Targeting a therapeutic LIF transgene to muscle via the immune system ameliorates muscular dystrophy.

Many potentially therapeutic molecules have been identified for treating Duchenne muscular dystrophy. However, targeting those molecules only to sites of active pathology is an obstacle to their clinical use. Because dystrophic muscles become extensively inflamed, we tested whether expressing a therapeutic transgene in leukocyte progenitors that invade muscle would provide selective, timely delivery to diseased muscle.

Clinical potential of ataluren in the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an autosomal dominant, X-linked neuromuscular disorder caused by mutations in dystrophin, one of the largest genes known to date. Dystrophin gene mutations are generally transmitted from the mother to male offspring and can occur throughout the coding length of the gene. The majority of the methodologies aimed at treating the disorder have focused on restoring a shorter, although partially functional, dystrophin protein.

Purloined mechanisms of bacterial immunity can cure muscular dystrophy.

Myriad strategies have been explored to compensate for the lack of dystrophin or to skip mutations that cause the lethal disease Duchenne muscular dystrophy (DMD). A new study shows that gene editing strategies used by bacteria can be applied in zygotes of a mouse model of DMD to correct the genetic defect that causes muscular dystrophy (Long et al., 2014).

High throughput screening in duchenne muscular dystrophy: from drug discovery to functional genomics.

Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD), the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases.

Emerging gene editing strategies for Duchenne muscular dystrophy targeting stem cells.

The progressive loss of muscle mass characteristic of many muscular dystrophies impairs the efficacy of most of the gene and molecular therapies currently being pursued for the treatment of those disorders. It is becoming increasingly evident that a therapeutic application, to be effective, needs to target not only mature myofibers, but also muscle progenitors cells or muscle stem cells able to form new muscle tissue and to restore myofibers lost as the result of the diseases or during normal homeostasis so as to guarantee effective and lost lasting effects. Correction of the genetic defect using oligodeoxynucleotides (ODNs) or engineered nucleases holds great potential for the treatment of many of the musculoskeletal disorders.

Ex vivo gene editing of the dystrophin gene in muscle stem cells mediated by peptide nucleic acid single stranded oligodeoxynucleotides induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level.

In utero delivery of oligodeoxynucleotides for gene correction.

Gene correction is attractive for single gene mutation disorders, such as Duchenne muscular dystrophy (DMD). The mdx mouse model of DMD is dystrophin deficient due to a premature chain-terminating point mutation in exon 23 of the dystrophin gene. Gene editing of genomic DNA using single-stranded oligodeoxynucleotides (ssODNs) offers the potential to change the DNA sequence to alter mRNA and protein expression in defined ways.

Methods for the assessment of ssODN-mediated gene correction frequencies in muscle cells.

The past decade has seen the development of new technologies capable of editing the genome that have naturally led to exploring their therapeutic application for the treatment of many disorders. Among those, Duchenne muscular dystrophy (DMD) represents an ideal candidate for gene editing primarily due to the large size of dystrophin, the gene responsible for the disease, which limits the use of gene replacement approaches. Critical in the evaluation of the efficacy of the treatment is the development of a method that can accurately quantitate the frequencies of gene repair obtained in the dystrophin gene at both the genomic level as well as the mRNA level.

Therapy development for neuromuscular diseases: translating hope into promise.

The Second Muscular Dystrophy Association Scientific Meeting was held on 21-24 April 2013 in Washington (DC, USA). The meeting provided an opportunity for research scientists, clinicians, government agencies and industry experts to highlight and discuss different aspects of therapy development for neuromuscular diseases, including novel targets, biomarkers, therapeutic approaches, animal models and clinical trials. With 500 participants, 66 presentations and 200 abstracts, the 3-day conference has become a central focus for scientists interested in translational research and moving potential therapies forward from the bench to the bedside.

A high-content, high-throughput siRNA screen identifies cyclin D2 as a potent regulator of muscle progenitor cell fusion and a target to enhance muscle regeneration.

Cell-mediated regenerative approaches using muscle progenitor cells hold promises for the treatment of many forms of muscle disorders. Their applicability in the clinic, however, is hindered by the low levels of regeneration obtained after transplantation and the large number of cells required to achieve an effect. To better understand the mechanisms that regulate the temporal switch of replicating muscle progenitor cells into terminally differentiated cells and to develop new strategies that could enhance muscle regeneration, we have developed and performed a high-throughput screening (HTS) capable of identifying genes that play active roles during myogenesis.

Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy.

Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD).

Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum.

Antisense morpholino oligonucleotides (AMOs) can reprogram pre-mRNA splicing by complementary binding to a target site and regulating splice site selection, thereby offering a potential therapeutic tool for genetic disorders. However, the application of this technology into a clinical scenario has been limited by the low correction efficiency in vivo and inability of AMOs to efficiently cross the blood brain barrier and target brain cells when applied to neurogenetic disorders such as ataxia-telangiecatasia (A-T). We previously used AMOs to correct subtypes of ATM splicing mutations in A-T cells; AMOs restored up to 20% of the ATM protein and corrected the A-T cellular phenotype.

Site-directed gene repair of the dystrophin gene mediated by PNA-ssODNs.

Permanent correction of gene defects is an appealing approach to the treatment of genetic disorders. The use of single-stranded oligodeoxynucleotides (ssODNs) has been demonstrated to induce single-point mutations in the dystrophin gene and to restore dystrophin expression in the skeletal muscle of models of Duchenne muscular dystrophy (DMD). Here we show that ssODNs made of peptide nucleic acids (PNA-ssODNs) can achieve gene repair frequencies more than 10-fold higher than those obtained using an older generation of targeting oligonucleotides.

Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides.

Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification.

Nonaminoglycoside compounds induce readthrough of nonsense mutations.

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)-enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS.

Clinical approaches in the treatment of Duchenne muscular dystrophy (DMD) using oligonucleotides.

Duchenne Muscular dystrophy (DMD) is one of the most severe forms of hereditary diseases in muscles. The identification and characterization of dystrophin, the gene responsible for the disease has lead to the development of potential gene therapy treatments for this disorder. The complex structure and size of the dystrophin gene represent a challenge for some gene therapy approaches such as gene replacement mediated by viral vectors.

Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage phiC31 integrase after nonviral DNA delivery.

Peripheral vascular disease (PVD), characterized by insufficient blood supply to extremities, can be a devastating illness. Although many gene therapy strategies for PVD using vascular endothelial growth factor (VEGF) have resulted in increased blood vessel formation, the vessels are often impermanent and regress after therapy, probably because of the short-lived VEGF expression mediated by gene therapy vectors (14 days or less). phiC31 integrase is a recombinase originally isolated from a bacteriophage of Streptomyces.

Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration.

Plasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse, a model of Duchenne muscular dystrophy. However, sufficient long-term expression and distribution of dystrophin remain a hurdle for translating this technology into a viable treatment for Duchenne muscular dystrophy. To improve plasmid-mediated gene therapy for muscle diseases, we studied the effects of targeted plasmid integration using a phage integrase (phiC31) that can mediate the integration of suitably modified plasmids into the mammalian genome.

Strand bias in oligonucleotide-mediated dystrophin gene editing.

Defects in the dystrophin gene cause the severe degenerative muscle disorder, Duchenne muscular dystrophy (DMD). Among the gene therapy approaches to DMD under investigation, a gene editing approach using oligonucleotide vectors has yielded encouraging results. Here, we extend our studies of gene editing with self-pairing, chimeric RNA/DNA oligonucleotides (RDOs) to the use of oligodeoxynucleotides (ODNs) to correct point mutations in the dystrophin gene.

Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping.

The most common types of dystrophin gene mutations that cause Duchenne muscular dystrophy (DMD) are large deletions that result in a shift of the translational reading frame. Such mutations generally lead to a complete absence of dystrophin protein in the muscle cells of affected individuals. Any therapeutic modality that could restore the reading frame would have the potential to substantially reduce the severity of the disease by allowing the production of an internally deleted, but partially functional, dystrophin protein as occurs in Becker muscular dystrophy (BMD).

Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides.

Point mutations in the dystrophin gene cause dystrophin deficiency and muscular dystrophy in the mdx mouse and a subset of patients with Duchenne muscular dystrophy. As an approach to gene therapy for muscular dystrophies due to point mutations, we have studied the ability of RNA-DNA chimeric oligonucleotides (chimeraplasts) to induce repair of the dystrophin gene in mdx mice. We have previously demonstrated that targeting chimeraplasts can repair the exon 23 point mutation in differentiated myofibers in vivo after intramuscular injection.