Design of precision small molecules targeting RNA repeating transcripts to manipulate and study disease biology

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PROJECT SUMMARY: An extraordinarily challenging problem is to develop general methods to target defective or malfunctioning RNAs that cause disease selectively. Current therapeutic strategies to target RNAs are based on specific sequence recognition by oligonucleotides. However, many human disorders are caused by highly structured RNAs not readily targetable by conventional base pairing, in particular RNA repeat expansions that cause or contribute to >30 incurable neuromuscular diseases and genetically defined dementia. Thus, allele- specific ASOs modalities for these microsatellite disorders have been developed by targeting polymorphisms outside of the repeating sequence. The consequences of this approach are that only patients with the polymorphisms benefit from treatment and that an ASO has to be developed for each disease, even if caused by the same repeating sequence. If the toxin in these diseases, the expanded repeat, could be targeted selectively with a structure-specific small molecule, then a single modality could be a therapeutic or chemical probe for multiple diseases and for all patients. Over the past 14 years, we have shown that RNA structures can be targeted selectively with small molecules in situ and in vivo, more selectively than oligonucleotides. Indeed, we have designed compounds against many RNA repeat expansions that selectively recognize the target’s structure and rescue disease-associated pathobiology in situ and in vivo. Further, these chemical probes have elucidated new mechanisms of disease, including a previously unknown RNA-mediated transcriptional silencing pathway that operates in fragile X syndrome. These studies, along with our innovative strategies to synthesize drugs at the site of disease and to engineer small molecules with novel activities, including antisense- or CRISPR-like modes of action, lay the foundation for our proposed research program. Herein, we propose a comprehensive strategy to study the molecular recognition of RNA repeat expansions by small molecules in situ and in vivo, enabling the establishment of new chemical biology frameworks to target RNA using small molecules and the development of preclinical candidates. Our studies span many types of repeat expansions, differing in both sequence and gene contexts (intron, untranslated region, or open reading frame). We have devised innovative strategies to: (i) exploit the structures of RNA repeats to coax the disease- causing RNA to synthesize its own drug; (ii) interface small molecules with natural RNA decay and QC pathways; and (iii) recruit endogenous nuclease to the repeats with small molecules. We will not only deliver proof-of- concept small molecules that rescue disease-associated defects in situ and in vivo, but make new discoveries about how to drug RNA using small molecules. Our proposed work would therefore be well supported by an R35 award, as the flexibility conferred by this award is truly necessary to ensure sustainable, long-term funding and the investment required to develop a new code for how molecules interface with RNA in health and disease.