Gene therapy delivered micro-dystrophins co-localize with transgenic utrophin in dystrophic skeletal muscle fibers

Abstract

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by the absence of functional dystrophin. There are multiple ongoing clinical trials for DMD that are testing gene therapy treatments consisting of adeno-associated viral (AAV) vectors carrying miniaturized versions of dystrophin optimized for function, termed micro-dystrophins (μDys). Utrophin, the fetal homolog of dystrophin, has repeatedly been reported to be upregulated in human DMD muscle as a compensatory mechanism, but whether µDys displaces full-length utrophin is unknown. In this study, dystrophin/utrophin-deficient mice with transgenic overexpression of full-length utrophin in skeletal muscles were systemically administered low doses of either AAV6-CK8e-Hinge3-µDys (μDysH3) or AAV6-CK8e-μDys5 (μDys5). We used immunofluorescence to qualitatively assess the localization of μDys with transgenic utrophin and neuronal nitric oxide synthase (nNOS) in quadriceps muscles. μDys protein resulting from both gene therapies co-localized at myofiber membranes with transgenic utrophin. We also confirmed the sarcolemmal co-localization of nNOS with μDys5, but not with transgenic utrophin expression or μDysH3. Transgenic utrophin expression and μDys proteins produced from both therapies stabilize the dystrophin-glycoprotein complex as observed by sarcolemmal localization of β-dystroglycan. This study suggests that µDys gene therapy will likely not inhibit any endogenous compensation by utrophin in DMD muscle.