Judith C.T. van Deutekom

Systemic Administration of PRO051 in Duchenne's Muscular Dystrophy

BACKGROUND Local intramuscular administration of the antisense oligonucleotide PRO051 in patients with Duchennes muscular dystrophy with relevant mutations was previously reported to induce the skipping of exon 51 during pre-messenger RNA splicing of the dystrophin gene and to facilitate new dystrophin expression in muscle-fiber membranes. The present phase 1-2a study aimed to assess the safety, pharmacokinetics, and molecular and clinical effects of systemically administered PRO051. METHODS We administered weekly abdominal subcutaneous injections of PRO051 for 5 weeks in 12 patients, with each of four possible doses (0.5, 2.0, 4.0, and 6.0 mg per kilogram of body weight) given to 3 patients. Changes in RNA splicing and protein levels in the tibialis anterior muscle were assessed at two time points. All patients subsequently entered a 12-week open-label extension phase, during which they all received PRO051 at a dose of 6.0 mg per kilogram per week. Safety, pharmacokinetics, serum creatine kinase levels, and muscle strength and function were assessed. RESULTS The most common adverse events were irritation at the administration site and, during the extension phase, mild and variable proteinuria and increased urinary α(1)-microglobulin levels; there were no serious adverse events. The mean terminal half-life of PRO051 in the circulation was 29 days. PRO051 induced detectable, specific exon-51 skipping at doses of 2.0 mg or more per kilogram. New dystrophin expression was observed between approximately 60% and 100% of muscle fibers in 10 of the 12 patients, as measured on post-treatment biopsy, which increased in a dose-dependent manner to up to 15.6% of the expression in healthy muscle. After the 12-week extension phase, there was a mean (±SD) improvement of 35.2±28.7 m (from the baseline of 384±121 m) on the 6-minute walk test. CONCLUSIONS Systemically administered PRO051 showed dose-dependent molecular efficacy in patients with Duchennes muscular dystrophy, with a modest improvement in the 6-minute walk test after 12 weeks of extended treatment. (Funded by Prosensa Therapeutics; Netherlands National Trial Register number, NTR1241.).

Entries in the Leiden Duchenne muscular dystrophy mutation database: An overview of mutation types and paradoxical cases that confirm the reading-frame rule

The severe Duchenne and milder Becker muscular dystrophy are both caused by mutations in the DMD gene. This gene codes for dystrophin, a protein important for maintaining the stability of muscle‐fiber membranes. In 1988, Monaco and colleagues postulated an explanation for the phenotypic difference between Duchenne and Becker patients in the reading‐frame rule: In Duchenne patients, mutations induce a shift in the reading frame leading to prematurely truncated, dysfunctional dystrophins. In Becker patients, in‐frame mutations allow the synthesis of internally deleted, but largely functional dystrophins. Currently, over 4700 mutations have been reported in the Leiden DMD mutation database, of which 91% are in agreement with this rule. In this study we provide an update of the mutational variability in the DMD gene, particularly focusing on genotype–phenotype correlations and mutations that appear to be exceptions to the reading‐frame rule. Muscle Nerve, 2006

Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations†‡

Antisense‐mediated exon skipping aiming for reading frame restoration is currently a promising therapeutic application for Duchenne muscular dystrophy (DMD). This approach is mutation specific, but as the majority of DMD patients have deletions that cluster in hotspot regions, the skipping of a small number of exons is applicable to relatively large numbers of patients. To assess the actual applicability of the exon skipping approach, we here determined for deletions, duplications and point mutations reported in the Leiden DMD mutation database, which exon(s) should be skipped to restore the open reading frame. In theory, single and double exon skipping would be applicable to 79% of deletions, 91% of small mutations, and 73% of duplications, amounting to 83% of all DMD mutations. Exon 51 skipping, which is being tested in clinical trials, would be applicable to the largest group (13%) of all DMD patients. Further research is needed to determine the functionality of different in‐frame dystrophins and a number of hurdles has to be overcome before this approach can be applied clinically. Hum Mutat 0, 1–7, 2009.

Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense.

Dystrophin deficiency, which leads to severe and progressive muscle degeneration in patients with Duchenne muscular dystrophy (DMD), is caused by frameshifting mutations in the dystrophin gene. A relatively new therapeutic strategy is based on antisense oligonucleotides (AONs) that induce the specific skipping of a single exon, such that the reading frame is restored. This allows the synthesis of a largely functional dystrophin, associated with a milder Becker muscular dystrophy phenotype. We have previously successfully targeted 20 different DMD exons that would, theoretically, be beneficial for >75% of all patients. To further enlarge this proportion, we here studied the feasibility of double and multiexon skipping. Using a combination of AONs, double skipping of exon 43 and 44 was induced, and dystrophin synthesis was restored in myotubes from one patient affected by a nonsense mutation in exon 43. For another patient, with an exon 46-50 deletion, the therapeutic double skipping of exon 45 and 51 was achieved. Remarkably, in control myotubes, the latter combination of AONs caused the skipping of the entire stretch of exons from 45 through 51. This in-frame multiexon skipping would be therapeutic for a series of patients carrying different DMD-causing mutations. In fact, we here demonstrate its feasibility in myotubes from a patient with an exon 48-50 deletion. The application of multiexon skipping may provide a more uniform methodology for a larger group of patients with DMD.

Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy is primarily caused by frame-disrupting mutations in the Duchenne muscular dystrophy gene which abort dystrophin synthesis. We have explored a gene correction therapy aimed at restoration of the reading frame in Duchenne muscular dystrophy patients. Through the binding of antisense oligoribonucleotides to exon-internal sequences in the pre-mRNA, the splicing can be manipulated in such a manner that the targeted exon is skipped and a slightly shorter, but in-frame, transcript is generated. We recently showed that antisense oligoribonucleotide-mediated skipping of exon 46 efficiently induced dystrophin synthesis in cultured muscle cells from Duchenne muscular dystrophy patients carrying an exon 45 deletion. In this study we have identified antisense oligoribonucleotides with which the skipping of 11 other Duchenne muscular dystrophy exons could be induced in cultured human muscle cells. The targeted skipping of only one particular exon may restore the reading frame in a series of patients with different mutations. Accordingly, these antisense oligoribonucleotides would allow correction of over 50% of deletions and 22% of duplications reported in the Leiden DMD-mutation Database.

In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping.

Antisense‐mediated exon skipping is a putative treatment for Duchenne muscular dystrophy (DMD). Using antisense oligonucleotides (AONs), the disrupted DMD reading frame is restored, allowing generation of partially functional dystrophin and conversion of a severe Duchenne into a milder Becker muscular dystrophy phenotype. In vivo studies are mainly performed using 2′‐O‐methyl phosphorothioate (2OMePS) or morpholino (PMO) AONs. These compounds were never directly compared.

Advances in Duchenne muscular dystrophy gene therapy

Since the initial characterization of the genetic defect for Duchenne muscular dystrophy, much effort has been expended in attempts to develop a therapy for this devastating childhood disease. Gene therapy was the obvious answer but, initially, the dystrophin gene and its product seemed too large and complex for this approach. However, our increasing knowledge of the organization of the gene and the role of dystrophin in muscle function has indicated ways to manipulate them both. Gene therapy for Duchenne muscular dystrophy now seems to be in reach.

Guidelines for Antisense Oligonucleotide Design and Insight Into Splice-modulating Mechanisms

Antisense oligonucleotides (AONs) can interfere with mRNA processing through RNase H-mediated degradation, translational arrest, or modulation of splicing. The antisense approach relies on AONs to efficiently bind to target sequences and depends on AON length, sequence content, secondary structure, thermodynamic properties, and target accessibility. We here performed a retrospective analysis of a series of 156 AONs (104 effective, 52 ineffective) previously designed and evaluated for splice modulation of the dystrophin transcript. This showed that the guanine-cytosine content and the binding energies of AON-target and AON-AON complexes were significantly higher for effective AONs. Effective AONs were also located significantly closer to the acceptor splice site (SS). All analyzed AONs are exon-internal and may act through steric hindrance of Ser-Arg-rich (SR) proteins to exonic splicing enhancer (ESE) sites. Indeed, effective AONs were significantly enriched for ESEs predicted by ESE software programs, except for predicted binding sites of SR protein Tra2beta, which were significantly enriched in ineffective AONs. These findings compile guidelines for development of AONs and provide more insight into the mechanism of antisense-mediated exon skipping. On the basis of only four parameters, we could correctly classify 79% of all AONs as effective or ineffective, suggesting these parameters can be used to more optimally design splice-modulating AONs.Antisense oligonucleotides (AONs) can interfere with mRNA processing through RNase H-mediated degradation, translational arrest, or modulation of splicing. The antisense approach relies on AONs to efficiently bind to target sequences and depends on AON length, sequence content, secondary structure, thermodynamic properties, and target accessibility. We here performed a retrospective analysis of a series of 156 AONs (104 effective, 52 ineffective) previously designed and evaluated for splice modulation of the dystrophin transcript. This showed that the guanine-cytosine content and the binding energies of AON-target and AON-AON complexes were significantly higher for effective AONs. Effective AONs were also located significantly closer to the acceptor splice site (SS). All analyzed AONs are exon-internal and may act through steric hindrance of Ser-Arg-rich (SR) proteins to exonic splicing enhancer (ESE) sites. Indeed, effective AONs were significantly enriched for ESEs predicted by ESE software programs, except for predicted binding sites of SR protein Tra2β, which were significantly enriched in ineffective AONs. These findings compile guidelines for development of AONs and provide more insight into the mechanism of antisense-mediated exon skipping. On the basis of only four parameters, we could correctly classify 79% of all AONs as effective or ineffective, suggesting these parameters can be used to more optimally design splice-modulating AONs.

Preclinical PK and PD Studies on 2 '-O-Methyl-phosphorothioate RNA Antisense Oligonucleotides in the mdx Mouse Model

Antisense oligonucleotides (AONs) are being developed as RNA therapeutic molecules for Duchenne muscular dystrophy. For oligonucleotides with the 2′-O-methyl-phosphorothioate (2OMePS) RNA chemistry, proof of concept has been obtained in patient-specific muscle cell cultures, the mouse and dog disease models, and recently by local administration in Duchenne patients. To further explore the pharmacokinetic (PK)/pharmacodynamic (PD) properties of this chemical class of oligonucleotides, we performed a series of preclinical studies in mice. The results demonstrate that the levels of oligonucleotides in dystrophin-deficient muscle fibers are much higher than in healthy fibers, leading to higher exon-skipping levels. Oligonucleotide levels and half-life differed for specific muscle groups, with heart muscle showing the lowest levels but longest half-life (~46 days). Intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) delivery methods were directly compared. For each method, exon-skipping and novel dystrophin expression were observed in all muscles, including arrector pili smooth muscle in skin biopsies. After i.v. administration, the oligonucleotide peak levels in plasma, liver, and kidney were higher than after s.c. or i.p. injections. However, as the bioavailability was similar, and the levels of oligonucleotide, exon-skipping, and dystrophin steadily accumulated overtime after s.c. administration, we selected this patient-convenient delivery method for future clinical study protocols.

Assessment of the feasibility of exon 45–55 multiexon skipping for duchenne muscular dystrophy

BackgroundThe specific skipping of an exon, induced by antisense oligonucleotides (AON) during splicing, has shown to be a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients. As different mutations require skipping of different exons, this approach is mutation dependent. The skipping of an entire stretch of exons (e.g. exons 45 to 55) has recently been suggested as an approach applicable to larger groups of patients. However, this multiexon skipping approach is technically challenging. The levels of intended multiexon skips are typically low and highly variable, and may be dependent on the order of intron removal. We hypothesized that the splicing order might favor the induction of multiexon 45–55 skipping.MethodsWe here tested the feasibility of inducing multiexon 45–55 in control and patient muscle cell cultures using various AON cocktails.ResultsIn all experiments, the exon 45–55 skip frequencies were minimal and comparable to those observed in untreated cells.ConclusionWe conclude that current state of the art does not sufficiently support clinical development of multiexon skipping for DMD.