Alberto Malerba

Chronic Systemic Therapy With Low-dose Morpholino Oligomers Ameliorates the Pathology and Normalizes Locomotor Behavior in mdx Mice

The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans.

Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity.

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.

Gene therapy for muscular dystrophy: current progress and future prospects

Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there is no effective treatment but new gene-based therapies are currently being developed with particular noted advances in using conventional gene replacement strategies, RNA-based approaches, or cell-based gene therapy with a main focus on Duchenne muscular dystrophy (DMD). DMD is the most common and severe form of muscular dystrophy and current treatments are far from adequate. However, genetic and cell-based therapies, in particular exon skipping induced by antisense strategies, and corrective gene therapy via functionally engineered dystrophin genes hold great promise, with several clinical trials ongoing. Proof-of-concept of exon skipping has been obtained in animal models, and most recently in clinical trials; this approach represents a promising therapy for a subset of patients. In addition, gene-delivery-based strategies exist both for antisense-induced reading frame restoration, and for highly efficient delivery of functional dystrophin mini- and micro-genes to muscle fibres in vivo and muscle stem cells ex-vivo. In particular, AAV-based vectors show efficient systemic gene delivery to skeletal muscle directly in vivo, and lentivirus-based vectors show promise of combining ex vivo gene modification strategies with cell-mediated therapies.

Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice.

Duchenne muscular dystrophy (DMD) is a myodegenerative disorder caused primarily by mutations that create premature termination of dystrophin translation. The antisense oligonucleotide approach for skipping dystrophin exons allows restoration of the correct reading frame in the dystrophin transcript, thus producing a shorter protein. A similar approach in humans would result in the conversion of DMD to the milder Becker muscular dystrophy. It has been demonstrated previously that repeated intravascular injection of phosphorodiamidate morpholino oligomers (PMOs) in the mdx mouse induces more dystrophin expression than a single injection, but this approach is costly, and data demonstrating the safety of high doses of systemically injected PMO are unavailable. Furthermore, several publications have demonstrated the efficacy of peptide-conjugated PMOs, but the clinical applicability of such compounds is unclear at this stage. Here, we report that multiple intravascular injections of low doses of naked PMO show significantly more dystrophin-positive fibers in a variety of muscle groups, 8 weeks after administration compared with a single dose of the same total amount. After administration of a total of 200 mg of PMO per kilogram, histological features, such as the cross-sectional area, centronucleation index, and expression of the dystrophin-associated protein complex, showed significant improvement in mice treated by repeated injection. Furthermore, four administrations of just 5 mg/kg induced a significant amount of dystrophin expression. These results clearly demonstrate the key role of the optimization of dosing regimen for the systemic administration of PMO in patients, and support the clinical feasibility of this approach with naked PMO.

Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Long-Term Systemic Administration of Unconjugated Morpholino Oligomers for Therapeutic Expression of Dystrophin by Exon Skipping in Skeletal Muscle: Implications for Cardiac Muscle Integrity

Duchenne muscular dystrophy (DMD) is a lethal X-linked inherited disease caused by mutations in the dystrophin gene and consequent lack of dystrophin in the skeletal, cardiac, and smooth musculature and in the nervous system. Patients die during their mid-twenties because of severe muscle loss and life-threatening respiratory and cardiac complications. The splicing modulation approach mediated by antisense oligonucleotides can restore the production of a partially functional quasi-dystrophin in skeletal muscles. We recently showed that a chronic, 12-month treatment with phosphorodiamidate morpholino oligomers efficiently restored dystrophin in widespread skeletal muscles and led to normal locomotor activity indistinguishable from that of dystrophin-expressing C57 mice. However, no detectable dystrophin expression was observed in the hearts of treated mice. In the present study, histological analyses show a more severe cardiac pathology compared with untreated animals in the face of enhanced locomotor behavior. This observation implies that the increase in locomotor activity of treated mdx mice may have a paradoxical detrimental effect on the dystrophic heart. In the context of skeletal muscle-centric therapies for DMD, our data suggest that particular vigilance should be instigated to monitor emergence of accelerated cardiac dysfunction.

Efficient delivery of human single fiber-derived muscle precursor cells via biocompatible scaffold

The success of cell therapy for skeletal muscle disorders depends upon two main factors: the cell source and the method of delivery. In this work we have explored the therapeutic potential of human muscle precursor cells (hMPCs), obtained from single human muscle fibers, implanted in vivo via micropatterned scaffolds. hMPCs were initially expanded and characterized in vitro by immunostaining and flow cytometric analysis. For in vivo studies, hMPCs were seeded onto micropatterned poly-lactic-glycolic acid 3D-scaffolds fabricated using soft-lithography and thermal membrane lamination. Seeded scaffolds were then implanted in predamaged tibialis anterior muscles of CD1 nude mice; hMPCs were also directly injected in contralateral limbs as controls. Similarly to what we previously described with mouse precursors cells, we found that hMPCs were able to participate in muscle regeneration and scaffold-implanted muscles contained a greater number of human nuclei, as revealed by immunostaining and Western blot analyses. These results indicate that hMPCs derived from single fibers could be a good and reliable cell source for the design of therapeutic protocols and that implantation of cellularized scaffolds is superior to direct injection for the delivery of myogenic cells into regenerating skeletal muscle.

Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO) led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO). Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA) muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD.

Captopril reduces the severity of bowel damage in a neonatal rat model of necrotizing enterocolitis

BACKGROUND/PURPOSE Selective mesenteric ischemia may result from activation of the renin-angiotensin system during periods of shock and is implicated in the pathogenesis of neonatal necrotizing enterocolitis (NEC). We investigated the effectiveness of captopril, an angiotensin-converting enzyme inhibitor, in reducing the severity of bowel damage in a neonatal rat model of NEC. METHODS Necrotizing enterocolitis was induced by a combination of gavage feeding of hypertonic formula, hypoxia, and oral lipopolysaccharide (LPS). Rats were randomly divided into 3 groups: group A, control (breast fed; n = 20); group B, NEC (gavage/hypoxia/LPS; n = 31); group C, NEC with captopril 20 mg/kg per dose with the formula for 4 days (gavage/hypoxia/LPS/captopril; n = 35). Pups were killed after 4 days. Incidence of NEC was evaluated microscopically. RESULTS Severity of bowel damage was higher in the NEC group compared to controls and was reduced by administration of captopril. Dilatation of the intestinal vasculature was observed in the captopril group. There were no cases of NEC in the controls; the incidence increased to 55% in NEC group and reduced to 29% by captopril. CONCLUSIONS In this model of neonatal NEC, captopril supplementation of formula reduces the severity of intestinal damage and the incidence of NEC, presumably by affecting mesenteric blood flow.

Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV–microdystrophin gene therapy in DMD patients.