A blood–brain penetrant RNA-targeted small molecule triggers elimination of r(G ₄ C ₂ ) ^exp in c9ALS/FTD via the nuclear RNA exosome

Abstract

A hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, or c9ALS/FTD. The RNA transcribed from the expansion, r(G 4 C 2 ) exp , causes various pathologies, including intron retention, aberrant translation that produces toxic dipeptide repeat proteins (DPRs), and sequestration of RNA-binding proteins (RBPs) in RNA foci. Here, we describe a small molecule that potently and selectively interacts with r(G 4 C 2 ) exp and mitigates disease pathologies in spinal neurons differentiated from c9ALS patient-derived induced pluripotent stem cells (iPSCs) and in two c9ALS/FTD mouse models. These studies reveal a mode of action whereby a small molecule diminishes intron retention caused by the r(G 4 C 2 ) exp and allows the liberated intron to be eliminated by the nuclear RNA exosome, a multi-subunit degradation complex. Our findings highlight the complexity of mechanisms available to RNA-binding small molecules to alleviate disease pathologies and establishes a pipeline for the design of brain penetrant small molecules targeting RNA with novel modes of action in vivo.

Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity

“…A repositioned small molecule drug, TPN101, is being developed for C9orf72 ALS (NCT04993755) with an emphasis on its potential to reduce central nervous system inflammation ( Zampatti et al, 2022 ). One challenge when developing these small molecule treatment approaches is careful consideration to avoid lowering levels of endogenous C9orf72 protein when targeting sequences adjacent to the hexanucleotide repeat, aiming to instead target repeat-containing RNAs ( Zhu et al, 2020 ; Bush et al, 2022 ). One small molecule developed with dual function was able to bind the expansion and recruit an endonuclease to remove the expansion from the RNA without effecting the levels of C9orf72 protein ( Bush et al, 2022 ).…”

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes

“…So far, candidate drugs have been developed to target protein–lead aggregation but have shown only partial effectiveness 58 , suggesting the need to explore alternative approaches. A recent study has shown potential for ALS treatment in a small molecule with the ability to bind to r(GGGGCC) n , stabilising the intermolecular hairpin conformation formed by the repeat 59 . This is in agreement with our observations and might suggest that depletion of mG4s by stabilisation of intermolecular hairpins could offer a starting point for a new therapeutical pathway for ALS and FTD 59 .…”

Aspects of degradation and translation of the expanded C9orf72 hexanucleotide repeat RNA

“…Moreover, the other ASO designated afinersen, which selectively targets C9orf72 V1 and V3 transcript, while sparing V2 transcript, demonstrated reduced poly‐GP level in CSF of an C9‐ALS patient, validating its target engagement effect (Tran et al, 2022). While ASO facilities degradation of target RNA via RNase H‐dependent pathway, recent RNA technology developed a small molecule binder to G 4 C 2 repeat RNA that recruits RNase L or nuclear RNA exosome and thus selectively degrade G 4 C 2 repeat RNA (Bush et al, 2021, 2022). These RNA‐targeting approach would reduce both repeat RNA and DPR, may thus theoretically favorable.…”