Current STRC gene therapy requires two AAV vectors because the gene (5325 bp) exceeds the single-AAV packaging limit (~4400 bp usable). AlphaFold structural analysis suggests a single-vector approach may be possible.
AlphaFold predicts stereocilin's structure with varying confidence along the protein. The N-terminal region (residues 1-615) has very low confidence (pLDDT < 50), indicating it is likely intrinsically disordered with no stable 3D structure. The functional core starts around residue 616.
All regions have pLDDT < 50 (no stable structure predicted)
3984 bp fits in single AAV (<4400 bp limit)
This approach has proven precedent. The dystrophin gene (11,000 bp) was too large for any AAV. Researchers created "micro-dystrophin" by removing non-essential spectrin-like repeats, fitting it into a single AAV. This is now in Phase 3 clinical trials (Sarepta SRP-9001). The same principle: identify the structural core, remove disordered/redundant regions, preserve function. Nobody has tried this for STRC yet.
Important: This is a computational hypothesis based on AlphaFold structural predictions. It requires experimental validation: does mini-stereocilin fold correctly? Does it localize to stereocilia tips? Does it form horizontal top connectors and tectorial membrane attachments? These questions need wet-lab work. But the structural data strongly suggests the N-terminal region is dispensable, and a single-AAV mini-STRC approach deserves investigation.
Systematic computational testing of the mini-STRC hypothesis and variant impact. 3D models rendered live from AlphaFold 3 CIF files. Drag to rotate, scroll to zoom.
Low confidence interaction. Best cross-chain PAE: 8.6 A at N-terminal.
N-terminal removal barely affects binding (0.43 vs 0.47). Confirms dispensable.
No structural damage. The fold is intact. E1659A affects function (charge loss), not structure.
Full protein. N-terminal drags score down (16% disordered).
Truncated protein folds excellently. 7% disordered. Key result.
Confirmed disordered. 38% unstructured. Safe to remove.
Positive control: validates the calcineurin-NFAT cascade. CnA-CnB ipTM 0.91 (known complex). NFAT-CnA ipTM 0.72 (substrate recognition). NFAT disorder-to-order transition confirmed.
Mini-STRC (without N-terminal) achieves pTM 0.81, significantly better than full-length wildtype (pTM 0.63). The removed N-terminal region scores only pTM 0.27 with 38% disorder. Removing the disordered N-terminal produces a better-folding protein that fits in a single AAV vector.