Hans Heemskerk

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.

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.

Phage display screening without repetitious selection rounds.

Phage display screenings are frequently employed to identify high-affinity peptides or antibodies. Although successful, phage display is a laborious technology and is notorious for identification of false positive hits. To accelerate and improve the selection process, we have employed Illumina next generation sequencing to deeply characterize the Ph.D.-7 M13 peptide phage display library before and after several rounds of biopanning on KS483 osteoblast cells. Sequencing of the naive library after one round of amplification in bacteria identifies propagation advantage as an important source of false positive hits. Most important, our data show that deep sequencing of the phage pool after a first round of biopanning is already sufficient to identify positive phages. Whereas traditional sequencing of a limited number of clones after one or two rounds of selection is uninformative, the required additional rounds of biopanning are associated with the risk of losing promising clones propagating slower than nonbinding phages. Confocal and live cell imaging confirms that our screen successfully selected a peptide with very high binding and uptake in osteoblasts. We conclude that next generation sequencing can significantly empower phage display screenings by accelerating the finding of specific binders and restraining the number of false positive hits.

Development of Antisense‐Mediated Exon Skipping as a Treatment for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe muscle‐wasting disease caused by frame shifting and nonsense mutations in the dystrophin gene. Through skipping of an (additional) exon from the pre‐mRNA, the reading frame can be restored. This can be achieved with antisense oligonucleotides (AONs), which induce exon skipping by binding to splice sites or splice enhancer sites. The resulting protein will be shorter but at least partially functional. So far, exon skipping has been very successful in cell cultures, in mouse and dog models, and even in a first exploratory study in patients. Current research mainly focuses on optimization of systemic AON delivery. Here we give an overview of the available mouse models. To obtain the most informative results for future clinical application, research may have to move from the currently preferred mdx mouse to mouse models more comparable to patients, such as the utrophin/dystrophin‐negative mouse and the hDMD mouse models. Further, we briefly discuss two AON backbone chemistries that are currently in clinical trials for DMD exon skipping. We propose that different chemistries should be further developed in parallel in order to hasten the transfer of the exon skipping therapy to the clinic.

Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD) is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs). Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype). Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK) levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

Accurate quantification of dystrophin mRNA and exon skipping levels in Duchenne Muscular Dystrophy

Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model.

O.12 Peptide-enhanced uptake and bioactivity of antisense oligonucleotides in muscle and heart of DMD and DM1 mouse models

RNA-modulation by antisense oligonucleotides (AONs) represents an interesting therapeutic approach for different neuromuscular disorders, such as Duchenne muscular dystrophy (DMD) and myotonic dystrophy type 1 (DM1). DMD is caused by out of frame mutations in the DMD gene. AON-mediated exon skipping, aimed to restore the disrupted reading frame of the dystrophin transcript, is currently in (pre-) clinical development. In DM1, DM protein kinase transcripts contain a toxic expanded (CUG) n repeat stretch. Suppression of these toxic transcripts by AONs is currently being explored as a potential molecular intervention for DM1. For both diseases, efficient body-wide delivery of the AONs to muscle tissue and heart would enhance their therapeutic effect. The lack of dystrophin in DMD muscle results in more permeable muscle fibers, which, in the mdx mouse model, was demonstrated to promote AON uptake in muscle but to a lesser extent in heart. As the muscle fiber membranes are not impaired in DM1, various delivery-enhancing complexes and ligands are being explored to obtain sufficient muscle and heart uptake of AONs. Here, we present results with a 7-amino acid linear peptide that, conjugated to 2’-O-methyl phosphorothioate AONs, seems to enhance their uptake and bioactivity in muscle and, particularly, heart, in both DMD and DM1 mouse models.

T.P.26 Low dystrophin levels increase survival and improve pathology and motor function in dystrophin/utrophin double knockout mice

Abstract In Duchenne Muscular Dystrophy (DMD) patients muscle fibers are susceptible to exercise-induced injury due to absence of functional dystrophin. No cure is available, but in the last decade major progress has been made in the challenge to restore dystrophin expression in DMD patients. It is unknown how much dystrophin is needed to slow or prevent disease progression. To elucidate this, we generated mdx-Xist Δhs utrn − / − mice in which skewed X-inactivation results in expression of variable, low dystrophin levels in a utrophin negative background. These mice ( n =20) underwent a 12week functional test regime after which histopathology was assessed. Dystrophin levels of 3–10% already significantly improved performance of two and four limb hanging wire tests and histopathology, while 10–17% further normalized this towards wild type. For improvement in grip strength higher dystrophin levels are needed. Most striking was the effect of already very modest dystrophin levels in maintenance of basic muscle function and protection against death from overall weakness. Whereas mdx/utrn − / − mice did not live beyond 12weeks, 62% of the mice expressing 3–10% dystrophin and all mice expressing 10–17% dystrophin survived 16weeks. A survival study in 42 mdx-Xist Δhs utrn − / − mice assessing skeletal muscle function and histopathology showed a median survival extension to 26weeks in mice with 3–10% dystrophin, while mice with 10–30% lived even longer. Biomarkers, skeletal muscle and heart function, and histopathology were significantly improved in mice with 3–10% dystrophin and further improvement was achieved with 10–30% dystrophin. These results suggest that even very low dystrophin levels already may have beneficial effects, and that survival and improvement of endurance efforts may be amongst the early effects of treatment. This underscores the urgency to develop better clinical readouts for the non-ambulatory phase.

Identification of peptides for tissue-specific delivery.

Antisense-mediated exon skipping has shown to be a promising therapeutic approach and is in clinical trials for Duchenne muscular dystrophy. However, after systemic treatment the majority of the injected antisense oligonucleotides (AONs) will not end up in the intended tissue. This mistargeting of AONs might have detrimental effects, especially with long-term treatment and continuous accumulation of AONs. Further, even when no detrimental effects occur, mistargeted AONs are lost for exon skipping in the intended tissue. One way to reduce the amount of mistargeted AONs is by adding a peptide that specifically binds to and is taken up by the intended tissue. Such peptides can be found by screening phage display libraries. With in silico, in vitro, and in vivo testing, the peptides that bind the intended tissue most efficiently and most specifically can be identified.