IF ABE8e-NRCH mRNA (the expanded-PAM adenine base editor variant demonstrated to enable noncanonical PAM targeting) co-encapsulated with a SMARCAL1 Exon 3-targeting sgRNA in ionizable lipid nanoparticles (ALC-0315:DSPC:cholesterol:PEG-2000-DMG at 50:10:38.5:1.5 molar ratio; N/P ratio = 6; 2 μg total nucleic acid per 100,000 cells in 24-well format) is delivered to ALT-positive U2OS osteosarcoma cells in vitro and as a weekly 1 mg/kg IV booster regimen in NSG subcutaneous xenograft mice,

THEN the following will be observed:

• ≥70% A-to-G editing efficiency at the target adenine(s) within SMARCAL1 Exon 3 (measured by targeted amplicon sequencing at 72 hours), specifically converting tryptophan codon TGG to a stop codon TAG/TGA via A•T→G•C transition at a locus inaccessible to standard NGG-PAM ABE8e but reachable by NRCH-PAM expansion;
• 2–5 fold increase in C-circle abundance (quantitative dot blot, normalized to genomic DNA) at days 5–10 post-delivery;
• Elevated APB frequency (>30% APB-positive nuclei per 200 counted vs. <10% in controls, by PML/TRF2 co-immunofluorescence);
• Pronounced G2/M arrest (>40% cells in G2/M by PI/BrdU flow cytometry) and mitotic catastrophe (≥3-fold increase in multinucleated/micronucleated cells by DAPI scoring);
• ≥50% reduction in xenograft tumor volume versus vehicle-treated controls at 8 weeks post-implantation,

BECAUSE the following causal chain operates:

1. The NRCH PAM variant of ABE8e expands the adenine editing window at SMARCAL1 Exon 3 beyond what canonical SpCas9 (NGG-PAM) ABE8e can access. The SCN8A therapeutic base editing study demonstrates that ABE8e-NRCH paired with an NRCH-compatible Cas variant (where N = any base, R = A or G, H = A/C/T) enables targeting of noncanonical PAM sequences, unlocking adenines in positions not reachable with standard PAM editors. (ABE8e-NRCH enables noncanonical PAM usage for therapeutic editing)[https://doi.org/10.1101/2025.04.09.647983] This is critical because TGG→TAG/TGA stop codon conversions in SMARCAL1 Exon 3 may lie in editing windows accessible only through non-NGG PAMs, meaning canonical ABE8e would miss these optimal stop-codon targets.

2. LNP-mediated mRNA delivery bypasses the nuclear entry bottleneck that limits plasmid-based editing in hard-to-transfect U2OS cells. The validated ionizable LNP formulation (50:10:38.5:1.5 molar ratio, N/P = 6) achieves >90% nucleic acid encapsulation, 60–80 nm particle diameter, and PDI <0.2, enabling efficient endosomal escape. The split-TadA-8e architecture demonstrated 42–93% editing with plasmid delivery in U2OS (Wang et al., 2023–2024), and LNP-mRNA delivery removes the rate-limiting nuclear entry step, predicting ≥70% efficiency when dosed at 300 ng total RNA per 100,000 cells. (Split-TadA-8e achieved 42–93% editing in U2OS with plasmid delivery)[Evidence Set: Wang et al. 2023-2024, reported in Literature Task Output]

3. **SMARCAL1 loss-of-function is synthetically lethal in ALT-positive cells but not normal...

SENS category: OncoSENS

Key references: • doi.org/10.1101/2025.04.09.647983] • doi.org/10.1101/2025.04.09.647983],