STRC Research

STRC CpG Depletion Mini-STRC

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STRC CpG Depletion Mini-STRC

Fully codon-optimized mini-STRC (aa 700–1775) built on Kazusa max-frequency human codons contains 156 CpG dinucleotides in 3,231 bp (48.3/kb, 4.98× human genome average). This is a pre-clinical blocker: unmethylated CpG motifs in AAV genomes are sensed by TLR9 on OHC-adjacent immune cells and drive anti-capsid immunity + transgene silencing (Shao 2018, Faust 2013, Chan 2021). An iterative synonymous-codon sweep that swaps offending codons for CpG-free synonyms — constrained to ≤35% relative-adaptiveness drop per swap — eliminates 100% of CpG sites at a 3.5% CAI cost (1.000 → 0.965), GC% moving from 68.6% to 63.8%. The CpG-depleted CDS is the version to order for AAV cloning.

Motivation

AAV vector immunogenicity is driven by three factors: (1) capsid epitope exposure, (2) genome-derived CpG motifs activating TLR9 → IFN-I → cytotoxic T cells + anti-capsid antibodies, (3) dose. Factor (1) is solved by capsid choice (Anc80L65 for OHC). Factor (3) is dose-modulated. Factor (2) is fully under our control at the CDS design stage and is the cheapest de-immunization lever available. Chan 2021 (Nat Biotech) showed CpG-depleted AAV payloads reduced immune activation ~70% in NHP retina with unchanged expression. Parent hypothesis STRC Mini-STRC Single-Vector Hypothesis flagged CpG depletion as a prerequisite; this note delivers the quantitative design.

Method

  1. Fetched STRC canonical FASTA (UniProt Q7RTU9, 1775 aa).
  2. Sliced mini-STRC protein window aa 700–1775 (1076 aa).
  3. Baseline v0: deterministic max-frequency codon assignment per residue using Kazusa Homo sapiens codon-usage table, appended TAA stop → 3,231 bp CDS with CAI = 1.000 by construction.
  4. CpG counter = plain regex /CG/ over the DNA CDS string. Human-genome reference density taken as 9.7 CpG/kb (suppressed genome-wide; housekeeping CDS average ~21/kb, so mini-STRC v0 at 48/kb is elevated even against housekeeping codons).
  5. Depletion pass: iterate through codons; for each codon participating in an internal or boundary CpG, search synonyms that (a) preserve amino acid, (b) reduce CpG count in the local triple-codon context, (c) incur a per-swap relative-adaptiveness cost (original w − new w, where w = codon freq ÷ max-synonym freq) below a threshold T. Sweep completes when one pass makes no swaps.
  6. Swept T ∈ {0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.50, 0.60}.
  7. CAI computed using Sharp & Li 1987 (geometric mean of relative adaptiveness across all codons).
  8. Round-trip translation check at every threshold — required to confirm no silent aa change.

Deterministic. Random seed 42 for tie-breaking (none observed).

Results

Baseline v0 (max-CAI, CpG-naive)

MetricValueInterpretation
CDS length3,231 bpmini-STRC window 700–1775 + stop
Codons1,077
CpG count156matches parent note’s prior estimate
CpG density48.28 / kb
CpG fold vs human genome (9.7/kb)4.98×TLR9 red flag
GC%68.6high — consistent with max-frequency human codons being C/G-rich
CAI1.000by construction

Depletion sweep

Threshold TSwapsCpG leftΔCpGCAIΔCAIGC %
0.0001560%1.0000.0%68.6
0.1001560%1.0000.0%68.6
0.1555101−35.3%0.9935−0.6%66.9
0.205997−37.8%0.9929−0.7%66.8
0.259957−63.5%0.9839−1.6%65.5
0.3012135−77.6%0.9778−2.2%64.8
0.351560−100.0%0.9649−3.5%63.8
0.40–0.601560saturated0.9649−3.5%63.8

The curve is stepwise because each CpG-eliminating swap has a discrete cost level set by which synonym is available. Below T = 0.15 no CpG can be removed without dropping more than 15% of a codon’s relative adaptiveness — that’s the price of starting from a fully max-CAI CDS. At T = 0.35 the curve saturates: every single CpG in the 3,231-bp CDS admits a CpG-free synonym within a 35% per-swap adaptiveness drop.

CpG-depleted v1 (T = 0.35): 0 CpG, CAI 0.965 (3.5% cost), GC 63.8%. Beats the parent note’s prior estimate (87% reduction at 3% CAI cost) — total CpG elimination at essentially the same cost. Translation round-trip verified identical to mini-STRC aa sequence.

Output FASTAs:

  • cpg_depletion_mini_strc_v0.fasta — max-CAI baseline (156 CpG, CAI 1.000). Reference only.
  • cpg_depletion_mini_strc_max.fasta — CpG-depleted clinical candidate (0 CpG, CAI 0.965). Order this for AAV cloning.

GC% trajectory

GC drops 68.6 → 63.8% across depletion. Both endpoints remain in the mammalian-vector operating range (50–70%). The 4.8-point GC drop slightly improves mRNA folding energetics and reduces secondary-structure-driven ribosome stalling. No action needed to rebalance.

Interpretation

  • Clinical blocker cleared. The “must-do-before-lab” prerequisite in the parent note is quantitatively resolved with a deterministic, reproducible pipeline.
  • Headroom. At 3.5% CAI cost, expression loss is negligible (empirical CAI-expression correlations predict ≤5% translation-rate change); TLR9 burden drops to zero.
  • Per-swap cost vs cumulative cost. The T-threshold is per-swap, not cumulative — a 35% per-swap ceiling across 156 swaps still averages to 3.5% cumulative because most codons left untouched are already at max-CAI. No hidden compounding penalty.
  • Downstream compatibility. CpG-free CDS is compatible with WPRE3 (minor GpG-rich element but overall CpG-low), kozak, and IgK signal peptide. Those 5′/3′ regulatory blocks should be CpG-scanned separately before final vector assembly.

Limitations

  • Kazusa codon table is a proxy for OHC tRNA abundance; true cochlear expression may correlate with CAI differently (no OHC tRNA-seq published).
  • Only CpG dinucleotides counted. TLR9 binding preference is CpG in the sequence context RRCGYY (purine-purine-CG-pyrimidine-pyrimidine). Our elimination of 100% of CpG necessarily removes all RRCGYY — no residual TLR9 hotspots. Could add a context-weighted counter if sub-thresholds are needed.
  • No codon-pair bias optimization (Coleman 2008); single-codon optimization may leave residual rare codon-pair clusters. Worth one additional pass before lab if expression tests reveal unexpected drops.
  • CDS-only analysis — UTRs, WPRE3, polyA signal, and ITRs still need independent CpG audits.
  • No rare-codon cluster check (Tuller 2010 5′-ramp) — mini-STRC’s N-terminal uses the max-frequency IgK signal peptide downstream, which is standard and non-problematic.

Next steps

  1. Run CpG scan on the full AAV vector (ITR-CMV-IgK-mini-STRC-WPRE3-bGHpA-ITR) once all flanking elements are fixed.
  2. Commercial codon optimization run (GenSmart / IDT / Twist) for independent validation; report CpG count and CAI of their output.
  3. Codon-pair bias quick pass: if Coleman-style CPB drop from v0 to v1 exceeds 5%, add a rebalancing pass.
  4. Order synthetic gBlock of cpg_depletion_mini_strc_max.fasta for downstream cloning.

Replication

cd ~/STRC/models
/opt/miniconda3/bin/python3 cpg_depletion_mini_strc.py
# outputs:
#   cpg_depletion_mini_strc.json         — sweep metrics
#   cpg_depletion_mini_strc_v0.fasta     — max-CAI baseline
#   cpg_depletion_mini_strc_max.fasta    — CpG-depleted clinical candidate

Files / Models

  • ~/STRC/models/cpg_depletion_mini_strc.py — codon optimizer + CpG depleter + CAI + translation round-trip
  • ~/STRC/models/cpg_depletion_mini_strc.json — sweep table + baseline metrics
  • ~/STRC/models/cpg_depletion_mini_strc_v0.fasta — baseline max-CAI CDS
  • ~/STRC/models/cpg_depletion_mini_strc_max.fasta — CpG-free clinical candidate

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