Cells Of Duchenne Muscular Dystrophy Patients Research Articles

Antisense PMO cocktails effectively skip dystrophin exons 45-55 in myotubes transdifferentiated from DMD patient fibroblasts.

Antisense-mediated exon skipping has made significant progress as a therapeutic platform in recent years, especially in the case of Duchenne muscular dystrophy (DMD). Despite FDA approval of eteplirsen–the first-ever antisense drug clinically marketed for DMD–exon skipping therapy still faces the significant hurdles of limited applicability and unknown truncated protein function. In-frame exon skipping of dystrophin exons 45–55 represents a significant approach to treating DMD, as a large proportion of patients harbor mutations within this “hotspot” region. Additionally, patients harboring dystrophin exons 45–55 deletion mutations are reported to have exceptionally mild to asymptomatic phenotypes. Here, we demonstrate that a cocktail of phosphorodiamidate morpholino oligomers can effectively skip dystrophin exons 45–55 in vitro in myotubes transdifferentiated from DMD patient fibroblast cells. This is the first report of substantive exons 45–55 skipping in DMD patient cells. These findings help validate the use of transdifferentiated patient fibroblast cells as a suitable cell model for dystrophin exon skipping assays and further emphasize the feasibility of dystrophin exons 45–55 skipping in patients.

T.I.2 Exon skipping and PRO044 in Duchenne muscular dystrophy: Extending the program

Patients with Duchenne muscular dystrophy (DMD) suffer from progressive muscle degeneration due to DMD gene mutations and resulting absence of dystrophin. Antisense oligonucleotides (AONs) have potential to modulate premRNA splicing so that a specific exon is skipped and the disrupted dystrophin reading frame restored. This allows production of internally deleted, partially functional dystrophins like those produced in Becker muscular dystrophy, which may lead to improvements in DMD skeletal muscle function through improved membrane stability and cell signaling. The first AON, currently in late phase clinical trials, is drisapersen (PRO051; GSK), a 2′-O-methyl phosphorothioate (2OMePS) RNA molecule inducing specific exon 51 skipping. Recently, a second AON was developed to target a different DMD patient population. PRO044, also a 2OMePS RNA molecule, applies to 6% of the DMD population and induces specific exon 44 skipping in dystrophin premRNA and dystrophin expression in cultured muscle cells of DMD patients. A Phase 1/2 open-label study of systemic PRO044 in 18 boys with DMD (5–15 years) is close to completion, exploring safety pharmacokinetics and molecular efficacy at different dose levels with five once-weekly sc administrations (0.5, 1.5, 5, 8, 12 mg/kg). Nine of these boys have also received 5 additional weekly doses at different dose levels by iv infusion (1.5, 5, 12 mg/kg). Preliminary data are encouraging. No clinical response was expected after 5 weeks’ treatment; however, the safety profile is consistent with the class of compound and a molecular response has been observed. Due to high degrees of spontaneous skipping, giving rise to revertant fibers in this DMD population, a new and robust, reproducible dystrophin detection methodology has been developed. Data will be presented to show effects on safety and plasma/tissue drug levels, putative biomarkers and muscle dystrophin expression, which will be used to support subsequent long-term PRO044 studies.

Exon‐skipping events in candidates for clinical trials of morpholino

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial. Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription-polymerase chain reaction. Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event. An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.

Enhanced Exon-skipping Induced by U7 snRNA Carrying a Splicing Silencer Sequence: Promising Tool for DMD Therapy

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. In most cases, the open-reading frame is disrupted which results in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and has recently been shown to correct the reading frame and restore dystrophin expression in vitro and in vivo. Specific exon skipping can be achieved using synthetic oligonucleotides or viral vectors encoding modified small nuclear RNAs (snRNAs), by masking important splicing sites. In this study, we demonstrate that enhanced exon skipping can be induced by a U7 snRNA carrying binding sites for the heterogeneous ribonucleoprotein A1 (hnRNPA1). In DMD patient cells, bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51, and thus restore dystrophin expression to near wild-type levels. Furthermore, we show the efficacy of these constructs in vivo in transgenic mice carrying the entire human DMD locus after intramuscular injection of adeno-associated virus (AAV) vectors encoding the bifunctional U7 snRNA. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications.

Diagnostic utility of skin biopsy in dystrophinopathies

Aims Muscle biopsy is an important diagnostic modality and screening test for the diagnosis of dystrophinopathies. Sometimes muscle biopsies are needed for the diagnosis when genetic tests are inconclusive and are also useful for immunoblotting assay of the dystrophin protein. However, the procedure is painful, requires anesthesia and sometimes needs to be repeated. This study was undertaken to elucidate the role of skin biopsy in the diagnosis of dystrophinopathies and to validate if it can be utilized as a useful adjunct/replacement for the muscle biopsy. Methods Paired skin and muscle biopsies were studied from 39 patients with Duchenne muscular dystrophy (DMD), 4 patients with Becker's muscular dystrophy (BMD) and 37 controls. Immunostaining for dystrophin and utrophin was done on frozen sections of the test group and controls and their staining pattern in skin biopsies was compared with corresponding muscle biopsies. Results Immunostaining for dystrophin was negative in the skin biopsies of all patients (39/39, 100%) with DMD and was only weakly expressed in skin of BMD patients (4/4, 100%). Dystrophin was strongly expressed on arrector pili muscle cells of all control patients (94.6%) except two cases in whom it was weakly expressed. Utrophin was expressed on the arrector pili muscle cells of DMD patients (39/39, 100%) as well as controls (30/37, 81.1%). Conclusion Our study suggests that skin biopsy is very useful for the diagnosis of dystrophinopathies and their differentiation from other muscle diseases. It has high degrees of sensitivity, specificity, and positive and negative predictive values. It can be a useful adjunct/replacement for the muscle biopsy especially when repeated biopsies are required for monitoring therapy or in patients with advanced DMD where extreme fibrosis, adipose tissue infiltration and inflammation make interpretation of the muscle biopsy difficult. Skin biopsy is a simple, cost effective, less invasive and less traumatic diagnostic procedure when compared with muscle biopsy. This is even more pertinent because patients with muscular dystrophies have a higher risk for any form of general anesthesia. A smaller scar and fewer chances of infection at the site of biopsy are other additional advantages of skin biopsy over muscle biopsy.

The free cytoplasmic Ca2+ levels in duchenne muscular dystrophy lymphocytes

An increased cellular Ca2+ content has been associated with Duchenne muscular dystrophy (DMD). However, estimates of the free cytoplasmic Ca2+ concentration ([Ca2+]i) in cells of DMD patients were not available. We compared the [Ca2+]i levels of normal and DMD peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using the novel probe, quin 2, an internally trapped fluorescent indicator. The [Ca2+]i levels of normal and DMD cells were not significantly different.