinked to original sources

bioRxiv2024

Suppression of Huntington's Disease Somatic Instability by Transcriptional Repression and Direct CAG Repeat Binding

Huntingtons disease (HD) arises from a CAG expansion in the huntingtin (HTT) gene beyond a critical threshold. A major thrust of current HD therapeutic development is lowering levels of mutant HTT mRNA (mHTT) and protein (mHTT) with the aim of reducing the toxicity of these product(s). Human genetic data also support a key role for somatic instability (SI) in HTTs CAG repeat - whereby it lengthens with age in specific somatic cell types - as a key driver of age of motor dysfunction onset. Thus, an attractive HD therapy would address both mHTT toxicity and SI, but to date the relationship between SI and HTT lowering remains unexplored. Here, we investigated multiple therapeutically-relevant HTT-lowering modalities to establish the relationship between HTT lowering and SI in HD knock-in mice. We find that repressing transcription of mutant Htt (mHtt) provides robust protection from SI, using diverse genetic and pharmacological approaches (antisense oligonucleotides, CRISPR-Cas9 genome editing, the Lac repressor, and virally delivered zinc finger transcriptional repressor proteins, ZFPs). However, we find that small interfering RNA (siRNA), a potent HTT-lowering treatment, lowers HTT levels without influencing SI and that SI is also normal in mice lacking 50% of total HTT levels, suggesting HTT levels, per se, do not modulate SI in trans. Remarkably, modified ZFPs that bind the mHtt locus, but lack a repressive domain, robustly protect from SI, despite not reducing HTT mRNA or protein levels. These results have important therapeutic implications in HD, as they suggest that DNA-targeted HTT-lowering treatments may have significant advantages compared to other HTT-lowering approaches, and that interaction of a DNA-binding protein and HTTs CAG repeats may provide protection from SI while sparing HTT expression.

neuroscience↗

bioRxiv2024

Identification of genetic modifiers of Huntington's disease somatic CAG repeat instability by in vivo CRISPR-Cas9 genome editing

Huntingtons disease (HD), one of >50 inherited repeat expansion disorders (Depienne and Mandel, 2021), is a dominantly-inherited neurodegenerative disease caused by a CAG expansion in HTT (The Huntingtons Disease Collaborative Research Group, 1993). Inherited CAG repeat length is the primary determinant of age of onset, with human genetic studies underscoring that the property driving disease is the CAG length-dependent propensity of the repeat to further expand in brain (Swami et al., 2009; GeM-HD, 2015; Hensman Moss et al., 2017; Ciosi et al., 2019; GeM-HD, 2019; Hong et al., 2021). Routes to slowing somatic CAG expansion therefore hold great promise for disease-modifying therapies. Several DNA repair genes, notably in the mismatch repair (MMR) pathway, modify somatic expansion in HD mouse models (Wheeler and Dion, 2021). To identify novel modifiers of somatic expansion, we have used CRISPR-Cas9 editing in HD knock-in mice to enable in vivo screening of expansion-modifier candidates at scale. This has included testing of HD onset modifier genes emerging from human genome-wide association studies (GWAS), as well as interactions between modifier genes, thereby providing new insight into pathways underlying CAG expansion and potential therapeutic targets.

genetics↗

bioRxiv2020

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

Somatic expansion of the CAG repeat tract that causes Huntingtons 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.

genetics↗