Therapeutic Potential of ASO-Mediated MSH3 Suppression in Huntington’s Disease

Academic Background

Huntington’s disease (HD) is a neurodegenerative disorder caused by abnormal expansion of the CAG repeat sequence in the huntingtin gene (HTT). This expanded CAG repeat continues to expand somatically over time, driving the onset and progression of the disease. MSH3, a DNA mismatch repair protein, influences the onset and progression of HD by driving the somatic expansion of the CAG repeat. Loss-of-function variants of MSH3 are relatively well-tolerated in humans, making it a potential therapeutic target. However, the specific mechanisms and therapeutic effects of MSH3 inhibition in HD remain unclear. This study aims to explore whether antisense oligonucleotide (ASO)-mediated suppression of MSH3 can effectively slow CAG repeat expansion in striatal neurons derived from induced pluripotent stem cells (iPSCs) of HD patients and to assess its safety.

Paper Source

This paper, authored by Emma L. Bunting and colleagues, involves researchers from multiple institutions, including the UCL Queen Square Institute of Neurology and UK Dementia Research Institute, the University of Cambridge, the University of Glasgow, and the Broad Institute of MIT and Harvard, among others. Published on February 12, 2025, in Science Translational Medicine, the paper is titled “Antisense oligonucleotide–mediated MSH3 suppression reduces somatic CAG repeat expansion in Huntington’s disease iPSC–derived striatal neurons.”

Research Process and Results

1. Research Design and Objectives

The primary objective of the study was to evaluate whether ASO-mediated MSH3 suppression can effectively slow CAG repeat expansion in striatal neurons derived from iPSCs of HD patients and to explore its therapeutic potential. The study was conducted in the following steps:
- iPSC-Derived Striatal Neuron Culture: iPSCs from an HD patient carrying 125 CAG repeats were differentiated into striatal neurons and cultured.
- ASO Design and Screening: ASOs targeting MSH3 mRNA were designed and screened, with the most effective ASO selected for further experiments.
- Assessment of MSH3 Suppression: The effects of ASO on MSH3 expression and CAG repeat expansion were evaluated at different doses.
- Transcriptomic Analysis: RNA sequencing (RNA-seq) was used to assess the impact of MSH3 suppression on the neuronal transcriptome.
- Animal Model Validation: A knock-in mouse model expressing the human MSH3 gene was constructed to validate the therapeutic effects of ASO in vivo.

2. Experimental Steps and Results

(1) iPSC-Derived Striatal Neuron Culture

Researchers differentiated iPSCs from an HD patient into striatal neurons and verified differentiation efficiency using immunofluorescence staining. Results showed that approximately 86% of cultured cells were neurons (MAP2+), 67% were striatal neurons (FOXP1+), and 30% were medium spiny neurons (DARPP32+), confirming the reliability of the model.

(2) ASO Design and Screening

Approximately 230 ASOs targeting human MSH3 mRNA were designed, and the six most effective ASOs were selected using quantitative reverse transcription PCR (qRT-PCR). The ASO named MSH3 ASO-1, which showed the strongest MSH3 suppression in A-431 cells, was chosen for subsequent experiments.

(3) Assessment of MSH3 Suppression

Dose-dependent experiments were conducted in iPSC-derived striatal neurons. Results showed that MSH3 ASO-1 dose-dependently suppressed MSH3 expression, with a reduction of over 95% at the highest dose (3 μM). ASO treatment significantly slowed CAG repeat expansion, nearly halting it at the highest dose. CRISPR-Cas9 knockout of MSH3 also validated these results, with MSH3-deficient neurons showing contraction of the CAG repeat.

(4) Transcriptomic Analysis

RNA-seq analysis revealed that MSH3 ASO-1 treatment significantly reduced MSH3 expression in neurons without dysregulating DNA repair pathways. Although ASO treatment affected the expression of some genes, these changes did not involve critical pathways, suggesting that MSH3 suppression is relatively safe in neurons.

(5) Animal Model Validation

Researchers constructed a knock-in mouse model expressing the human MSH3 gene and administered MSH3 ASO-1 via intracerebroventricular (ICV) injection. Results showed that ASO treatment significantly reduced MSH3 expression in the brain and spinal cord of mice, with similar suppression effects observed in multiple brain regions. Animal experiments demonstrated that ASO treatment was well-tolerated, with no significant toxic side effects observed.

3. Conclusions and Significance

This study systematically evaluated the effects of ASO-mediated MSH3 suppression on CAG repeat expansion in human iPSC-derived striatal neurons for the first time and demonstrated its therapeutic potential for HD. The results indicate that MSH3 suppression can effectively slow or even halt CAG repeat expansion, with minimal impact on the neuronal transcriptome. Furthermore, validation in a mouse model demonstrated the feasibility and safety of ASO treatment in vivo. These findings provide critical experimental evidence for developing MSH3 suppression-based therapeutic strategies for HD.

4. Research Highlights

• Innovative Therapeutic Strategy: Suppressing MSH3 expression via ASO offers a novel approach to HD treatment.
  
• Comprehensive Experimental Validation: The study systematically validated the effects and safety of MSH3 suppression from in vitro cell models to in vivo animal models.
  
• Clinical Translation Potential: The results lay a strong scientific foundation for future clinical trials in HD.
  
• Multidisciplinary Collaboration: The research team leveraged expertise in genetics, molecular biology, neuroscience, and other fields to advance the study.
  

5. Other Valuable Information

The human MSH3 knock-in mouse model developed in this study provides an essential experimental tool for future MSH3-targeted drug development. Additionally, the study found that MSH3 suppression remains effective in neurons lacking FAN1 (a gene associated with HD onset age), suggesting that this therapeutic strategy may be applicable to HD patients with high-risk genetic variants.

Summary

Through multi-level experimental validation, this study systematically assessed the therapeutic potential of ASO-mediated MSH3 suppression in HD and provided critical scientific evidence for its clinical application. Future research will further explore the efficacy of this therapeutic strategy in patients and optimize ASO designs to enhance effectiveness and safety.