U1 snRNA as a novel RNA-based therapeutic approach to modulate C9ORF72 pathology in patient-derived iPSC-motoneurons

A novel class of RNA-based therapeutic molecules is represented by modified spliceosomal U1 small nuclear RNAs (snRNAs) which, by acting on pre-mRNA splicing, have already proved to be effective in mice models of spinal muscular atrophy and Familial dysautonomia. Aim of this work is to test whether modified U1 snRNAs, designed to bind the C9ORF72 hexanucleotide repeat expansion (HRE), correct C9ORF72-associated pathology as a valuable and alternative RNA-based therapeutic strategy to antisense oligonucleotides in preclinical ALS patient-derived cell models such as iPSC-motoneurons (iPSC-MN). Two different modified U1 snRNAs (U1C and U1G) were preliminarily transfected in HEK293T cells overexpressing 66 hexanucleotide repeats. FISH analysis showed that both U1 constructs significantly decreased the mean number of pathological RNA foci per cell. To assess whether U1C and U1G had an effect also on RAN translation of the HRE-containing transcripts, we used a nonATG-plasmid with the 66-hexanucleotide repeat upstream a GFP tag. A significant reduction also in the formation of polyGP-GFP proteins was observed upon transfection with both modified U1s. We then tested the efficacy of U1 snRNAs in reducing RNA foci formation in C9ORF72 patient-derived iPSC-MN carrying 1200 repeats by lentiviral-mediated delivery of U1s. FISH analysis showed a significant decrease both in the percentage of cells containing pathological RNA foci and in the number of RNA foci per cell. We are currently confirming our data in other C9ORF72 iPSC lines carrying different HRE size (150 and 670 units) to assess if U1s efficacy in reducing RNA foci is length-dependent. Our results suggest that modified U1 snRNAs targeting the pathological HRE represent an innovative therapeutic strategy to treat ALS and FTD patients carrying C9ORF72 mutation.