Engineering Duplex RNAs for Challenging Targets: Recognition of GGGGCC/CCCCGG Repeats at the ALS/FTD C9orf72 Locus

Abstract

SUMMARY A GGGGCC expansion within an intronic region of the C9orf72 gene forms RNA foci that are associated with one third of familial amyotrophic lateral sclerosis (ALS) and one quarter of frontotemporal dementia (FTD). The C9orf72 locus also expresses an antisense transcript with a CCCCGG expansion that forms foci and may contribute to disease. Synthetic agents that bind these hexanucleotide repeats and block foci would be leads for therapeutic discovery. We have engineered duplex RNAs to enable them to recognize difficult C/G targets. Recognition inhibits foci formed by both GGGGCC and CCCCGG RNA. Our findings show that a single duplex RNA can be used to recognize both disease-related C9orf72 transcripts. More broadly, we extend RNAi to previously inaccessible C/G sequences and provide another example of target recognition in human cells by nuclear RNAi.

c9orf72 Disease-Related Foci Are Each Composed of One Mutant Expanded Repeat RNA

“…These methods do not provide insights into whether the distribution among individual cells is homogeneous or heterogeneous. In our previous studies we had visualized foci and noted that there were only a few foci per cell (Hu et al, 2015). This low number of foci, together with the low RNA copy number observed by ddPCR and qPCR, suggested that each focus may represent a single RNA molecule.…”

“…Approaches to treatment include the use of antisense oligonucleotides (Donnelly et al, 2012; Sareen et al, 2012; Jiang et al, 2016; Lagier-Tourenne et al, 2013), duplex RNAs (Hu et al, 2015), or small molecules (Su et al, 2014) that either block the expanded repeat within intronic c9or72 RNA or that target the intron outside the expansion. The small number of target transcripts suggests that relatively low numbers of inhibitors are needed to achieve the excess compound needed for potent inhibition of the delirious effects of mutant c9orf72 .…”

“…Healthy control fibroblast cell line GM02153 was obtained from Coriell institute (Camden, NJ, USA). Cells were cultured as described (Hu et al, 2015). Astrocyte and motor neuron differentiated from ALS patient-derived ips cell lines were prepared as described (Zhang et al, 2015; Li et al, 2015).…”

“…Nuclear and cytoplasmic fractions were prepared as described (Gagnon et al, 2014). In situ hybridization was performed as described (Hu et al, 2015). …”

Insights into disease mechanisms and potential therapeutics for C9orf72-related amyotrophic lateral sclerosis/frontotemporal dementia

“…Despite the unclear pathogenic role of RNA-mediated toxicity, a therapy targeting the repeat-containing transcripts could potentially reduce RNA foci (Hu et al, 2015;Martier et al, 2019a), alleviating disease progression.…”