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Potential Huntington's Disease Therapeutic Target Identified

Scientists have identified a potential new therapeutic target for Huntington's Disease (HD) – MutS Homolog 3 (MSH3). HD is an uncommon neurodegenerative disease that affects cognitive and motor function, and currently, there are no disease-modifying treatments available.

HD is caused by an expansion of the CAG repeat tract in the huntingtin gene (HTT), and age of disease onset is strongly driven by the number of CAG repeats. Somatic repeat expansion occurs when repetitive DNA codons misalign during transcription, creating a slipped loop intermediate that recruits mismatch repair (MMR) machinery to cleave the opposite strand. The slipped loop is then used as a template to add new nucleotides that further expand the locus. A recent genome-wide association study identified several MMR genes as major modifiers of HD onset, expansion of the CAG repeat tract, and clinical HD progression, suggesting this pathway as a potential therapeutic target for HD. MSH3 forms a complex with MSH2, called MutSβ, that selectively recognizes large DNA loops created by expanded CAG repeats and is not involved in other pathways essential for maintenance of DNA integrity. Genetic knockout of Msh3 blocks somatic repeat expansion in HdhQ111 mice, and exploration of pharmaceutical approaches that selectively lower MSH3 expression in the brain is warranted.

Scientists have identified fully chemically stabilized siRNAs targeting human, non-human primate, and mouse Msh3, and show that di-valent siRNA mediated silencing of Msh3 results in blockage of somatic repeat expansion over two and four months in two HD mouse models. These findings provide evidence that silencing MSH3 with siRNA is a promising therapeutic approach for HD patients.

In this study, the researchers aimed to identify chemically modified siRNA sequences that could silence MSH3 mRNA in human, mouse, and NHP cells in vitro, and test the in vivo efficacy of the most promising sequences. They used a modified siRNA efficacy prediction algorithm to design 60 high-scoring siRNA sequences, which were synthesized in an entirely modified asymmetric scaffold with an optimized 2'-O-Methyl RNA/2'-Fluoro RNA pattern and a 3'-cholesterol conjugate on the sense strand to enable passive internalization into all cell types following addition to culture media.

The entire siRNA panel (60 compounds) was screened in HeLa cells, and all twelve cross-reactive siRNAs were additionally screened in the mouse neuronal cell line, N2a. MSH3 and Msh3 mRNA levels were evaluated by QuantiGene Assay at 72 hours post-transfection. In HeLa cells, twelve human-targeting and six cross-reactive compounds induced >75% silencing of MSH3 mRNA. The level of Msh3 silencing in N2a cells was less pronounced. However, the researchers identified five compounds that achieved >50% silencing of Msh3 mRNA.

The two cross-reactive compounds with the highest silencing efficacy in both human and mouse cells were siMSH3_1000 and siMSH3_1468. These two compounds induced dose-dependent silencing in HeLa, N2a, and LLC-MK2 NHP cell lines (IC50s from 15-479 nM). Injection of di-valent siMSH3_1000 potently silences Msh3 and blocks somatic repeat expansion in the striatum of HdhQ111 mice.

To test the in vivo efficacy of siMSH3_1000 and siMSH3_1468, each compound, along with an siRNA with a non-targeting control sequence (NTC), were synthesized in the di-valent scaffold with a 5'-Vinylphosphonate to chemically stabilize the 5' phosphate. Lead or control compounds (10 nmol, or 125 µg dose) were delivered to 12-week-old HdhQ111 mice via intracerebroventricular (ICV) injection and euthanized at 20 weeks of age to evaluate Msh3 protein silencing and somatic repeat expansion. At two months post-injection, di-valent siMSH3_1000, but not di-valent siMSH3_1468, showed potent silencing.

For full study documentation click here.

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