Restoring circadian amplitude in liver, adipose, and skeletal muscle via AAV-delivered BMAL1 and CLOCK re-synchronizes systemic NAD+ cycles, boosting SIRT1 activity and mitigating age-related mitochondrial decline and inflammation.

We hypothesize that a triple‑tissue AAV cocktail—AAV9‑BMAL1 to liver, AAV1‑BMAL1 to skeletal muscle, and AAV6‑BMAL1 to adipose—will reinstate coherent circadian transcription of NAMPT and NAD+‑dependent deacetylases across these metabolic hubs. Elevated NAD+ will activate SIRT1 and SIRT3, enhancing mitochondrial oxidative capacity, reducing ROS production, and suppressing NF‑κB‑driven inflammasome signaling. Consequently, treated aged mice should show improved glucose tolerance, increased endurance, and lower circulating IL‑6 and TNF‑α compared with controls receiving single‑tissue or empty vectors.

To test this, we will inject 20‑month‑old C57BL/6J mice with the triple‑tissue AAV mix or appropriate controls and monitor circadian gene expression (Bmal1, Clock, Per2) in each tissue via qPCR at 4‑hour intervals over 24 h. NAD+ levels will be measured by LC‑MS, SIRT1 activity by fluorometric assay, and mitochondrial respiration in isolated myofibers and hepatocytes using Seahorse. Systemic phenotypes will be assessed by intraperitoneal glucose tolerance test, treadmill exhaustion time, and plasma cytokine multiplex. A successful outcome is defined as ≥20 % increase in NAD+ amplitude, ≥15 % improvement in glucose clearance, and ≥30 % reduction in pro‑inflammatory cytokines relative to baseline aged controls. Failure to observe any of these changes despite confirmed transgene expression will falsify the hypothesis that circadian gene rescue via AAV drives systemic geroprotection.

Mechanistically, we propose that circadian‑driven NAD+ oscillations act as a timing cue for SIRT1‑mediated deacetylation of PGC‑1α and FOXO1, aligning mitochondrial biogenesis with feeding‑fasting cycles. When this temporal coupling is lost, SIRT1 activity becomes constitutive and less effective, leading to acetyl‑protein accumulation and mitochondrial inefficiency. By reinstating rhythmic NAD+, AAV‑BMAL1 restores the pulsatile activation of SIRT1, thereby re‑establishing proper protein acetylation cycles and downstream antioxidant defenses. This mechanism explains why targeting downstream effectors such as FGF21 or GDE yields limited benefits—they correct metabolic output but not the upstream temporal disorder that governs multiple pathways simultaneously.

The approach leverages well‑established AAV tropism: AAV9 shows superior liver transduction 1, AAV1/7 excel in skeletal muscle 1, and AAV2/6 achieve highest adipocyte transduction with 1.5‑2‑fold advantages over alternatives 2. Prior work demonstrates that AAV8‑FGF21 reduces adiposity 3 and that AAV9 gene therapy synergizes with rapamycin 4, supporting the feasibility of combining AAV platforms with other interventions. Moreover, AAV’s non‑pathogenic profile and low genomic integration risk make it suitable for chronic, multi‑tissue delivery 5.

If validated, this strategy would redefine aging as a loss of temporal coherence that can be pharmacologically repaired, shifting focus from isolated tissue repair to whole‑organism clock restoration.