M6229 Protects against Extracellular-Histone-Induced Liver Injury, Kidney Dysfunction, and Mortality in a Rat Model of Acute Hyperinflammation.

Extracellular histones have been shown to act as DAMPs in a variety of inflammatory diseases. Moreover, they have the ability to induce cell death. In this study, we show that M6229, a low-anticoagulant fraction of unfractionated heparin (UFH), rescues rats that were challenged by continuous infusion of calf thymus histones at a rate of 25 mg histones/kg/h. Histone infusion by itself induced hepatic and homeostatic dysfunction characterized by elevated activity of hepatic enzymes (ASAT and ALAT) and serum lactate levels as well as by a renal dysfunction, which contributed to the significantly increased mortality rate. M6229 was able to restore normal levels of both hepatic and renal parameters at 3 and 9 mg M6229/kg/h and prevented mortality of the animals. We conclude that M6229 is a promising therapeutic agent to treat histone-mediated disease.

Bis(maltolato)oxovanadium(IV) Induces Angiogenesis via Phosphorylation of VEGFR2.

VEGFR2 and VEGF-A play a pivotal role in the process of angiogenesis. VEGFR2 activation is regulated by protein tyrosine phosphatases (PTPs), enzymes that dephosphorylate the receptor and reduce angiogenesis. We aim to study the effect of PTPs blockade using bis(maltolato)oxovanadium(IV) (BMOV) on in vivo wound healing and in vitro angiogenesis. BMOV significantly improves in vivo wound closure by 45% in C57BL/6JRj mice. We found that upon VEGFR2 phosphorylation induced by endogenously produced VEGF-A, the addition of BMOV results in increased cell migration (45%), proliferation (40%) and tube formation (27%) in HUVECs compared to control. In a mouse ex vivo, aortic ring assay BMOV increased the number of sprouts by 3 folds when compared to control. However, BMOV coadministered with exogenous VEGF-A increased ECs migration, proliferation and tube formation by only 41%, 18% and 12% respectively and aortic ring sprouting by only 1-fold. We also found that BMOV enhances VEGFR2 Y951 and p38MAPK phosphorylation, but not ERK1/2. The level of phosphorylation of these residues was the same in the groups treated with BMOV supplemented with exogenous VEGF-A and exogenous VEGF-A only. Our study demonstrates that BMOV is able to enhance wound closure in vivo. Moreover, in the presence of endogenous VEGF-A, BMOV is able to stimulate in vitro angiogenesis by increasing the phosphorylation of VEGFR2 and its downstream proangiogenic enzymes. Importantly, BMOV had a stronger proangiogenic effect compared to its effect in coadministration with exogenous VEGF-A.

The Pharmacokinetics of 2'-O-Methyl Phosphorothioate Antisense Oligonucleotides: Experiences from Developing Exon Skipping Therapies for Duchenne Muscular Dystrophy.

Delivery to the target site and adversities related to off-target exposure have made the road to clinical success and approval of antisense oligonucleotide (AON) therapies challenging. Various classes of AONs have distinct chemical features and pharmacological properties. Understanding the similarities and differences in pharmacokinetics (PKs) among AON classes is important to make future development more efficient and may facilitate regulatory guidance of AON development programs. For the class of 2'-O-methyl phosphorothioate (2OMe PS) RNA AONs, most nonclinical and clinical PK data available today are derived from development of exon skipping therapies for Duchenne muscular dystrophy (DMD). While some publications have featured PK aspects of these AONs, no comprehensive overview is available to date. This article presents a detailed review of absorption, distribution, metabolism, and excretion of 2OMe PS AONs, compiled from publicly available data and previously unpublished internal data on drisapersen and related exon skipping candidates in preclinical species and DMD patients. Considerations regarding drug-drug interactions, toxicokinetics, and pharmacodynamics are also discussed. From the data presented, the picture emerges of consistent PK properties within the 2OMe PS class, predictable behavior across species, and a considerable overlap with other single-stranded PS AONs. A level of detail on muscle as a target tissue is provided, which was not previously available. Furthermore, muscle biopsy samples taken in DMD clinical trials allowed confirmation of the applicability of interspecies scaling approaches commonly applied in the absence of clinical target tissue data.

Placebo-controlled Phase 2 Trial of Drisapersen for Duchenne Muscular Dystrophy.

OBJECTIVE: This double-blind, randomized, placebo-controlled Phase 2 study (NCT01462292) assessed the 24-week efficacy, safety, tolerability, and pharmacokinetics of two different subcutaneous drisapersen doses, and the 24-week off-dose persistent effect, in ambulant Duchenne muscular dystrophy (DMD) patients. METHODS: Male DMD patients (≥5 years; time to rise from floor ≤15 s) were randomized to drisapersen 3 mg/kg/week, 6 mg/kg/week or placebo. The primary efficacy endpoint was change from baseline in 6-minute walking distance (6MWD) at week 24. Secondary endpoints included changes in timed function tests, muscle strength, and pulmonary function tests. RESULTS: Fifty-one patients were randomized to placebo (N = 16), drisapersen 3 mg/kg/week (N = 17) or 6 mg/kg/week (N = 18). All but 2 patients had baseline rise from floor time <7 s. This study was exploratory and not prospectively powered; however, a difference in mean 6MWD versus placebo in favor of drisapersen 6 mg/kg/week was observed at week 24 (27.1 m; P = 0.069) and maintained 24 weeks off-treatment (27.9 m; P = 0.177). The 3 mg/kg/week group showed no statistically significant difference in mean 6MWD versus placebo. For some secondary endpoints, a more positive response in favor of drisapersen 6 mg/kg/week compared to placebo was shown. Drisapersen had a long half-life with steady state reached after approximately 36 weeks. Most common adverse events in both drisapersen groups were related to injection site reactions and subclinical proteinuria. INTERPRETATION: Drisapersen 6 mg/kg/week for 24 weeks resulted in a treatment benefit in 6MWD, largely maintained 24 weeks off-treatment. This study provided insights for further studies to optimize dosage regimen.

Long-Term Efficacy, Safety, and Pharmacokinetics of Drisapersen in Duchenne Muscular Dystrophy: Results from an Open-Label Extension Study.

BACKGROUND: Drisapersen induces exon 51 skipping during dystrophin pre-mRNA splicing and allows synthesis of partially functional dystrophin in Duchenne muscular dystrophy (DMD) patients with amenable mutations. METHODS: This 188-week open-label extension of the dose-escalation study assessed the long-term efficacy, safety, and pharmacokinetics of drisapersen (PRO051/GSK2402968), 6 mg/kg subcutaneously, in 12 DMD subjects. Dosing was once weekly for 72 weeks. All subjects had a planned treatment interruption (weeks 73-80), followed by intermittent dosing (weeks 81-188). RESULTS: Subjects received a median (range) total dose of 5.93 (5.10 to 6.02) mg/kg drisapersen. After 177 weeks (last efficacy assessment), median (mean [SD]) six-minute walk distance (6MWD) improved by 8 (-24.5 [161]) meters for the 10 subjects able to complete the 6MWD at baseline (mean age [SD]: 9.5 [1.9] years). These statistics include 2 subjects unable to complete the test at later visits and who scored "zero". When only the 8 ambulant subjects at week 177 were taken into account, a median (mean [SD]) increase of 64 (33 [121]) meters in 6MWD was observed. Of 7 subjects walking ≥330 m at extension baseline, 5 walked farther at week 177. Of 3 subjects walking <330 m, 2 lost ambulation, while 1 declined overall but walked farther at some visits. Over the 188 weeks, the most common adverse events were injection-site reactions, raised urinary α1-microglobulin and proteinuria. Dystrophin expression was detected in all muscle biopsies obtained at week 68 or 72. CONCLUSION: Drisapersen was generally well tolerated over 188 weeks. Possible renal effects, thrombocytopenia and injection-site reactions warrant continued monitoring. Improvements in the 6MWD at 12 weeks were sustained after 3.4 years of dosing for most patients. For a small, uncontrolled study, the outcomes are encouraging, as natural history studies would anticipate a decline of over 100 meters over a 3-year period in a comparable cohort. TRIAL REGISTRATION: ClinicalTrials.gov NCT01910649.

Preclinical studies on intestinal administration of antisense oligonucleotides as a model for oral delivery for treatment of duchenne muscular dystrophy.

Antisense oligonucleotides (AONs) used to reframe dystrophin mRNA transcripts for Duchenne muscular dystrophy (DMD) patients are tested in clinical trials. Here, AONs are administered subcutaneously and intravenously, while the less invasive oral route would be preferred. Oral delivery of encapsulated AONs supplemented with a permeation enhancer, sodium caprate, has been successfully used to target tumor necrosis factor (TNF)-α expression in liver. To test the feasibility of orally delivered AONs for DMD, we applied 2'-O-methyl phosphorothioate AONs (with or without sodium caprate supplementation) directly to the intestine of mdx mice and compared pharmacokinetics and -dynamics with intravenous, intraperitoneal, and subcutaneous delivery. Intestinally infused AONs were taken up, but resulted in lower plasma levels compared to other delivery routes, although bioavailability could be largely improved by supplementation of sodium caprate. After intestinal infusion, AON levels in all tissues were lower than for other administration routes, as were the ratios of target versus nontarget organ levels, except for diaphragm and heart where comparable levels and ratios were observed. For each administration route, low levels of exon skipping in triceps was observed 3 hours post-AON administration. These data suggest that oral administration of naked 2'-O-methyl phosphorothioate AONs may be feasible, but only when high AON concentrations are used in combination with sodium caprate.

A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2-17% and intra-assay precision, CV 2-10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.

Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study.

BACKGROUND: Duchenne muscular dystrophy is caused by dystrophin deficiency and muscle deterioration and preferentially affects boys. Antisense-oligonucleotide-induced exon skipping allows synthesis of partially functional dystrophin. We investigated the efficacy and safety of drisapersen, a 2'-O-methyl-phosphorothioate antisense oligonucleotide, given for 48 weeks. METHODS: In this exploratory, double-blind, placebo-controlled study we recruited male patients (≥5 years of age; time to rise from floor ≤7 s) with Duchenne muscular dystrophy from 13 specialist centres in nine countries between Sept 1, 2010, and Sept 12, 2012. By use of a computer-generated randomisation sequence, we randomly allocated patients (2:2:1:1; block size of six; no stratification) to drisapersen 6 mg/kg or placebo, each given subcutaneously and either continuously (once weekly) or intermittently (nine doses over 10 weeks). The primary endpoint was change in 6-min walk distance (6MWD) at week 25 in patients in the intention-to-treat population for whom data were available. Safety assessments included renal, hepatic, and haematological monitoring and recording of adverse events. This trial is registered with ClinicalTrials.gov, number NCT01153932. FINDINGS: We recruited 53 patients: 18 were given continuous drisapersen, 17 were given intermittent drisapersen, and 18 were given placebo (continuous and intermittent groups combined). At week 25, mean 6MWD had increased by 31·5 m (SE 9·8) from baseline for continuous drisapersen, with a mean difference in change from baseline of 35·09 m (95% CI 7·59 to 62·60; p=0·014) versus placebo. We recorded no difference in 6MWD changes from baseline between intermittent drisapersen (mean change -0·1 [SE 10·3]) and placebo (mean difference 3·51 m [-24·34 to 31·35]) at week 25. The most common adverse events in drisapersen-treated patients were injection-site reactions (14 patients given continuous drisapersen, 15 patients given intermittent drisapersen, and six given placebo) and renal events (13 for continuous drisapersen, 12 for intermittent drisapersen, and seven for placebo), most of which were subclinical proteinuria. None of the serious adverse events reported (one for continuous, two for intermittent, and two for placebo) resulted in withdrawal from the study. INTERPRETATION: Continuous drisapersen resulted in some benefit in 6MWD versus placebo at week 25. The safety findings are similar to those from previous studies. Ambulation improvements in this young population with early-stage Duchenne muscular dystrophy are encouraging but need to be confirmed in larger studies. FUNDING: GlaxoSmithKline, Prosensa Therapeutics BV (a subsidiary of Prosensa Holding NV).

The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice.

Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD) to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides) will be required. To investigate the dynamics and persistence of antisense 2'-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours-24 weeks). Oligonucleotide half-life was longer in heart (~65 days) compared with that in skeletal muscle, liver, and kidney (~35 days). Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days). Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3-8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2'-O-methyl phosphorothioate oligos used for the treatment of DMD.Molecular Therapy-Nucleic Acids (2014) 3, e148; doi:10.1038/mtna.2014.1; published online 18 February 2014.

Species-specific inflammatory responses as a primary component for the development of glomerular lesions in mice and monkeys following chronic administration of a second-generation antisense oligonucleotide.

Chronic administration of drisapersen, a 2'-OMe phosphorothioate antisense oligonucleotide (AON) to mice and monkeys resulted in renal tubular accumulation, with secondary tubular degeneration. Glomerulopathy occurred in both species with species-specific characteristics. Glomerular lesions in mice were characterized by progressive hyaline matrix accumulation, accompanied by the presence of renal amyloid and with subsequent papillary necrosis. Early changes involved glomerular endothelial hypertrophy and degeneration, but the chronic glomerular amyloid and hyaline alterations in mice appeared to be species specific. An immune-mediated mechanism for the glomerular lesions in mice was supported by early inflammatory changes including increased expression of inflammatory cytokines and other immunomodulatory genes within the renal cortex, increased stimulation of CD68 protein, and systemic elevation of monocyte chemotactic protein 1. In contrast, kidneys from monkeys given drisapersen chronically showed less severe glomerular changes characterized by increased mesangial and inflammatory cells, endothelial cell hypertrophy, and subepithelial and membranous electron-dense deposits, with ultrastructural and immunohistochemical characteristics of complement and complement-related fragments. Lesions in monkeys resembled typical features of C3 glomerulopathy, a condition described in man and experimental animals to be linked to dysregulation of the alternative complement pathway. Thus, inflammatory/immune mechanisms appear critical to glomerular injury with species-specific sensitivities for mouse and monkey. The lower observed proinflammatory activity in humans as compared to mice and monkeys may reflect a lower risk of glomerular injury in patients receiving AON therapy.

Dose-dependent pharmacokinetic profiles of 2'-O-methyl phosphorothioate antisense oligonucleotidesin mdx mice.

Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy. It aims to restore the dystrophin open reading frame by skipping exons with antisense oligonucleotides (AONs) to allow production of partly functional proteins. The approach is currently tested in phase 3 clinical trials, but dosing and maintenance regimens have not yet been well studied. This study compared pharmacokinetic and pharmacodynamic effects of different 2'-O-methyl phosphorothioate RNA AON dosing and maintenance regimens in the preclinical mdx mouse model. When comparing different dosing regimens over a period of 8 weeks, higher levels of AON, exon skipping, and protein were observed in muscle after low daily doses compared with large weekly doses. Secondly, after receiving a high loading dose (1,250 mg/kg) in the first week, mice treated with maintenance injections twice weekly for 8 weeks showed higher preservation of therapeutic effects than mice receiving less or no maintenance injections. In both cases, the regimen resulting in the highest AON and exon skipping levels in muscle also resulted in high AON levels in liver and kidneys. These studies underline the importance of balancing optimal AON efficacy and tolerable levels in non-target organs, which may be fine-tuned by further optimization of AON treatment regimens.

Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP).

The cytokine interleukin 1(IL-1) initiates a wide range of proinflammatory cascades and its inhibition has been shown to decrease inflammation in a variety of diseases. IL-1 receptor accessory protein (IL-1RAcP) is an indispensible part of the IL-1R complex that stabilizes IL-1/IL-1R interaction and plays an important role in the signal transduction of the receptor complex. The soluble form of IL-1RAcP (sIL-1RAcP) contains only the extracellular domain and serves as a natural inhibitor of IL-1 signaling. Therefore, increasing sIL-1RAcP levels might be an attractive therapeutic strategy to inhibit IL-1-driven inflammation. To achieve this we designed specific antisense oligonucleotides (AON), to redirect pre-mRNA IL-1RAcP splicing by skipping of the transmembrane domain encoding exon 9. This would give rise to a novel Δ9IL-1RAcP mRNA encoding a soluble, secreted form of IL-1RAcP, which might have similar activity as natural sIL-1RAcP. AON treatment resulted in exon 9 skipping both in vitro and in vivo. A single dose injection of 10 mg AON/kg body weight induced 90% skipping in mouse liver during at least 5 days. The truncated mRNA encoded for a secreted, soluble Δ9IL-1RAcP protein. IL-1RAcP skipping resulted in a substantial inhibition of IL-1 signaling in vitro. These results indicate that skipping of the transmembrane encoding exon 9 of IL-1RAcP using specific AONs might be a promising therapeutic strategy in a variety of chronic inflammatory diseases.Molecular Therapy - Nucleic Acids (2013) 2, e66; doi:10.1038/mtna.2012.58; published online 22 January 2013.

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.Molecular Therapy-Nucleic Acids (2012) 1, e44; doi:10.1038/mtna.2012.38; published online 4 September 2012.

Systemic administration of PRO051 in Duchenne's muscular dystrophy.

BACKGROUND: Local intramuscular administration of the antisense oligonucleotide PRO051 in patients with Duchenne's 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 Duchenne's 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.).

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 (approximately 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.

Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast-myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2'-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

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

BACKGROUND: 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. METHODS: mdx and humanized (h)DMD mice were injected intramuscularly and intravenously with short versus long 2OMePS and PMO for mouse exon 23 and human exons 44, 45, 46 and 51. RESULTS: Intramuscular injection showed that increasing the length of 2OMePS AONs enhanced skipping efficiencies of human exon 45, but decreased efficiency for mouse exon 23. Although PMO induced more mouse exon 23 skipping, PMO and 2OMePS were more comparable for human exons. After intravenous administration, exon skipping and novel protein was shown in the heart with both chemistries. Furthermore, PMO showed lower intramuscular concentrations with higher exon 23 skipping levels compared to 2OMePS, which may be due to sequestration in the extracellular matrix. Finally, two mismatches rendered 2OMePS but not PMO AONs nearly ineffective. CONCLUSIONS: The results obtained in the present study indicate that increasing AON length improves skipping efficiency in some but not all cases. It is feasible to induce exon skipping and dystrophin restoration in the heart after injection of 2OMePS and unconjugated PMO. Furthermore, differences in efficiency between PMO and 2OMePS appear to be sequence and not chemistry dependent. Finally, the results indicate that PMOs may be less sequence specific than 2OMePS.

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.

Local dystrophin restoration with antisense oligonucleotide PRO051.

BACKGROUND: Duchenne's muscular dystrophy is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. By inducing specific exon skipping during messenger RNA (mRNA) splicing, antisense compounds were recently shown to correct the open reading frame of the DMD gene and thus to restore dystrophin expression in vitro and in animal models in vivo. We explored the safety, adverse-event profile, and local dystrophin-restoring effect of a single, intramuscular dose of an antisense oligonucleotide, PRO051, in patients with this disease. METHODS: Four patients, who were selected on the basis of their mutational status, muscle condition, and positive exon-skipping response to PRO051 in vitro, received a dose of 0.8 mg of PRO051 injected into the tibialis anterior muscle. A biopsy was performed 28 days later. Safety measures, composition of mRNA, and dystrophin expression were assessed. RESULTS: PRO051 injection was not associated with clinically apparent adverse events. Each patient showed specific skipping of exon 51 and sarcolemmal dystrophin in 64 to 97% of myofibers. The amount of dystrophin in total protein extracts ranged from 3 to 12% of that found in the control specimen and from 17 to 35% of that of the control specimen in the quantitative ratio of dystrophin to laminin alpha2. CONCLUSIONS: Intramuscular injection of antisense oligonucleotide PRO051 induced dystrophin synthesis in four patients with Duchenne's muscular dystrophy who had suitable mutations, suggesting that further studies might be feasible.