STRC h03 Clinical-Vector Blockers — Follow-Up 2026-04-24
Extends STRC h03 Parameter Provenance Audit 2026-04-25 with targeted retrieval for the 3 hard blockers preventing clinical vector ordering. Triggered by pending lit_audit: partial status on the S-tier h03 hypothesis.
Blocker 1 — B8 Exact Size (Zhao 2025 Neuron)
Finding: Zhao 2025 is NOT a Cell paper — it was published in Neuron (113(10):1579-1596). DOI: 10.1016/j.neuron.2025.03.023. PMID: 40262614. The phantom cite “Yoshimura 2018” is fully replaced.
From MinerU parse of Zhao 2025 PDF (retrieved via Anna’s Archive):
- E1P3 module (most conserved E1 fragment): 93 bp (main text p.1586, Fig S6A)
- E2P2 module (most conserved E2 fragment): 132 bp (main text p.1586, Fig S6B)
- E2P3 module (second conserved E2 fragment): 128 bp (main text p.1586, Fig S6B)
- B8 = E1P3×2 + E2P2×2 + E2P3×2 (confirmed, text p.1587 + Fig 6A)
- Back-calculated B8 size: 2(93) + 2(132) + 2(128) = 706 bp
Script error: ultra_mini_promoter_shortlist.py and ultramini_vector_cpg_audit.py use 587 bp. This is a 119 bp discrepancy relative to the back-calculated 706 bp.
Vector budget impact: If B8 = 706 bp (not 587 bp), the Ultra-Mini construct is 119 bp larger than currently modeled. The B8+WPRE3 candidate (02) would be 706 + 247 + other elements. Recalculating against the 4700 bp budget (−300 ITR −450 KOZAK/SP/polyA −2106 CDS = 1844 bp regulatory budget): 706 (B8) + 247 (WPRE3) = 953 bp < 1844 bp. Still fits comfortably with 891 bp spare. The 587→706 correction does NOT threaten vector fit for the Ultra-Mini design. Budget remains safe.
Remaining blocker: Table S2 (Zhao 2025 supplementary Excel) was not extracted by MinerU — it’s an Excel file in the SI, not a PDF page. The main text does not explicitly state “706 bp” or “587 bp” for B8. The 706 bp is a back-calculation from module sizes. Need to retrieve SI directly from Neuron journal or Zenodo if deposited. Scripts updated with WARNING flag.
Paper note: 2025-zhao-arbiter-b8-enhancer-ohc
Blocker 2 — Myo15 Promoter Source (956/1157 bp)
Finding: Source paper identified and confirmed. The 956 bp and 1157 bp Myo15 promoter variants are from:
Hu SW et al. “Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear.” Research (Wash D C). 2024;7:0341. DOI: 10.34133/research.0341. PMID: 38665848.
This is the Shu lab (Yilai Shu, Huawei Li) — same group responsible for the Shanghai Shu Yilai c.4976 knock-in mouse model relevant to Misha’s case. The paper truncates the native 1,611 bp Myo15 promoter by removing 88 bp and 201 bp UTR elements.
Confirmed numbers:
- Native Myo15 promoter: 1,611 bp
- Mid-Myo15: 1,157 bp
- Mini-Myo15: 956 bp
- Mini-Myo15 OHC efficiency: ~77%/74%/69% (apical/middle/basal)
- Mini-Myo15 IHC efficiency: ~98%/91%/76%
- AAV serotype: AAV-PHP.eB; injection via RWM at P0-P2
Note: Myo15 is NOT OHC-exclusive (also infects IHCs). B8 (OHC-exclusive, ~100%) remains superior for STRC delivery. Myo15 variants remain valid for DFNB9 (OTOF) rescue where IHC targeting is needed.
Scripts corrected: Phantom cite “Zhao 2024” replaced with “Hu et al. 2024 Research (AAAS); PMID 38665848” in both ultra_mini_promoter_shortlist.py MODULES dict and docstring.
Also confirmed: Myo15 1611 bp native promoter source is Wang H et al., Mol Ther Nucleic Acids 35:102135, 2024; PMID 38404504 (first author Wang H, not “Liu 2024” as previously cited in scripts).
Paper note: 2024-hu-myo15-promoter-engineering
Blocker 3 — WPRE3 Compact Verification (Choi 2014)
Finding: Choi 2014 is NOT in Cell 157 — it is in Molecular Brain (7:17). DOI: 10.1186/1756-6606-7-17. PMID: 24618276. Open access via PMC (PMC3975461).
Confirmed numbers from PMC full text:
- WPRE3 size: 247 bp (confirmed — gamma + alpha regions of woodchuck hepatitis PRE)
- Full-length WPRE: 600 bp
- WPRE3 expression (CW3B cassette): 83.4% of full WPRE
- Best compact cassette (CW3SL with WPRE3 + SV40 late pA): 103.4% of original expression
- Total size saved by CW3SL vs CWB: ~399 bp
Script update: ultramini_vector_cpg_audit.py ELEMENTS dict corrected from “Choi 2014 Cell 157” to “Choi et al. 2014, Mol Brain 7:17; PMID 24618276.” The 247 bp value in scripts is confirmed correct.
Paper note: 2014-choi-wpre3-aav-expression-cassette
Additional Literature — PKPD Calibration
DB-OTO clinical titer identification:
The script dual_vector_otof_calibration.py cites “Lustig et al. 2024 (NEJM Evidence)” — this paper was actually published in October 2025 in NEJM (DOI: 10.1056/NEJMoa2400521). The first author of the trial is not Lustig (Lustig is a collaborator). Titer: 7.2×10¹² GC/ear (confirmed from press releases). PDF not yet retrieved; paper note deferred.
“Sun 2024 Lancet” identification: The script cites “Sun et al. 2024 (Lancet)” as cohort 3. The matching Lancet 2024 OTOF paper is Lv et al. (Fudan University): “AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial.” Lancet 2024;403:2317-2325. PMID 38280389. First author is Lv, not Sun. Exact titer per cohort requires full paper access.
Paper note: 2024-lv-aav1-otof-lancet-trial
B8 Size Delta — Vector Budget Recalculation
| Element | Current script | Zhao 2025 back-calc | Delta |
|---|---|---|---|
| B8 enhancer | 587 bp | 706 bp | +119 bp |
| B8 + WPRE3 total | 587 + 247 = 834 bp | 706 + 247 = 953 bp | +119 bp |
| Regulatory budget (Ultra-Mini) | 1,844 bp | 1,844 bp | unchanged |
| Headroom (B8+WPRE3 candidate) | 1,844 − 834 = 1,010 bp | 1,844 − 953 = 891 bp | −119 bp |
Conclusion: Even if B8 = 706 bp, the Ultra-Mini single-vector construct still fits with 891 bp spare regulatory budget. The vector architecture is not invalidated. The 587→706 correction requires updating the script’s displayed total vector size (currently reported as 3,446 bp; corrected would be ~3,565 bp), still well under 4,700 bp.
h03 ranking is unaffected by this correction. The clinical-grade Ultra-Mini B8+WPRE3 architecture remains viable.
Remaining Blockers
| Blocker | Status | Action needed |
|---|---|---|
| B8 exact bp | PARTIAL — back-calc 706 bp, Table S2 needed | Retrieve Zhao 2025 SI Excel from Neuron journal (supplementary download page) |
| Myo15 source | CLOSED | Hu 2024 Research; PMID 38665848 |
| WPRE3 source | CLOSED | Choi 2014 Mol Brain; PMID 24618276 |
| DB-OTO titer | PARTIAL — 7.2×10¹² GC/ear confirmed | Retrieve Lustig 2025 NEJM PDF for full cohort data |
| ”Sun 2024” Lancet | PARTIAL — Lv 2024 identified | Retrieve Lv 2024 Lancet for exact titer per cohort |
Ranking Delta
h03: S HELD. No tier/score change.
This proof:
- Closes the Myo15 phantom cite blocker (was preventing vector ordering)
- Closes the WPRE3 citation error (247 bp confirmed)
- Establishes 706 bp as the best available B8 estimate (vs 587 bp in scripts)
- Confirms vector budget remains safe even with 706 bp B8
- Does NOT change the fundamental therapeutic strategy — B8+WPRE3+Ultra-Mini remains the design
One partial blocker remains (B8 Table S2 exact bp), but it does NOT block vector ordering since the budget is safe at both 587 and 706 bp.
lit_audit status: pending user decision to move from partial to near-fixed pending Table S2 retrieval.
Connections
[part-of]h03 hub[see-also]STRC h03 Parameter Provenance Audit 2026-04-25[source]2025-zhao-arbiter-b8-enhancer-ohc[source]2024-hu-myo15-promoter-engineering[source]2014-choi-wpre3-aav-expression-cassette[source]2024-lv-aav1-otof-lancet-trial[see-also]STRC B8 Enhancer Selection[see-also]STRC Ultra-Mini Promoter Shortlist[part-of]STRC Hypothesis Ranking