IF a codon-optimized fusion protein comprising Pyrococcus furiosus RNase HII (PfuRNaseHII) C-terminally linked via a flexible (Gly₄Ser)₃ linker to a tandem dual-copy TRF2 TRFH-docking peptide (YRLSP×2) and an SV40 nuclear localization signal (NLS), packaged within an AAV2/9 hybrid capsid, is delivered by intratumoral injection at 1×10¹¹ vector genomes per mouse into established subcutaneous U2OS or Saos-2 xenografts (100–150 mm³) in male NSG mice aged 6–8 weeks,

THEN over 28 days the following will be observed: (1) ≥50% reduction in C-circle abundance by rolling-circle amplification qPCR (baseline sensitivity 0.1 attomoles circular template), (2) ≥40% reduction in ALT-associated PML body (APB) frequency per nucleus (scoring ≥200 nuclei per condition by PML/TRF2 co-immunofluorescence), and (3) ≥3-fold increase in telomere dysfunction-induced foci (TIF) measured by γH2AX/telomeric FISH co-localization, relative to AAV2/9-GFP control-injected xenografts,

BECAUSE the following causal chain operates:

1. ATRX loss elevates TERRA transcription and R-loop accumulation at telomeres. In ATRX-null U2OS and Saos-2 cells, failure to deposit histone H3.3 at telomeres abolishes heterochromatic repression, leading to pervasive TERRA upregulation and the chronic formation of telomeric RNA:DNA hybrids that are the mechanistic substrate of the ALT pathway (Arora et al., 2014, Science, as reviewed in Evidence Set).

2. TERRA R-loops directly nucleate the BIR-based recombination events that produce C-circles and sustain APBs. R-loop formation at telomeres loads RAD52 and drives break-induced replication, generating extrachromosomal C-circles and assembling APBs as recombination hubs; enzymatic resolution of these hybrids by RNase H1 overexpression markedly reduces both C-circle abundance and APB frequency, confirming causal dependence (Arora et al., 2014; Clynes et al., 2015; RNase H1 overexpression abolishes TERRA-induced telomeric fragility)[https://pmc.ncbi.nlm.nih.gov/articles/PMC12361433/].

3. The YRLSP peptide docks with nanomolar affinity into the hydrophobic groove of the TRF2 TRFH domain. Crystallographic data demonstrate that the TIN2-derived YRLSP sequence adopts an extended conformation with its Tyr and Leu side chains buried in the TRFH hydrophobic pocket, while the Arg engages surface acidic residues; this interaction is structurally orthogonal to TRF1 TRFH, preventing shelterin crosstalk (Fairall et al., Molecular Cell, 2008, as reviewed in Evidence Set). A tandem YRLSP×2 configuration [SPECULATIVE: valency expected to increase telomeric dwell time] will concentrate PfuRNaseHII at the very chromosomal compartment where TERRA R-loops accumulate.

4. PfuRNaseHII cleaves specifically at RNA-DNA junctions rather than processively along the hybrid, conferring substrate discrimination that limits off-target nuclear RNA:DNA hybrid cleavage. Unlike Type I RNases H (including human RNase H1 and TthRNH1), which degrade RNA moieties proc...

SENS category: OncoSENS