Hypothesis

We hypothesize that targeting senescence-associated lncRNAs to guide CRISPR-dCas9-KRAB epigenetic silencing of inflammatory SASP genes (e.g., IL6, IL8) while sparing reparative SASP components (VEGF, TGFB1) will decouple harmful inflammation from tissue repair when combined with intermittent NAD+ boosting to sustain sirtuin activity.

Rationale

SASP heterogeneity drives both pathology and healing; current senolytics/senomorphics lack selectivity Senolytics improve physical function Senomorphics dampen inflammation. Epigenetic regulators like BRD4 can uncouple SASP transcription from cell-cycle arrest Epigenetic regulation via BRD4. Senescence-associated lncRNAs are upregulated in senescent cells and retain chromatin specificity, offering a guide for locus-specific repression. Meanwhile, intermittent NAD+ precursors elevate SIRT1 activity, enhancing autophagy and mitochondrial resilience without chronic mTOR inhibition AMPK activation boosting NAD+. Metformin’s ROS‑lowering action via NADPH oxidase 4 shows that senostatic effects can be separated from AMPK signaling Metformin senostatic via NOX4. Combining lncRNA‑dCas9‑KRAB with pulsed NAD+ should therefore silence deleterious SASP while preserving beneficial secretome.

Predictions

1. In human fibroblast cultures induced senescent by irradiation, lncRNA‑dCas9‑KRAB will reduce IL6 and IL8 secretion by >70% without decreasing VEGF or TGFB1 levels (measured by ELISA).
2. Adding intermittent NAD+ (e.g., nicotinamide riboside 250 mg/kg every 3 days) will further increase SIRT1-dependent deacetylation of p65, lowering NF‑κB activity without affecting mTORC2‑dependent Akt phosphorylation.
3. In aged mice, topical delivery of the lncRNA‑dCas9‑KRAB vector plus NAD+ cycling will improve wound closure rate by 30% compared with senolytic dasatinib+quercetin alone, while serum IL‑6 remains low.
4. Off‑target RNA‑seq will show no global transcriptional dysregulation, confirming specificity.

Experimental Design

• Identify senescence-upregulated lncRNAs via RNA‑seq of IR‑induced senescent fibroblasts; select candidates with nuclear enrichment and SASP-proximal chromatin loops (Hi‑C data).
• Clone dCas9‑KRAB under a doxycycline‑inducible promoter; fuse to MS2 coat protein and engineer lncRNA to contain MS2 stem loops for recruitment.
• Treat cells with doxycycline to induce repression; collect supernatant at 24, 48, 72 h for cytokine profiling.
• Parallel groups receive NAD+ precursor (NR) on a 3-days-on/4-days-off cycle; measure SIRT1 activity, autophagic flux (LC3-II), and mitochondrial membrane potential (JC-1).
• In vivo, use progeroid Ercc1-/- mice; deliver AAV9 carrying the lncRNA‑dCas9‑KRAB construct intravenously; administer NR via drinking water on the same intermittent schedule.
• Assess wound healing (full‑thickness excisional wounds), serum SASP biomarkers, and histology for senescence (p16, SA-β-gal) over 8 weeks.
• Include control groups: vehicle, dasatinib+querticin senolytic, continuous NAD+, and scrambled lncRNA guide.
• Statistical analysis: two‑way ANOVA with post‑hoc Tukey; power calculation to detect 20% difference with n=8 per group.

If predictions hold, the approach offers a precision SASP‑modulating strategy that separates detrimental inflammation from regenerative capacity, addressing a key translational gap in senescence therapy.