Protein coding messenger RNAs (mRNA) undergo extensive processing before their translation into protein (1). Precursor mRNA (pre-mRNA) transcripts, copied directly from the genome, contain both coding (exons) and intervening sequences (introns) (2, 3). mRNA biogenesis requires intron removal and exon splicing. When this process fails, genes produce a toxic RNA encoding a faulty protein (4, 5). Our goal is to design antisense oligonucleotides that can rescue damaged exons.
The spliceosome is an ensemble of five small nuclear ribonucleoprotein particles (snRNPs) and hundreds of protein co-factors that catalyze the splicing reaction (6, 7). This complex assembles de novo on every intron (8). Conserved dinucleotide sequences (splice sites) mark the beginning and end of introns (9, 10). Auxiliary RNA elements including the polypyrimidine tract, influence recognition of these simple landmarks.
Recognition of exon and intron boundaries is a critical initial step in spliceosome assembly (11). Splicing enhancers and silencers are important determinants of exon identity (12–16). Located near splice sites, these cis-elements engage regulatory RNA binding proteins (RBPs). These splicing factors influence recognition of the 5' and 3' splice sites by the U1 snRNP and U2 snRNP auxiliary factor (U2AF), respectively (17–19). Together, this exon definition complex specifies sequences destined for the message (20).
Shortly after the discovery of introns, connections between pre-mRNA splicing and human disease emerged (21, 22). We now understand that ~10% of all disease-causing mutations ablate splice sites (30). A further 18-50% of pathogenic variants disrupt sequences required for exon definition (31–33). These data underscore the impact of aberrant splicing on mechanisms of human inherited disease.
Can restoring accurate splicing could rescue disease phenotypes? A proven approach to rescue normal RNA splicing in the context of human disease is antisense oligonucleotides (ASOs) (23). Their facile design exploits the chemical language of nucleic acid base pairing interactions. Yet, many problems including toxicity, delivery and stability blocked translation to the clinic (24). Excitingly, these challenges are solvable as evidenced by FDA approval of multiple ASO therapeutics.
There are currently ten FDA approved ASOs for neuro-developmental and neuromuscular diseases (25). Notable examples include the splice modulating drugs Nusinersin and Milasen. 15 years in development, Nusinersin was the first FDA approved cure for spinal muscular atrophy (26–28). By contrast, Milasen is a patient-specific ASO for treatment of Batten's disease, developed in only 16 months (29).
In theory the search for active ASOs is simple. Yet in practice the process is both laborious and expensive. The impact of OpenASO will be to understand how RNA structure informs principles of ASO design. This challenge will accelerate ASO design and improve the economics of rare disease drug development.
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