Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

In vivo selection of synthetic nucleocapsids for tissue targeting.

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.

Duchenne muscular dystrophy models show their age.

The lack of appropriate animal models has hampered efforts to develop therapies for Duchenne muscular dystrophy (DMD). A new mouse model lacking both dystrophin and telomerase (Sacco et al., 2010) closely mimics the pathological progression of human DMD and shows that muscle stem cell activity is a key determinant of disease severity.

Comparison of dystrophin expression following gene editing and gene replacement in an aged preclinical DMD animal model.

Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6-8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.

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

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.

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

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.

AAV-based gene therapies for the muscular dystrophies.

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.

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

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.

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

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/mTRG2 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.

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

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 ß-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.

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

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.

Development of Novel Micro-dystrophins with Enhanced Functionality.

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 mdx4cv 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.

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

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.

Progress toward Gene Therapy for Duchenne Muscular Dystrophy.

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.

Muscle structure influences utrophin expression in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin-/- mice. An ∼ 2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼ 91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin-/-, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.

Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

Phosphorylation within the cysteine-rich region of dystrophin enhances its association with ß-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin.