Carmen Bertoni

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. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein.

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. For long-term benefit to patients with muscular dystrophy, any gene therapy technology must target not only differentiated myofibers but also undifferentiated muscle precursor cells that are involved in ongoing muscle repair. The focus of the current studies was to test whether chimeraplasts could repair the dystrophin mutation in mdx muscle precursor cells. Initial studies were done by transfecting a targeting chimeraplast into mdx myoblasts in vitro. Gene repair was demonstrated at the DNA, RNA, and protein levels in these cells, whereas treatment of the cells with a control chimeraplast resulted in no gene correction. After differentiation of mdx cells that had been treated with a targeting chimeraplast, immunoblot analysis demonstrated full-length dystrophin expression. By quantitative analysis of independent cultures, the amount of dystrophin expressed ranged from 2 to 15% of that expressed in wild-type cells, providing a measure of the efficacy of gene conversion in vitro. To extend the assessment to muscle precursor cells in vivo, we injected targeting and control chimeraplasts into muscles of mdx mice. When muscle precursor cells were subsequently derived from muscles injected with a targeting chimeraplast, we found that gene repair had occurred in these cells as well. These results, taken together, further demonstrate that chimeraplast-mediated gene repair may be effective as an approach to gene therapy for muscular dystrophies due to point mutations.

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). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for 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. In this study, we demonstrate that an arginine-rich cell-penetrating peptide, (RXRRBR)(2)XB, dramatically improved ATM splicing correction efficiency when conjugated with AMOs, and almost fully corrected aberrant splicing. The restored ATM protein was close to normal levels in cells with homozygous splicing mutations, and a gene dose effect was observed in cells with heterozygous mutations. A significant amount of the ATM protein was still detected 21 days after a single 5 µm treatment. Systemic administration of an fluorescein isothiocyanate-labeled (RXRRBR)(2)XB-AMO in mice showed efficient uptake in the brain. Fluorescence was evident in Purkinje cells after a single intravenous injection of 60 mg/kg. Furthermore, multiple injections significantly increased uptake in all areas of the brain, notably in cerebellum and Purkinje cells, and showed no apparent signs of toxicity. Taken together, these results highlight the therapeutic potential of (RXRRBR)(2)XB-AMOs in A-T and other neurogenetic disorders.

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. Others, including oligonucleotide-mediated gene therapies have allowed forms of manipulation in the dystrophin gene not possible with other disorders. The use of oligonucleotides to modulate gene expression has shown to be a feasible alternative treatment to DMD. Antisense-mediated technologies have made outstanding progress in the last decade and two phase I clinical trials for exon skipping in DMD are already in progress. Gene correction mediated by oligonucleotides faces much greater obstacles, but the outcome of the approach, permanent correction of the gene defect, represents an ideal treatment to the disease. Gene therapy mediated by antisense oligonucleotides or oligonucleotide mediated gene editing have the potential to have a primary role in gene therapy applications to muscles, but they are still far from representing an effective cure. Factors like safety and sustained beneficial effects in patients will have to be considered in detail before this technology can become applicable to the treatment of muscles disorders. Ultimately the need for production of oligonucleotides in large scale and the cost of treatment for each individual patient will play a big role in the feasibility of these approaches in DMD.

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. Correction was demonstrated in muscles cells isolated from mdx(5cv) mice and was stably inherited over time. Direct intramuscular injection of PNA-ssODNs targeting the mdx(5cv) mutation resulted in a significant increase in dystrophin-positive fibers when compared with muscles that received the ssODNs designed to correct the dystrophin gene but made of unmodified bases. Correction was demonstrated at both the mRNA and the DNA levels using quantitative PCR and was confirmed by direct sequencing of amplification products. Analysis at the protein level demonstrated expression of full-length dystrophin in vitro as well as in vivo. These results demonstrate that oligonucleotides promoting strand invasion in the DNA double helix can significantly enhance gene repair frequencies of the dystrophin gene. The use of PNA-ssODNs has important implications in terms of both efficacy and duration of the repair process in muscles and may have a role in advancing the treatment of DMD.

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. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested.

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. The encouraging results obtained by studying in vitro systems and model organisms have set the groundwork for what is likely to become an emerging field in the area of molecular and regenerative medicine. Furthermore, the ability to isolate and expand from patients various types of muscle progenitor cells capable of committing to the myogenic lineage provides the opportunity to establish cell lines that can be used for transplantation following ex vivo manipulation and expansion. The purpose of this article is to provide a perspective on approaches aimed at correcting the genetic defect using gene editing strategies and currently under development for the treatment of Duchenne muscular dystrophy (DMD), the most sever of the neuromuscular disorders. Emphasis will be placed on describing the potential of using the patient own stem cell as source of transplantation and the challenges that gene editing technologies face in the field of regenerative biology.

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. Secondary and tertiary screens were used to confirm the effects of RNAi in vitro and in vivo and to select for candidate hits that significantly increase regeneration into skeletal muscles. Downregulation of cyclin D2 (CCND2) was shown to dramatically enhance myogenic differentiation of muscle progenitor cells and to induce a robust regeneration after cell transplantation into skeletal muscles of dystrophin-deficient mice. Protein interaction network and pathway analysis revealed that CCND2 directly interacts with the cyclin-dependent kinase Cdk4 to inhibit phosphorylation of the retinoblastoma protein (pRb), thus blocking the activation of the myogenic switch during fusion. These studies identify CCND2 as a new key regulator of terminal differentiation in muscle progenitor cells and open new possibilities for the treatment of many forms of muscle disorders characterized by impaired regeneration and loss of muscle mass.

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. Key issues covered by the meeting included the need to identify new drugs to treat patients with neuromuscular diseases and the importance of establishing collaborations between government, academic and industry sectors toward an efficient and rigorous translational path for neuromuscular diseases.