Monitoring disease activity noninvasively in the <em>mdx</em> model of Duchenne muscular dystrophy

Antonio Filareto

Katie Maguire-Nguyen

Qiang Gan

Garazi Aldanondo

Léo Machado

Jeffrey Chamberlain

Thomas Rando

Supplementary Material

Supplementary File

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^{Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, 94305;}

^{Paul F. Glenn Center for the Biology of Aging, Stanford University School of Medicine, Stanford, CA, 94305;}

^{Center for Tissue Regeneration, Repair and Restoration, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, 90304;}

^{Neuroscience Area, Biodonostia Research Institute, 20014 San Sebastian, Spain;}

^{Faculte de Medecine, Universite Paris Est Creteil, 94000 Creteil, France;}

^{Department of Neurology, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, WA, 98195}

^{To whom correspondence should be addressed. Email:}ude.drofnats@odnar.

Edited by Louis M. Kunkel, Boston Children’s Hospital, Harvard Medical School, Boston, MA, and approved June 15, 2018 (received for review February 20, 2018)

Author contributions: A.F., K.M.-N., and T.A.R. designed research; A.F., K.M.-N., Q.G., G.A., and L.M. performed research; A.F., K.M.-N., Q.G., G.A., and T.A.R. analyzed data; J.S.C. provided the rAAV vectors and critical input throughout the project and in the writing of the manuscript; and A.F., K.M.-N., and T.A.R. wrote the paper.

^{A.F. and K.M.-N. contributed equally to this work.}

Edited by Louis M. Kunkel, Boston Children’s Hospital, Harvard Medical School, Boston, MA, and approved June 15, 2018 (received for review February 20, 2018)

Published under the PNAS license.

Significance

Duchenne muscular dystrophy (DMD) is characterized by a catastrophic progression of muscle degeneration that leads to early death. Currently, there is neither a cure for DMD nor any treatments that effectively halt muscle degeneration. Novel methods that assess disease activity non-invasively would greatly accelerate the development of effective therapies. Here we present a novel preclinical animal model that uses bioluminescence imaging as a read-out of muscle degeneration, therefore constituting a non-invasive method to assess disease progression and response to experimental gene therapy in the mdx model of DMD. This model should be widely applicable for monitoring disease activity and responses to therapy in mouse models of muscular dystrophy.

Keywords: monitoring disease activity, DMD, BLI, muscle degeneration, muscle regeneration

Significance

Abstract

Duchenne muscular dystrophy (DMD) is a rare, muscle degenerative disease resulting from the absence of the dystrophin protein. DMD is characterized by progressive loss of muscle fibers, muscle weakness, and eventually loss of ambulation and premature death. Currently, there is no cure for DMD and improved methods of disease monitoring are crucial for the development of novel treatments. In this study, we describe a new method of assessing disease progression noninvasively in the mdx model of DMD. The reporter mice, which we term the dystrophic Degeneration Reporter strains, contain an inducible CRE-responsive luciferase reporter active in mature myofibers. In these mice, muscle degeneration is reflected in changes in the level of luciferase expression, which can be monitored using noninvasive, bioluminescence imaging. We monitored the natural history and disease progression in these dystrophic report mice and found that decreases in luciferase signals directly correlated with muscle degeneration. We further demonstrated that this reporter strain, as well as a previously reported Regeneration Reporter strain, successfully reveals the effectiveness of a gene therapy treatment following systemic administration of a recombinant adeno-associated virus-6 (rAAV-6) encoding a microdystrophin construct. Our data demonstrate the value of these noninvasive imaging modalities for monitoring disease progression and response to therapy in mouse models of muscular dystrophy.

Abstract

Duchenne muscular dystrophy (DMD) is the most common muscle genetic disorder, with an incidence of ∼1 in 5,000 live male births (1, 2). The disease is caused by mutations in the gene coding for dystrophin (3). The lack of dystrophin protein leads to membrane damage associated with fiber necrosis and inflammation, ultimately resulting in progressive muscle degeneration and weakness (4 –7). Symptom onset begins in early childhood, usually between the ages of 3 and 5 (3, 8). Boys with DMD present with inability to run and difficulty in rising from the floor, in the first 5 y of life. The combination of muscle weakness, degeneration, and contractures leads to loss of independent walking (9, 10).

There is currently no cure for DMD, nor is there any effective treatment to reverse or halt the muscle degeneration. Since 1987, when the genetic defect was identified (3, 11), many promising therapeutic strategies, including pharmacological, genetic, and cell-based approaches, have been tested in several animal models of DMD (12 –17). Of the hundreds of experimental therapies that have been tested, only a few have been approved by the Food and Drug Administration and are currently in phase III clinical trials (18).

One of the hurdles in developing cures or treatments for DMD is the lack of efficient, time-saving, and simple assays that assess the effectiveness of a therapeutic intervention. Most commonly, the outcomes of therapeutic interventions are determined by histologic analysis of acute or cumulative muscle damage, such as areas of necrosis, regeneration, and fibrosis; serum markers of muscle breakdown, such as creatine kinase levels; or muscle function in either isolated muscles or living animals, such as grip strength. Although all of these methods have been used extensively, they present disadvantages in that they are labor intensive, poorly sensitive to changes in disease activity and therefore of limited quantitative value, terminal for the experimental animals, or some combination of these. Thus, the development of new methods to monitor disease progression is crucial for the assessment of novel treatments for DMD.

Noninvasive imaging modalities have a number of distinct advantages, including offering the potential to monitor disease activity in living animals. This allows for repetitive serial measurements without having to sacrifice animals at each time point, and it permits longitudinal monitoring of response to a therapeutic intervention. To date, there are a very limited number of approaches for monitoring disease activity in dystrophic muscles in living animals. Among the noninvasive imaging modalities that have been used to follow disease progression in the muscular dystrophies are magnetic resonance imaging (MRI) and fluorescence optical imaging (19 –23). Bioluminescence imaging (BLI) has been applied extensively to the monitoring of many different diseases, particularly cancer, in living animals because of its remarkable sensitivity combined with relative special resolution. Although requiring transgenesis, this technology has been widely applied to studies of temporal progression of disease and it offers great advantages over the other approaches. It is accessible, accurate, relatively inexpensive, and easy to use (24, 25). Furthermore, BLI offers fast, sensitive imaging, even detecting microscopic activity (26, 27). This thereby permits disease activity to be tracked temporally and noninvasively.

Recently, we developed a transgenic reporter mouse strain (which we termed the “regeneration reporter” strain) to test whether we could assess muscle disease activity and progression noninvasively in living animals over time (26). By conditionally expressing luciferase in muscle stem cells (MuSCs), or satellite cells, of the SJL strain [a dysferlin-deficient mouse strain that is an animal model for the human disease, limb girdle muscular dystrophy 2B (LGMD2B)], we could monitor muscle regeneration as a surrogate marker of disease activity using BLI of MuSC activity. Indeed, the BLI readout correlated with the temporal progression and spatial distribution of muscle degeneration. This strain is thus a reliable and quantitative reporter, even if indirect, of disease activity.

In this report, we describe a model, which we term the “degeneration reporter” strain, in which muscle degeneration can be directly monitored using BLI by conditionally expressing luciferase in the myofiber compartment of muscle. Crossing this strain with a dystrophic mouse strain (the mdx mouse), we were able to monitor disease activity directly using BLI. Furthermore, we validated the value of this strain (as well as the previously described regeneration reporter) to faithfully report changes in disease progression in response to experimental therapy. By delivering microdystrophin to the muscle using an rAAV vector (28), we found that the decline (for the degeneration reporter) or increase (for the regeneration reporter) of the BLI signal ceased following effective gene therapy. These data demonstrate that these reporter strains are reliable and are quantitative tools to monitor the efficacy of experimental therapeutics for mouse models of muscular dystrophy noninvasively in living mice.

Supplementary File

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Acknowledgments

We thank Dr. Martin Guess for technical assistance with the hydrodynamic tail vein injections. This work was supported by grants from the Muscular Dystrophy Association and the Duchenne Parent Project Netherlands (to T.A.R.).

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802425115/-/DCSupplemental.

Footnotes

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