Small Molecule-Mediated Cleavage of RNA In Cells. Application to Incurable Human Genetic Disease

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Microsatellite disorders are devastating diseases caused by specific nucleotide repeats present within the genome of affected patients. Whereas these repeat expansions are not pathogenic within a certain length, they can become pathogenic when exceeding a precise threshold. When transcribed, they initiate various toxic mechanisms which are the basis of more than 20 neuromuscular disorders including myotonic dystrophy type 1 (DM1) (caused by CUG repeats) and chromosome 9p21-linked amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) (triggered by GGGGCC repeats), for which at present no effective treatment exists. The current research proposal presents two therapeutic strategies meant to improve the symptoms of DM1 and c9ALS/FTD by employing chemical nucleases (without turnover!) that specifically degrade toxic RNA repeats. A chemical nuclease consists of three modules: (1) RNA binding module, a multivalent construct composed of monomeric units previously shown to interact with the repeats; (2) linker unit; (3) RNA cleavage module, responsible for the degradation of toxic RNA expansions. We will employ (-)-Pyrimidoblamic acid, the cleavage unit of a structurally complex antibiotic Bleomycin, which was previously shown to efficiently cleave ribonucleic acids. The first strategy will focus on DM1 and consists of assembling the chemical nuclease modules in vitro using standard amide coupling reactions. The second approach addresses c9ALS/FTD and attempts to generate the chemical nuclease in cellulo from monomer units functionalized with alkyne and azide moieties. The assembly process occurs within the diseased cells by means of in situ click chemistry catalyzed by the structure of the toxic RNA which acts as a template bringing the monomeric units in close proximity. These approaches offer a generalized platform to approach other microsatellite diseases that were so far not efficiently addressed. Moreover, the cleaving approach via chemical nucleases could be applied to a host of other RNAs, outside of repeats, such as cancer-causing RNAs. Altogether, the results of this research will: (1) provide a small molecule-based therapeutic alternative to antisense technology; (2) define basic rules and principles for effective targeting of toxic RNA in cellulo and potentially in animal model; (3) reveal structural features of the small molecules or small molecule assemblies required to promote efficient and selective RNA cleavage in cellulo; (4) help transform RNA into a druggable target.