Direct targeting of C9ORF72 repeat RNA with fluorinated antisense oligonucleotides.

Hexanucleotide repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. These expansions give rise to pathogenic sense and antisense repeat RNAs that form nuclear foci and undergo repeat-associated non-AUG translation, producing dipeptide repeat proteins with cellular toxicity. Directly targeting the causative repeat RNAs with antisense oligonucleotides represents a promising therapeutic strategy. One barrier to further development is the propensity of this G-rich repeat-containing RNA target to form stable secondary structures, which may hinder efficient hybridization. In this study, we designed a panel of fluorine-modified ASOs that target the sense repeat expansions. We identified C-rich F-ASO gapmers that reduced translation from sense repeat RNAs in a cell-based reporter assay and lowered the RNA foci burden in patient-derived cells. Structural analyses in vitro revealed that the 2'F-RNA gapmer formed a stable hairpin structure. Our results demonstrate that structural properties of fluorine modifications can be leveraged for effective binding of repeat RNA and highlight the potential for F-ASOs to serve as therapeutic tools when targeting toxic repeat RNAs in C9ORF72-mediated FTD/ALS and other repeat expansion diseases.