IF a computationally re-engineered CCR9-FOXN1-TAT-XTEN fusion protein — designated αTEC-FOXN1v2 — in which RFdiffusion-generated rigid inter-domain linkers sterically shield FOXN1's intrinsically disordered regions (IDRs) during systemic transit while a nuclear-activatable XTEN half-life extension module is proteolytically released upon importin-β-mediated nuclear entry, is administered intraperitoneally (3×/week, 200 µg/dose) to 20-month-old C57BL/6J male mice,

THEN a measurable restoration of thymic cortical and medullary TEC compartments — defined as ≥40% recovery of EpCAM⁺MHCII⁺ TEC number, ≥2-fold increase in naïve CD4⁺CD8⁻ recent thymic emigrant (RTE) output, and re-establishment of Dll4, Ccl25, and Psmb11 expression in bulk thymic RNA-seq — will be observed within 8 weeks,

BECAUSE:

1. Thymic involution in aged mice is driven by accumulated loss of functional FOXN1 protein in TECs, a damage state not reversed by systemic mTOR inhibition or senolytic treatment alone, because FOXN1 protein levels — not merely its mRNA — collapse in aged TECs, leaving the transcriptional program governing TEC identity irreversibly dormant (FOXN1 downregulation drives thymic involution)[https://pmc.ncbi.nlm.nih.gov/articles/PMC9934747/].

2. The N-terminal extracellular domain of CCR9 (residues ~1–25; UniProt murine Q9WUT7, human P51686) retains high-affinity binding to CCL25, which is constitutively expressed by thymic stromal cells, enabling the fusion protein to preferentially accumulate at the thymic microenvironment following systemic administration (CCR9 N-terminus mediates CCL25-dependent thymic homing)[https://pmc.ncbi.nlm.nih.gov/articles/PMC9934747/].

3. The HIV-1 TAT protein transduction domain (GRKKRRQRRRPQ) mediates size-dependent but receptor-independent membrane translocation into TECs after CCR9-mediated thymic accumulation; however, large, structurally disordered cargoes reduce transduction efficiency, and the existing CCR9-FOXN1-TAT construct suffers from cytoplasmic aggregation before nuclear entry (TAT transduction efficiency inversely related to cargo disorder and aggregation propensity)[https://pmc.ncbi.nlm.nih.gov/articles/PMC9934747/].

4. FOXN1 (UniProt human O15353, murine Q61575) executes its transcriptional program not through isolated DNA binding alone but through liquid-liquid phase separation (LLPS) driven by its C- and N-terminal IDRs, which form condensates at super-enhancers of thymopoietic target genes including Dll4, Ccl25, and Psmb11; mutations or truncations disrupting IDR hydrophobicity abolish condensate formation and transcriptional output even when the Forkhead DBD is intact, meaning any stabilization strategy that truncates or rigidly buries these IDRs will produce a transcriptionally inert protein (FOXN1 condensate formation by IDRs is essential for transactivation)[https://www.science.org/doi/10.1126/sciadv.abj9247].

5. [SPECULATIVE] RFdiffusion can scaffold novel protein backbones around fixed fun...

SENS category: RepleniSENS

Key references: • doi.org/10.1038/mt.2015.77],