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

INTRODUCTION – AIM A novel class of RNA-based therapeutic molecules is represented by modified spliceosomal U1 small nuclear RNA (snRNA) which, by acting on pre-mRNA splicing, have already proved to be effective in mice models of SMA 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. MATERIALS AND METHODS Induced pluripotent stem cell (iPSC) derived from C9ORF72 patients were differentiated into MNs. RESULTS Two modified U1 snRNAs (U1C and U1G) were co-transfected in HEK293T cells overexpressing 66 hexanucleotide repeats. By FISH analysis we observed that both U1 constructs significantly decreased the number of cells forming C9ORF72 RNA foci. 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 repeats upstream the GFP tag and a significant reduction also in the formation of polyGP-GFP protein was observed upon co-transfection with both modified U1s. We then tested the efficacy of U1 snRNA in reducing RNA foci formation in C9ORF72 patient-derived iPSC-motoneurons 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 (40% vs 15%) and in the number of pathological RNA foci per cell. CONCLUSION We are currently measuring the impact of U1s on polyGP synthesis and confirming our data in other C9ORF72 iPSC-motoneurons 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 that target the pathological HRE represent an innovative therapeutic strategy to treat ALS and FTD patients carrying C9ORF72 mutation.