Abstract 4370250: Regulatory Profiling of LMNA Identifies Therapeutic Targets for LMNA-Associated Dilated Cardiomyopathy

Introduction/Background: LMNA (encoding gene lamin A/C) cardiomyopathy is associated with high morbidity and mortality. LMNA loss-of-function variants frequently lead to dilated cardiomyopathy with high arrhythmic burden, necessitating transplantation or resulting in heart failure. While in vivo genome editing holds promise for preventing development of cardiomyopathy, correction of each rare variant requires custom-designed editors, limiting general applicability. Mapping the regulatory landscape of LMNA by identifying transcriptional activators and repressors and modulating LMNA expression may enable a broader therapeutic approach. Research Question/Hypothesis: We hypothesize that chromatin accessibility data in cardiomyocytes can be used to locate LMNA promoters and enhancers, facilitating identification of key transcription factors (TFs) involved in regulating LMNA expression and restoring normal Lamin A/C levels. Methods/Approach: After aligning ATAC-seq data from iPSC-derived cardiomyocytes, we identified 12 candidate regulatory peaks near the LMNA transcription start site (TSS): 3 promoters, 6 enhancers, and 4 CTCF binding sites. To prioritize these regions, we integrated ChIA-PET and Hi-C data. Functional validation was performed using CRISPR interference (CRISPRi: dCas9-KRAB) and CRISPR activation (CRISPRa: dCas9-VP64) with gRNAs targeting these candidate loci. Additionally, we trained a gapped k-mer support vector machine (gkm-SVM) model on our ATAC-seq data to predict key TFs based on chromatin accessibility in iPSCs and iPSC-derived cardiomyocytes, cross-referencing predictions with ENCODE heart tissue datasets. Results: CRISPRi targeting of several candidate sites resulted in significant downregulation of LMNA expression in vitro. The gkm-SVM model identified TFs active in both iPSCs and iPSC-derived cardiomyocytes, including TBX2, SP1, AP1, SREBP1 and NFY as putative regulators of LMNA. siRNA knockdown of these TFs confirmed their role, each leading to a significant decrease in LMNA expression. Conclusion: We identified and functionally validated key regulatory elements and transcription factors governing LMNA expression in cardiomyocytes. Both CRISPRi and siRNA experiments indicate SP1, AP1, SREBP1, and NFY as key regulators of LMNA expression. These findings define a regulatory framework for targeted LMNA modulation to build a potential therapeutic strategy for treating LMNA-related dilated cardiomyopathy.