Virginia Arechavala-Gomeza

Biocruces Health Research Institute

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) netw...

Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45 – 55 DMD exons (del45 – 55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation...

Preclinical evaluation of nucleic acid therapeutics (NATs) in relevant experimental model systems is essential for NAT drug development. As part of COST Action "DARTER" (Delivery of Antisense RNA ThERapeutics), a network of researchers in the field of RNA therapeutics, we have conducted a survey on the experimental model systems routinely used by o...

The nucleic acid therapeutics field has made tremendous progress in the past decades. Continuous advances in chemistry and design have led to many successful clinical applications, eliciting even more interest from researchers including both academic groups and drug development companies. Many preclinical studies in the field focus on improving the...

Myotonic dystrophy type I (DM1) is a complex disease characterized by multisystemic impairment. The wide range of symptoms suggests a broad tissue involvement, leading to multiple cell type candidates for pre-clinical drug evaluation, such as patient-derived fibroblasts and myoblasts. Nevertheless, DM1 hallmarks are not homogenous among tissues, wh...

Satellite cells (SCs), muscle stem cells, display functional heterogeneity and dramatic changes linked to their regenerative capabilities are associated with muscle wasting diseases. SCs behaviour is related to endogenous expression of the myogenic transcription factor MYF5 and the propensity to enter into cell cycle. Here, we report a role for miR...

Several exon skipping antisense oligonucleotides (eteplirsen, golodirsen, viltolarsen, and casimersen) have been approved for the treatment of Duchenne muscular dystrophy, but many more are in development targeting an array of different DMD exons. Preclinical screening of the new oligonucleotide sequences is routinely performed using patient-derive...

Nucleic acid therapeutics is a growing field aiming to treat human conditions that has gained special attention due to the successful development of mRNA vaccines against SARS-CoV-2. Another type of nucleic acid therapeutics is antisense oligonucleotides, versatile tools that can be used in multiple ways to target pre-mRNA and mRNA. While some year...

Eteplirsen is a medicine developed for the treatment of Duchenne muscular dystrophy, a neuromuscular disorder that affects 1 in 5000 newborn boys. It was only the fourth drug of its kind ever approved (a splice switching antisense oligonucleotide) and the first with a morpholino chemistry. Its developmental path was constrained by these factors and...

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immort...

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin‐deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co‐localize in human fetal mu...

Nucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for bo...

Since the gene responsible for Duchenne muscular dystrophy was first described in 1987 [...]

MicroRNAs (miRNAs) are mostly known for their gene regulation properties, but they also play an important role in intercellular signaling. This means that they can be found in bodily fluids, giving them excellent biomarker potential. Myotonic Dystrophy type I (DM1) is the most frequent autosomal dominant muscle dystrophy in adults, with an estimate...

Myotonic dystrophy type I (DM1) is the most common form of adult muscular dystrophy, caused by expansion of a CTG triplet repeat in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase (DMPK) gene. The pathological CTG repeats result in protein trapping by expanded transcripts, a decreased DMPK translation and the disruption...

Scientific advance is based on reproducibility, corroboration, and availability of research results. However, large numbers of experimental results that contradict previous work do not get published and many research results are not freely available as they are hidden behind paywalls. As part of COST Action "DARTER", a network of researchers in the...

CRISPR/Cas9-mediated gene editing may allow treating and studying rare genetic disorders by respectively, correcting disease mutations in patients, or introducing them in cell cultures. Both applications are highly dependent on Cas9 and sgRNA delivery efficiency. While gene editing methods are usually efficiently applied to cell lines such as HEK29...

Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, o...

Background Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify...

Exon skipping levels of DMDΔ48–50 and DMDΔ52 cells obtained by qPCR (1st transfection experiment). Error bars represent standard deviation. (TIF)

Examples of raw data to determine exon skipping levels by ddPCR (A), bioanalyzer (B) and densitometry (C) of Δ48–50 cells treated with an AON to skip exon 51. A. The 1D amplitude plot shows positive (blue) and negative dots (grey) for the skipped and the non-skipped assays. B. Results of the electrophoresis run of the high sensitivity DNA assay sho...

Intra-laboratory variation of exon 51 skipping levels in DMDΔ48–50 (A-C) and DMDΔ52 cells (D-E). Three different protocols were repeated by the same operator (n = 2/3). Error bars represent standard deviation. (TIF)

Sequences of primers, probes and AON used. FAM = 6-carboxyfluorescein label, 2’OMePS = 2'-O-methyl-modified bases on a phosphorothioate backbone. (DOCX)

Exon 51 skipping percentages obtained by the different laboratories for the different technologies. Three biological replicates of each sample were measured. SD = standard deviation. (DOCX)

Detailed description of cell culture conditions, transfection and RNA purification. (DOCX)

Eteplirsen, a compound designed to restore dystrophin in patients with Duchenne muscular dystrophy, controversially received approval by the FDA in 2016. Owing to limited clinical data, the approval was based on eteplirsen’s effect on dystrophin expression. Now, the dystrophin quantification results have been published, and although low levels of d...

Aims: New therapies for neuromuscular disorders are often mutation-specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are being developed but, as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate d...

The use of splice-switching antisense therapy is highly promising, with a wealth of pre-clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleot...

Selection of reference genes for normalisation of dystrophin mRNA RT-qPCR data Iker Garcia-Jimenez, Estíbaliz Ruiz-Del-Yerro, Virginia Arechavala-Gomeza Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Spain Accurate quantification of either total levels of dystrophin mRNA or dystrophin exon skipping is vital not onl...

Duchenne muscular dystrophy is a rare, progressive, muscle-wasting disease leading to severe disability and premature death. Treatment is currently symptomatic, but several experimental therapies are in development. Implemented care standards, validated outcome measures correlating with clinical benefit, and comprehensive information about the natu...

Duchenne muscular dystrophy is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the full translation of its protein product, dystrophin. Restoration of the open reading frame and dystrophin production can be achieved by exon skipping using antisense oligonucleotides targeted to splicing elements. This approach aim...

Multicenter quantification of dystrophin exon 51 skipping

Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splici...

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis...

Importance: In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than ot...

Restoration of the open reading frame of the DMD gene and dystrophin protein production in Duchenne muscular dystrophy (DMD) can be achieved by exon skipping using antisense oligomers (AOs) targeted to splicing elements. Several such RNA-based gene therapy approaches are in clinical development where to date, all studies have assessed AO efficacy b...

Exon skipping using antisense oligonucleotides (AONs) is a promising therapy for Duchenne muscular dystrophy (DMD) which aims to genocopy the milder Becker muscular dystrophy (BMD) by restoring the reading frame of an out-of-frame deletion. The less severe phenotype of BMD patients suggests that internally truncated dystrophin retains some function...

An article in the March issue (A novel imaging method to quantify low levels of dystrophin in Duchenne muscular dystrophy. Nat. Rev. Neurol. 8, 120; 2012

Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death....

Duchenne muscular dystrophy is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the full translation of its protein product, dystrophin. Restoration of the open reading frame and dystrophin production can be achieved by exon skipping using antisense oligonucleotides targeted to splicing elements. This approach aim...

We previously conducted a proof of principle; dose escalation study in Duchenne muscular dystrophy (DMD) patients using the morpholino splice-switching oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon 51 in patients with relevant deletions, restores the open reading frame and induces dystrophin protein expression after...

We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duch...

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...

There are currently no effective treatments to halt the muscle breakdown in Duchenne muscular dystrophy (DMD), although genetic-based clinical trials are being piloted. Most of these trials have as an endpoint the restoration of dystrophin in muscle fibers, hence requiring sufficiently well-preserved muscle of recruited patients. The choice of the...

In mice, both AVI-4658 and AVI-4225 were well tolerated at doses including 960 mg/kg/injection, with no adverse effects. Findings were generally limited to the kidney, and were generally reversible, as shown in the 28 day recovery groups. No evidence of kidney function change was detected. In cynomolgus monkeys, AVI-4658 was also well tolerated at...

Duchenne muscular dystrophy (DMD) is characterised by the absence of dystrophin in muscle biopsies, although residual dystrophin can be present, either as dystrophin-positive (revertant) fibres or traces. As restoration of dystrophin expression is the end point of clinical trials, such residual dystrophin is a key factor in recruitment of patients...

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dy...

Stem cell therapy holds promise for treating muscle diseases. Although satellite cells regenerate skeletal muscle, they only have a local effect after intra-muscular transplantation. Alternative cell types, more easily obtainable and systemically-deliverable, were therefore sought. Human synovial stem cells (hSSCs) have been reported to regenerate...

Table S1 Primer pairs used to amplify overlapping regions of cDNA from the index case (1-1 to 1-12 and 2-1 to 2-10), genomic DNA to demonstrate the mutation (3-1) and for the RFLP (3-2) and nested primer pairs for the exon skipping (4-1 and 4-2). For primer pair 4-2, a 666 bp product (in parentheses) is the size of the product expected with deletio...

Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resu...

The quantification of protein levels in muscle biopsies is of particular relevance in the diagnostic process of neuromuscular diseases, but is difficult to assess in cases of partial protein deficiency, particularly when information on protein localization is required. The combination of immunohistochemistry and Western blotting is often used in th...

Background Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading...

Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and...

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in the absence of functional protein. In the majority of cases these are out-of-frame deletions that disrupt the reading frame. Several attempts have been made to restore the dystrophin mRNA reading frame by modulation of pre-mRNA splicing with antisense oli...

This chapter discusses the epidemiology, clinical phenotype, and genetic basis of the heritable forms of motor neuron disorder (MND) that have been classified as amyotrophic lateral sclerosis (ALS). All of these conditions are due to single gene defects that result in a progressive degeneration of upper motor neurons (UMNs) in the precentral gyrus...