Genomic Engineering of Gene Dosage: A Generalizable Framework for Modeling Haploinsufficiency-Mediated Human Disorders through Splicing Modulation

Abstract

Heterozygous loss-of-function variants causing gene dosage reduction underlie many human genetic disorders, yet preclinical mouse models frequently fail to recapitulate human disease phenotypes due to post-translational compensation. Here, we present a generalizable framework to modulate gene dosage by alternative splicing via genome editing. By shifting proportions of transcripts toward nonsense-mediated decay, this approach enables precise titration of functional protein levels. Applying this concept to Mybpc3, we combined hypomorphic splice-altering alleles to overwhelm post-translational buffering in mice, successfully reproducing hallmark structural and functional features of human hypertrophic cardiomyopathy. Extending this concept, we engineered a portable Modulated Alternative Splice Cassette (MASC) inserted into Dsp, yielding patient-level protein reductions and characteristic arrhythmogenic cardiomyopathy pathologies, including subepicardial cardiac fibrosis and immune cell infiltration. These novel haploinsufficient models of Mybpc3 hypertrophic cardiomyopathy and Dsp arrhythmogenic cardiomyopathy will critically enable mechanistic and therapeutic testing under protein stoichiometry conditions that closely replicate the patient disorders. Finally, systematic in silico predictive modeling across 645 human haploinsufficiency-associated genes demonstrated the generalizability of MASC insertion effects on splicing across diverse tissue types. Notably, the framework developed here leaves endogenous gene expression regulatory logic intact, allowing these models to be used to develop and validate therapeutic approaches that target transcription. Overall, this strategy provides a scalable roadmap for engineering high-fidelity animal models for human diseases caused by haploinsufficiency.