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ASO Exon Skipping

NEW RNA THERAPEUTICS

Instead of replacing the entire STRC gene, what if we could make the cell skip the broken part? Antisense oligonucleotides (ASOs) bind to pre-mRNA and force the splicing machinery to skip specific exons, producing a shorter but potentially functional protein. This approach has 4 FDA-approved drugs for Duchenne muscular dystrophy.

Proven precedent: DMD exon skipping

Dystrophin (DMD gene, 79 exons) is the closest analogue to STRC's situation. Too large for AAV, with many patients having exon-specific mutations. The solution: ASOs that skip mutant exons, producing a shorter "Becker-like" dystrophin that still works. Four drugs are FDA-approved, treating exons 51, 53, 45, and 44.

4
FDA-approved exon-skipping ASOs
DMD
Target gene (79 exons, like STRC's 29)
Weekly
Dosing schedule (repeatable, dose-adjustable)

Eteplirsen (exon 51), Golodirsen (exon 53), Viltolarsen (exon 53), Casimersen (exon 45). All use phosphorodiamidate morpholino oligomers (PMOs). Accelerated approval based on dystrophin expression; clinical benefit debated but mechanism validated.

Application to STRC

STRC has 29 exons. Many DFNB16 patients carry mutations in specific exons. If we can identify which exons are dispensable for stereocilin function, ASOs could skip the mutant exon, producing a functional truncated protein.

ASO design
Skip mutant exon
Shorter mRNA
Truncated but functional protein

Key principle: Stereocilin is a large extracellular protein composed mainly of leucine-rich repeats (LRRs). LRR proteins are inherently modular: removing one repeat often shifts the structure by one unit without catastrophic misfolding. This is exactly the principle behind micro-dystrophin.

For Misha's mutation (c.4976A>C / p.E1659A in exon 27): Could exon 27 be skipped entirely? If the resulting protein (missing one LRR module) retains its ability to form horizontal top connectors and TM attachment crowns, this could be a viable non-viral, repeatable therapy. AF3 modeling of the exon-27-skipped variant would provide the first structural prediction.

ASO vs AAV gene replacement

Advantages of ASO approach
No viral vector needed, no immune barrier
Repeatable and dose-adjustable (weekly/monthly injections)
Reversible if problems arise (stop dosing)
Shorter regulatory path (ASO platform well-established)
Limitations
Requires repeated intratympanic or intracochlear delivery
Only works for exon-specific mutations, not whole-gene deletions
Must prove the skipped protein retains function (needs in silico + animal data)

Open questions for STRC exon skipping

1. Which STRC exons are dispensable? Need systematic AF3 modeling of each single-exon deletion.

2. Can ASOs be delivered to OHCs efficiently? Intratympanic injection reaches the round window, but OHC uptake of naked ASOs is unknown.

3. What dosing schedule maintains therapeutic levels? DMD uses weekly IV infusions; cochlear delivery would need a different approach (depot, sustained release, or LNP-encapsulated ASO).

Eteplirsen (Exondys 51), Golodirsen (Vyondys 53), Viltolarsen (Viltepso), Casimersen (Amondys 45) - FDA-approved exon-skipping ASOs for DMD.

Leclere et al. (2024) "Gene therapy for hearing loss: Advances and prospects of non-viral vectors." Hear Res.

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