Evaluation of calcium handling and dwarf open reading frame AAV gene therapy in Duchenne muscular dystrophy
Abstract
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease. Increased concentrations of cytosolic calcium in dystrophic muscle cells has been implicated in DMD pathogenesis. In healthy muscle, calcium from within the sarcoplasmic reticulum (SR) is released to the cytosol to initiate muscle contraction and is returned to the SR by the sarcoendoplasmic reticulum calcium ATPase (SERCA) during relaxation. In DMD, SR calcium release is elevated, while calcium uptake by SERCA is reduced. Strategies that improve SERCA function hold promise to reduce cytosolic calcium levels and treat DMD. In the present work, we explored the role of a small peptide SERCA enhancer, dwarf open reading frame (DWORF), in dystrophic muscle. We found DWORF was significantly reduced in the heart and diaphragm of the mdx mouse model of DMD. Following this discovery, we delivered 6 x 1012 vg particles/mouse of an AAV9.DWORF vector to 6-week-old mdx mice by tail vein injection. Because SERCA over-activation has been reported to reduce muscle function, we also injected wild-type (WT) mice, whose DWORF levels are already significantly higher than in mdx mice. We found that AAV DWORF gene therapy (1) improved SERCA uptake, heart function, and fibrosis in mdx hearts, (2) reduced SERCA uptake, worsened heart function, and induced expression of the SERCA inhibitor sarcolipin in WT hearts, and (3) did not improve muscle force production but slowed maximum rates of contraction and relaxation in mdx diaphragm. Additionally, we assessed the transcript levels of several calcium handling proteins in the canine model of DMD. We found transcriptional differences between normal and dystrophic muscles and between male and female muscles. Our findings are the first to show DWORF downregulation plays a role in SERCA dysfunction in dystrophic muscle and that AAV DWORF gene therapy is a promising technique to treat DMD, especially DMD cardiomyopathy. Our findings also highlight the importance of considering animal age and sex in dystrophic canine studies.
Degree
Ph. D.