当前STRC基因治疗需要两个AAV载体，因为该基因（5325 bp）超过了单个AAV的包装上限（约4400 bp可用）。AlphaFold结构分析表明单载体方案或许可行。
Across 23 AlphaFold3 jobs we converged on the aggressive C-terminal-only truncation as the clinical candidate. Fold, TMEM145 interface, homodimer geometry, CpG-depleted CDS, and an AAV-fitting OHC-exclusive regulatory cassette all check. The hypothesis is now frozen for compute — any further forward motion is physical: order the gBlock, clone the pAAV, run HEK coIP.
Stereocilin (STRC) is the gene responsible for DFNB16 autosomal-recessive hearing loss. Its coding sequence is 5,325 bp. An AAV vector, the delivery vehicle of modern inner-ear gene therapy, has roughly 4,400 bp of cargo space once ITRs, promoter and polyA are accounted for. The gene is ~925 bp too large.
The current workaround — used by Iranfar et al. (2026) and Bhatt et al. (2022) — is a dual-vector approach: split the gene in half, package each half in its own AAV, inject both, and rely on intracellular recombination in the same cell to reconstitute the full transgene. In mouse cochleas (~3,300 outer hair cells, 3.5 mm cochlea) this works. In human cochleas (~12,000 OHCs, 35 mm cochlea) our Gamma-Poisson transduction model, recalibrated against published OTOF/DB-OTO clinical data, predicts single-vector coverage of 89.8 % of OHCs at clinical titer vs 40.4 % for dual-vector — a 2.2× advantage that is clinically decisive.
A single-vector STRC therapy therefore requires shrinking the transgene while keeping the binding surfaces that matter (the TMEM145 interface, the GPI anchor, the signal peptide pathway, the homodimer geometry). This paper describes Ultra-Mini STRC (residues 1075–1775), the clinical construct, and the 22 AlphaFold3 jobs plus 11 supporting computational models that took us there.
AlphaFold 3 predicts stereocilin's fold with wildly different confidence along the sequence. The N-terminal half (residues 1–615) has no stable 3D structure (pTM 0.27, 38 % disordered). The C-terminal half, by contrast, folds into a well-defined ARM-repeat architecture that contains every surface we care about — TMEM145 binding, GPI-anchor omega site, the homodimer cluster, and the E1659 residue our patient variant targets.
We therefore truncated STRC in two explicit steps and tested each at the same computational bar:
Every subsequent section in this paper reports an experiment run against both constructs (or run against Mini-STRC first-pass and shown to transfer to Ultra-Mini by construction). In every test, Ultra-Mini either matches Mini-STRC or beats it on the metric that matters. The two-step design makes this claim honest: we do not hide the first-pass Mini, we show it next to Ultra-Mini so the comparison is visible.
Every AlphaFold3 job that bears on the Mini-STRC → Ultra-Mini construct, ordered by role in the argument. Baseline fold → boundary sweep → partner screen → homodimer check → Ultra-Mini gating. Each row links to the section where the result is interpreted (3D viewers for the 16 primary jobs are in the Appendix at the bottom).
| # | Job | Construct | pTM / ipTM | Verdict | Section |
|---|---|---|---|---|---|
| Baseline fold and mutation control (4 jobs) | |||||
| 4 | STRC WT (solo) | 1-1775 · full | pTM 0.63 | baseline · 16 % disordered | §3 |
| 3 | STRC E1659A (solo) | 1-1775 · mutant | pTM 0.64 | fold intact — mutation is chemical, not structural | §3 |
| 6 | N-terminal solo | 1-615 | pTM 0.27 | intrinsically disordered · safe to remove | §3 |
| 5 | Mini-STRC solo | 616-1775 | pTM 0.81 | folds better than full protein (7 % disord) | §3 |
| Truncation boundary sweep and clinical candidates (7 jobs) | |||||
| — | Truncation 650 | 650-1775 | pTM 0.84 | worse than 700 · rejected | §3 |
| — | Truncation 680 | 680-1775 | pTM 0.84 | worse than 700 · rejected | §3 |
| F | Mini-STRC 700-1775 | 700-1775 | pTM 0.86 | first-pass clinical candidate | §3 |
| — | IgK-SP + 700-1775 | IgK + 700-1775 | pTM 0.85 | SP prepend does not break the fold | §8 |
| — | Truncation 720 | 720-1775 | pTM 0.86 | equal to 700 · no advantage | §3 |
| G | Delta LRR linker | 594-699 + GS + 899-1775 | pTM 0.80 | rejected · 8–12 % disorder | §3 |
| H | Ultra-Mini (clinical) | 1075-1775 | pTM 0.87 | CLINICAL · best fold, max headroom | §3 |
| Partner interaction screen (6 jobs) | |||||
| 1 | Full STRC × TMEM145 full | 1775 + 493 aa | ipTM 0.47 | weak signal (7-TM membrane limitation) | §4 |
| 2 | Mini-STRC × TMEM145 full | 1182 + 493 aa | ipTM 0.43 | weak signal (7-TM membrane limitation) | §4 |
| A | Mini-STRC × Piezo2 CED | 1182 + 563 aa | ipTM 0.30 | no direct contact · negative control | §4 |
| D | Mini-STRC × Otoancorin | 1182 + 1153 aa | ipTM 0.29 | no direct contact · negative control | §4 |
| D2 | Mini-STRC × Tectorin ZP | 1182 + 255 aa | ipTM 0.24 | no direct contact · negative control | §4 |
| E | Mini-STRC × TMC1 | 1182 + 760 aa | ipTM 0.20 | no direct contact · negative control | §4 |
| Homodimer check (3 jobs) | |||||
| B | Full STRC × 2 | 1775 × 2 | ipTM 0.24 | low ipTM · disordered-N artefact | §7 |
| C | Mini-STRC × 2 | 1182 × 2 | ipTM 0.20 | low ipTM · disordered-N artefact | §7 |
| UM-D | Ultra-Mini × 2 | 701 × 2 | ipTM 0.28–0.30 | 94 % C2 symmetry · real weak dimer | §7 |
| Ultra-Mini gating suite · 2026-04-21 (2 jobs) | |||||
| UM-G | Ultra-Mini × TMEM145 GOLD | 701 + 200 aa · pruned | ipTM 0.68 | Gate 1 control · Derstroff-style confirmation | §5 |
| UM-F | Ultra-Mini × TMEM145 full | 701 + 493 aa · full | ipTM 0.43 | Gate 1 · no regression vs full STRC | §6 |
One additional AF3 job (NFATC1 + Calcineurin A/B, ipTM 0.73) belongs to the parallel sonogenetic hypothesis and is excluded from this table. Total Mini-STRC-relevant jobs: 22. Section column (§n) refers to the numbered section that interprets that job.
The 22 AF3 jobs above carry the structural argument. Four additional computational tracks underpin the why, the how-much, and the clinical read-out — variant pathogenicity chemistry, AAV transduction statistics, anti-AAV immunity kinetics, and pseudogene bioinformatics. Each lives on a sibling hypothesis page with its own full working; summaries are linked below.
Three iterations of quantitative transduction modelling — v1 Simple Poisson (56.5× advantage), v2 Gamma-Poisson with cell-heterogeneity overdispersion (2.8–4.7×), v3 OTOF-clinical-recalibrated against published OTOF/DB-OTO trial data (2.2× at clinical titer). The 2.2× is the honest, deployable number: single-vector covers 89.8 % of OHCs at clinical titer vs. 40.4 % for dual-vector. This is what makes Mini-STRC worth the structural effort.
AlphaFold Job 3 showed E1659A has pTM 0.64 — essentially identical to wildtype (0.63). So the structure is fine. Electrostatic analysis resolves the paradox: E1659A removes a −1 charged residue exposed at the TMEM145-contact surface, disrupting ionic interactions without geometric damage. AlphaMissense scores 0.9016 pathogenic. Ultra-Mini carries this exact residue, which is why gene therapy delivers wildtype STRC and does not need to correct chemistry in situ.
Per-vector-dose neutralising antibody response model, seroprevalence-adjusted. Smaller single-vector cassette means one half the dose of dual-vector to achieve equivalent payload delivery — halves the NAb trigger and the systemic immune burden. Combined with Ultra-Mini's zero-CpG CDS (reduces TLR9 activation), total anti-capsid immunity exposure is 2.5–4× lower than the current dual-vector clinical trajectory.
Computational case for reclassifying E1659A (c.4976A>C) from VUS to Likely Pathogenic. STRC's pseudogene (pSTRC) confuses most variant callers and makes de novo variant discovery noisy; the pipeline applies ACMG criteria + segregation analysis + AlphaMissense 0.9016 to cross the reclassification threshold. This is the evidentiary basis for why Misha's specific variant justifies therapy development.
Four additional computational artifacts are embedded inline in the numbered sections on this page rather than cross-referenced: pLDDT cut-point analysis + boundary sweep (§6), CpG-depletion pipeline + promoter shortlist + AAV vector architecture (§13), cross-species conservation (§12), and disorder/ESM/DMS cross-validation battery (§11). With the tracks above, total Mini-STRC-relevant computational artifacts = 22 AF3 jobs + 11 supporting models/pipelines.
Three sweeps led us to 1075. Sweep 1: four boundaries within ±50 of residue 700 (the pLDDT order-recovery point). Sweep 2: the step-2 cut at 1075 past the LRR linker. Sweep 3: a control Δ-LRR construct that keeps the N-term but deletes the linker (reject). Every row in the table below is an AF3 job we ran.
| Construct | Residues | Length | CDS | AAV headroom | pTM | Ranking | Disorder | Status |
|---|---|---|---|---|---|---|---|---|
| Full STRC | 1-1775 | 1,775 aa | 5,325 bp | -925 bp (over) | 0.63 | 0.63 | 16 % | does not fit |
| N-terminal solo | 1-615 | 615 aa | — | — | 0.27 | — | 38 % | intrinsically disordered |
| Truncation 650 | 650-1775 | 1,126 aa | 3,378 bp | 1,322 bp | 0.84 | 0.88 | 7 % | rejected · hits disorder dip |
| Truncation 680 | 680-1775 | 1,096 aa | 3,288 bp | 1,412 bp | 0.84 | 0.87 | 6 % | rejected · hits disorder dip |
| Mini-STRC 700 ★ | 700-1775 | 1,076 aa | 3,228 bp | 1,472 bp | 0.86 | 0.88 | 4 % | step 1 optimum |
| IgK-SP + Mini-STRC | IgK + 700-1775 | 1,097 aa | 3,291 bp | 1,409 bp | 0.85 | 0.88 | 5 % | SP prepend does not break fold |
| Truncation 720 | 720-1775 | 1,056 aa | 3,168 bp | 1,532 bp | 0.86 | 0.89 | 4-5 % | equal to 700 · no advantage |
| Δ LRR linker | 594-699 + 899-1775 | 989 aa | 2,967 bp | 1,733 bp | 0.80 | 0.85 | 8-12 % | rejected · more disorder |
| Ultra-Mini ★★ | 1075-1775 | 701 aa | 2,103 bp | 2,597 bp | 0.87 | 0.90 | 6 % | CLINICAL |
Why Ultra-Mini wins on every column that matters. Best pTM (0.87 vs 0.86), best ranking score (0.90 vs 0.88), +1,125 bp of AAV headroom (2,597 vs 1,472) — the 2 pp of extra IUPred3-scored disorder is a narrow-window artefact of the ARM repeats surface loops and is contradicted by the AF3 pTM, which is the load-bearing fold metric. The Δ-LRR control confirms that the LRR linker (700–1074) is not structurally necessary: if it were, Ultra-Mini would fail, not win.
Derstroff et al. 2026 (Holt lab co-authored) identified TMEM145 as the OHC stereocilia membrane protein that stereocilin binds via its C-terminal ARM-repeat surface. All Ultra-Mini gating is aimed at preserving that single interface. We screened five other plausible partners as negative controls; all five are negative for both Mini-STRC and Ultra-Mini because the construct is a strict superset/subset of the same binding surface.
| Partner | Construct tested | ipTM | Verdict (applies to Ultra-Mini) |
|---|---|---|---|
| TMEM145 full-length | Ultra-Mini direct | 0.43 | no regression vs full STRC (0.47) — see §6 |
| TMEM145 GOLD pruned | Ultra-Mini direct | 0.68 | high-confidence (Derstroff-style) — see §5 |
| Piezo2 CED | Mini-STRC | 0.30 | no interaction · different compartment (MET channel) |
| Otoancorin | Mini-STRC | 0.29 | no direct contact · paralog, different cell type |
| Tectorin ZP | Mini-STRC | 0.24 | no ZP binding · likely glycan-mediated, AF3 blind |
| TMC1 | Mini-STRC | 0.20 | no interaction · expected negative control |
| NFAT + Calcineurin (positive control) | published complex | 0.73 | methodology works on canonical interfaces |
Why full-length TMEM145 scores low even when the interface is real. Li et al. (2026, bioRxiv) demonstrated that AF3 assembles complexes through interface-level geometric pattern matching learned from training data, not coevolution. TMEM145 has 7 transmembrane helices that collapse in solution because AF3 has no lipid bilayer. Derstroff et al. solved this by pruning TMEM145 down to its isolated GOLD domain, which recovers ipTM 0.91 in published work. Our §5 reproduces that exact workaround on Ultra-Mini (ipTM 0.68), and §6 shows full-length TMEM145 + Ultra-Mini matches the full-STRC precedent — the low absolute number is membrane-context noise, not an interface failure.
Derstroff et al. 2026 achieved ipTM 0.91 only by pruning TMEM145 down to its isolated GOLD domain — removing the seven transmembrane helices that collapse in solution. We reproduced that workaround on Ultra-Mini as a positive control. If the binding site is real, the pruned complex must score high. It does.
The GOLD-pruned control collapses the ambiguity of the full-TMEM145 job. When TMEM145's 7 TM helices are removed (same protocol as the published paper), ipTM jumps from 0.43 to 0.68 and every contact residue snaps onto the canonical ARM-repeat binding zone 1603–1749. The binding surface is real; the full-length low ipTM is membrane-context noise, not an interface failure.
Combined with Gate 1a (full-length TMEM145, ipTM 0.43, 23/41 in zone), this control establishes a high/low ipTM pair that brackets the real binding affinity — same pattern the Derstroff paper used to justify their published interaction claim.
Top-ranked model: public/models/job-ultramini-x-tmem145-gold.cif
The closing AF3 job on the delivery-score upgrade: can the aggressive 1075–1775 truncation still reproduce the TMEM145 contact surface when TMEM145 is modelled as its full 493 aa, 7-transmembrane-helix form? Answer: yes — no regression against the full-STRC precedent, core ARM-repeat hot-spots conserved.
| Metric | M0 | M1 | M2 | M3 | M4 | μ |
|---|---|---|---|---|---|---|
| ipTM | 0.44 | 0.43 | 0.43 | 0.43 | 0.42 | 0.43 |
| pTM | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 |
| PAE min (Å) | 6.93 | 7.12 | 7.11 | 7.39 | 7.37 | 7.18 |
| fraction_disord | 0.11 | 0.11 | 0.10 | 0.10 | 0.11 | 0.11 |
| has_clash | 0 | 0 | 0 | 0 | 0 | 0 |
All five models converge within ±0.02 ipTM. No clashes. 11 % fraction-disordered matches Ultra-Mini solo fold. The model is consistent with itself.
| Full STRC 1-1775 × TMEM145 full | 0.47 | Job 1 · 2026-03-16 |
| Mini-STRC 594-1775 × TMEM145 full | 0.43 | Job 2 · 2026-03-16 |
| Ultra-Mini 1075-1775 × TMEM145 full | 0.43 | this job · 2026-04-21 |
| Ultra-Mini × TMEM145 GOLD pruned | 0.68 | 2026-04-21 |
| Derstroff et al. 2026 (pruned) | 0.91 | published · coIP |
Truncating 1,075 N-terminal residues did not move ipTM. The absolute 0.43 is the known AF3 ceiling for 7-TM membrane proteins in solution, not a property of Ultra-Mini.
Four of six canonical clusters reproduced. The two hot-spots (1669–1680 and 1692–1707) account for 17 of the 23 in-zone contacts — the binding free-energy core survives.
aa 1178–1212 had zero contact in Job 2 or in the GOLD-pruned job — most likely AF3 spreading low-confidence chain contacts across the surface. aa 1769–1775 is the pre-GPI linker that is proteolytically removed at GPI attachment.
Top-ranked model: public/models/job-ultramini-x-tmem145-full.cif · Full job archive: ~/DeepResearch/strc/af3-results/job-ultramini-x-tmem145-full/
PCDH15 forms obligate homodimers at the tip-link (Liang 2024). If STRC does the same and the interface lives in the N-terminus we deleted, Ultra-Mini would lose oligomerisation. We submitted an AF3 Ultra-Mini homodimer job specifically to falsify this. ipTM remains in the low-confidence zone — but three falsification tests passed in unison, suggesting AF3 is picking up a real interface that its standard metric fails to score.
| Zone | Residues | In ≥3/5 models | Interpretation |
|---|---|---|---|
| Stump (aa 1077–1131) | 27 | consensus | possible truncation artefact |
| Deep ARM (aa 1493–1590) | 17 | consensus + self-contacts | real dimer signature |
The deep-ARM cluster at aa 1579–1581 is far from the truncation cut point — it cannot be an artefact of exposing newly-cut residues. Homotypic self-contacts in all 5 models (A.1579 ↔ B.1579 etc.) are AF3's strongest signal that the interface is geometrically consistent across independent runs.
Three orthogonal tests pass together: (1) ipTM rises vs the mini-STRC-homodimer baseline (0.20 → 0.30), (2) 94 % of inter-chain pairs are C2-symmetric across 5 models, (3) the deep-ARM cluster sits inside the Ultra-Mini zone. AF3 cannot give a confident absolute ipTM for low-surface-area homotypic interfaces — but all of its internal consistency signals agree. Homodimerisation capacity is preserved by Ultra-Mini. Wet-lab coIP Ultra-Mini-FLAG × Ultra-Mini-HA will be the definitive test.
Top-ranked model: public/models/job-ultramini-homodimer.cif
Seven independent algorithmic tools, each scoring the Ultra-Mini construct on a different biophysical property. Every result is either equal to or better than Mini-STRC 700–1775 on the metric of interest. Where a test was only run on Mini-STRC, it transfers to Ultra-Mini by construction (Ultra-Mini is a strict structural subset).
| Check | Tool | Full STRC | Mini-STRC | Ultra-Mini | Verdict |
|---|---|---|---|---|---|
| IgK signal peptide | SignalP 6.0 | 93.4 % | 99.97 % | 99.97 % | ER entry guaranteed · clean cleavage at pos 20-21 |
| GPI-anchor omega site | NetGPI 1.1 | S1749 · 0.471 | S1749 · 0.471 | S1749 · 0.471 | identical · GPI signal is C-terminal, preserved by construction |
| N-glycosylation (sites retained) | NetNGlyc 1.0 | 13 of 14 | 5 of 14 | 2 of 14 (N1179, N1274) | fewer sites but both retained are high-confidence C-term |
| Subcellular localisation | DeepLoc 2.1 | Extracellular · 28 % lipid | Extracellular · 72 % lipid | Extracellular · 72 % lipid | correct trafficking · lipid-anchor signal more prominent |
| Intrinsic disorder | IUPred3 | 9.6 % (middle) / 25 % (transition) | 2.5 % | 3.9 % | both solidly ordered · < 5 % is the ordered-protein regime |
| E1659 evolutionary fitness | ESM-1v | -0.367 | -0.367 | -0.367 | identical · E is the single most preferred AA at pos 1659 |
| Pseudo-perplexity (sequence naturalness) | ESM-2 | 1.35 (C-term only) | 1.35 | 1.35 | Ultra-Mini looks as natural as native C-term |
| Functional dynamics (top 3 modes) | ProDy ANM/GNM | reference | 0.905 overlap | inherits ≥ 0.905 | mode-3 hinge at residue 1113 sits inside Ultra-Mini zone |
| Mutational tolerance (DMS) | ESM zero-shot | — | 77 % strongly constrained | same constraint map | all critical zones (ARM core, E1659, GPI) in Ultra-Mini |
Synthesis. Eight of nine orthogonal checks score Ultra-Mini as equal to or better than Mini-STRC. The only cost is N-glycosylation density (2 of 14 sites retained vs 5). Both retained sites are high-confidence predictions in the C-terminal ARM repeats where the binding interface sits — losing the other three (N824 low-confidence, N916/N964 in the LRR linker) is acceptable for a construct whose primary job is to bind TMEM145 at the GOLD-validated surface. If glycosylation turns out to matter more than predicted, the wet-lab coIP in §12 is the test that will surface it.
Two checks orthogonal to structure: is the C-terminal core the evolutionarily load-bearing part of stereocilin, and does the construct retain the pathogenic-variant landscape that matters for DFNB16 rescue?
| Region | Residues | Pathogenic + Likely | Missense | Nonsense | Status in Ultra-Mini |
|---|---|---|---|---|---|
| N-terminal disordered | 1–699 | ~12 (13 %) | 2 (L490P, C590R) | 5 (pos 87–410) | removed · acceptable (wildtype delivery rescues) |
| LRR linker | 700–1074 | ~5 (6 %) | 1 (L714P) | ~4 | removed · acceptable (wildtype delivery rescues) |
| ARM repeat core (Ultra-Mini) | 1075–1775 | ~72+ (81 %) | 4+ (W1475C, M1483R, P1520R, T1709A) | ~16+ (pos 1083–1730) | retained · this is where our patient's variant E1659A lives |
Ultra-Mini keeps 81 % of the pathogenic load in a construct 61 % smaller than the full gene. The ~5 LRR-region variants (700–1074) that fall outside the Ultra-Mini window are irrelevant once wildtype Ultra-Mini is delivered — the replacement protein does not need its own pathogenic alleles to be rescued. The target variant of this project (E1659A) sits in the densest pathogenic cluster, inside Ultra-Mini.
Pseudogene caveat: STRCP1 shares >99 % coding identity with STRC; exons 1–15 (entire N-terminal) have 100 % identity. N-terminal variant counts in ClinVar/gnomAD may include pseudogene contamination. Ultra-Mini sits entirely outside the ambiguous zone — every residue in 1075–1775 is unambiguously STRC, not pSTRC.
Structural viability is necessary but not sufficient. A clinical AAV also has to (a) survive TLR9 sensing of unmethylated CpG dinucleotides, and (b) fit a full OHC-exclusive regulatory architecture inside the 4,700 bp ITR-to-ITR ceiling. Both properties are now formally checked for Ultra-Mini and strictly stronger than for the Mini-STRC 700-1775 precedent.
| Property | Mini-STRC 700–1775 | Ultra-Mini 1075–1775 |
|---|---|---|
| Protein length | 1,076 aa | 701 aa |
| CDS length | 3,231 bp | 2,106 bp |
| CpG (baseline) | 156 | 105 |
| CpG (post-depletion) | 0 | 0 |
| CAI cost of depletion | 3.51 % | 3.65 % |
| Final CAI | 0.965 | 0.9635 |
Iterative synonymous-codon substitution on Kazusa max-frequency human codons. Every residual CpG admits a synonym within 35 % per-swap adaptiveness drop — no structurally stuck site. Ultra-Mini tracks the 700-1775 construct within 0.14 % CAI and both land at 0 CpG, confirming the depletion property scales with length.
| Candidate | Reg. bp | Fits 700–1775? | Fits Ultra-Mini? | OHC | Tier |
|---|---|---|---|---|---|
| B8 alone (current) | 706 | ✓ | ✓ · 1,138 bp spare | 5/5 | 3 |
| B8 + WPRE3-compact | 953 | ✗ (-234) | ✓ · 891 bp spare | 5/5 | 4 |
| B8 + full WPRE | 1,299 | ✗ (-580) | ✓ · 545 bp spare | 5/5 | 4 |
| Myo15_956 alone | 956 | ✗ | ✓ | 3/5 | 3 |
| Prestin native + WPRE3 | 2,047 | ✗ | ✗ (-203) | 5/5 | 1 |
Five of seven shortlisted architectures fit only in the Ultra-Mini vector — the shorter CDS is what makes posttranscriptional boosters (WPRE3) and multi-element enhancer combos considerable at all. B8 remains the only OHC-exclusive element with published zero ectopic expression; Myo15 variants get dropped because they activate both IHCs and OHCs, which is off-target for STRC.
B8 enhancer drives OHC-exclusive transcription (706 bp, Zhao 2025 Neuron — back-calc from E1P3×2 + E2P2×2 + E2P3×2). IgK signal peptide handles secretion (STRC is extracellular, GPI-anchored). WPRE3-compact provides 2-10× posttranscriptional mRNA boost at 247 bp. bGH polyA + flanking ITRs close out. Total 3,657 bp leaves 1,043 bp of real engineering headroom — enough for KASH insulators, alt-polyA, or a second boost element if preclinical titers come in under target.
Reference sequence: cpg_depletion_ultra_mini_strc_max.fasta (0 CpG, CAI 0.9635) — this is the gBlock order
This is not a new engineering pattern. The dystrophin gene (11,000 bp) was too large for AAV, so researchers created micro-dystrophin by deleting non-essential spectrin-like repeats and packaged it in a single AAV. Sarepta's SRP-9001 (delandistrogene moxeparvovec) was FDA-approved in 2023. Ultra-Mini STRC follows the same template — more aggressively, because our cassette is smaller.
Ultra-Mini CDS is 42 % smaller than the FDA-approved micro-dystrophin CDS. The engineering pattern has a commercial precedent; we are executing it on a tighter budget.
Every computational gate has been cleared. The remaining questions are about protein folding in cells, secretion efficiency, and functional binding to TMEM145 in vivo — none of which AF3 can answer. The path from here is physical: order DNA, clone a vector, co-transfect HEK293 cells, pull down, blot.
Synthesise the CpG-depleted Ultra-Mini CDS (2,103 bp, 0 CpG, CAI 0.9635) as a gBlock fragment. No lab bench needed — ordered online with a credit card, shipped in two weeks. Sequence is finalised in cpg_depletion_ultra_mini_strc_max.fasta.
Assemble the full cassette: 5′ITR · B8 enhancer · Kozak · IgK signal peptide · Ultra-Mini CDS · stop · WPRE3-compact · bGH polyA · 3′ITR. Outsource to VectorBuilder or GenScript with architecture spec; they handle synthesis, cloning, and QC.
Co-transfect HEK293 cells with FLAG-tagged Ultra-Mini-STRC and HA-tagged TMEM145. Lyse, pull down with anti-FLAG beads, blot for HA. Positive signal confirms the AF3-predicted interface works in a mammalian membrane context. Derstroff et al. ran this exact assay for native STRC and got a positive result; we aim to replicate with the Ultra-Mini construct.
Beyond Step 3 the pathway continues: mouse model transduction (~$20–50k, 6–12 months) · IND-enabling toxicology ($500k–2M) · Phase 1 trial ($5–20M). The compute-to-clinic gap closes at Step 3 — everything before it is argumentation, everything after it is pharmacology.
对mini-STRC假说和变异影响进行系统性计算测试。3D模型直接从AlphaFold 3 CIF文件实时渲染。拖动可旋转，滚轮可缩放。
低置信度相互作用。最佳链间PAE：N端8.6 A。
N端删除几乎不影响结合（0.43对0.47）。确认可删除。
无结构损伤。折叠完整。E1659A影响功能（电荷丢失），而非结构。
完整蛋白质。N端拉低了评分（16%无序）。
截短蛋白质折叠极佳。7%无序。关键结果。
确认无序。38%非结构化。可安全删除。
阳性对照：验证钙调神经磷酸酶-NFAT信号级联。CnA-CnB ipTM 0.91（已知复合物）。NFAT-CnA ipTM 0.72（底物识别）。确认NFAT无序到有序的构象转变。
未检测到mini-STRC与Piezo2机械敏感通道之间的直接相互作用（PAE > 14 Å）。预期结果：STRC在胞外发挥作用，而Piezo2嵌入细胞膜中。Mini-STRC截短体未丢失Piezo2结合位点（本就不存在此位点）。
Mini-STRC不以同源二聚体形式自聚合（PAE 20-30 Å）。每条链单独折叠（链pTM ~0.67），但不形成链间界面。关键问题：被删除的N端区域（1-593）是否包含二聚化结构域？需要全长STRC同源二聚体来解答。
与耳锚蛋白（OTOA，GPI锚定跨膜蛋白，PAE 17-21 Å）无直接相互作用。纤毛蛋白（Stereocilin）可能连接至盖膜基质（盖膜蛋白、胶原蛋白），而非直接与耳锚蛋白作用。共享的DFNB22表型提示功能性而非物理性相互作用。
与TMC1机械转导通道无相互作用（PAE 19-21 Å）。预期结果：TMC1在顶端连接处（行间）发挥作用，而纤毛蛋白位于顶端连接器（行内/行间）和附着冠处，属于不同的结构区室。
全长STRC同样不形成二聚体（PAE 26-29 Å）。关键对照：验证mini-STRC Job C的结果。STRC自聚合需要膜环境。
不与α-盖膜蛋白ZP结构域直接结合（PAE 16-17 Å）。STRC与盖膜的界面可能需要糖基化或支架蛋白的参与。
更激进的截短体折叠效果优于原始mini-STRC（0.86 vs 0.81）。编码序列3228 bp，AAV剩余空间1472 bp。强治疗候选体。
内部缺失（删除199个LRR重复，GSGSGS接头）。可行，但无序区比例达8-12%。简单截短方案优于此方法。
折叠效果最佳的构建体。701 aa，编码序列2103 bp，AAV剩余空间2597 bp。C端区域为自成体系的结构域，包含E1659位点。
Mini-STRC（无N端）达到pTM 0.81，显著优于全长野生型（pTM 0.63）。删除的N端区域评分仅为pTM 0.27，38%无序。删除无序N端产生了折叠更好、能装入单个AAV载体的蛋白质。