Direct targeting of C9ORF72 repeat RNA with fluorinated antisense oligonucleotides

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Hexanucleotide repeat expansions in the C9ORF72 gene are the primary genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
• These expansions produce toxic repeat RNAs and dipeptide repeat proteins, leading to cellular dysfunction and disease pathology.
• Antisense oligonucleotides (ASOs) are a potential therapeutic strategy, but targeting the G-rich repeat RNA is challenging due to its stable secondary structures.

Purpose of the Study:

• To design and evaluate fluorine-modified antisense oligonucleotides (F-ASOs) for targeting C9ORF72 repeat expansions.
• To assess the efficacy of F-ASOs in reducing toxic RNA and protein production in cellular models.
• To investigate the structural properties of F-ASOs and their impact on RNA binding.

Main Methods:

• Design and synthesis of a panel of fluorine-modified ASOs targeting C9ORF72 sense repeat expansions.
• Cell-based reporter assays to measure the reduction of translation from repeat RNAs.
• Analysis of RNA foci burden in patient-derived cells.
• In vitro structural analyses of F-ASO-RNA interactions.

Main Results:

• Identified C-rich F-ASO gapmers that significantly reduced translation from sense repeat RNAs.
• Demonstrated a reduction in RNA foci burden in patient-derived cells treated with F-ASOs.
• Structural analysis revealed that a 2'F-RNA gapmer formed a stable hairpin structure, facilitating effective binding.

Conclusions:

• Fluorine modifications can be leveraged to overcome structural barriers in targeting toxic repeat RNAs.
• F-ASOs show potential as therapeutic agents for C9ORF72-mediated FTD/ALS and other repeat expansion diseases.
• The structural properties of F-ASOs contribute to their efficacy in binding and neutralizing pathogenic repeat RNAs.