IF a codon-optimized fusion protein comprising the thermostable Thermus thermophilus RNase H1 (TthRNH1) appended C-terminally to the TRF2 TRFH-domain minimal binding peptide (YRLSP), delivered via intratumoral injection of AAV2/9 hybrid capsid vector at 1×10¹¹ vector genomes per animal, is administered to NSG mice bearing established subcutaneous U2OS or Saos-2 osteosarcoma xenografts (100–150 mm³ volume at treatment initiation),

THEN over a 28-day window the following quantifiable outcomes will be observed: (1) ≥50% reduction in extrachromosomal C-circle abundance measured by φ29-based rolling circle amplification qPCR (baseline sensitivity 0.1 attomoles); (2) ≥40% reduction in ALT-associated PML body (APB) frequency per nucleus scored by dual PML/TRF2 immunofluorescence across ≥200 nuclei per condition (from a baseline of 8–12 APBs per APB-positive nucleus); and (3) ≥3-fold increase in telomere dysfunction-induced foci (TIFs) measured by γH2AX / telomeric FISH co-localization, reflecting synthetic lethality in ALT-dependent cells specifically deprived of their R-loop scaffold,

BECAUSE the following mechanistic chain operates:

1. The YRLSP peptide (consensus YxLxP) docks into the hydrophobic groove of the TRF2 TRFH domain through a high-affinity protein–protein interaction, physically anchoring the TthRNH1 catalytic domain at shelterin-coated telomeric chromatin in ATRX-null cells where TERRA R-loops are pathologically elevated. (TRF2 TRFH domain minimal binding motif mediates telomere localization)[https://doi.org/10.1038/ncomms6220]

2. In ATRX-null ALT cells (U2OS, Saos-2), loss of ATRX-mediated G-quadruplex remodeling permits TERRA transcripts to co-invade the complementary telomeric DNA template, forming stable RNA:DNA hybrids exceeding the ≥4-consecutive-ribonucleotide threshold required by prokaryotic Type-1 RNase H enzymes. TthRNH1 therefore specifically targets these extended TERRA R-loops rather than single ribonucleotide misincorporations, providing a substrate-selectivity advantage over mammalian RNase H1, which processes both. (RNase H1 depletion strongly stabilizes telomeric RNA–DNA hybrids in U2OS but not HeLa cells)[https://doi.org/10.1038/ncomms6220]

3. TthRNH1, a thermostable prokaryotic Type-1 endonuclease, retains significant hydrolytic activity at 37°C despite optimal activity above 60°C, and its elevated structural rigidity compared to mesophilic homologs confers resistance to proteasomal and lysosomal unfolding within the tumor microenvironment. [SPECULATIVE: the assumption that thermal stability translates to enhanced intracellular half-life in a rapidly proliferating tumor cell has not been directly measured but follows from the protein stability literature on thermophilic enzyme engineering.]

4. Enzymatic degradation of TERRA R-loops by the anchored TthRNH1-YRLSP fusion collapses the RNA:DNA hybrid scaffold that is required to initiate break-induced replication (BIR) at ALT telomeres; without this scaffold, the R...

SENS category: OncoSENS

Key references: • doi.org/10.1038/ncomms6220] • doi.org/10.1101/2025.01.09.632133] • doi.org/10.1038/ncomms6220 • doi.org/10.1101/2025.01.09.632133 • doi.org/10.1038/ncomms6220].