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Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option.

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1(Prx1-/-) mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1(Prx1-/-) mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1(Prx1-/-) mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin-1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1(Prx1-/-) limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches.

Objective— Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of noncanonical (Erk-mediated) TGFβ signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFβ neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFβ neutralization and AT1r antagonism in mice with progressively severe MFS ( Fbn1 mgR/mgR mice). Approach and Results— Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1 mgR/mgR mice were compared after a ≈3-month-long treatment with placebo and either the AT1r antagonist losartan or the TGFβ-neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFβ neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (postnatal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence-related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFβ hyperactivity that was in part AT1r dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1 mgR/mgR mice from ruptured TAA. Conclusions— By demonstrating that promiscuous AT1r and TGFβ drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways although sparing the early protective role of TGFβ.

ObjectiveTo describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen.MethodsClinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen.ResultsReverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001).ConclusionTaken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression.Classification of evidenceThis study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

Expansions of CUG trinucleotide sequences in RNA transcripts provide the basis for toxic RNA gain-of-function that leads to detrimental changes in RNA metabolism. A CTG repeat element normally resides in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, but when expanded it is the genetic lesion of myotonic dystrophy type 1 (DM1), a hereditary neuromuscular disease. The pathogenic DMPK transcript containing the CUG expansion is retained in ribonuclear foci as part of a complex with RNA-binding proteins such as muscleblind-like 1 (MBNL1), resulting in aberrant splicing of numerous RNA transcripts and consequent physiological abnormalities including myotonia. Herein, we demonstrate molecular and physiological amelioration of the toxic effects of mutant RNA in the HSA(LR) mouse model of DM1 by systemic administration of peptide-linked morpholino (PPMO) antisense oligonucleotides bearing a CAG repeat sequence. Intravenous administration of PPMO conjugates to HSA(LR) mice led to redistribution of Mbnl1 protein in myonuclei and corrections in abnormal RNA splicing. Additionally, myotonia was completely eliminated in PPMO-treated HSA(LR) mice. These studies provide proof of concept that neutralization of RNA toxicity by systemic delivery of antisense oligonucleotides that target the CUG repeat is an effective therapeutic approach for treating the skeletal muscle aspects of DM1 pathology.

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUG(exp)) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUG(exp) RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

Respiratory function is the main cause of mortality in patients with Duchenne muscular dystrophy (DMD). Elevated levels of TGF-β play a key role in the pathophysiology of DMD. To determine whether therapeutic attenuation of TGF-β signaling improves respiratory function, mdx mice were treated from 2 weeks of age to 2 months or 9 months of age with either 1D11 (a neutralizing antibody to all three isoforms of TGF-β), losartan (an angiotensin receptor antagonist), or a combination of the two agents. Respiratory function was measured in nonanesthetized mice by plethysmography. The 9-month-old mdx mice had elevated Penh values and decreased breathing frequency, due primarily to decreased inspiratory flow rate. All treatments normalized Penh values and increased peak inspiratory flow, leading to decreased inspiration times and breathing frequency. Additionally, forelimb grip strength was improved after 1D11 treatment at both 2 and 9 months of age, whereas, losartan improved grip strength only at 2 months. Decreased serum creatine kinase levels (significant improvement for all groups), increased diaphragm muscle fiber density, and decreased hydroxyproline levels (significant improvement for 1D11 only) also suggested improved muscle function after treatment. For all endpoints, 1D11 was equivalent or superior to losartan; coadministration of the two agents was not superior to 1D11 alone. In conclusion, TGF-β antagonism may be a useful therapeutic approach for treating DMD patients.

Adult human skeletal muscle-derived cells (HuSkMC) propagated in vitro are under investigation as a cell-based therapy for the treatment of myocardial infarction. We have characterized HuSkMC with respect to cell identity and state of differentiation as a prerequisite to their clinical use. Flow cytometric analysis of propagated HuSkMC revealed a population of cells that expressed the myoblast markers CD56 and desmin. The presence of myoblasts in these cultures was further confirmed by their capacity to form myotubes and increase creatine kinase activity when cultured in low serum conditions. The non-myoblast fraction of these propagated cells expressed TE7, a marker associated with the fibroblast phenotype. Spontaneous differentiation of myoblasts occurred during serial propagation of HuSkMC, as judged by myotube formation, thereby reducing the myoblast representative fraction with continued cell expansion. We examined transforming growth factor beta2 (TGF-beta2) for its utility in controlling this spontaneous differentiation of adult human myoblasts in vitro. Propagation of HuSkMC in the presence of 1 ng/ml TGF-beta2 for 5 days decreased desmin expression within the myoblast population and caused a parallel reduction of creatine kinase activity. CD56 expression was unaffected, indicating a differential regulation of these myoblast markers. The reduction in desmin expression and creatine kinase activity was, however, reversible upon the removal of TGF-beta. These data collectively indicate that TGF-beta2 restrained differentiation of adult human skeletal myoblasts during propagation without causing irreversible loss of the myoblast phenotype, demonstrating the potential utility of using TGF-beta2 during cultivation and expansion of HuSkMC intended for therapeutic implantation.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α -glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

The Epicel ASAProgram service generates autologous keratinocyte grafts used for the closure of full-thickness wounds in moderately and severely burned patients. The manufacturing process used to generate Epicel service autografts (ESA) is based upon the keratinocyte co-culture technique described by Rheinwald and Green which employs murine Swiss 3T3/J2 fibroblasts as feeder cells. Recently, a technique has been described that employs a polyurethane wound dressing, HydroDerm (HD, Innovative Technologies, Ltd), as a delivery vehicle for cultured keratinocytes intended for autologous grafting. We have examined the practical feasibility of this technique and report on testing the ability of HD to support keratinocyte growth and epithelium formation in vitro, at the air-liquid interface (ALI), and in vivo, after grafting to full-thickness wounds created on the backs of athymic (Swiss Nu/Nu) mice. The results demonstrate that keratinocytes grow on the HD dressing in Gibco SFM at a rate that is approximately 15 per cent of that observed when cells are cultivated on tissue culture (TC) plastic using standard techniques, yet the cells retain their proliferative capacity and form an epithelium in vitro when cultivated at the ALI on a dermal substrate. Keratinocyte-seeded HD membranes were also transferred to full-thickness wounds in athymic mice. Animals grafted with cells seeded to HD developed human epithelium, as revealed by species-specific detection of involucrin and evolved a normal attachment to the wound substratum, as demonstrated through the expression of dermally opposed laminin and alpha 6 beta 4 integrin. The ability of keratinocytes to maintain proliferative potential after seeding onto HD and their ability to form a properly oriented epithelium in vitro and in vivo suggests that this wound dressing may be useful as a vehicle for autologous keratinocyte grafting and help to provide earlier epithelial coverage to the burned patient. However, because of the slow proliferation rate of keratinocytes on HydroDerm, timely graft delivery would be best achieved by combining cell expansion via the Rheinwald and Green culture system, followed by the seeding of cells onto HydroDerm in a reduced calcium medium for subsequent autologous grafting.

The therapeutic mechanism of mesenchymal stromal/stem cells (MSC) for the treatment of acute myocardial infarction is not well understood. Our goal was to get insights into this mechanism by analyzing the survival kinetics of allogeneic and syngeneic cell transplants under different tissue conditions. Two MSC cell banks, stably and equally expressing the luciferase reporter construct, were developed for these studies and injected directly to the myocardium of Lewis rat recipients under syngeneic or allogeneic transplantation conditions. Cell survival was monitored by real-time fashion for up to 2 weeks, using optical imaging device (IVIS, Xenogen Corp.). We found that both syngeneic and allogeneic grafts reduced significantly in size during the first week of transplantation, either in the normal or in the late infarcted heart (5 days after MI) and allotransplants became always smaller than syngeneic grafts during this period. Low dose of cyclosporine A treatment had a benefit on both allo- and syngeneic graft sizes, suggesting that multiple mechanisms play a role in early graft reduction. The MSC characteristic factors IL-6, IL-8, MCP-1, and VEGF were well above the control level in the heart tissue at 4 days after cell injection, suggesting that the peak therapeutic effect of MSC can be expected during the first week of the administration. Although allogeneic cells induced immunoglobulin production, their biological effects (cell survival, factor productions) are very similar to the syngeneic transplants and therefore they could deliver the same therapeutic effect as the syngeneic cells. Finally, freshly infarcted tissue (30 min) supported better the survival of MSC than late postischemic tissue (5 days) but only “off the shelf” allogeneic cell transplants fits with this treatment strategy.