Duchenne muscular dystrophy: From diagnosis to therapy

Duchenne muscular dystrophy (DMD) is an X-linked inherited neuromuscular disorder due to mutations in the dystrophin gene. It is characterized by progressive muscle weakness and wasting due to the absence of dystrophin protein that causes degeneration of skeletal and cardiac muscle. The molecular diagnostic of DMD involves a deletions/duplications analysis performed by quantitative technique such as microarray-based comparative genomic hybridization (array-CGH), Multiple Ligation Probe Assay MLPA. Since traditional methods for detection of point mutations and other sequence variants require high cost and are time consuming, especially for a large gene like dystrophin, the use of next-generation sequencing (NGS) has become a useful tool available for clinical diagnosis. The dystrophin gene is large and finely regulated in terms of tissue expression, and RNA processing and editing includes a variety of fine tuned processes. At present, there are no effective treatments and the steroids are the only fully approved drugs used in DMD therapy able to slow disease progression. In the last years, an increasing variety of strategies have been studied as a possible therapeutic approach aimed to restore dystrophin production and to preserve muscle mass, ameliorating the DMD phenotype. RNA is the most studied target for the development of clinical strategies and Antisense Oligonucleotides (AONs) are the most used molecules for RNA modulation. The identification of delivery system to enhance the efficacy and to reduce the toxicity of AON is the main purpose in this area and nanomaterials are a very promising model as DNA/RNA molecules vectors. Dystrophinopathies therefore represent a pivotal field of investigation, which has opened novel avenues in molecular biology, medical genetics and novel therapeutic options

Translational and Regulatory Challenges for Exon Skipping Therapies

Several translational challenges are currently impeding the therapeutic development of antisense-mediated exon skipping approaches for rare diseases. Some of these are inherent to developing therapies for rare diseases, such as small patient numbers and limited information on natural history and interpretation of appropriate clinical outcome measures. Others are inherent to the antisense oligonucleotide (AON)-mediated exon skipping approach, which employs small modified DNA or RNA molecules to manipulate the splicing process. This is a new approach and only limited information is available on long-term safety and toxicity for most AON chemistries. Furthermore, AONs often act in a mutation-specific manner, in which case multiple AONs have to be developed for a single disease. A workshop focusing on preclinical development, trial design, outcome measures, and different forms of marketing authorization was organized by the regulatory models and biochemical outcome measures working groups of Cooperation of Science and Technology Action: "Networking towards clinical application of antisense-mediated exon skipping for rare diseases." The workshop included participants from patient organizations, academia, and members of staff from the European Medicine Agency and Medicine Evaluation Board (the Netherlands). This statement article contains the key outcomes of this meeting.status: publishe

A patient with limb girdle muscular dystrophy carries a TRIM32 deletion, detected by a novel CGH array, in compound heterozygosis with a nonsense mutation

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Tamoxifen induces oxidative stress and apoptosis in oestrogen receptor-negative human cancer cell lines

Recent data have demonstrated that the anti-oestrogen tamoxifen (TAM) is able to facilitate apoptosis in cancer cells not expressing oestrogen receptor (ER). In an attempt to identify the biochemical pathway for this phenomenon, we investigated the role of TAM as an oxidative stress agent. In two ER-negative human cancer cell lines, namely T-leukaemic Jurkat and ovarian A2780 cancer cells, we have demonstrated that TAM is able to generate oxidative stress, thereby causing thiol depletion and activation of the transcriptional factor NF-κB. As described for other oxidative agents, TAM was able to induce either cell proliferation or apoptosis depending on the dose. When used at the lowest dose tested (0.1 μM), a slight proliferative effect of TAM was noticed in terms of cell counts and DNA synthesis rate, whereas at higher doses (10 μM) a consistent occurrence of apoptosis was detected. Importantly, the induction of apoptosis by TAM is not linked to down-regulation or functional inactivation by phosphorylation of the antiapoptotic bcl-2 protein. © 1999 Cancer Research Campaig

Antitumour activity of novel taxanes that act at the same time as cytotoxic agents and P-glycoprotein inhibitors

Taxanes antitumour agents such as paclitaxel and docetaxel represent a successful family of chemotherapeutic drugs. Unfortunately, acquired and innate resistance represents a clinical problem for these drugs. We investigated, on a panel of 7 human cancer cell lines, the growth inhibition effect of 3 newly developed taxanes (SB-T-1213, SB-T-1250 and SB-T-101187) with modification at the C10 and C3′ positions of the taxane framework. These positions have been previously characterized as critical to make taxanes highly active against cells overexpressing the efflux pump P-glycoprotein (P-gp). Paclitaxel and docetaxel were used as reference compounds. Results unambiguously indicate the exceptional activity of the novel taxanes toward P-gp positive cells (up to >400 fold higher potency than that of paclitaxel). SB-T-1213 and SB-T-1250 are also substantially more active than the reference compounds against P-gp negative cells. To better understand the mechanisms underlying the enhanced activity of the newly developed taxanes, we performed cell cycle and apoptosis analysis. This study demonstrates that the striking growth inhibition effect exhibited by the novel taxanes is ascribed to their increased ability in inducing apoptosis and G 2/M cell cycle block. SB-T-1213 and SB-T-1250 are also more active than reference compounds in inducing intracellular accumulation of the beta-tubulin subunits. Finally, it is revealed that these novel taxanes have ability to inhibit the function of the P-gp efflux pump on the basis of the Rhodamine 123 assay. These findings strongly suggest that SB-T-1213, SB-T-1250 and SB-T-101187 represent a new tool to overcome innate or acquired P-gp mediated taxane-resistance. © 2000 Cancer Research Campaign http://www.bjcancer.co