Publications by authors named "Jeffrey S Chamberlain"

138 Publications

Micro-dystrophin gene therapy prevents heart failure in an improved Duchenne muscular dystrophy cardiomyopathy mouse model.

JCI Insight 2021 Apr 8;6(7). Epub 2021 Apr 8.

Department of Physiology & Cell Biology and Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio, USA.

Gene replacement for Duchenne muscular dystrophy (DMD) with micro-dystrophins has entered clinical trials, but efficacy in preventing heart failure is unknown. Although most patients with DMD die from heart failure, cardiomyopathy is undetectable until the teens, so efficacy from trials in young boys will be unknown for a decade. Available DMD animal models were sufficient to demonstrate micro-dystrophin efficacy on earlier onset skeletal muscle pathology underlying loss of ambulation and respiratory insufficiency in patients. However, no mouse models progressed into heart failure, and dog models showed highly variable progression insufficient to evaluate efficacy of micro-dystrophin or other therapies on DMD heart failure. To overcome this barrier, we have generated the first DMD mouse model to our knowledge that reproducibly progresses into heart failure. This model shows cardiac inflammation and fibrosis occur prior to reduced function. Fibrosis does not continue to accumulate, but inflammation persists after function declines. We used this model to test micro-dystrophin gene therapy efficacy on heart failure prevention for the first time. Micro-dystrophin prevented declines in cardiac function and prohibited onset of inflammation and fibrosis. This model will allow identification of committed pathogenic steps to heart failure and testing of genetic and nongenetic therapies to optimize cardiac care for patients with DMD.
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http://dx.doi.org/10.1172/jci.insight.146511DOI Listing
April 2021

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

PLoS Genet 2020 11 11;16(11):e1009179. Epub 2020 Nov 11.

Department of Neurology, University of Washington, Seattle, Washington, United States of America.

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 (μUtrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-μUtrn 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 μUtrn 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 μUtrn. As such, we found that μUtrn 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 μUtrn, μdystrophin (μDys) expression did not show this fiber type preference. Interestingly, μUtrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv 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 & μUtrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.
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http://dx.doi.org/10.1371/journal.pgen.1009179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682874PMC
November 2020

Dystrophin Gene-Editing Stability Is Dependent on Dystrophin Levels in Skeletal but Not Cardiac Muscles.

Mol Ther 2021 03 5;29(3):1070-1085. Epub 2020 Nov 5.

Department of Neurology, University of Washington School of Medicine, Seattle, WA 98109-8055, USA; Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine, Seattle, WA 98109-8055, USA; Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98109-8055, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA 98109-8055, USA.

Gene editing is often touted as a permanent method for correcting mutations, but its long-term benefits in Duchenne muscular dystrophy (DMD) may depend on sufficiently high editing efficiencies to halt muscle degeneration. Here, we explored the persistence of dystrophin expression following recombinant adeno-associated virus serotype 6 (rAAV6):CRISPR-Cas9-mediated multi-exon deletion/reframing in systemically injected 2- and 11-week-old dystrophic mice and show that induction of low dystrophin levels persists for several months in cardiomyocytes but not in skeletal muscles, where myofibers remain susceptible to necrosis and regeneration. Whereas gene-correction efficiency in both muscle types was enhanced with increased ratios of guide RNA (gRNA)-to-nuclease vectors, obtaining high dystrophin levels in skeletal muscles via multi-exon deletion remained challenging. In contrast, when AAV-microdystrophin was codelivered with editing components, long-term gene-edited dystrophins persisted in both muscle types. These results suggest that the high rate of necrosis and regeneration in skeletal muscles, compared with the relative stability of dystrophic cardiomyocytes, caused the rapid loss of edited genomes. Consequently, stable dystrophin expression in DMD skeletal muscles will require either highly efficient gene editing or the use of cotreatments that decrease skeletal muscle degeneration.
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http://dx.doi.org/10.1016/j.ymthe.2020.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934576PMC
March 2021

Micro-dystrophin AAV Vectors Made by Transient Transfection and Herpesvirus System Are Equally Potent in Treating mdx Mouse Muscle Disease.

Mol Ther Methods Clin Dev 2020 Sep 9;18:664-678. Epub 2020 Jul 9.

Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USA.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 10 (high), 5 × 10 (medium), or 5 × 10 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.
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http://dx.doi.org/10.1016/j.omtm.2020.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403893PMC
September 2020

Correction to: Expressing a Z-disk nebulin fragment innebulin-deficient mouse muscle: effects on muscle structure and function.

Skelet Muscle 2020 Apr 20;10(1). Epub 2020 Apr 20.

Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, 85721, USA.

Following the publication of this paper [1], it was brought to the authors' attention that one of the contributing authors was left off of the paper. The authors apologize for the unfortunate oversight. In this correction paper, they have included Dr. Paola Tonino in the author list section.
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http://dx.doi.org/10.1186/s13395-020-00223-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168831PMC
April 2020

Gene Therapy Rescues Cardiac Dysfunction in Duchenne Muscular Dystrophy Mice by Elevating Cardiomyocyte Deoxy-Adenosine Triphosphate.

JACC Basic Transl Sci 2019 Nov 2;4(7):778-791. Epub 2019 Oct 2.

Department of Neurology, University of Washington, Seattle, Washington.

Mutations in the gene encoding for dystrophin leads to structural and functional deterioration of cardiomyocytes and is a hallmark of cardiomyopathy in Duchenne muscular dystrophy (DMD) patients. Administration of recombinant adeno-associated viral vectors delivering microdystrophin or ribonucleotide reductase (RNR), under muscle-specific regulatory control, rescues both baseline and high workload-challenged hearts in an aged, DMD mouse model. However, only RNR treatments improved both systolic and diastolic function under those conditions. Cardiac-specific recombinant adeno-associated viral treatment of RNR holds therapeutic promise for improvement of cardiomyopathy in DMD patients.
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http://dx.doi.org/10.1016/j.jacbts.2019.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978556PMC
November 2019

Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function.

Skelet Muscle 2020 01 28;10(1). Epub 2020 Jan 28.

Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, 85721, USA.

Background: Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease.

Methods: We studied whether sarcomeric structure and function can be improved by introducing nebulin's Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied.

Results: Intramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression.

Conclusions: This study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.
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http://dx.doi.org/10.1186/s13395-019-0219-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986074PMC
January 2020

Surrogate gene therapy for muscular dystrophy.

Nat Med 2019 10;25(10):1473-1474

University of Washington School of Medicine, Departments of Neurology, Medicine and Biochemistry, Wellstone Muscular Dystrophy Specialized Research Center, Seattle, Washington, USA.

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http://dx.doi.org/10.1038/s41591-019-0604-2DOI Listing
October 2019

High levels of AAV vector integration into CRISPR-induced DNA breaks.

Nat Commun 2019 09 30;10(1):4439. Epub 2019 Sep 30.

Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.
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http://dx.doi.org/10.1038/s41467-019-12449-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769011PMC
September 2019

AAV-based gene therapies for the muscular dystrophies.

Hum Mol Genet 2019 10;28(R1):R102-R107

Department of Neurology, University of Washington, Seattle, WA, USA.

Muscular dystrophy (MD) is a group of progressive genetic diseases affecting the musculature that are characterized by inflammatory infiltrates, necrosis and connective tissue and fat replacement of the affected muscles. Unfortunately, treatments do not exist for the vast majority of MD patients. Adeno-associated viral vector (AAV)-based gene therapy is thus emerging as a potential treatment for many types of MD. Treatments strategies based on AAV are being adapted for replacement of mutant disease-causing genes, knockdown of dominant disease-causing genes using antisense oligonucleotides or inhibitory RNAs, delivery of gene editing tools such as clustered regularly interspaced short palindromic repeats/Cas9 and effecting alterations in pre-mRNA splicing and by manipulating expression levels of modifier genes. Translational and clinical trial work focused on these types of AAV treatments for Duchenne MD, various limb girdle MDs, myotonic dystrophy 1, facioscapulohumeral MD, dysferlinopathies and congenital MDs are discussed here, with a focus on recent studies, pre-clinical large animal work and many promising ongoing and upcoming AAV clinical trials.
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http://dx.doi.org/10.1093/hmg/ddz128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796995PMC
October 2019

Stabilization of the cardiac sarcolemma by sarcospan rescues DMD-associated cardiomyopathy.

JCI Insight 2019 04 30;5. Epub 2019 Apr 30.

Department of Integrative Biology & Physiology and.

In the current preclinical study, we demonstrate the therapeutic potential of sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN transgenic mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography reveals SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN transgenic mdx mice by hemodynamic pressure-volume methods highlights enhanced systolic performance compared to mdx controls. SSPN restores cardiac sarcolemma stability, the primary defect in DMD disease, reduces fibrotic response and improves contractile function. We demonstrate that SSPN ameliorates more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and β1D integrin that mitigate disease severity and partially restores responsiveness to β-adrenergic stimulation. Overall, our current and previous findings suggest SSPN overexpression in DMD mouse models positively impacts skeletal, pulmonary and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.
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http://dx.doi.org/10.1172/jci.insight.123855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629091PMC
April 2019

Development of Novel Micro-dystrophins with Enhanced Functionality.

Mol Ther 2019 03 1;27(3):623-635. Epub 2019 Feb 1.

Molecular and Cellular Biology Program, University of Washington School of Medicine, Seattle, WA 98195, USA; Department of Neurology, University of Washington School of Medicine, Seattle, WA 98195, USA; Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Seattle, WA 98195, USA; Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA. Electronic address:

Gene therapies using adeno-associated viral (AAV) vectors have advanced into clinical trials for several diseases, including Duchenne muscular dystrophy (DMD). A limitation of AAV is the carrying capacity (∼5 kb) available for genes and regulatory cassettes (RCs). These size constraints are problematic for the 2.2-Mb dystrophin gene. We previously designed a variety of miniaturized micro-dystrophins (μDys) that displayed significant, albeit incomplete, function in striated muscles. To develop μDys proteins with improved performance, we explored structural modifications of the dystrophin central rod domain. Eight μDys variants were studied that carried unique combinations of between four and six of the 24 spectrin-like repeats present in the full-length protein, as well as various hinge domains. Expression of μDys was regulated by a strong but compact muscle-restricted RC (CK8e) or by the ubiquitously active cytomegalovirus (CMV) RC. Vectors were evaluated by intramuscular injection and systemic delivery to dystrophic mdx mice, followed by analysis of skeletal muscle pathophysiology. Two μDys designs were identified that led to increased force generation compared with previous μDys while also localizing neuronal nitric oxide synthase to the sarcolemma. An AAV vector expressing the smaller of these (μDys5) from the CK8e RC is currently being evaluated in a DMD clinical trial.
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http://dx.doi.org/10.1016/j.ymthe.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403485PMC
March 2019

Micro-utrophin Improves Cardiac and Skeletal Muscle Function of Severely Affected D2/ Mice.

Mol Ther Methods Clin Dev 2018 Dec 16;11:92-105. Epub 2018 Oct 16.

Oxford Neuromuscular Centre at the University of Oxford, Department of Physiology, Anatomy and Genetics, Oxford OX1 3PT, UK.

Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disease caused by mutations in the dystrophin gene. DMD boys are wheelchair-bound around 12 years and generally survive into their twenties. There is currently no effective treatment except palliative care, although personalized treatments such as exon skipping, stop codon read-through, and viral-based gene therapies are making progress. Patients present with skeletal muscle pathology, but most also show cardiomyopathy by the age of 10. A systemic therapeutic approach is needed that treats the heart and skeletal muscle defects in all patients. The dystrophin-related protein utrophin has been shown to compensate for the lack of dystrophin in the mildly affected BL10/ mouse. The purpose of this investigation was to demonstrate that AAV9-mediated micro-utrophin transgene delivery can not only functionally replace dystrophin in the heart, but also attenuate the skeletal muscle phenotype in severely affected D2/ mice. The data presented here show that utrophin can indeed alleviate the pathology in skeletal and cardiac muscle in D2/ mice. These results endorse the view that utrophin modulation has the potential to increase the quality life of all DMD patients whatever their mutation.
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http://dx.doi.org/10.1016/j.omtm.2018.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216100PMC
December 2018

Non-invasive tracking of disease progression in young dystrophic muscles using multi-parametric MRI at 14T.

PLoS One 2018 26;13(10):e0206323. Epub 2018 Oct 26.

Department of Radiology, University of Washington, Seattle, WA, United States of America.

In this study, multi-parametric magnetic resonance imaging (MRI) was conducted to monitor skeletal muscle changes in dystrophic (mdx4cv) and age-matched control (C57BL/6J) mice starting at 3 weeks of age. The objective of this study was to evaluate and characterize changes in muscle tissue characteristics of hind limbs in young, dystrophic mice using MRI. Mdx4cv (n = 25) and age-matched C57BL/6J (n = 5) were imaged at 3, 5, 7, 9, and 11 weeks of age. Multiple MR measurements were taken from the tibialis anterior, gastrocnemius, and soleus muscles. There were significant differences between dystrophic and control groups for all three muscle types when comparing transverse relaxation times (T2) in lower hind limb muscles. Additionally, fractional anisotropy, radial diffusivity, and eigenvalue analysis of diffusion tensor imaging also demonstrated significant differences between groups. Longitudinal relaxation times (T1) displayed no significant differences between groups. The earliest time points in the magnetization transfer ratio measurements displayed a significant difference. Histological analysis revealed significant differences in the tibialis anterior and gastrocnemius muscles between groups with the mdx mice displaying greater variability in muscle fiber size in later time points. The multi-parametric MRI approach offers a promising alternative for future development of a noninvasive avenue for tracking both disease progression and treatment response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206323PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203357PMC
April 2019

Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy.

Proc Natl Acad Sci U S A 2018 09 4;115(39):E9182-E9191. Epub 2018 Sep 4.

Department of Neurology, Stanford University, Stanford, CA 94304;

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTR mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.
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http://dx.doi.org/10.1073/pnas.1808648115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166850PMC
September 2018

Cas9 immunity creates challenges for CRISPR gene editing therapies.

Nat Commun 2018 08 29;9(1):3497. Epub 2018 Aug 29.

Department of Neurology, University of Washington, Seattle, WA, 98195-7720, USA.

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http://dx.doi.org/10.1038/s41467-018-05843-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115392PMC
August 2018

Monitoring disease activity noninvasively in the model of Duchenne muscular dystrophy.

Proc Natl Acad Sci U S A 2018 07 9;115(30):7741-7746. Epub 2018 Jul 9.

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

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 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.
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http://dx.doi.org/10.1073/pnas.1802425115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6064991PMC
July 2018

Syntrophin binds directly to multiple spectrin-like repeats in dystrophin and mediates binding of nNOS to repeats 16-17.

Hum Mol Genet 2018 09;27(17):2978-2985

Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290, USA.

Mutation of the gene encoding dystrophin leads to Duchenne and Becker muscular dystrophy (DMD and BMD). Currently, dystrophin is thought to function primarily as a structural protein, connecting the muscle cell actin cytoskeleton to the extra-cellular matrix. In addition to this structural role, dystrophin also plays an important role as a scaffold that organizes an array of signaling proteins including sodium, potassium, and calcium channels, kinases, and nitric oxide synthase (nNOS). Many of these signaling proteins are linked to dystrophin via syntrophin, an adapter protein that is known to bind directly to two sites in the carboxyl terminal region of dystrophin. A search of the dystrophin sequence revealed three additional potential syntrophin binding sites (SBSs) within the spectrin-like repeat (SLR) region of dystrophin. Binding assays revealed that the site at SLR 17 bound specifically to the α isoform of syntrophin while the site at SLR 22 bound specifically to the β-syntrophins. The SLR 17 α-SBS contained the core sequence known to be required for nNOS-dystrophin interaction. In vitro and in vivo assays indicate that α-syntrophin facilitates the nNOS-dystrophin interaction at this site rather than nNOS binding directly to dystrophin as previously reported. The identification of multiple SBSs within the SLR region of dystrophin demonstrates that this region functions as a signaling scaffold. The signaling role of the SLR region of dystrophin will need to be considered for effective gene replacement or exon skipping based DMD/BMD therapies.
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http://dx.doi.org/10.1093/hmg/ddy197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097012PMC
September 2018

Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins.

Hum Mol Genet 2018 06;27(12):2090-2100

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

Delivery of miniaturized dystrophin genes via adeno-associated viral vectors is one leading approach in development to treat Duchenne muscular dystrophy. Here we directly compared the functionality of five mini- and micro-dystrophins via skeletal muscle-specific transgenic expression in dystrophin-deficient mdx mice. We evaluated their ability to rescue defects in the microtubule network, passive stiffness and contractility of skeletal muscle. Transgenic mdx mice expressing the short dystrophin isoform Dp116 served as a negative control. All mini- and micro-dystrophins restored elevated detyrosinated α-tubulin and microtubule density of mdx muscle to values not different from C57BL/10, however, only mini-dystrophins restored the transverse component of the microtubule lattice back to C57BL/10. Passive stiffness values in mdx muscles expressing mini- or micro-dystrophins were not different from C57BL/10. While all mini- and micro-dystrophins conferred significant protection from eccentric contraction-induced force loss in vivo and ex vivo compared to mdx, removal of repeats two and three resulted in less protection from force drop caused by eccentric contraction ex vivo. Our data reveal subtle yet significant differences in the relative functionalities for different therapeutic constructs of miniaturized dystrophin in terms of protection from ex vivo eccentric contraction-induced force loss and restoration of an organized microtubule lattice.
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http://dx.doi.org/10.1093/hmg/ddy113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985723PMC
June 2018

AAV6 Vector Production and Purification for Muscle Gene Therapy.

Methods Mol Biol 2018 ;1687:257-266

Department of Neurology, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington, Seattle, WA, 98195-7720, USA.

Vectors derived from adeno-associated viruses (AAV) have been generated using numerous naturally occurring and synthetic serotypes of the virus. Such vectors have proven to be extremely useful for a variety of gene transfer studies, both in vitro and in vivo, and are increasingly being used in gene therapy protocols for a variety of human disorders. Methods to produce AAV vectors typically rely on co-transfection of several different plasmid vectors that carry the transgene of interest (the gene to be delivered , in a "transfer plasmid") and helper genes needed for AAV vector replication and packaging (helper plasmids). While the methods used to generate AAV are conceptually simple, minor variations in a variety of steps can result in significant differences in the overall yield of vector. Here we describe protocols for generating vectors derived from AAV6, which are particularly useful for gene transfer to muscle tissues.
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http://dx.doi.org/10.1007/978-1-4939-7374-3_18DOI Listing
June 2018

A Five-Repeat Micro-Dystrophin Gene Ameliorated Dystrophic Phenotype in the Severe DBA/2J-mdx Model of Duchenne Muscular Dystrophy.

Mol Ther Methods Clin Dev 2017 Sep 27;6:216-230. Epub 2017 Jul 27.

Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.

Micro-dystrophins are highly promising candidates for treating Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. Here, we report robust disease rescue in the severe DBA/2J-mdx model with a neuronal nitric oxide synthase (nNOS)-binding micro-dystrophin vector. 2 × 10 vector genome particles/mouse of the vector were delivered intravenously to 10-week-old mice and were evaluated at 6 months of age. Saturated micro-dystrophin expression was detected in all skeletal muscles and the heart and restored the dystrophin-associated glycoprotein complex and nNOS. In skeletal muscle, therapy substantially reduced fibrosis and calcification and significantly attenuated inflammation. Centronucleation was significantly decreased in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles but not in the quadriceps. Muscle function was normalized in the TA and significantly improved in the EDL muscle. Heart histology and function were also evaluated. Consistent with the literature, DBA/2J-mdx mice showed myocardial calcification and fibrosis and cardiac hemodynamics was compromised. Surprisingly, similar myocardial pathology and hemodynamic defects were detected in control DBA/2J mice. As a result, interpretation of the cardiac data proved difficult due to the confounding phenotype in control DBA/2J mice. Our results support further development of this microgene vector for clinical translation. Further, DBA/2J-mdx mice are not good models for Duchenne cardiomyopathy.
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http://dx.doi.org/10.1016/j.omtm.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596503PMC
September 2017

Translation of Cardiac Myosin Activation with 2-deoxy-ATP to Treat Heart Failure via an Experimental Ribonucleotide Reductase-Based Gene Therapy.

JACC Basic Transl Sci 2016 Dec;1(7):666-679

Department of Bioengineering, University of Washington, Seattle, Washington, USA.

Despite recent advances, chronic heart failure remains a significant and growing unmet medical need, reaching epidemic proportions carrying substantial morbidity, mortality, and costs. A safe and convenient therapeutic agent that produces sustained inotropic effects could ameliorate symptoms, and improve functional capacity and quality of life. We discovered small amounts of 2-deoxy-ATP (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin crossbridge cycling with greater force generation during each contraction. We describe the rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy that upregulates ribonucleotide reductase, the rate-limiting enzyme for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with no toxicity or safety concerns identified to-date. Further animal studies are being conducted with the goal of testing this agent in patients with heart failure.
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http://dx.doi.org/10.1016/j.jacbts.2016.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444879PMC
December 2016

Progress toward Gene Therapy for Duchenne Muscular Dystrophy.

Mol Ther 2017 05 15;25(5):1125-1131. Epub 2017 Apr 15.

Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington, Seattle, WA 98195, USA; Department of Neurology, University of Washington, Seattle, WA 98195, USA. Electronic address:

Duchenne muscular dystrophy (DMD) has been a major target for gene therapy development for nearly 30 years. DMD is among the most common genetic diseases, and isolation of the defective gene (DMD, or dystrophin) was a landmark discovery, as it was the first time a human disease gene had been cloned without knowledge of the protein product. Despite tremendous obstacles, including the enormous size of the gene and the large volume of muscle tissue in the human body, efforts to devise a treatment based on gene replacement have advanced steadily through the combined efforts of dozens of labs and patient advocacy groups. Progress in the development of DMD gene therapy has been well documented in Molecular Therapy over the past 20 years and will be reviewed here to highlight prospects for success in the imminent human clinical trials planned by several groups.
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http://dx.doi.org/10.1016/j.ymthe.2017.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417844PMC
May 2017

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

Nat Commun 2017 02 14;8:14454. Epub 2017 Feb 14.

Department of Neurology, University of Washington, Seattle, Washington 98195-7720, USA.

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.
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http://dx.doi.org/10.1038/ncomms14454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316861PMC
February 2017

Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell homeostasis improving muscle regeneration.

Skelet Muscle 2016 4;6:34. Epub 2016 Oct 4.

Department of Cellular, Molecular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309 USA.

Background: The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells, required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressed in satellite cells, supports communication with the niche, providing cell interactions and signals to maintain quiescent satellite cells.

Results: Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophic mice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscle aging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulate Pax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellite cell homeostasis and satellite cell homing to the niche.

Conclusions: Manipulating syndecan-3 provides a promising target for development of therapies to enhance muscle regeneration in muscular dystrophies and in aged muscle.
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http://dx.doi.org/10.1186/s13395-016-0104-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064903PMC
October 2017

Prosurvival Factors Improve Functional Engraftment of Myogenically Converted Dermal Cells into Dystrophic Skeletal Muscle.

Stem Cells Dev 2016 10 7;25(20):1559-1569. Epub 2016 Sep 7.

1 Department of Neurology, University of Washington , Seattle, Washington.

In Duchenne muscular dystrophy (DMD) and other muscle wasting disorders, cell therapies are a promising route for promoting muscle regeneration by supplying a functional copy of the missing dystrophin gene and contributing new muscle fibers. The clinical application of cell-based therapies is resource intensive, and it will therefore be necessary to address key limitations that reduce cell engraftment into muscle tissue. A pressing issue is poor donor cell survival following transplantation, which in preclinical studies limits the ability to effectively test the impact of cell-based therapy on whole muscle function. We, therefore, sought to improve engraftment and the functional impact of in vivo myogenically converted dermal fibroblasts (dFbs) using a prosurvival cocktail (PSC) that includes heat shock followed by treatment with insulin-like growth factor-1, a caspase inhibitor, a Bcl-XL peptide, a K channel opener, basic fibroblast growth factor, Matrigel, and cyclosporine A. Advantages of dFbs include compatibility with the autologous setting, ease of isolation, and greater proliferative potential than DMD satellite cells. dFbs expressed tamoxifen-inducible MyoD and carried a mini-dystrophin gene driven by a muscle-specific promoter. After transplantation into muscles of mdx mice, a 70% reduction in donor cells was observed by day 5, and a 94% reduction by day 28. However, treatment with PSC gave a nearly three-fold increase in donor cells in early engraftment, and greatly increased the number of donor-contributed muscle fibers and total engrafted area in transplanted muscles. Furthermore, dystrophic muscles that received dFbs with PSC displayed reduced injury with eccentric contractions and an increase in maximum isometric force. Thus, enhancing survival of myogenic cells increases engraftment and improves structure and function of dystrophic muscle.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035915PMC
http://dx.doi.org/10.1089/scd.2016.0136DOI Listing
October 2016

Gene Therapy for Duchenne muscular dystrophy.

Expert Opin Orphan Drugs 2015;3(11):1255-1266. Epub 2015 Oct 6.

University of Washington, Wellstone Muscular Dystrophy Research Center, Department of Neurology, Seattle, WA, 98195-7720, USA.

Introduction: Duchenne muscular dystrophy (DMD) is a relatively common inherited disorder caused by defective expression of the protein dystrophin. The most direct approach to treating this disease would be to restore dystrophin production in muscle. Recent progress has greatly increased the prospects for successful gene therapy of DMD, and here we summarize the most promising developments.

Areas Covered: Gene transfer using vectors derived from adeno-associated virus (AAV) has emerged as a promising method to restore dystrophin production in muscles bodywide, and represents a treatment option applicable to all DMD patients. Using information gleaned from PubMed searches of the literature, attendance at scientific conferences and results from our own lab, we provide an overview of the potential for gene therapy of DMD using AAV vectors including a summary of promising developments and issues that need to be resolved prior to large-scale therapeutic implementation.

Expert Opinion: Of the many approaches being pursued to treat DMD and BMD, gene therapy based on AAV-mediated delivery of microdystrophin is the most direct and promising method to treat the cause of the disorder. The major challenges to this approach are ensuring that microdystrophin can be delivered safely and efficiently without eliciting an immune response.
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http://dx.doi.org/10.1517/21678707.2015.1088780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651452PMC
October 2015

Progress and prospects of gene therapy clinical trials for the muscular dystrophies.

Hum Mol Genet 2016 Apr 8;25(R1):R9-17. Epub 2015 Oct 8.

Department of Neurology and

Clinical trials represent a critical avenue for new treatment development, where early phases (I, I/II) are designed to test safety and effectiveness of new therapeutics or diagnostic indicators. A number of recent advances have spurred renewed optimism toward initiating clinical trials and developing refined therapies for the muscular dystrophies (MD's) and other myogenic disorders. MD's encompass a heterogeneous group of degenerative disorders often characterized by progressive muscle weakness and fragility. Many of these diseases result from mutations in genes encoding proteins of the dystrophin-glycoprotein complex (DGC). The most common and severe form among children is Duchenne muscular dystrophy, caused by mutations in the dystrophin gene, with an average life expectancy around 25 years of age. Another group of MD's referred to as the limb-girdle muscular dystrophies (LGMDs) can affect boys or girls, with different types caused by mutations in different genes. Mutation of the α-sarcoglycan gene, also a DGC component, causes LGMD2D and represents the most common form of LGMD. Early preclinical and clinical trial findings support the feasibility of gene therapy via recombinant adeno-associated viral vectors as a viable treatment approach for many MDs. In this mini-review, we present an overview of recent progress in clinical gene therapy trials of the MD's and touch upon promising preclinical advances.
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http://dx.doi.org/10.1093/hmg/ddv420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802376PMC
April 2016