Author(s): Alexis R. Demonbreun, Northwestern University; Katherine S. Fallon, Northwestern University; Mattia Quattrocelli, Northwestern University; Patrick G.T. Page, Northwestern University; Michele Hadhazy, Northwestern University; Carl Morris, Solid Biosciences; Elizabeth M. McNally, Northwestern University //

ABSTRACT: Background and Methods:    Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by dystrophin mutations.  DMD is treated with glucocorticoid steroids to delay disease progression. Antisense oligonucleotides have now been approved to treat certain DMD mutations, and gene replacement therapy is being evaluated in clinical trials.  A genomewide scan was conducted in a mouse model of muscular dystrophy and identified a powerful modifier locus, which was ultimately identified as Ltbp4, encoding Latent TGF- Binding Protein 4.  LTBP4 protein localizes to the myofiber exterior where it binds and sequesters all three forms of TGF-beta, regulating latent TGF-beta release and activation.  Excess TGF-beta activation is a pathological finding in many forms of neuromuscular disease, especially DMD, the limb girdle muscular dystrophies and the congenital muscular dystrophies.  In the muscular dystrophies, excess or hyper-activated TGF-beta is linked to fibrotic infiltration of muscle and impaired muscle regeneration. The genetic data from mice identified an insertion/deletion polymorphism in the hinge region of LTBP4 as critical to latent TGF-beta release and activation. LTBP4’s hinge region can be proteolytically cleaved, and this cleavage promotes release of latent TGF-beta which is then fully activated by additional steps. The genetically protective form of LTBP4 in mice contains 12 amino acids inserted into the hinge, rendering it less susceptible to protease cleavage, correlating with a reduction in the hyperactive TGF-beta state in muscular dystrophy.  Similarly, human protective LTBP4 polymorphisms encode a protein that results in tighter binding to latent TGF-beta leading to reduced TGF-beta signaling.  In humans, the protective effect of LTBP4 correlated with longer ambulation in three independent DMD cohorts, illustrating the strong modifying effect of this pathway.     Results:    We developed antibodies to stabilize the LTBP4 hinge and limit latent TGF-beta release. The anti-LTBP4 antibodies have a high affinity for the target (LTBP4 hinge) and inhibit LTBP4 proteolysis thereby reducing TGF-beta activation. In isolated live myofibers, anti-LTBP4 antibody stabilized the sarcolemma from injury, while in vivo anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by muscle contraction. In a long-term in vivo study, anti-LTBP4 treatment reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, we show that co-administration of anti-LTBP4 antibody in combination with glucocorticoid acts synergistically to reduce the susceptibility of dystrophic muscle injury-induced force impairment.    Conclusions:  Together, these data demonstrate that genomic signals can be used to define mechanisms of action of genetic modifiers and to drive development of biologic agents to reduce muscle fibrosis and increase muscle performance in muscular dystrophy.

Source of Funding: This work was supported by National Institutes of Health HL140938 (EMM), AR052646 (EMM), DK121875 (MQ), Parent Project Muscular Dystrophy (EMM), the Muscular Dystrophy Association (MQ), Department of Defense W81XWH-17-DMDRP-IIRA (EMM) and a sponsored research agreement from Solid Biosciences to Northwestern University (EMM).