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

Abstract

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.

Therapeutic validation of MMR-associated genetic modifiers in a humanex vivomodel of Huntington’s disease

“…This may suggest MSH3 has a higher affinity than MSH6 for MSH2, resulting in a bias towards MutSβ formation where MSH2 is lowered. Regardless, the net reduction in MutSβ here is still sufficient to attenuate expansion rates, as seen in heterozygous Msh3 knock-out or Msh3 siRNA treated mice (Dragileva et al, 2009; O’Reilly et al, 2023). The reciprocal effect was seen upon lowering either MSH3 or MSH6, however MSH2 levels were more substantially reduced where MSH6 levels were targeted, fitting with the higher abundance of MutSα than MutSβ (Reyes et al, 2015).…”

“…Although lowering MSH2 or MSH3 resulted in a robust slowing of repeat expansion in iPSCs, lowering of MSH2 in vivo is unlikely to be a viable long-term approach due its oncogenic potential (Dominguez-Valentin et al, 2020; Therkildsen et al, 2015). In contrast, loss of MSH3 is substantially less oncogenic, particularly in the context of CNS cancers and multiple approaches to therapeutic MSH3 lowering are currently under investigation (Flower et al, 2019; O’Reilly et al, 2023). We instead chose to focus on characterising the effect of lowering the less well understood MutL factors – MLH1, PMS1, PMS2 and MLH3.…”

“…Therefore, targeted expression modulation of these genes could also likely be tolerated in individuals. For example, functionally deleterious polymorphisms in MSH3 slow repeat expansion and HD progression (Flower et al, 2019), and lowering MSH3 expression is currently being pursued therapeutically (O'Reilly et al, 2023).…”

Nucleic acid-based therapeutics for the treatment of central nervous system disorders

“…A di-siRNA consists of two siRNAs connected by a linker. The administration of di-valent siRNAs in vivo has resulted in increased brain distribution, target silencing, and duration of silencing (Alterman et al, 2019;Conroy et al, 2022;O'Reilly et al, 2023). Specifically, a single high dose of 475ug of di-siRNA targeting the HTT gene was administered to BACHD-ΔN17 mice via intracerebroventricular (ICV) injection and resulted in significant HTT silencing and guide strand accumulation in key brain regions such as the cortex and striatum for up to 6 months (Alterman et al, 2019).…”

mRNA nuclear clustering leads to a difference in mutant huntingtin mRNA and protein silencing by siRNAsin vivo

“…We recently developed an siRNA scaffold for delivery to the central nervous system (CNS), termed di-valent siRNA, which supports broad distribution and modulation of target gene expression in mouse and non-human primate CNS for up to six months following intracerebroventricular (ICV) injection ( 13 ). When programmed with an siRNA sequence complementary to both wild-type Htt and mutant HTT , di-valent siRNA silenced wild-type Htt protein and mutant HTT protein with similar efficacy in multiple HD mouse modes ( 14 , 15 ).…”