IF a panel of 8–12 AI-engineered MEF2C MADS-box variants — generated by RFdiffusion motif-scaffolding anchored to the crystallographic DNA-reading helix geometry from PDB structures of MEF2–DNA complexes (PDB: 1EGW, PDB: 6BYY), followed by ProteinMPNN surface-sequence optimization that amplifies positive electrostatic potential at the DNA-contacting face (residues Arg3, Arg15, Lys23, Arg24 and neighboring shell) while preserving wild-type dimerization geometry — are cloned as the MEF2C component of the MΔGΔT polycistronic cassette, packaged in AAV9 under the cardiac fibroblast-selective Postn promoter, and delivered intramyocardially at 5×10¹¹ vg to 22-month-old male and female C57BL/6J mice 7 days after isoproterenol (ISO, 85 mg/kg s.c., ×2)-induced myocardial fibrosis,

THEN the top-performing variant will yield ≥2.5-fold more α-actinin⁺/cTnT⁺ induced cardiomyocytes (iCMs) per unit myocardial area compared with wild-type MΔGΔT-AAV9-Postn at 8 weeks post-injection (quantified by flow cytometry and confocal sarcomere scoring), accompanied by ≥8% absolute improvement in left-ventricular ejection fraction (LVEF) by echocardiography and ≥30% reduction in picrosirius-red-positive fibrotic area, with RNA-seq of sorted iCMs revealing <500 differentially expressed genes (DEGs, FDR<0.05) relative to wild-type MΔGΔT iCMs — indicating preserved cardiac transcriptional specificity rather than global dysregulation —

BECAUSE the following causal chain links the structural engineering to the functional outcome in the aged, epigenetically hostile cardiac fibroblast:

1. Wild-type MEF2C has insufficient pioneer-factor potency in aged fibroblast chromatin. In 22-month-old ISO-injured mice, cardiac fibroblasts harbour compacted heterochromatin and elevated H3K27me3 marks at loci including Myh6, Nppa, and Tnnt2. Wild-type MEF2C's MADS-box binds the canonical YTA(A/T)₄TAR motif with moderate affinity (Kd ~10–50 nM for naked DNA), but this is insufficient to displace the nucleosomal barrier in aged epigenetic contexts. [SPECULATIVE on exact Kd shift in aged fibroblasts, but consistent with reports that wild-type GMT yields drop sharply with donor age]

2. The MEF2–DNA interface is structurally tractable for affinity enhancement without specificity loss. Crystal structures of MEF2–DNA complexes (PDB: 1EGW; PDB: 6BYY) reveal that the MADS-box alpha-helix H1 inserts into the major groove, making direct phosphate-backbone contacts via Arg24 and adenine-specific contacts via Lys23, while Arg3 and Arg15 traverse the minor groove. The electrostatic surface along the concave DNA-binding face is strongly positive and is the primary determinant of association rate — a parameter that is rate-limiting for chromatin invasion. The MEF2 domain (residues ~58–86) does not contact DNA but rigidly positions H1, meaning sequence changes restricted to the electrostatic shell around the DNA interface will not perturb the fold's dimerization geometry. (Evid...

SENS category: GlycoSENS