IF a dual-vector AAV.cc84 system — Vector-1 carrying GHMT (Gata4, Hand2, Mef2c, Tbx5) under a synthetic TREE-Postn hybrid promoter (injury-responsive enhancer fused to Periostin minimal promoter) and Vector-2 carrying CCNA2 under a TREE-cTnT hybrid promoter — is delivered systemically (tail vein, 1×10¹¹ vg each vector) to aged (20–24 month) male C57BL/6J mice within 72 hours of experimental myocardial infarction (LAD ligation), co-administered with a single intramyocardial injection of the ALK5 inhibitor A83-01 (10 µM, 50 µL),

THEN at 12 weeks post-infarction, treated aged mice will demonstrate: (i) ≥25% reduction in scar fibrosis area (Masson trichrome), (ii) ≥8% absolute improvement in left ventricular ejection fraction (echocardiography) versus AAV-GFP controls, (iii) measurable de novo cardiomyocytes in the border zone co-expressing GHMT-lineage markers (Gata4+/Mef2c+) and CM maturation markers (cTnT+/sarcomeric α-actinin+), and (iv) CCNA2-driven cardiomyocyte cytokinesis events detected by Aurora B kinase midbody immunostaining at 4 weeks — without ventricular arrhythmia by telemetric ECG monitoring,

BECAUSE the following mechanistic chain operates in sequence:

1. Post-MI vascular permeabilization creates a defined 3-day systemic delivery window during which cardiac AAV uptake is transiently enhanced across multiple serotypes (AAV1, AAV6, AAV9) independent of promoter identity, maximizing myocardial vector penetrance at the exact moment fibroblast activation peaks (Myocardial infarction creates a critical time window for AAV gene therapy)[https://doi.org/10.1101/2024.06.10.597311].

2. The AAV.cc84 engineered capsid eliminates hepatic transgene sequestration, redirecting vector dose toward cardiac tissue; liver de-targeting was validated with the AAV.cc84 capsid precisely because systemic AAV delivery of therapeutic payloads is otherwise dose-limited by first-pass hepatic transduction (Spatial and longitudinal tracking of enhancer-AAV vectors)[https://doi.org/10.1101/2025.04.28.651096].

3. TREE (Transcriptional Regulatory Element for Enhancer) sequences provide bioluminescence-trackable, spatially confined, injury-responsive transgene expression limited to the infarct zone, preventing off-target GHMT expression in remote myocardium and non-cardiac organs — a validated injury-gated switch confirmed by longitudinal imaging (Spatial and longitudinal tracking of enhancer-AAV vectors)[https://doi.org/10.1101/2025.04.28.651096].

4. The Postn (Periostin) promoter element, fused downstream of the TREE enhancer in Vector-1, restricts GHMT expression exclusively to activated myofibroblasts within the scar border zone; Periostin is negligible in healthy adult heart but strongly upregulated in injury-activated fibroblasts, providing dual-layer cell-type specificity on top of the injury-responsive TREE element — thereby preventing GHMT-driven dedifferentiation of resident cardiomyocytes (Literature Review Section: Fibroblast-Spec...

SENS category: GlycoSENS

Key references: • doi.org/10.1101/2024.06.10.597311]. • doi.org/10.1101/2025.04.28.651096]. • doi.org/10.1101/2024.07.12.603277]. • doi.org/10.1101/2024.03.01.583057]. • doi.org/10.1038/s41467-018-05059-x].