Proteolysis-targeting chimeras (PROTACs) are an increasingly established modality to induce protein degradation by bridging the protein target to proteolytic machinery. Ribonuclease-targeting chimeras (RIBOTACs) perform a similar function, bringing RNA target molecules to RNases for degradation. Writing in Science Translational Medicine, a team led by Matthew Disney design a RIBOTAC to degrade the disease-causing RNA in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In patient-derived spinal neurons and a mouse model of ALS, their molecule induced degradation of the pathogenic mRNA and reduced the associated pathology.
ALS and FTD are progressive neurodegenerative conditions that result in motor and cognitive impairment. These diseases are usually sporadic; the most commonly associated mutation is a hexanucleotide repeat expansion (HRE) in an intron, usually intron 1, of chromosome 9 open reading frame 72 (C9orf72). This subset of the disease is termed c9ALS/FTD.
The HRE-containing RNA is translated into a protein that can contain one of five different dipeptide repeats (depending on the reading frame of the HRE), often poly(GP) or poly(GA).
The HRE-containing RNA and the resulting dipeptide repeat protein are both thought to promote neuronal death. Both the RNA and the protein form toxic aggregates. The RNA also sequesters gene expression machinery.
The authors reasoned that removing the mRNA itself could be of particular therapeutic value, because it would eliminate both of these toxic species. The HRE mRNA forms a particular 3D structure, and targeting this structure, rather than the primary sequence, could have fewer off-target effects from targeting other, non-pathogenic mRNAs that contain shorter HREs.
Using structure–activity relationships combined with biophysical and structural analyses, the authors first designed a small molecule dimeric compound that would bind to the 3D RNA structure. Each monomer bound within the internal loops of the RNA hairpins. The dimer bound with a Kd of 4±0.7 nM, and had a long residence time on the target RNA.
In a pull-down assay, the dimer associated with the target RNA dose-dependently in cells, including patient-derived lymphoblastoid cell lines and induced pluripotent stem cells (iPSCs). In multiple cell lines, including those derived from patients, the molecule reduced ribosome loading and translation of RNAs containing the expanded C9orf72 intron 1.
Building on that dimer, they added an RNase L recruiter to turn the molecule into a RIBOTAC. In HEK293T cells, their RIBOTAC inhibited translation and dose-dependently reduced levels of the HRE-containing RNA in a manner that required RNase L.
Their RIBOTAC rescued pathological hallmarks in c9ALS/FTD patient-derived cell lines, too. This molecule reduced the abundance of C9orf72 intron 1 in patient-derived lymphoblastoid cell lines and iPSCs, and reduced levels of the dipeptide repeat protein in iPSCs. No effects on other transcripts were observed.
These same reductions were seen in iPSC-derived spinal neurons (iPSNs), which recapitulate many of the genetic, transcriptional and biochemical signatures of brain tissue from patients with c9ALS/FTD. The RIBOTAC dose-dependently reduced levels of C9orf72 intron 1 and poly(GP), without altering the transcription of other transcripts that contain short, non-pathogenic (G4C2) repeats.
Nuclear pore proteins have previously been found to be reduced in patients with c9ALS/FTD. By super-resolution structured illumination microscopy, the authors found the RIBOTAC restored levels of Nup98, a key nuclear pore protein.
The mouse model for c9ALS/FTD contains a C9orf72 bacterial artificial chromosome that expresses 500 r(G4C2) repeats, resulting in foci containing the aberrant RNA or poly(GP) proteins. Treatment of these mice with the RIBOTAC by a single intracerebroventricular injection reduced r(G4C2)-containing mRNA, r(G4C2)-containing foci, and poly(GP)-containing proteins. These effects were observed as early as 1 week after injection, and persisted until at least 6 weeks after treatment (the earliest and latest time points analysed). The RIBOTAC reduced known hallmarks of c9ALS/FTD, including poly(GP) and poly(GA) aggregates, as well as transactivation response DNA-binding protein 43 (TDP-43) inclusions, detected by immunohistochemical analysis.
The authors note that further optimization of the medicinal chemistry and physicochemical properties would be required to translate their RIBOTAC to the clinic. However, this work demonstrates that targeting RNA is feasible, and could be an optimal modality for diseases in which RNA plays a key pathogenic role.
Bush, J. A. et al. Ribonuclease recruitment using a small molecule reduced c9ALS/FTD r(G4C2) repeat expansion in vitro and in vivo ALS models. Sci. Transl Med. 13, eabd5991 (2021)
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