Recent advances in AAV engineering have yielded capsids with enhanced cardiomyocyte transduction and liver de‑targeting (AAV2‑THGTPAD, AAV2‑NLPGSGD) [1] and hepatocyte‑tropic variants that evade anti‑AAV8 antibodies [2]. Machine‑learning platforms now generate variants with up to 31‑fold increased muscle tropism [3], while carboxypeptidase D (AAVR2) provides an alternative receptor enabling dose reduction [4]. Immune barriers—pre‑existing neutralizing antibodies, TLR9 sensing of CpG motifs, and capsid‑specific CD8+ T‑cell clearance—remain the chief obstacles to systemic rejuvenation therapies [5]. Strategies such as IdeS-mediated IgG cleavage, low‑CpG genomes, and complement inhibition have shown promise in mitigating these responses [6].

We hypothesize that combining a rationally designed capsid that simultaneously engages AAVR2 and a muscle‑enhanced tropic motif, a synthetic promoter containing liver‑detargeting motifs and heart‑specific enhancers, and a genome engineered with reduced CpG content and microRNA‑122/1‑206 target sites will produce durable, multi‑tissue expression of Klotho without triggering innate or adaptive immune activation in aged mice. The mechanistic basis is threefold: (1) AAVR2 usage lowers the required vector dose, reducing innate immune stimulation; (2) incorporation of TLR9‑silencing sequence modifications diminishes plasmacytoid dendritic cell interferon production; (3) tissue‑restrictive promoter and miRNA detargeting limits Klotho expression to cardiomyocytes, skeletal muscle, and hepatocytes, lowering the antigenic load presented to capsid‑specific CD8+ T cells.

To test this hypothesis we will produce four vectors: (A) wild‑type AAV9‑Klotho (control), (B) AAV‑R2‑muscle‑heart capsid with standard promoter, (C) same capsid with low‑CpG synthetic promoter, and (D) capsid plus promoter plus miRNA detargeting sites. Each will be administered intravenously to 20‑month‑old C57BL/6 mice at a dose of 1e11 vg/kg. Primary outcomes measured at 4 and 12 weeks post‑injection include: (i) vector genome copies in heart, skeletal muscle, and liver by qPCR; (ii) Klotho protein levels via ELISA and western blot; (iii) serum cytokines (IFN‑α, IL‑6) and anti‑AAV IgG titers; (iv) cardiac echocardiography, grip strength, and liver function tests. A successful hypothesis will show that vector D achieves ≥2‑fold higher Klotho protein in all three tissues compared to control, with no significant increase in IFN‑α or anti‑AAV titers relative to baseline. Falsification occurs if vector D fails to elevate Klotho above control levels or if it provokes a ≥2‑fold rise in innate immune markers or transgene‑specific CD8+ T‑cell infiltration, indicating that the combined strategies do not overcome immune barriers in aged hosts.