Thursday, April 3, 2025
4/3/2025
Developing DWORF gene therapy to treat heart failure and muscular dystrophy - PROJECT SUMMARY Calcium is an essential regulator of muscle function and its dysregulation contributes significantly to the common pathogenic signaling cascades that drive multiple striated muscle diseases including heart failure and muscular dystrophy. The sarcoplasmic reticulum (SR) is the major calcium storage site in muscle and SR calcium loading is achieved by the SR calcium-ATPase (SERCA). Cardiomyocytes from human heart failure patients show robust calcium defects including decreased systolic calcium transients, increased diastolic calcium, and SR calcium leak, which contributes to impaired contraction/relaxation and can induce arrhythmias. Muscular dystrophy is a family of genetic disorders caused by mutations in genes that serve a structural role in stabilizing the membrane of skeletal muscle myofibers and cardiomyocytes. These mutations result in membrane instability, rupture, and unregulated calcium influx leading to mitochondrial calcium overload and myocyte necrosis. Numerous studies have shown that in heart failure and muscular dystrophy, calcium homeostasis can be restored by enhancing SERCA activity or expression, thereby reducing cytosolic calcium and increasing SR calcium stores, resulting in significant protection from disease. We recently discovered a novel SR membrane microprotein, DWORF, that binds to SERCA and potently activates its calcium transport activity. In mice, DWORF overexpression in the heart leads to robust SERCA activation, increased SR calcium uptake and SR calcium stores, faster cytosolic calcium clearance, and increased cardiomyocyte contractility. Additionally, DWORF overexpression by cardiac- specific transgene or adeno-associated virus (AAV9)-mediated gene delivery has proven to be cardioprotective in experimental and genetic mouse models of heart failure. Notably, a significant limitation to previous AAV9- DWORF gene therapy studies in mice has been the reliance on gene delivery in neonates to achieve meaningful cardiomyocyte infectivity, as adult cardiac and skeletal muscle are not efficiently targeted by AAV9. A recent breakthrough in the field was achieved with the development of the muscle-tropic MyoAAV capsid, which is significantly more potent than any current AAV in transducing cardiac and skeletal muscle. Here we will leverage the optimized MyoAAV system to comprehensively assess the therapeutic potential of DWORF gene therapy in mouse models of striated muscle disease with diverse etiologies where calcium dysregulation plays a primary role in disease pathogenesis. Aim 1 will use two distinct mouse models of heart failure, myocardial infarction and pressure overload, which mimic different clinically relevant scenarios. Aim 2 will employ a robust model of muscular dystrophy caused by combined loss of dystrophin and utrophin, and analysis will focus on both cardiac and skeletal muscle. Aim 3 will utilize a newly developed humanized mouse model of monogenic phospholamban disease, which presents with calcium defects that lead to ventricular arrhythmias. In all 3 Aims, we expect that MyoAAV-DWORF gene therapy will protect from cardiac and skeletal muscle disease pathogenesis and these findings will inform on future translational studies in large animals and human gene therapy trials.
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