In theory, any stage of the RNA life cycle could be targeted with small molecules (Fig. 1). Many companies are targeting pre-mRNAs. Following the success of PTC Therapeutics’ and Roche’s Evrysdi, RNA processing is now wide open for drug intervention. In eukaryotes, gene transcription generates a pre-mRNA containing introns, which the cell’s splicing machinery removes when ligating exons into mature mRNA. This transcript, after further modification, is exported to the cytoplasm for translation into protein. Evrysdi binds to a pre-mRNA–snRNP (small nuclear ribonucleoprotein) complex to drive the splicing and expression of a missing exon in the SMN2 gene2. In SMA, the SMN1 gene is mutated or missing, and the SMN2 gene is nonfunctional. By splicing an exon into SMN2, Evrysdi enables expression of a functional SMN2 protein that compensates for the loss of SMN1. The drug’s mechanism “opens up a whole new area of therapeutic research on modulating gene expression by targeting splicing,” says PTC co-founder Stuart Peltz, who retired as CEO in March.

Source: Concept and graphics courtesy ReviR Therapeutics.

Evrysdi “really shows it’s possible to do this,” says Smith. A major Remix focus, besides neurodegeneration, is cancer, where mutations in splicing factors affect roughly 20% of acute myeloid leukemias, 10–15% of chronic lymphocytic leukemias, and smaller percentages of several solid tumors. Remix is seeking RNA-binding small molecules that can correct or compensate for these splicing defects. Skyhawk Therapeutics, like Remix, is working on cancer and neurodegeneration. DNA and mRNA nucleotide repeat expansions are hallmarks of Huntington’s disease and a common form of amyotrophic lateral sclerosis (ALS), among other diseases. Skyhawk’s RNA-binding compounds work by “either reducing or increasing RNA levels, changing the functionality of the proteins,” says Sergey Paushkin, Skyhawk’s senior vice president of discovery biology. PTC, the splicing pioneer, has “ten or fifteen programs that are ongoing where we’re looking for selective molecules that modulate different aspects of splicing,” says Peltz.

This is not classic rational design — splicing is probably too complicated for that to be practical yet, says Paushkin. Both companies screen for compounds using cell-based assays to read out the splicing outcome they seek, then optimize hits for efficacy, potency and other qualities using structural biology tools to visualize the drug–RNA interaction. They then try to nail down the mechanism, determine specificity (using RNA sequencing to identify altered transcripts, and sometimes proteomics), and further optimize chemical leads for druglike properties, before going into animals for efficacy and safety studies.

The biology is complex, but not intractable. RNA splicing is performed by an RNA–protein complex called the spliceosome. It contains snRNPs that initiate the process when they bind to the pre-mRNA. PTC and Roche have reported that Evrysdi acts as a molecular glue that stabilizes the interaction between the 5′ splice site of the SMN2 mRNA’s exon 7 and the U1 snRNP, enhancing splicing. This general mechanism can, say Peltz and Paushkin, be exploited in mRNAs implicated in other diseases. That’s “the best of all worlds,” says Weeks. “You’re targeting a pretty well-defined complex in splicing, you are targeting a specific RNA. And then in addition splicing is the kind of molecular event that lends itself to functional assays.”

Another splicing strategy is to insert pseudoexons, or ‘poison exons’, into mRNA. These sequences, which encode premature stop codons, can be found in introns and, if somehow spliced into coding regions, engage nonsense-mediated decay pathways to degrade the mRNA. This story gained traction when Novartis reported that branaplam, an experimental small-molecule drug for treating SMA, induces a pseudoexon in HTT (huntingtin), the gene that, when mutated, causes Huntington’s disease3. This pseudoexon reduced levels of HTT in patient-derived cells. Novartis suspended its Huntington’s clinical last August because branaplam “might be causing peripheral neuropathy,” and has since discontinued the program. PTC is testing its own small-molecule pseudoexon inducer, more specific than branaplam, in a global phase 2 Huntington’s trial. (The FDA paused the US portion of this trial last October pending more data from PTC.) Several other companies are in the hunt for poison exon inducers. Some are also looking for small molecules capable of boosting, instead of reducing, expression of proteins in diseases where poison exons are already causing transcript degradation. “It could be either way,” says Paushkin. “You could induce or inhibit poison exon inclusion.”

The potential of small molecules to remove transcripts this way extends beyond the suite of rare diseases caused by aberrant splicing. “We’re looking for being able to do this in common diseases as well,” says Peltz.

Whereas rare diseases are a clear opportunity, there’s doubt about the ultimate efficacy of splicing drugs in common diseases with splicing mutations, like cancers. One hypothesis posits that such mutations are not individually driving the disease but that accumulated abnormal splice products create a state of ‘splicing sickness’. If that’s the case, targeting individual splicing defects may not be that effective. “It’s still an open question,” says Smith. “Are [splicing mutations] truly driving the disease, or are they contributing in a broad sense?” The fact that many mutations are ‘hot spot’ clustered mutations and occur early in the disease, he says, suggests that they’re driver events and that drugs that induce poison exons should be effective. “All of the signals to me point towards it being an important event that should still be targeted therapeutically,” he says.