IF a two-component AAV9 vector system encoding (1) a computationally redesigned MEF2C variant (hereafter "sMEF2C"), generated via RFdiffusion motif-scaffolding of the MADS-MEF2 domain (residues 1–86, anchored on PDB: 1EGW/6BYY backbone) followed by ProteinMPNN sequence optimization and AlphaFold2 structural validation — with mutations targeting the DNA-recognition α-helix for increased affinity at the CTA(A/T)₄TAG consensus and the hydrophobic cofactor-docking groove for enhanced p300/CBP recruitment and steric exclusion of class IIa HDACs — and (2) a dCas9-p300 fusion guided by a pool of three sgRNAs targeting H3K27me3-silenced promoter/enhancer regions of MYH6, TNNT2, and ACTC1 respectively, is delivered by direct intramyocardial injection (10¹¹ vg per construct) into aged C57BL/6J mice (18–22 months, both sexes) 72 hours after surgically induced myocardial infarction (LAD ligation), alongside wildtype GATA4 and TBX5 (GHMT backbone),

THEN the efficiency of direct cardiac fibroblast-to-induced cardiomyocyte (iCM) conversion will increase from the current 3–9% baseline observed with wildtype GMT/GHMT to ≥25% cTnT⁺/αMHC⁺ cells at 8 weeks post-delivery, with durable (≥12-week) genome-wide H3K27ac deposition and reciprocal H3K27me3 eviction confirmed at the three targeted loci, measurable by CUT&RUN and ATAC-seq, and accompanied by a ≥15% improvement in left ventricular ejection fraction (LVEF) by echocardiography relative to wildtype GMT/GHMT controls,

BECAUSE the following causal chain is supported by the evidence:

1. H3K27me3 marks deposited by EZH2 at MYH6, TNNT2, and ACTC1 promoters constitute the primary chromatin-level barrier that physically prevents wildtype GMT/GHMT pioneer factors from binding and activating the cardiac transcriptional program, with TGFβ/BRD4 signaling in the post-infarct milieu further sequestering H3K27 demethylases away from these cardiac promoters, locking silencing even as exogenous TFs are delivered — a self-reinforcing loop that explains why efficiency plateaus below 9% regardless of wildtype factor dosing (Direct cardiac reprogramming efficiency is limited by H3K27me3 and EZH2/TGFβ signaling)[https://pmc.ncbi.nlm.nih.gov/articles/PMC10347886/].

2. dCas9-p300 fusion proteins, when guided to specific loci, deposit H3K27ac and catalyze local chromatin opening at previously silenced genes in a locus-specific, programmable fashion; unlike systemic EZH2 inhibitors (e.g., GSK126) or pan-HDAC blockers, this approach achieves permanent, locus-resolved desilencing that persists after dCas9 protein turnover because the deposited H3K27ac mark is propagated through subsequent cell divisions via BRD4 reader recruitment — the distinction between transient pharmacological derepression and stable epigenetic editing is the critical unmet gap in the field (No CRISPR epigenome editing has been applied to permanently desilence cardiac loci in the reprogramming context)[https://pmc.ncbi.nlm.nih.gov/articles/PM...

SENS category: GlycoSENS