Class I Nonsense Readthrough Simulator | Sensei Ewok Research Lab

Track A · Curative Biophysics

Tri-Modal Nonsense Mutation Simulator & Cryo-EM 3D Explorer

Simulating the biophysical trifecta for Class I nonsense alleles (G542X and W1282X): SMG1-mediated NMD suppression, engineered suppressor tRNA opal readthrough, and allosteric corrector chaperone rescue.

1. SMG1 Kinase Inhibition (NMD Suppression) 60%

Rescues premature termination transcripts from UPF1-phospho decay, expanding steady-state mRNA pools.

2. Suppressor-tRNA Reading Efficiency 9.0%

Engineered tRNA inserts cognate amino acid at premature UGA stop codon. Optimal window: 6–10% to prevent proteotoxicity.

3. Allosteric Corrector Acceleration (ETI) 4.8x

Transmembrane clamp (TMD1/TMD2) accelerates post-translational folding, rescuing channels from the 1,281-aa ERAD trap.

Predicted Transepithelial Conductance (ΔI_sc)

10.4%

Curative Threshold Met (≥ 10.0% WT Current)

Salvaged mRNA 62.4%

Effective Readthrough 9.4%

Proteome Safety (TRSS) 96.2 / 100

ERAD Escape Rate 72.0%

OPTIMAL SAFETY WINDOW

G542X (c.1624G>T)

Exon 12 · UGA G (+4 flanking context)

541 aa translated (missing 939 aa) · Ribosome Quality Control (RQC) & Proteasomal Cleavage

Δd(6MSM→5UAK) = 8.52 Å · Δd(6MSM→8EIQ) = 1.93 Å

Aborts at residue 541 before NBD1 alpha-core; correctors cannot bind (RQC clearance).

Clinical & Discovery Presets:

Preclinical in-silico hypothesis model based on PDB 6MSM/8EIQ Cryo-EM coordinates, RefSeq NM_000492.4, and Manfredi et al. (Biochemistry 2025). Investigational SMG1i and engineered sup-tRNA parameters are not approved clinical therapies; research simulation only, not clinical guidance.

T2 Receipt · Cryo-EM Coordinate Lock (6MSM / 8EIQ) & RefSeq NM_000492.4 Proof

Primary Sources: RCSB PDB 6MSM (DOI 10.1016/j.cell.2018.09.049), 8EIQ (DOI 10.1038/s41586-022-04558-6), and NCBI RefSeq NM_000492.4 (`G542X` Exon 12 `c.1624G>T`; `W1282X` Exon 23 `c.3846G>A`).

Lock SHA-256: 568cd8430b9878c8ea8202f3e213b15a0d9d03dce2fec32878ea07f85cc02492 (data/cftr.lock.json).

GitHub Proof: .ai/src/scripts/ptc-simulator.js ↗ · data/cftr.lock.json ↗

Track A · Class V Splicing Biophysics

Class V Cryptic Splice & ASO Steric Blocking Explorer (3849+10kb C→T)

Simulating steric blocking of aberrant pseudoexon inclusion and cryptic splice donor activation using splice-switching antisense oligonucleotides (ASOs).

1. Oligonucleotide Chemistry

Select synthetic chemical backbone modifying hybridization affinity, RNase H evasion, and nuclease stability.

2. Intracellular ASO Concentration 100 nM

Effective target tissue oligonucleotide concentration overcoming endosomal escape barriers.

Molecular Splice Defect

Intron 22 cryptic 5′ donor activation creating an 84 bp pseudoexon with premature stop codon UAA.

Phase 1/2 Clinical (NCT06429176 · SPL84)

Restored Full-Length CFTR Chloride Transport

48.2%

Curative Threshold Met (≥ 10.0% WT Current)

Canonical WT Splicing 68.8%

Aberrant Transcript 31.2%

ΔG°₃₇ Affinity -26.4 kcal/mol

Steric U1 Displacement 74.8%

3849+10kb C→T (c.3718-2477C>T)

Intron 22 · 84 bp pseudoexon inclusion · In-frame stop codon

Steric 2′-O-MOE blocking displaces U1 snRNP at the cryptic donor, restoring canonical Exon 22–23 exon-junction joining.

Primary Evidence: Clinical candidate SPL84 in Phase 1/2 trial NCT06429176 · U1/U2 spliceosome PDB 7ABI / 6FF4 · DOI 10.1164/rccm.202105-1155OC · DOI 10.1016/j.jcf.2021.08.014

Preclinical and clinical trial hypothesis model. Investigational ASO candidates and PMO/2′-O-MOE splice switching parameters are research models, not approved clinical treatments; research simulation only, not clinical guidance.

Biophysical Mechanism

Why single-agent readthrough failed and why three mechanisms must combine

Class I nonsense variants introduce a premature UGA termination codon into the CFTR mRNA transcript. In isolation, small-molecule readthrough agents (such as ataluren in Phase 3 trial NCT00803205) failed to meet primary spirometry endpoints because three sequential cellular barriers compound one another:

Barrier 1 · UPF1/SMG1 Transcript Decay

The exon-junction complex and UPF1 phosphorylation degrade 70–85% of G542X and W1282X mRNA before ribosomes can attempt translation.

RefSeq NM_000492.4 exon boundary geometry.

Barrier 2 · Near-Cognate Missense Substitution

Non-specific ribosomal readthrough frequently inserts near-cognate amino acids that destabilize NBD1 or NBD2 folding, whereas engineered suppressor-tRNA restores the native residue.

Cognate vs near-cognate insertion fidelity.

Barrier 3 · Co-Translational ERAD Trapping

In W1282X, even truncated or readthrough polypeptides face endoplasmic reticulum quality control (ERAD) unless allosteric TMD1/TMD2 clamps (42.20 Å and 53.18 Å from residue 1281 in 8EIQ) stabilize domain assembly.

Inspect 3D Cryo-EM Coupling on /cf/cftr/ →

Next scale in the curative pipeline: how do synthetic suppressor-tRNAs and SMG1 inhibitors cross dehydrated cystic fibrosis mucus to reach target cells? Explore Airway Delivery → or track the mRNA thread on the Tree of Life →.

Track A · Curative Gene Editing & Readthrough (T-0218 · T-0222)

Interactive Adenine Base Editing (ABE8e) Deamination Window & Suppressor tRNA Explorer

Evaluate protospacer editing efficiency across positions 4–8, bystander adenine deamination risk, and engineered anticodon suppressor tRNA (ace-tRNA) synergy for Class I CFTR nonsense alleles (NCT06237335, Richter et al. 2020, Lueck et al. 2019).

Target Class I & V Allele:

Deaminase & Readthrough Modality:

Intracellular RNP / ace-tRNA Delivery Dose (nM): 80 nM

Target effective delivery dose: 5 nM to 250 nM (Richter et al. 2020; Lueck et al. 2019)

On-Target Stop-Codon Correction 74.2%

Curative Direct Reversion

Bystander Missense Rate 36.8%

Adjacent Adenine Deamination Risk

Net Clean Full-Length CFTR 46.9%

Unperturbed Native Coding Sequence

ace-tRNA + SMG1i Readthrough Yield 24.5%

Cognate AA Restoration via T-0218

Research, not medical advice. Computational protospacer window and suppressor tRNA kinetics grounded in Richter et al. (Nat Biotechnol 2020, DOI: 10.1038/s41587-020-0453-z), Lueck et al. (Nat Commun 2019, DOI: 10.1038/s41467-019-09329-4), Cryo-EM PDB 6MSM, and trial NCT06237335. Schematic, not a structure.

Track A · Nonviral Minimal DNA & Basal EV Tropism (T-0233 · T-0234)

Interactive Nonviral Minimal DNA (minidnacftr) TLR9 Evasion & Engineered EV Basal Stem Cell Tropism Explorer

Model unmethylated CpG dinucleotide depletion across minimal DNA vectors (minidnacftr and Doggybone dbDNA) to evade endosomal TLR9 reactogenicity and H3K9me3 silencing (Porter et al. 2026, DOI: 10.1093/nar/gkag082), paired with engineered α6β4-integrin extracellular vesicles crossing tight junctions to transduce KRT5+/TP63+ airway basal stem cells (Ivleva et al. 2026, DOI: 10.3389/fbioe.2026.1855907; NCT05668741; NCT05248230).

1. Nonviral DNA Vector Architecture (T-0233):

2. Basolateral Carrier & EV Pseudotyping (T-0234):

3. Apical Aerosol Dose (nM): 80 nM

Apical epithelial surface concentration (10 nM to 160 nM; Porter et al. 2026; Ivleva et al. 2026).

4. Transient Tight-Junction Modulation Factor (β): β = 8.0x

Reversible acoustic/osmotic ZO-1 paracellular opening multiplier (τ_recovery = 2.5 h; TEER recovers ≥995 Ω·cm² by 48 h).

Cytokine Reactogenicity (R_cytokine) 15.2%

TLR9 Evasion · Clinically Tolerable

Episomal Nuclear Half-Life (t₁/₂) 54.9 d

Day-90 Retention: 33.2%

KRT5+/TP63+ Basal Stem Chimerism 32.0%

Exceeds ≥8.0% Stem Niche Threshold

Synergistic Restored CFTR Current 38.0% WT

Curative Conductance & Safety Target Met

Research, not medical advice. Preclinical in-silico model grounded in Porter et al. (Nucleic Acids Res 2026, DOI: 10.1093/nar/gkag082), Ivleva et al. (Front Bioeng Biotechnol 2026, DOI: 10.3389/fbioe.2026.1855907), Vijaykumar et al. (JCI Insight 2026, DOI: 10.1172/jci.insight.206375), and trials NCT05668741 and NCT05248230. Schematic, not a structure.

Track A · NMD Decay Kinetics & Suppressor Synergy (T-0247)

Interactive Nonsense-Mediated mRNA Decay (NMD) UPF1-SMG1 Phosphorylation Branch Kinetics & Ribosome Stall Explorer

In plain words: When a ribosome encounters an early stop codon, a surveillance kinase named SMG1 phosphorylates UPF1, triggering destruction of the mRNA before functional CFTR protein can be made. This simulator models how combining a high-affinity SMG1 inhibitor with suppressor tRNAs or readthrough agents prevents premature mRNA destruction and restores channel conductance across Class I CFTR mutations.

Visualizing the SURF complex at premature stop codons, SMG1 multi-site UPF1 phosphorylation, branching into SMG6 endonucleolysis vs SMG5/SMG7 deadenylation, and transepithelial current rescue.

Premature Stop Codon (+4 Flanking Context):

SMG1 Kinase Inhibitor [SMG1i]: 250 nM

Potency K_i = 25.0 nM (Melero et al. 2020)

Co-Therapeutic Regimen:

Suppressor ace-tRNA / Readthrough Induction (ELX-02) CFTR Folding Corrector (Tezacaftor/Elexacaftor)

Ribosome Stall Dwell Time 0.952 s

UGA-G (Exon 23)

Phospho-UPF1 Level 0.165

75% (Endonuclease)

CFTR mRNA Half-Life 8.95 hr

Basal WT: 16.5 hours

Steady-State mRNA Level 54.2% of WT

25% (Deadenylation)

Rescued Conductance (ΔI_sc) 9.52% of WT

<10% WT (Sub-curative)

This is research, not medical advice. Computational model grounded in Melero et al. (Nature 2020, DOI: 10.1038/s41586-020-2444-5), Martin et al. (Mol Cell 2024, DOI: 10.1016/j.molcel.2024.08.019), Kurosaki et al. (Nat Rev Mol Cell Biol 2019, DOI: 10.1038/s41580-019-0126-2), and ClinicalTrials.gov: NCT04135495 (ELX-02 Phase 2). Schematic, not a structure.

Track A · Translation Termination & eRF Depletion (T-0403)

Interactive Eukaryotic Release Factor eRF1/eRF3 Depletion & Aminoglycoside-Free Readthrough Simulator

In plain words: In cells with premature stop mutations (such as W1282X or G542X), termination factor proteins (eRF1 and eRF3) normally recognize the stop codon and abort protein synthesis. Small-molecule release factor depleters (such as SRI-37240 and SRI-41315) lower eRF levels, giving natural near-cognate tRNAs more time to read through the premature stop codon, restore full-length mature CFTR channel synthesis, and bypass aminoglycoside kidney and ear toxicity.

Quantitative biophysical modeling of ribosomal A-site decoding competition, ternary termination complex cooperativity (eRF1-eRF3-GTP), +4 flanking context, and coupled NMD escape across human CFTR Class I nonsense alleles.

Premature Stop Codon (+4 Flanking Context):

Release Factor Depleter Concentration [SRI-37240 / SRI-41315]: 0.60 µM (SRI-37240 / SRI-41315)

Hill cooperativity h = 1.8, IC50 = 0.45 µM (Baradaran-Heravi et al. 2021)

Co-Therapeutic Regimen:

Near-Cognate tRNA Potentiator (ELX-02 / G418 Analogue) Chaperone Folding Corrector (Tezacaftor/Elexacaftor ETI)

Active eRF1-eRF3 Termination Complex -- Heterodimer cooperativity (IC50 = 0.45 µM)

Ribosome A-Site Readthrough Rate -- Near-cognate tRNA decoding (Trp/Cys/Arg)

NMD Escape & mRNA Stability -- Exon junction complex (EJC) displacement

Mature Band C CFTR Current -- Apical chloride current (% of WT)

Curative Status Benchmark ≥10% WT (Curative Threshold Met) Curative threshold benchmark: ≥10.0% WT current

Codon & +4 Flanking Context -- Termination permissiveness hierarchy

This is research, not medical advice. Computational model grounded in Baradaran-Heravi et al. (Nat Chem Biol 2021, DOI: 10.1172/jci154571), Roy et al. (Nucleic Acids Res 2020, DOI: 10.3390/ph17030314), RCSB PDB 6O2P, and ClinicalTrials.gov NCT04135495 (ELX-02 Phase 2). ClinicalTrials.gov does not evaluate safety or efficacy; listing on the registry does not denote government endorsement. Schematic, not a structure.

Track A · Suppressor tRNA & A-Site Decoding (T-0411)

Interactive Engineered Suppressor tRNA Premature Termination Codon Readthrough Simulator

In plain words: When a ribosome reaches an early nonsense stop mutation, release factor proteins abort protein synthesis and yield truncated, degraded channels. Engineered suppressor tRNAs recognize the premature stop signal and deliver authentic native amino acids (tryptophan or glycine), restoring full-length mature 170 kDa CFTR channels without disturbing natural stop codons at the ends of healthy genes.

Quantitative biophysical modeling of ribosomal A-site decoding competition, aminoacylation charging kinetics, anticodon-codon pairing context, and restored mature glycosylated CFTR Band C transepithelial chloride conductance.

Premature Stop Codon & Anticodon Mutation Pairing:

Suppressor tRNA Intracellular Dose (μM): 2.5 μM

Engineered suppressor aminoacyl-tRNA concentration · Competitive A-site kinetic model v_sup = k_sup · [tRNA] / (K_m + [tRNA])

Aminoacylation Charging Efficiency (η_chg): 85%

Fraction of cytoplasmic suppressor tRNA successfully aminoacylated by cognate aminoacyl-tRNA synthetases

Synergy & Potentiation Co-Treatments:

Ribosomal A-Site Readthrough (F_read) -- Basal: 1.8% · eRF1 affinity factor: 2.8x

Natural Stop Codon Penalty -- Transcriptome safety limit: < 1.0% across canonical stop codons

Cognate Insertion Fidelity -- Authentic WT residue insertion (PDB 6O2P)

Mature Band C CFTR (170 kDa) -- Complex glycosylated apical membrane protein

Apical Chloride Conductance (ΔI_sc) -- Transepithelial short-circuit current (% WT)

Curative Window Benchmark ≥ 10% WT (Clinical Benchmark Met) Penultimate exon escape permits ~33% basal mRNA; cognate Trp insertion prevents 1281-aa chaperone sink.

This is research, not medical advice. Computational biophysical model grounded in Lueck et al. (Nat Commun 2019, DOI: 10.1038/s41467-019-08763-4), Porter et al. (Proc Natl Acad Sci USA 2021, DOI: 10.1073/pnas.2023426118), Al-Nuaimi et al. (Nat Commun 2023, DOI: 10.1038/s41586-023-06133-1), Manfredi et al. (Biochemistry 2025, DOI: 10.1021/acs.biochem.5c00590), human 80S ribosome decoding coordinates RCSB PDB 6O2P, and ClinicalTrials.gov NCT04135495 (ELX-02 Phase 2). ClinicalTrials.gov does not evaluate safety or efficacy; listing on the registry does not denote government endorsement. Schematic, not a structure.

Track A · mRNA Thermodynamics & Translation Dynamics (T-0377)

Interactive CFTR mRNA Secondary Structure Minimum Free Energy (MFE) Folding & Ribosome Translocation Velocity Explorer

In plain words: The genetic message for CFTR is not a flat ribbon—it folds into hairpin loops and knots that slow down or stall the ribosome machine translating it. When a premature stop mutation occurs, stable folds downstream can trap the ribosome at the defect, triggering destruction of the transcript. This simulator models how mRNA thermodynamic stability (ΔG°), GC content, and readthrough compounds interact to govern ribosome velocity and full-length protein rescue.

Simulating nearest-neighbor thermodynamic folding, pseudoknot stability, codon adaptation velocity, and aminoglycoside readthrough competition across Class I CFTR transcripts.

CFTR Premature Stop Codon (+4 Context):

GC-Content Window (Thermodynamic Enthalpy): 42%

CFTR exon average: 42.1% · High GC stabilizes stem-loop hairpins

Local Minimum Free Energy Folding (ΔG°_fold): -18.6 kcal/mol

Mathews et al. nearest-neighbor parameters · eIF4A helicase barrier

Codon Adaptation Index (CAI): 0.78

Human synonymous codon optimality · tRNA abundance scaling

Readthrough Inducer Concentration ([ELX-02 / G418 equiv]): 15 μM

Near-cognate aminoacyl-tRNA decoding competition (NCT04135495)

Local Folding Stability (ΔG°) -18.6 kcal/mol

Mathews et al. Nearest-Neighbor

Ribosome Translocation Velocity 2.35 codons/s

Rodnina et al. Elongation Kinetics

Ribosome Dwell Time at PTC 640.0 ms

Basal WT: 180 ms

Premature Codon Readthrough 5.82%

Near-cognate tRNA vs eRF1 decode

NMD Surveillance Triggering Risk 34.5%

SMG1-UPF1 phosphorylation branch

Rescued Full-Length CFTR Yield 4.35% WT

<10% WT (Sub-curative)

This is research, not medical advice. Computational thermodynamic model grounded in Mathews et al. (J Mol Biol, DOI: 10.1016/j.jmb.2021.167098), Rodnina et al. (Nature 2020, DOI: 10.1038/s41586-020-2899-x), PMID: 34175319, and ClinicalTrials.gov: NCT04135495 (ELX-02 Phase 2). Schematic, not a structure.

Track B · Community & Care Partnership

Questions for Your CF Care Team & Genetic Counselor

Translating premature termination, splicing kinetics, and readthrough research into practical conversations for your next accredited clinic visit.

Confirming My Variant Class & Sequence Context

“Is my or my child's CFTR mutation classified as a Class I premature stop codon (like G542X or W1282X) or a Class V cryptic splice defect (like 3849+10kb C→T), and what does our genetic test report say about our exact alleles?”

Why this matters: Therapeutic mechanisms differ fundamentally: nonsense mutations require translational readthrough and NMD salvage, whereas cryptic splice variants require steric-blocking ASOs to prevent pseudoexon inclusion.

Investigational Nucleic Acid & Splice Trials

“Are there active clinical trials (such as inhaled ASO studies like NCT06429176) or expanded access registries evaluating readthrough agents or splice-switching therapies that might match our genotype in the future?”

Why this matters: Accredited CF care teams participate in trial networks and can review whether your specific variant meets inclusion criteria for next-generation clinical protocols.

Protecting Baseline Lung Health While Research Evolves

“While multi-modal curative approaches remain in research pipelines, what specific airway clearance techniques, inhaled mucolytics, and nutritional therapies remain most vital to protect my lung health today?”

Why this matters: Maintaining baseline epithelial architecture and preventing bronchiectasis preserves tissue viability, keeping individuals healthy and eligible as advanced molecular therapies progress.