Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects.

Author summary Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. Utrophin is structurally similar to dystrophin and can potentially be utilized to prevent muscle necrosis in preclinical models of DMD. Consequently, utrophin-mediated therapies are a primary target for treating DMD, particularly as it may circumvent immune responses to dystrophin-mediated therapies. One promising therapeutic option is to utilize recombinant adeno-associated viral vectors (rAAV) to deliver a rationally designed miniaturized utrophin (mu Utrn) to striated muscles to prevent necrosis. Here, we found that rAAV-mu Utrn can profoundly prevent skeletal muscle necrosis in the mdx(4cv) mouse model of DMD. mu Utrn was also able to replace many functions of dystrophin at the neuromuscular synapse and myotendinous junctions. However, we provide several lines of in vivo evidence that steric hindrance between mu Utrn and the endogenous full-length utrophin on the sarcolemma impacts the longevity of the therapy in a muscle fiber-type selective manner. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy. Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (mu Utrn) therapy in the mdx(4cv) mouse model of DMD. We found that rAAV-mu Utrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for mu Utrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed mu Utrn. As such, we found that mu Utrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike mu Utrn, mu dystrophin (mu Dys) expression did not show this fiber type preference. Interestingly, mu Utrn was better able to protect 2a and 2d fibers in mdx:utrn(-/-) mice than in mdx(4cv) mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & mu Utrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.