IF a bicistronic LNP-mRNA construct encoding (1) codon-humanized Deinococcus radiodurans TDP1 (DrTDP1) fused to a nuclear localization signal (NLS-DrTDP1) and (2) human APE1 — formulated as a single ionizable-lipid nanoparticle at a total mRNA dose of 0.5 mg/kg — is administered intravenously to 24-month-old male C57BL/6 mice weekly for 12 weeks,

THEN hepatic 8-oxo-dG lesion burden will decrease by ≥40% from aged baseline (measured by ID-LC-MS/MS), DNA-protein crosslink (DPC) levels will decline by ≥30% (K-SDS precipitation/MS), and senescence marker expression (p16INK4a, p21) will fall by ≥25% (RT-qPCR/immunofluorescence) without serum ALT/AST exceeding 3× baseline, compared to empty-LNP controls and to a monocistronic NLS-DrTDP1-only arm,

BECAUSE the following causal chain is predicted:

1. In the aged C57BL/6 liver, endogenous BER efficiency declines and mitochondrial ROS rises, driving 8-oxo-dG to 1.5–2.5 lesions per 10⁶ dG (a 2–3-fold increase over young controls) and elevating DPC burden ~1.5-fold, with accumulation concentrated at transcriptionally active loci — creating a substrate pool that endogenous human TDP1 cannot clear at the rate it accumulates. (Hamilton et al., Proc. Natl. Acad. Sci. USA, 2001, as summarized in Evidence Set)

2. IV-administered ionizable-LNPs at 0.5 mg/kg adsorb serum ApoE and are taken up by hepatocytes via LDLR-mediated endocytosis, delivering >85–90% of payload to the liver, with translation initiating within 1–2 hours and peak protein expression between 6–12 hours post-injection — a pharmacokinetic window fully compatible with weekly dosing cycles. (Akinc et al., Nature Biotechnology, 2010; Pardi et al., Journal of Controlled Release, 2015, as summarized in Evidence Set)

3. DrTDP1 (UniProtKB: Q9RZE6), via its conserved HKD dual-histidine catalytic motif, executes a two-step phosphoamide hydrolysis: a nucleophilic histidine attacks the 3'-phosphotyrosyl DPC bond, forming a covalent phospho-enzyme intermediate, which is hydrolyzed by a second activated histidine to release the protein adduct and restore a clean 3'-phosphate terminus. Crucially, DrTDP1 evolved in an extremophilic context and demonstrates broader 3'-adduct specificity than human TDP1 — efficiently processing 3'-phosphoglycolate and 3'-phosphoglycaldehyde termini generated by oxidative strand breaks near 8-oxo-dG sites, substrates the endogenous enzyme handles sub-optimally. (Interthal et al., Proc. Natl. Acad. Sci. USA, 2001; Lehtio et al., J. Mol. Biol., 2008, as summarized in Evidence Set)

4. [SPECULATIVE] The NLS fusion directs translated DrTDP1 to the nucleus, the compartment of highest age-accumulated DPC density and 8-oxo-dG-proximal strand-break burden. Without an NLS, the compact bacterial enzyme (lacking the extended N-terminal regulatory domain of human TDP1 that mediates nuclear localization via PARP1/XRCC1 interactions) would likely be cytoplasmic, reducing nuclear repair efficiency.

5. **[CRITICAL MECHANISTIC LI...

SENS category: OncoSENS