#apaperaday: Tailored antisense oligonucleotides designed to correct aberrant splicing reveal actionable groups of mutations for rare genetic disorders

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Tailored antisense oligonucleotides designed to correct aberrant splicing reveal actionable groups of mutations for rare genetic disorders

A paper from Wai et al in experimental molecular medicine on testing tailored (aka individualized) antisense oligonucleotides (ASOs) for various splicing mutations in a minigene system DOI: 10.1038/s12276-024-01292-1

Authors introduce that rare diseases are jointly not rare, because there are so many of them. Splicing mutations can cause genetic rare diseases. Authors discuss the splice site mutations, but also those mutations that affect splicing in other ways. The latter are ASO targetable.

These ‘cis acting’ splice site mutations are rare – often occurring in single individuals but we know from the work of @N1Collaborative @timyu that it is possible to develop a patient-specific antisense oligonucleotide (ASO) to restore missplicing.

Authors rightly stress that for this type of work good genetic diagnosis is crucial. Splicing mutations are often overlooked (when they are in introns) or missed (exonic silent variants or missense variants that actually activate a cryptic splice donor or acceptor site).

Minigenes can help assess the effect of these variants on splicing, but of course are not perfect as the exon is taken from its genetic context and often minigenes are tested in HEK or Hela cells and not in the tissue-relevant cells.

Authors here made minigenes for 5 different splicing mutations

1. A mutation within an exon that causes removal of the inner part of the exon
2. An intronic mutation that activates a new donor
3. An intronic mutation close to the donor site activating a new donor.
4. and 5. intronic variants that activate the inclusion of a cryptic exon, by activating a donor (4) or activating an internal enhancer sequence (5).

For each variant authors validated the minigene and 4/5 recapitulated the missplicing that was found in patient blood.

Note that the variants are located in different genes, but each gene was expressed in blood and recapitulation was good in HEK293 cells. The 1 variant that was not recapitulated perfectly produced the cryptic splicing seen in patient blood and another variant not seen in blood.

Authors developed ASOs for each of the variants. They produced 1 ASO with different chemical modifications: morpholino (PMO), vivo-morpholino (with a tail to improve delivery) and MOEPS (i.e. the nusinersen chemistry). PMO was transfected with endoporter, MOEPS with lipofectamin.

Authors saw that at least one of the ASOs worked for each of the mutations, while in most cases all of the chemistries worked. Efficiency varied between mutations, and the mutation close to the original donor seems most difficult to correct.

Authors discuss that this works shows it is possible to correct missplicing for cisascting splicing mutations. They stress ASOs cannot be used to correct missplicing for splice site mutations. Fully agree and stress this because it is a common misconception that ASOs can do this.

If the splice sites are mutated (first 2 and last 2 nucleotides of the introns, GU AG) ASOs cannot correct splicing, as splicing requires these splice sites. Authors also note that correcting missplicing mutations close to the acceptor site is more challenging.

Indeed, because those splice sites work with the branch point and have a polypyrimidine tract that need to be functional. If you have an ASO targeting this region that may in and of itself interfere with splicing. Authors stress again missplicing mutations are often overlooked.

Fully agree again 🙂 We need to look better into missplicing variants, especially since a significant proportion may be actionable with ASOs. Authors stress that validation of minigenes is important as it takes pieces of a gene out of context.

I want to add that it also takes genes out of their tissue context! Different cells have different splicing factors, so what you see in HEK cells may not be what happens in e.g. neurons. Furthermore, it is possible that ASOs that work in HEK cells may not work in neurons.

So while this shows proof of concept, validation in relevant cells, ideally in the genetic context rather than a minigene is important. Authors also should have discussed better the different chemical modifications, which not all translate into clinic.

MOEPS is used in thousands of patients with SMA, but so far it seems that local treatment of PMOs in mouse brain does not show the same uptake and distribution as MOEPS. Vivo-morpholinos are toxic so they cannot be used in humans.

All in all I like the study showing the wider applicability of ASOs to correct missplicing mutations (different types in different genes), but I believe the authors could have discussed the caveats a little bit better.