The CARDIOREPAIR consortium achieved major technical and scientific progress toward functional variant interpretation and therapeutic development for RBM20-associated dilated cardiomyopathy. In objective one, we achieved comprehensive RBM20 mutagenesis libraries covering more than 4,500 single variants across the RRM, RS-rich, and E-rich domains and robustly identified all known RS-domain mislocalization hotspots and uncovered additional unknown variants that drive cytoplasmic RBM20 translocation. Furthermore, we also identified all variants that cause mis-splicing by establishing a high-throughput splicing dependent cell sorting approach. This revealed that all strongly mislocalized variants are splicing-deficient, but also identified variants with impaired splicing despite normal localization. A newly discovered E-rich domain hotspot, including clinically annotated pathogenic variants, suggests an additional RBM20 functional region potentially involved in protein–protein interactions or spliceosome assembly. The resulting screening scores clearly separated (likely) pathogenic from (likely) benign clinical variants and help to reclassify variants of uncertain significance.
In objective two, our genome-editing strategies, the mutation-agnostic RS-stretch replacement and NLS insertion approaches using paired prime editing proved mechanistically feasible but currently lack sufficient precision, delivery efficiency, and therapeutic robustness. These findings led to a strategic pivot toward mutation-specific base or prime editing, with optimized ENVLPE-based delivery achieving up to 60% precise editing in iPSC-derived cardiomyocytes. We also advanced our therapeutic strategy to a proof-of-concept by developing a RBM20 specific nuclear localization approach, that simultaneously dissolved cytoplasmic RBM20 granules, partially rescued splicing, improved ejection fraction, reduced mortality, and promoted reverse remodelling in RBM20-DCM mouse models.
Lastly, we incorporated our first HopOn grant partner to the consortium to study the mechanical regulation of RBM20 in the human heart.
