Prevention of Dystrophic Pathology in Severely Affected Dystrophin/Utrophin-deficient Mice by Morpholino-oligomer-mediated Exon-skipping

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon-skipping is one of the most promising approaches for the treatment of DMD because of its capacity to correct the reading frame and restore dystrophin expression, which has been demonstrated in vitro and in vivo. In particular, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been shown to induce widespread high levels of dystrophin expression in the mdx mouse model. Here, we report the efficiency of the PPMO-mediated exon-skipping approach in the utrophin/dystrophin double-knockout mouse (dKO) mouse, which is a much more severe and progressive mouse model of DMD. Repeated intraperitoneal (i.p.) injections of a PPMO targeted to exon 23 of dystrophin pre-mRNA in dKO mice induce a near-normal level of dystrophin expression in all muscles examined, except for the cardiac muscle, resulting in a considerable improvement of their muscle function and dystrophic pathology. These findings suggest great potential for PPMOs in systemic treatment of the DMD phenotype.     

In-frame Dystrophin Following Exon 51-Skipping Improves Muscle Pathology and Function in the Exon 52–Deficient mdx Mouse

A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52–deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5′ and 3′ splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20–30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.     

Rescue of a Dystrophin-like Protein by Exon Skipping In Vivo Restores GABAA-receptor Clustering in the Hippocampus of the mdx Mouse

Dystrophin, the cytoskeletal protein whose defect is responsible for Duchenne muscular dystrophy (DMD), is normally expressed in both muscles and brain. Genetic loss of brain dystrophin in the mdx mouse model of DMD reduces the capacity for type A γ-aminobutyric acid (GABA_A)-receptor clustering in central inhibitory synapses, which is thought to be a main molecular defect leading to brain and cognitive alterations in this syndrome. U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors have proven efficient after intramuscular injection to induce skipping of the mutated exon 23 and rescue expression of a functional dystrophin-like product in muscle tissues of mdx mice in vivo. Here, we report that intrahippocampal injection of a single dose of rAAV2/1-U7 can rescue substantial levels of brain dystrophin expression (15–25%) in mdx mice for months. This is sufficient to completely restore GABA_A-receptor clustering in pyramidal and dendritic layers of CA1 hippocampus, suggesting exon-skipping strategies offer the prospect to investigate and correct both brain and muscle alterations in DMD. This provides new evidence that in the adult brain dystrophin is critical for the control of GABA_A-receptor clustering, which may have an important role in activity-dependent synaptic plasticity in hippocampal circuits.     

Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector

Dystrophin gene transfer using helper-dependent adenoviral vectors (HDAd) deleted of all viral genes is a promising option to treat muscles in Duchenne muscular dystrophy (DMD). Previously, we reported high-level dystrophin expression and functional correction of dystrophin-deficient (mdx) mouse muscle 60 days after gene transfer with an HDAd encoding two full-length murine dystrophin cDNAs (referred to as HDCBDysM). In the present study, we tested the long-term efficacy of HDCBDysM by examining muscle contractility parameters and the stability of dystrophin expression 1 year after injection into neonatal mdx muscles. At this point, HDCBDysM-treated muscles averaged 52% dystrophin-positive fibers. Treated muscles also displayed significantly greater isometric force production as well as greater resistance to the force deficits and damage caused by eccentric contractions. The level of protection against eccentric contraction-induced force deficits correlated with the percentage of dystrophin-positive fibers. Furthermore, HDCBDysM treatment restored the dystrophin-glycoprotein complex (DGC) to the sarcolemma and improved other aspects of mdx muscle histopathology examined (central nucleation, muscle hypertrophy, and mononuclear [phagocytic] cell infiltration). These improvements occurred despite the induction of a humoral response against murine dystrophin. Our results indicate that major therapeutic benefits of HDCBDysM are maintained for a long period of the animals' lifespan and suggest that HDCBDys holds promise for treating DMD by gene therapy.     

Dystrophin delivery in dystrophin-deficient DMDmdx skeletal muscle by isogenic muscle-derived stem cell transplantation

Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.     

Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice

The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD.     

One-year Treatment of Morpholino Antisense Oligomer Improves Skeletal and Cardiac Muscle Functions in Dystrophic mdx Mice

Antisense therapy has been successful to skip targeted dystrophin exon with correction of frameshift and nonsense mutations of Duchenne muscular dystrophy (DMD). Systemic production of truncated but functional dystrophin proteins has been achieved in animal models. Furthermore, phase I/II clinical trials in United Kingdom and the Netherlands have demonstrated dystrophin induction by local and systemic administrations of antisense oligomers. However, long-term efficacy and potential toxicity remain to be determined. The present study examined 1-year systemic effect of phosphorodiamidate morpholino oligomers (PMO) treatment targeting mutated dystrophin exon 23 in mdx mice. PMO induced dystrophin expression dose-dependently and significantly improved skeletal muscle pathology and function with reduced creatine kinase (CK) levels by a regimen of 60 mg/kg biweekly administration. This regimen induced <2% dystrophin expression in the heart, but improved cardiac functions demonstrated by hemodynamics analysis. The results suggest that low levels of dystrophin induction may be able to provide detectable benefit to cardiac muscle with limited myopathy. Body weight, serum enzyme tests, and histology analysis showed no sign of toxicity in the mice treated with up to 1.5 g/kg PMO for 6 months. These results indicate that PMO could be used safely as effective drugs for long-term systemic treatment of DMD.     

Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.     

Galectin-1 Protein Therapy Prevents Pathology and Improves Muscle Function in the mdx Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by mutations in the dystrophin gene, leading to the loss of a critical component of the sarcolemmal dystrophin glycoprotein complex. Galectin-1 is a small 14 kDa protein normally found in skeletal muscle and has been shown to be a modifier of immune response, muscle repair, and apoptosis. Galectin-1 levels are elevated in the muscle of mouse and dog models of DMD. Together, these findings led us to hypothesize that Galectin-1 may serve as a modifier of disease progression in DMD. To test this hypothesis, recombinant mouse Galectin-1 was produced and used to treat myogenic cells and the mdx mouse model of DMD. Here we show that intramuscular and intraperitoneal injections of Galectin-1 into mdx mice prevented pathology and improved muscle function in skeletal muscle. These improvements were a result of enhanced sarcolemmal stability mediated by elevated utrophin and α7β1 integrin protein levels. Together our results demonstrate for the first time that Galectin-1 may serve as an exciting new protein therapeutic for the treatment of DMD.     

Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose�Cderived Stem Cells With Forced Expression of MyoD

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose–derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2^−/−γC^−/− mice resulted in a substantial increase in the number of hMADS cell–derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.     

Muscle Function Recovery in Golden Retriever Muscular Dystrophy After AAV1-U7 Exon Skipping

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.     

Matching host muscle and donor myoblasts for myosin heavy chain improves myoblast transfer therapy

Intensive efforts have been made to develop an effective therapy for Duchenne muscular dystrophy (DMD). Although myoblast transplantation has been found capable of transiently delivering dystrophin and improving the strength of the injected dystrophic muscle, this approach has been hindered by the immune rejection problems as well as the poor survival and limited spread of the injected cells. In the present study, we have investigated whether the careful selection of donor myoblasts and host muscle for the myosin heavy chain expression (MyHCs) plays a role in the success of myoblast transfer. Highly purified normal myoblasts derived from the m. soleus and m. gastrocnemius white of normal mice were transplanted into the m. soleus (containing 70% of type I fibers) and gastrocnemius white (100% of type II fibers) of dystrophin deficient mdx mice. At several time-points after injection (10, 20 and 30 days), the number of dystrophin-positive fibers was monitored and compared among the different groups. A significantly higher number and better persistence of dystrophin-positive myofibers were observed when the injected muscle and donor myoblasts expressed a similar MyHC in comparison with myoblast transfer between host muscle and donor myoblasts that were not matched for MyHC. These results suggest that careful matching between the injected myoblasts and injected muscle for the MyHC expression can improve the efficiency of myoblast-mediated gene transfer to skeletal muscle. Gene Therapy (2000) 7, 428-437.     

Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in the absence of functional protein. In the majority of cases these are out-of-frame deletions that disrupt the reading frame. Several attempts have been made to restore the dystrophin mRNA reading frame by modulation of pre-mRNA splicing with antisense oligonucleotides (AOs), demonstrating success in cultured cells, muscle explants, and animal models. We are preparing for a phase I/IIa clinical trial aimed at assessing the safety and effect of locally administered AOs designed to inhibit inclusion of exon 51 into the mature mRNA by the splicing machinery, a process known as exon skipping. Here, we describe a series of systematic experiments to validate the sequence and chemistry of the exon 51 AO reagent selected to go forward into the clinical trial planned in the United Kingdom. Eight specific AO sequences targeting exon 51 were tested in two different chemical forms and in three different preclinical models: cultured human muscle cells and explants (wild type and DMD), and local in vivo administration in transgenic mice harboring the entire human DMD locus. Data have been validated independently in the different model systems used, and the studies describe a rational collaborative path for the preclinical selection of AOs for evaluation in future clinical trials.