IF a somatic mosaic murine model is first established by low-efficiency (target ~12–18% hepatocyte transduction) AAV8-CRISPR delivery of the Trp53 R249S allele (AGG→AGT at codon 249) in 10-month-old male C57BL/6J mice — allowing 8 weeks of clonal expansion under natural competitive conditions to generate defined chimeric livers with measurable R249S VAF prior to treatment — and IF this model then receives sequential ABE8e-LNP therapy (MC3 ionizable lipid formulation, 1.5 mg/kg intravenous tail-vein injection on days 0 and 28, encoding ABE8e mRNA + sgRNA targeting the AGT adenine within the A4–A8 editing window) followed by navitoclax senolytic treatment (50 mg/kg oral gavage daily for 14 days, days 42–56),

is administered to this controlled chimeric liver system in 12-month-old C57BL/6J male mice (n=15 per arm),

THEN the sequential combination will achieve ≥55% reduction in hepatocyte Trp53 R249S variant allele frequency (measured by droplet digital PCR and single-cell whole-genome amplification sequencing of 500 hepatocytes per animal at 12 weeks post-treatment), exceed the VAF reduction achieved by either ABE8e-LNP alone (predicted ≤35%) or navitoclax alone (predicted ≤20%), maintain serum ALT below 2× baseline, and produce ≥2-fold upregulation of p21/MDM2 transcripts following 5 mg/kg doxorubicin challenge confirming functional p53 restoration,

BECAUSE the following mechanistic chain operates synergistically:

1. R249S-bearing hepatocytes accumulate Bcl-2 family dependence via oncogene-induced senescence (OIS). Cells harbouring gain-of-function TP53 mutations that impair apoptotic signaling paradoxically upregulate Bcl-2 and Bcl-xL to survive chronic oncogenic stress, rendering them preferentially vulnerable to navitoclax-mediated apoptosis. (Navitoclax senolytic clearance of Bcl-2-dependent senescent hepatocytes is established in aged murine liver) (Chang et al., 2016, Nature Medicine, PMID: 26657143)

2. ABE8e-LNP achieves high on-target A-to-G conversion at the Trp53 codon 249 locus in the first editing window. MC3 ionizable LNP formulations exploit ApoE-mediated LDLR targeting in hepatocytes; a single 1.5 mg/kg IV dose of ABE8e mRNA achieves >60% on-target editing efficiency in murine liver. Repeated dosing at day 28 is predicted to raise cumulative editing to 70–80% of the transiently edited fraction, directly reducing R249S VAF in the corrected subset. (ABE8e-LNP editing efficiency exceeding 60% in murine liver demonstrated) (Richter et al., 2020, PMID: 32661439)

3. The A4 adenine at codon 249 (5′-...NAGT...-3′) falls within the canonical ABE8e editing window (positions 4–8 of the protospacer), enabling precise AGT→AGC conversion to serine (wild-type TP53 at this locus) without requiring the AGG arginine codon present in the human hotspot context. [SPECULATIVE: The murine Trp53 codon 249 PAM landscape requires computational verification with Cas-OFFinder to confirm NG or NGG PAM availability within ≤4 bp...

SENS category: GlycoSENS

Key references: • PMID: 26657143 • PMID: 32661439 • PMID: 28609658 • PMID: 18451130