Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice.

Duchenne muscular dystrophy (DMD) is a severe childhood muscle disease primarily caused by the lack of functional dystrophin at the muscle fiber membranes. Multiple therapeutic approaches are currently in (pre)clinical development, aimed at restoring expression of (truncated) dystrophin. Key questions in this phase relate to route of drug administration, dose regimen, and levels of dystrophin required to improve muscle function. A series of studies applying antisense oligonucleotides (AONs) in the mdx mouse model for DMD has been reported over the last two decades, claiming a variable range of exon skipping and increased dystrophin levels correlated to some functional improvement. The aim of this study was to compare the efficacy of subcutaneous (SC) versus intravenous (IV) dosing routes of an mdx-specific AON at both the molecular and functional level, using state-of-the-art quantitative technologies, including digital droplet polymerase chain reaction, capillary Western immunoassay, magnetic resonance imaging, and automated kinematic analysis. The majority of all readouts we quantified, both molecular and functional, showed that IV dosing of the AON had a more pronounced beneficial effect than SC dosing in mdx mice. Last, but not least, the more quantitative molecular and functional data obtained in this study suggest that low levels of dystrophin protein of at least 2.5% of wild type may already have a beneficial effect on muscle leakiness and may improve motor performance of mdx mice.

Suppression of Mutant Protein Expression in SCA3 and SCA1 Mice Using a CAG Repeat-Targeting Antisense Oligonucleotide.

Spinocerebellar ataxia type 3 (SCA3) and type 1 (SCA1) are dominantly inherited neurodegenerative disorders that are currently incurable. Both diseases are caused by a CAG-repeat expansion in exon 10 of the Ataxin-3 and exon 8 of the Ataxin-1 gene, respectively, encoding an elongated polyglutamine tract that confers toxic properties to the resulting proteins. We have previously shown lowering of the pathogenic polyglutamine protein in Huntington's disease mouse models using (CUG)7, a CAG repeat-targeting antisense oligonucleotide. Here we evaluated the therapeutic capacity of (CUG)7 for SCA3 and SCA1, in vitro in patient-derived cell lines and in vivo in representative mouse models. Repeated intracerebroventricular (CUG)7 administration resulted in a significant reduction of mutant Ataxin-3 and Ataxin-1 proteins throughout the brain of SCA3 and SCA1 mouse models, respectively. Furthermore, in both a SCA3 patient cell line and the MJD84.2 mouse model, (CUG)7 induced formation of a truncated Ataxin-3 protein species lacking the polyglutamine stretch, likely arising from (CUG)7-mediated exon 10 skipping. In contrast, skipping of exon 8 of Ataxin-1 did not significantly contribute to the Ataxin-1 protein reduction observed in (CUG)7-treated SCA1154Q/2Q mice. These findings support the therapeutic potential of a single CAG repeat-targeting AON for the treatment of multiple polyglutamine disorders.

Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.

Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

The expanded CAG repeat in the huntingtin gene as target for therapeutic RNA modulation throughout the HD mouse brain.

The aim of these studies was to demonstrate the therapeutic capacity of an antisense oligonucleotide with the sequence (CUG)7 targeting the expanded CAG repeat in huntingtin (HTT) mRNA in vivo in the R6/2 N-terminal fragment and Q175 knock-in Huntington's disease (HD) mouse models. In a first study, R6/2 mice received six weekly intracerebroventricular infusions with a low and high dose of (CUG)7 and were sacrificed 2 weeks later. A 15-60% reduction of both soluble and aggregated mutant HTT protein was observed in striatum, hippocampus and cortex of (CUG)7-treated mice. This correction at the molecular level resulted in an improvement of performance in multiple motor tasks, increased whole brain and cortical volume, reduced levels of the gliosis marker myo-inositol, increased levels of the neuronal integrity marker N-aceyl aspartate and increased mRNA levels of the striatal marker Darpp-32. These neuroanatomical and neurochemical changes, together with the improved motor performance, suggest that treatment with (CUG)7 ameliorates basal ganglia dysfunction. The HTT-lowering was confirmed by an independent study in Q175 mice using a similar (CUG)7 AON dosing regimen, further demonstrating a lasting reduction of mutant HTT protein in striatum, hippocampus and cortex for up to 18 weeks post last infusion along with an increase in motor activity. Based on these encouraging results, (CUG)7 may thus offer an interesting alternative HTT-lowering strategy for HD.

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.

Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy.

Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0-100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD.

Cell membrane integrity in myotonic dystrophy type 1: implications for therapy.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.

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.

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.

Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.

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.

Prednisolone treatment does not interfere with 2'-O-methyl phosphorothioate antisense-mediated exon skipping in Duchenne muscular dystrophy.

In Duchenne muscular dystrophy (DMD), dystrophin deficiency leading to progressive muscular degeneration is caused by frame-shifting mutations in the DMD gene. Antisense oligonucleotides (AONs) aim to restore the reading frame by skipping of a specific exon(s), thereby allowing the production of a shorter, but semifunctional protein, as is found in the mostly more mildly affected patients with Becker muscular dystrophy. AONs are currently being investigated in phase 3 placebo-controlled clinical trials. Most of the participating patients are treated symptomatically with corticosteroids (mainly predniso[lo]ne) to stabilize the muscle fibers, which might affect the uptake and/or efficiency of AONs. Therefore the effect of prednisolone on 2'-O-methyl phosphorothioate AON efficacy in patient-derived cultured muscle cells and the mdx mouse model (after local and systemic AON treatment) was assessed in this study. Both in vitro and in vivo skip efficiency and biomarker expression were comparable between saline- and prednisolone-cotreated cells and mice. After systemic exon 23-specific AON (23AON) treatment for 8 weeks, dystrophin was detectable in all treated mice. Western blot analyses indicated slightly higher dystrophin levels in prednisolone-treated mice, which might be explained by better muscle condition and consequently more target dystrophin pre-mRNA. In addition, fibrotic and regeneration biomarkers were normalized to some extent in prednisolone- and/or 23AON-treated mice. Overall these results show that the use of prednisone forms no barrier to participation in clinical trials with AONs.

Targeting several CAG expansion diseases by a single antisense oligonucleotide.

To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)(7), also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

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.).

Exonic sequences provide better targets for antisense oligonucleotides than splice site sequences in the modulation of Duchenne muscular dystrophy splicing.

Antisense-mediated exon skipping is currently the most promising therapeutic approach for Duchenne muscular dystrophy (DMD). The rationale is to use antisense oligonucleotides (AONs) to hide exons from the splicing machinery, causing them to be skipped from the mature mRNA. Thus, the mutated, out-of-frame dystrophin transcripts as seen in DMD are reframed, allowing the generation of internally deleted, partly functional dystrophin proteins, rather than prematurely truncated, nonfunctional ones. This approach is mutation specific, so multiple AONs targeting all internal DMD exons have been designed and tested. Here, we have retrospectively compared our own set of 156 exon-internal AONs and 256 AONs as present in patents and publications from Dr. Wilton (Australia), which includes exon-internal as well as splice site-targeting AONs. Effective AONs are significantly more often exon-internal and, as anticipated, have better thermodynamic properties. Comparison of splice site and exon-internal AONs revealed that exon-internal AONs are more efficient and target more predicted exonic splicing enhancer and less predicted exon splicing silencer sites, but also have better thermodynamic properties. This suggests that exons may be better AON targets than introns per se, because of their higher GC content, which generally will result in improved AON binding.

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.

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.

Antisense-mediated exon skipping to correct IL-12Rbeta1 deficiency in T cells.

Patients with Mendelian susceptibility to mycobacterial disease have severe, recurrent life-threatening infections with otherwise poorly pathogenic mycobacteria and salmonellae. The extreme susceptibility is the result of genetic defects in the interleukin-12/interferon-gamma (IL-12/IFN-gamma) pathway. The infections are difficult to treat, and therapeutic options are limited. We explored the feasibility of antisense-mediated exon skipping as therapy for Mendelian susceptibility to mycobacterial disease with cells from a complete IL-12Rbeta1(-/-) patient. Expression constructs were first studied to determine whether IL12RB1 lacking exon 2 encodes a functional protein. The IL-12Rbeta1 expression construct lacking exon 2 was expressed on T cells. On IL-12 or IL-23 stimulation, this construct phosphorylated similar amounts of STAT1, STAT3, and STAT4 and induced similar amounts of IFN-gamma compared with a normal IL-12Rbeta1 construct. Antisense oligonucleotides (AONs) directed at exon 2 resulted in transcripts lacking exon 2 in both controls' and patients' T cells. In IL-12Rbeta1(-/-) cells, skipping of exon 2 led to expression of IL-12Rbeta1 on the cell surface and responsiveness to IL-12. We showed that IL12RB1 lacking exon 2 encodes a functional IL-12Rbeta1. We demonstrated that T cells can be highly efficiently transduced with AONs and are amenable to antisense-mediated exon skipping. Furthermore, we showed that exon skipping (partly) corrects the IL-12Rbeta1 deficiency in patients' cells.

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.