IF a sequential triple-modality protocol is administered to 20–22 month C57BL/6J male mice, consisting of:

1. Senolytic preconditioning — dasatinib (5 mg/kg, oral gavage) + quercetin (50 mg/kg, oral gavage) on days −7/−6 and −3/−2 relative to injection,
2. Intrathymic STING pathway silencing — small-molecule STING inhibitor H-151 (0.75 µM, 5 µL bilateral intrathymic injection) on day −1, and
3. AAV9-FOXN1 delivery — 1×10¹¹ vector genomes bilaterally intrathymic on day 0,

THEN by day 45 post-injection, treated mice will demonstrate:

• ≥60% increase in total thymocyte count versus AAV9-FOXN1 alone (flow cytometry, CD3/CD4/CD8 panels),
• ≥3-fold increase in FOXN1⁺ EpCAM⁺ thymic epithelial cells versus AAV9-FOXN1 alone (immunofluorescence and scRNA-seq),
• ≥70% reduction in p16^INK4a⁺ cells versus untreated aged controls (SA-β-galactosidase, p16 immunohistochemistry),
• ≥2.5-fold higher AAV9 vector genome copies per EpCAM⁺ TEC versus AAV9-FOXN1-alone controls (ddPCR on sorted cells),
• ≥50% reduction in intrathymic type-I interferon signature genes (IFN-β, Ifit1, Ifit3, Mx1) versus untreated aged thymus (bulk RNA-seq),

BECAUSE the following causal chain operates:

1. Aged thymic epithelial cells (TECs) in 20–22 month C57BL/6J mice accumulate high p16^INK4a burden, as Cdkn2a expression increases consistently and robustly with age across tissues including thymus (p16^INK4a accumulates with age across tissues)[https://doi.org/10.3389/fgene.2018.00059], generating senescent TECs that release cytoplasmic mitochondrial DNA (cf-mtDNA) as part of their SASP.
2. Senescent cells in aged tissues are a principal source of elevated cf-mtDNA, and D+Q senolytic treatment in aged C57BL/6 mice reduces cf-mtDNA levels, attenuates inflammatory T-cell responses, and improves tissue graft survival — directly linking senescent cell burden to cf-mtDNA-driven inflammation (D+Q reduces cf-mtDNA and inflammatory responses in aged organs)[https://doi.org/10.1038/s41467-020-18039-x].
3. [SPECULATIVE] cf-mtDNA released by senescent TECs activates the cGAS-STING innate immune sensing axis in neighboring healthy TECs and thymic macrophages, triggering a sustained type-I interferon (IFN-I) response within the thymic microenvironment. This IFN-I state simultaneously suppresses FOXN1 transcription (via STAT1-IRF3 binding at FOXN1 promoter elements — a mechanistic link established in epithelial interferon biology but not yet formally demonstrated in TEC context) and upregulates antiviral restriction factors (TRIM5α, APOBEC3, MX1) that degrade incoming single-stranded AAV9 genomes before second-strand synthesis.
4. Senescent-like cells with active SASP suppress tissue-regenerative programs in neighboring cells: senescent microglia expressing p16^INK4a and SASP factors actively limit remyelination in adjacent progenitor compartments (SASP-producing senescent cells suppress tissue regeneration in neighboring progenitors)[https://doi.org/10.1101/2024.05.23.595605]....

SENS category: GlycoSENS

Key references: • doi.org/10.3389/fgene.2018.00059], • doi.org/10.1038/s41467-020-18039-x]. • doi.org/10.1101/2024.05.23.595605]. • doi.org/10.1111/acel.13103]; • doi.org/10.1016/j.ebiom.2017.04.013],