IF a single intra-thymic bolus injection of lipid-nanoparticle-formulated FOXN1 mRNA (estimated 20–50 µg mRNA-LNP per mouse, delivered stereotactically into the involuted thymic remnant under ultrasound guidance) is administered to 20-month-old male and female C57BL/6 mice (n ≥ 10 per group),

THEN in situ epigenetic reprogramming of surviving but transcriptionally silenced resident thymic epithelial cells (TECs) within the involuted remnant will restore cortical-medullary zonation and increase peripheral naive CD4⁺CD62L⁺CD44ˡᵒ T-cell output by ≥ 40% relative to vehicle-injected aged controls within 16 weeks post-treatment, without evidence of autoimmune infiltration (H&E score ≤ 1 across thyroid, salivary gland, kidney, and liver sections),

BECAUSE the following causal chain applies:

1. FOXN1 is the master transcription factor governing TEC identity and thymic organogenesis: its expression declines irreversibly with age-related involution, but its re-introduction into non-thymic cells—including embryonic fibroblasts—is sufficient to generate functional TEC-like cells that restore central tolerance mechanisms and suppress systemic age-related inflammation. (FOXN1 reprogramming of fibroblasts restores thymic function)[https://doi.org/10.1172/jci.insight.140313]

2. The aged thymic remnant retains residual, involuted but not fully ablated, TEC populations: these cells are transcriptionally suppressed rather than entirely eliminated, meaning they retain nuclei, cytoskeletal structure, and potentially accessible chromatin at TEC-lineage loci. Transient FOXN1 mRNA delivery could reactivate these dormant progenitor programs without requiring exogenous cell engraftment. [SPECULATIVE — supported by analogy to partial reprogramming literature and the presence of a stromal remnant in aged C57BL/6 thymus]

3. mRNA-LNP delivery enables transient, non-integrating FOXN1 protein expression with a defined pulse duration (48–72 h), avoiding the oncogenic risk of constitutive FOXN1 overexpression or lentiviral integration while still providing a sufficient transcriptional burst to reactivate downstream TEC differentiation programs (e.g., DLL4, CXCL12, CCL25, AIRE). [SPECULATIVE — delivery route adapted from mRNA vaccine platforms; no intra-thymic mRNA data yet published]

4. Restored cortical TEC (cTEC) function will re-establish MHC-II-restricted positive selection niches, increasing thymic output of naive CD4⁺ T cells that have undergone normal positive selection. (iPSC-derived TEPs can generate both cTEC and mTEC-like populations and support T-cell development)[https://doi.org/10.1016/j.jaci.2021.07.021]

5. Restored medullary TEC (mTEC) function, including AIRE expression, will re-establish central tolerance checkpoints, reducing the probability of autoreactive T-cell escape and systemic autoimmunity — the key safety endpoint. (FOXN1-driven TEC reprogramming counteracts disruption of central tolerance in the aged involuted thymu...

SENS category: RepleniSENS

Key references: • doi.org/10.1172/jci.insight.140313] • doi.org/10.1016/j.jaci.2021.07.021] • doi.org/10.1172/jci.insight.140313]. • doi.org/10.1016/j.jaci.2021.07.021],