A NOVEL MULTI-TARGET COMBINATION THERAPY FOR C9ORF72-RELATED AMYOTROPHIC LATERAL SCLEROSIS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting both upper and lower motor neurons. The most common genetic cause of ALS is a hexanucleotide repeat expansion within the first intron of the C9orf72 gene. This expansion drives disease progression through multiple mechanisms, including formation of toxic RNA expansions that lead to RNA foci and accumulation of aberrant dipeptide repeat proteins (DPRs).
The aim of this study was to identify a novel therapeutic strategy simultaneously targeting the two principal disease drivers in ALS: expansion-containing RNA and pathogenic DPRs. We developed a multi-target therapy exploiting non-viral nanovectors for delivery of GapmeR antisense oligonucleotides - complementary to the first intronic region of C9orf72, promoting selective RNA degradation and preventing RNA aggregation - combined with a pharmacological agent modulating the unfolded protein response to mitigate DPR-induced proteotoxic stress. The therapy was tested in fibroblasts from ALS patients and age-matched controls, and the efficacy was assessed via RNAscope, immunofluorescence, and molecular analyses. The strategy was also tested in vivo in a Caenorhabditis elegans model of C9orf72-ALS, where locomotor function was measured by the thrashing assay, and neuron degeneration analysed by counting cholinergic motor neurons expressing GFP.
Our results show that the multi-target therapy significantly reduced RNA foci and DPR accumulation, while restoring autophagic function in ALS fibroblasts. In vivo, GapmeR was well tolerated, rescued motor deficits, and reduced motor neuron loss with a dose-response relationship.
These findings provide a proof-of-concept for a combinatorial therapy with translational potential towards clinical application in C9orf72-ALS patients.