RNAi-Based Degradation of C9ORF72 Variants as a Potential Therapy for ALS

Abstract

Amyotrophic, Lateral Sclerosis (ALS), a progressive and fatal disease, is characterized by loss of neurons specifically in the motor cortex, brainstem, and spinal cord. Current FDA approved therapeutics provide limited benefit to patients; thus a large unmet medical need exists. A G4C2 hexanucleotide expansion mapped to chromosome 9 (C9ORF72) presents as the leading genetic cause of familial (~40%), and sporadic ALS (~6%). Oligonucleotide therapeutics for C9ORF72 are currently in clinical trials. However, early results are not promising. My thesis work employed extensive screening of C9ORF72 to identify regions amenable to siRNA directed mRNA degradation. Importantly, we identify siRNA capable of reducing all C9ORF72 mRNA transcript variants, or selectively reducing repeat-containing mRNA variants while sparing non-repeat variants. This represents two possible therapeutic strategies for C9ORF72 mediated ALS. Using an siRNA chemical scaffold with widespread central nervous system activity, we show efficacy of siRNA in a mouse model of ALS. Importantly, we are able to significantly reduce C9ORF72 mRNA, as well as RNA foci and di-peptide repeat proteins, which are key disease hallmarks of C9ORF72-driven ALS. Our studies also demonstrate that C9ORF72 RNA localization is split between the nuclear and cytoplasmic compartments and that disease-associated RNA foci are primarily comprised of non-repeat-containing RNA. Additionally, our work supports the intron retention model of C9ORF72 biology which has been previously debated. My thesis work demonstrates the promise of siRNA for the treatment of C9ORF72 driven ALS and highlights key biological features of C9ORF72 RNA which may drive future therapeutic development.