Hypothesis

We hypothesize that AAV capsids modified with a senescence‑activated, cleavable polyethylene glycol (PEG) shield will exhibit reduced liver transduction and increased transduction of senescent‑rich tissues (skeletal muscle, brain) in aged organisms, thereby improving the efficacy of systemically delivered partial reprogramming factors (OSK) without raising vector dose.

Mechanistic Rationale

Aged tissues accumulate senescent cells that overexpress senescence‑associated β‑galactosidase (SA‑β‑gal) and remodel the extracellular matrix with increased heparan sulfate proteoglycans and fibronectin. Conventional liver‑detargeted AAVs (e.g., AAV9‑PHP.B) still sequester a substantial fraction in the liver due to remnant affinity for hepatocyte surface receptors and scavenger uptake in sinusoidal endothelial cells. By grafting a short PEG chain onto the capsid surface via a peptide linker cleaved specifically by SA‑β‑gal, we create a "stealth‑to‑senescent" switch: in young or non‑senescent tissues the PEG remains intact, shielding the capsid from nonspecific interactions and preserving low liver uptake; in senescent microenvironments SA‑β‑gal removes the PEG, exposing the underlying capsid and allowing enhanced binding to upregulated integrins (αvβ3) and heparan sulfate motifs present in damaged matrix.

This design leverages two recent advances: (1) machine‑learning‑driven capsid optimization that can incorporate peptide linkers while maintaining structural fidelity [1, 2], and (2) immune‑modulation strategies such as IdeS/imlifidase that enable repeat dosing [3]. Manufacturing scalability remains addressable through high‑density perfusion bioreactors capable of >1.6×10¹⁵ VG/L [5].

Testable Predictions

1. In vitro: Incubation of PEG‑shielded AAV with lysates from senescent fibroblasts will result in measurable PEG loss (Western blot for PEG) and a ≥2‑fold increase in binding to integrin αvβ3‑coated plates compared with non‑senescent lysates.
2. Biodistribution in young mice (3‑month): Liver‑detargeted AAV‑PEG and standard liver‑detargeted AAV will show comparable low liver transduction (<5 % of total vector genomes) and negligible differences in muscle/brain transduction.
3. Biodistribution in aged mice (24‑month): PEG‑shielded AAV will achieve a ≥1.8‑fold higher vector genome copy number in gastrocnemius and cortex relative to standard liver‑detargeted AAV, while liver transduction remains unchanged or further reduced (<3 % of total).
4. Functional outcome: Delivery of OSK via PEG‑shielded AAV in aged mice will yield a ≥30 % reduction in p16^Ink4a^‑positive senescent cells in muscle and brain after 8 weeks, accompanied by improved grip strength and spontaneous activity, whereas standard liver‑detargeted AAV‑OSK will produce <10 % reduction.
5. Falsifiability: If PEG‑shielded AAV does not show increased transduction in senescent tissues or liver transduction remains elevated relative to controls, the hypothesis is refuted.

Experimental Plan

• Capsid production: Use AAVGen generative models to design an AAV9 backbone with a surface‑exposed SA‑β‑gal cleavable linker (Gly‑Ser‑Ser‑Lys‑PEG₅). Produce vectors via optimized dual‑plasmid transfection in HEK293F cells and purify by iodixanol gradient followed by anion‑exchange chromatography.
• In vitro assays: Confirm PEG attachment via MALDI‑TOF, verify SA‑β‑gal cleavage using purified enzyme and senescent fibroblast lysate, quantify integrin binding via ELISA.
• In vivo studies: Cohorts of young (n=10) and aged (n=10) C57BL/6 mice receive intravenous infusion of 1×10¹¹ vg of either PEG‑shielded AAV‑GFP or control liver‑detargeted AAV‑GFP. Harvest tissues at 2 weeks for qPCR, immunohistochemistry for GFP, and senescence markers (SA‑β‑gal, p16^Ink4a^).
• Therapeutic test: Separate cohorts receive AAV‑OSK (PEG‑shielded vs control) at the same dose; assess senescence, fibrosis, and functional endpoints at 8 and 16 weeks.
• Statistical analysis: Two‑way ANOVA with factors age and vector type; significance set at p<0.05.

Implications

If validated, this approach would provide a generalized strategy to redirect AAV therapeutics toward pathological tissues characterized by senescence‑linked enzymatic activity, enabling lower vector doses, reducing hepatic toxicity, and facilitating repeated administration of complementary longevity payloads (senolytics, telomerase, etc.). It directly addresses the translational barriers of immunogenicity and manufacturing by leveraging existing high‑yield production platforms while adding a microenvironment‑responsive targeting layer that is absent from current liver‑detargeted designs.