Therapeutic strategies for microsatellite expansion diseases using RNA targeting

PROJECT SUMMARY Microsatellites, also known as simple sequence repeats or short tandem repeats (STRs), are 2-10 bp tandem sequence repeats that occur throughout the human genome. Germ-line expansions of these repeats beyond a critical threshold are associated with more than 50 neurological, neurodegenerative and neuromuscular disorders, including Huntington disease, C9orf72-linked amyotrophic lateral sclerosis/ frontotemporal dementia, several types of spinocerebellar ataxias, as well as myotonic dystrophy types 1 and 2 (DM1/2). Although the complete range of molecular features associated with these diseases varies among these conditions (and often are unknown), cellular and mouse models demonstrate that RNA-mediated toxicity is a major factor. Thus, suppression of mutant RNA levels is expected to address all associated pathologies. While numerous strategies exist to attenuate gene expression via targeted destruction of RNA, all have limitations relating to their mode of administration and tissue targeting (e.g. antisense oligonucleotides, siRNAs), or carry the risk of toxicity caused by adaptive immune responses or pre-existing immunity to the therapeutic agent (systems based delivery of RNA-targeting Cas proteins). Here we develop novel non-immunogenic RNA targeting system compatible with delivery by recombinant adeno-associated viral vectors (rAAVs) that support safe and long-lasting expression. Our platform is based on human spliceosomal RNAs, which in preliminary data we show can be engineered to target and degrade STR-containing transcripts. As a proof-of principle for the therapeutic potential of this system, we focus on DM1, the most common form of adult-onset most common adult-onset muscular dystrophy. We have developed a novel human stem cell based organoid model that, for the first time, provides insight into the molecular basis of the severe neurocognitive deficits associated with DM1. We will use this model to test the safety and efficacy of our RNA-targeting systems in conjunction with a novel mouse model that we generate and that we hope will recapitulate the multi-tissue pathology of DM1. If successful, our work will provide a flexible therapeutic RNA-targeting based platform for treatment of STR-associated diseases.