STRC Ultra-Mini Promoter Shortlist

Regulatory-architecture shortlist for Ultra-Mini STRC (aa 1075-1775, 2,103 bp CDS) that leverages the 1,125 bp of AAV headroom unlocked by the shorter truncation. Seven candidate architectures scored on size fit, OHC specificity, expression strength, and AAV track record. Winner: B8 enhancer + WPRE3-compact (834 bp, 1,010 bp spare) — tier-4, OHC-exclusive specificity preserved, posttranscriptional expression boost added. Critical property: this combo does not fit in the clinical 700-1775 vector (-115 bp over capacity), so the upgrade is unique to Ultra-Mini. Second-place: B8 + full WPRE (1,180 bp) for max expression if clinical titer is below target. Myo15 promoter variants come in as tier-3 despite published in-vivo rescue because they broaden expression to both IHCs and OHCs — off-target for STRC, which is OHC-specific.

Question

If Ultra-Mini is validated by the pending AF3 multimer job, what regulatory architecture should the therapeutic vector use? The current mini-STRC design uses B8 alone (587 bp synthetic enhancer, OHC-exclusive, zero ectopic expression — see STRC B8 Enhancer Selection). Ultra-Mini’s extra 1,125 bp of AAV headroom enables additional regulatory elements. Which combination dominates?

Constraints:

• AAV ITR-to-ITR capacity: 4,700 bp.
• ITR overhead: ~300 bp (2 × ~150 bp).
• Non-promoter CDS overhead (Kozak + IgK SP + stop + bGH polyA + misc): ~450 bp.
• Ultra-Mini CDS: 2,106 bp. Regulatory budget: 1,844 bp (vs 719 bp for mini 700-1775).

Candidate architectures scored

Rubric: size fit / OHC specificity / expression strength / AAV track record, each 1-5, tier = min axis.

#	Candidate	Regulatory bp	Ultra spare	Fits mini 700-1775?	Size	OHC	Expr	AAV	Tier
1	B8 alone (current)	587	1,257	✅ (132 spare)	5	5	3	4	3
2	B8 + WPRE3-compact	834	1,010	❌ (-115)	5	5	4	4	4
3	B8 + WPRE_full	1,180	664	❌ (-461)	5	5	5	4	4
4	Myo15_956 alone	956	888	❌ (-237)	5	3	4	5	3
5	Myo15_1611 full	1,611	233	❌ (-892)	3	3	5	5	3
6	B8 + Myo15_core + WPRE3	1,134	710	❌ (-415)	5	5	5	3	3
7	Prestin_native + WPRE3	2,047	-203	❌	1	5	4	3	1 (doesn’t fit)

Candidates exclusively enabled by Ultra-Mini

Five of the seven architectures would not fit in the clinical 700-1775 vector: #2, #3, #4, #5, #6. The shorter Ultra-Mini CDS is the reason any of them becomes considerable in the first place.

Winner analysis: B8 + WPRE3-compact (834 bp)

• OHC specificity (5/5): B8 is already validated as OHC-exclusive with zero ectopic expression per the ARBITER synthetic-enhancer panel used in STRC B8 Enhancer Selection. WPRE3 is a posttranscriptional element (mRNA stability + nuclear export), cell-type-agnostic — it boosts whatever the promoter drives without broadening expression.
• Expression strength (4/5): WPRE elements consistently increase steady-state transgene mRNA and protein levels 2-10× in AAV systems. Compact WPRE3 (Choi 2014, 247 bp) retains ~70% of full WPRE’s boost in a third of the footprint.
• Size fit (5/5): 834 bp uses 45% of Ultra-Mini regulatory budget, leaves 1,010 bp spare for future-proofing (KASH insulators, tissue-specific 3’ UTR elements, alternative polyA architectures).
• AAV track record (4/5): B8 validated in mouse cochlea for prestin rescue; WPRE3 used broadly in AAV gene therapy; the specific combo B8 + WPRE3 for hair-cell STRC is novel, but both components are individually proven.
• Tier 4 (limited by AAV track record — novel combo). The path to tier 5 is the first successful STRC rescue trial using this architecture.

Runner-up: B8 + WPRE_full (1,180 bp)

• Same tier 4 as the compact variant but with a max-strength expression axis (5/5 vs 4/5).
• Costs 346 bp of additional regulatory budget vs WPRE3-compact, buying the last ~30% of WPRE boost.
• Recommended fallback if preclinical expression data (mouse cochlea transduction measurements) show Ultra-Mini per-OHC protein levels below WT-equivalent threshold.

Dropped: Myo15 variants

The Myo15 promoter is a well-published hair-cell-restricted promoter (Zhao 2024 Research; Liu 2024 Mol Therapy Nucleic Acids rescue of DFNB9). It scores 5/5 on AAV track record. But it directs expression in both inner and outer hair cells — off-target for STRC, which is OHC-exclusive. Ectopic STRC expression in IHCs could perturb IHC physiology (STRC has no physiological role there) and creates a novel toxicity risk the B8 enhancer avoids by design. The hybrid option (#6) that uses B8 as the enhancer and Myo15 only as the minimal core promoter would retain OHC restriction (B8 dominant) while gaining Myo15’s strong basal transcription — but without published data on this exact combination, AAV track record drops to 3.

Dropped: Prestin_native

Does not fit in Ultra-Mini (-203 bp over budget at 2,047 bp with WPRE3). Slc26a5 endogenous regulatory region offers true OHC-exclusive expression matching STRC’s natural pattern, but its size makes it infeasible even at Ultra-Mini scale. Would require further CDS truncation (below the 1075-1775 structural validation) or a novel sub-kilobase Prestin-derived enhancer.

Recommendation

Clinical architecture for Ultra-Mini vector (pending AF3 multimer validation):

ITR — B8 (587 bp) — Kozak — IgK SP (63 bp) — Ultra-Mini CDS (2,103 bp) —
      stop (3 bp) — WPRE3-compact (247 bp) — bGH polyA (225 bp) — ITR

Total: ~4,543 bp in 4,700 bp AAV capacity. Spare: 157 bp, reserved for ITR variability and optional 5’/3’ UTR scaffolds.

Alternative if preclinical expression is subthreshold: swap WPRE3-compact for WPRE_full (+346 bp), consuming reserve.

Limitations

• Module sizes are canonical literature estimates; actual cloning may vary ±20 bp per element.
• Expression-strength scoring (1-5) is qualitative; no cross-construct direct comparison in cochlear tissue exists for all seven candidates.
• AAV track record axis treats mouse-cochlea in-vivo rescue and NHP in-vivo expression as equivalent grade-5 evidence; NHP data is stronger but no STRC-specific NHP trial has been run.
• Prestin endogenous regulatory region size (1,800 bp est) is rough — literature reports vary from ~1,200 to ~2,500 bp depending on definition boundaries. If a compact Slc26a5-derived enhancer exists at ~700 bp, candidate #7 could become a tier-5 contender.
• No codon-pair-bias or full-vector CpG audit performed at this architecture level; assumes flanking elements contribute negligible additional CpG burden (checked for B8 previously).

Next steps

1. If AF3 Ultra-Mini × TMEM145 validates (submitted 2026-04-21), this architecture is ordered.
2. Full-vector CpG audit on B8 + Kozak + IgK + Ultra-Mini-depleted CDS + WPRE3-compact + bGH polyA — regulatory elements may reintroduce CpGs not counted in STRC Ultra-Mini CpG Depletion (CDS-only scan).
3. Commercial codon-pair bias check (Coleman 2008 metric) on final assembled vector before gBlock synthesis.
4. Consider Prestin enhancer literature scan — if a sub-700-bp Slc26a5 cis-regulatory element exists, it would match STRC’s native OHC-exclusive pattern better than B8 and may score tier-5.

Replication

cd ~/STRC/models
python3 ultra_mini_promoter_shortlist.py
# outputs: ultra_mini_promoter_shortlist.json

Files / Models

• ~/STRC/models/ultra_mini_promoter_shortlist.py — module-size scoring + candidate ranking
• ~/STRC/models/ultra_mini_promoter_shortlist.json — scored tradeoff matrix

Ranking delta

• STRC Mini-STRC Single-Vector Hypothesis: S-tier, no change; evidence +1 on Ultra-Mini branch. This proof formalizes the vector-architecture upgrade path enabled by Ultra-Mini’s 1,125 bp of extra AAV headroom. The B8 + WPRE3-compact combo is uniquely Ultra-Mini-enabled and preserves OHC exclusivity. Delivery score upgrade 4→5 remains gated on the pending AF3 multimer; this architecture assumes that job passes.
• STRC B8 Enhancer Selection: no change. B8 remains the chosen enhancer; this proof adds WPRE3 to the architecture, does not replace B8.
• STRC AAV Vector Design: implicit update — recommended architecture shifts from “B8 alone” to “B8 + WPRE3-compact” for Ultra-Mini variant; parent vector design note should be updated when AF3 validation lands.
• All other hypotheses: no change.

Connections

• [part-of] STRC Mini-STRC Single-Vector Hypothesis
• STRC B8 Enhancer Selection — keeps B8, adds WPRE3
• [part-of] STRC AAV Vector Design
• STRC Mini-STRC Truncation Interface Validation — Ultra-Mini’s headroom makes this upgrade possible
• [see-also] STRC Ultra-Mini CpG Depletion — CDS-level clinical validation already done
• [see-also] STRC Hypothesis Ranking
• [about] Misha