7',5'-alpha-bicyclo-DNA: new chemistry for oligonucleotide exon splicing modulation therapy.

Antisense oligonucleotides are small pieces of modified DNA or RNA, which offer therapeutic potential for many diseases. We report on the synthesis of 7',5'-α-bc-DNA phosphoramidite building blocks, bearing the A, G, T and MeC nucleobases. Solid-phase synthesis was performed to construct five oligodeoxyribonucleotides containing modified thymidine residues, as well as five fully modified oligonucleotides. Incorporations of the modification inside natural duplexes resulted in strong destabilizing effects. However, fully modified strands formed very stable duplexes with parallel RNA complements. In its own series, 7',5'-α-bc-DNA formed duplexes with a surprising high thermal stability. CD spectroscopy and extensive molecular modeling indicated the adoption by the homo-duplex of a ladder-like structure, while hetero-duplexes with DNA or RNA still form helical structure. The biological properties of this new modification were investigated in animal models for Duchenne muscular dystrophy and spinal muscular atrophy, where exon splicing modulation can restore production of functional proteins. It was found that the 7',5'-α-bc-DNA scaffold confers a high biostability and a good exon splicing modulation activity in vitro and in vivo.

Safety issues and harmful pharmacological interactions of nutritional supplements in Duchenne muscular dystrophy: considerations for Standard of Care and emerging virus outbreaks.

At the moment, little treatment options are available for Duchenne muscular dystrophy (DMD). The absence of the dystrophin protein leads to a complex cascade of pathogenic events in myofibres, including chronic inflammation and oxidative stress as well as altered metabolism. The attention towards dietary supplements in DMD is rapidly increasing, with the aim to counteract pathology-related alteration in nutrient intake, the consequences of catabolic distress or to enhance the immunological response of patients as nowadays for the COVID-19 pandemic emergency. By definition, supplements do not exert therapeutic actions, although a great confusion may arise in daily life by the improper distinction between supplements and therapeutic compounds. For most supplements, little research has been done and little evidence is available concerning their effects in DMD as well as their preventing actions against infections. Often these are not prescribed by clinicians and patients/caregivers do not discuss the use with their clinical team. Then, little is known about the real extent of supplement use in DMD patients. It is mistakenly assumed that, since compounds are of natural origin, if a supplement is not effective, it will also do no harm. However, supplements can have serious side effects and also have harmful interactions, in terms of reducing efficacy or leading to toxicity, with other therapies. It is therefore pivotal to shed light on this unclear scenario for the sake of patients. This review discusses the supplements mostly used by DMD patients, focusing on their potential toxicity, due to a variety of mechanisms including pharmacodynamic or pharmacokinetic interactions and contaminations, as well as on reports of adverse events. This overview underlines the need for caution in uncontrolled use of dietary supplements in fragile populations such as DMD patients. A culture of appropriate use has to be implemented between clinicians and patients' groups.

Low dystrophin levels in heart can delay heart failure in mdx mice.

Duchenne muscular dystrophy is caused by mutations that prevent synthesis of functional dystrophin. All patients develop dilated cardiomyopathy. Promising therapeutic approaches are underway that successfully restore dystrophin expression in skeletal muscle. However, their efficiency in the heart is limited. Improved quality and function of only skeletal muscle potentially accelerate the development of cardiomyopathy. Our study aimed to elucidate which dystrophin levels in the heart are required to prevent or delay cardiomyopathy in mice. Heart function and pathology assessed with magnetic resonance imaging and histopathological analysis were compared between 2, 6 and 10-month-old female mdx-Xist(Δhs) mice, expressing low dystrophin levels (3-15%) in a mosaic manner based on skewed X-inactivation, dystrophin-negative mdx mice, and wild type mice of corresponding genetic backgrounds and gender. With age mdx mice developed dilated cardiomyopathy and hypertrophy, whereas the onset of heart pathology was delayed and function improved in mdx-Xist(Δhs) mice. The ejection fraction, the most severely affected parameter for both ventricles, correlated to dystrophin expression and the percentage of fibrosis. Fibrosis was partly reduced from 9.8% in mdx to 5.4% in 10 month old mdx-Xist(Δhs) mice. These data suggest that mosaic expression of 4-15% dystrophin in the heart is sufficient to delay the onset and ameliorate cardiomyopathy in mice.

DMD transcript imbalance determines dystrophin levels.

Duchenne and Becker muscular dystrophies are caused by out-of-frame and in-frame mutations, respectively, in the dystrophin encoding DMD gene. Molecular therapies targeting the precursor-mRNA are in clinical trials and show promising results. These approaches will depend on the stability and expression levels of dystrophin mRNA in skeletal muscles and heart. We report that the DMD gene is more highly expressed in heart than in skeletal muscles, in mice and humans. The transcript mutated in the mdx mouse model shows a 5' to 3' imbalance compared with that of its wild-type counterpart and reading frame restoration via antisense-mediated exon skipping does not correct this event. We also report significant transcript instability in 22 patients with Becker dystrophy, clarifying the fact that transcript imbalance is not caused by premature nonsense mutations. Finally, we demonstrate that transcript stability, rather than transcriptional rate, is an important determinant of dystrophin protein levels in patients with Becker dystrophy. We suggest that the availability of the complete transcript is a key factor to determine protein abundance and thus will influence the outcome of mRNA-targeting therapies.

Circulating specific antibodies enhance systemic cross-priming by delivery of complexed antigen to dendritic cells in vivo.

Increasing evidence suggests that antibodies can have stimulatory effects on T-cell immunity. However, the contribution of circulating antigen-specific antibodies on MHC class I cross-priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross-presentation of antigen to CD8(+) T cells. Mice with hapten-specific circulating antibodies, but naϊve for the T-cell antigen, were infused with haptenated antigen and CD8(+) T-cell induction was measured. Mice with circulating hapten-specific antibodies showed significantly enhanced cross-presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross-presentation in mice with circulating antigen-specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c(+) APCs were responsible for the enhanced and sustained cross-presentation, although CD11c(-) APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen-antibody immune complexes improves MHC class I cross-presentation, and CD8(+) T-cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor-associated antigens intensively used in the clinic nowadays.

Gene therapy for Duchenne muscular dystrophy.

PURPOSE OF REVIEW: Duchenne muscular dystrophy is a severe neuromuscular disorder for which there is currently no cure. Years of research have come to fruition during the past 18 months with publications on clinical trials for several gene therapy approaches for Duchenne muscular dystrophy. This review covers the present status of these approaches. RECENT FINDINGS: The exon skipping approach is most advanced in the process of clinical application. Encouraging results have been obtained in two systemic clinical trials and further optimization has increased delivery to the heart in animal models. Limitations of the approach are the mutation-specificity and the anticipated requirement for lifelong treatment. Gene therapy by means of gene transfer holds the promise of more long-lasting effects. Results of a first, early-stage gene therapy trial, using viral vectors to deliver a minidystrophin gene, were reported. Animal studies suggest that it may be possible to overcome the main challenges currently facing gene therapy (immunogenicity of the vector and systemic body-wide delivery). SUMMARY: Significant steps have been made in the development of gene therapy approaches for Duchenne muscular dystrophy. These approaches aim to slow down disease progression, requiring robust outcome measures to assess efficacy.

Simvastatin Treatment Does Not Ameliorate Muscle Pathophysiology in a Mouse Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is an X-linked, recessive muscular dystrophy in which the absence of the dystrophin protein leads to fibrosis, inflammation and oxidative stress, resulting in loss of muscle tissue. Drug repurposing, i.e. using drugs already approved for other disorders, is attractive as it decreases development time. Recent studies suggested that simvastatin, a cholesterol lowering drug used for cardiovascular diseases, has beneficial effects on several parameters in mdx mice. To validate properly the effectiveness of simvastatin, two independent labs tested the effects of 12-week simvastatin treatment in either young (starting at 4 weeks of age) or adult (starting at 12 weeks of age) mdx mice. In neither study were benefits of simvastatin treatment observed on muscle function, histology or expression of genes involved in fibrosis, regeneration, oxidative stress and autophagy. Unexpectedly, although the treatment protocol was similar, simvastatin plasma levels were found to be much lower than observed in a previous study. In conclusion, in two laboratories, simvastatin did not ameliorate disease pathology in mdx mice, which could either be due to the ineffectiveness of simvastatin itself or due to the low simvastatin plasma levels following oral administration via the food.

Therapeutic developments for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein. Improvements in patient care and disease management have slowed down disease progression, but current treatments cannot stop the relentless loss of muscle tissue and function, which leads to premature death. Research is ongoing to develop effective therapies for DMD. Gene-addition, exon-skipping, stop codon readthrough and genome-editing therapies can restore the expression of partially functional dystrophin protein, whereas other therapeutic approaches aim to improve muscle function and quality by targeting pathways involved in the pathogenesis of DMD. This Review outlines important developments in these research areas and specifically focuses on new therapies that are in the clinical trial phase or have already been approved.

Cross-sectional study into age-related pathology of mouse models for limb girdle muscular dystrophy types 2D and 2F.

Limb girdle muscular dystrophy (LGMD) types 2D and 2F are caused by mutations in the genes encoding for α- and δ-sarcoglycan, respectively, leading to progressive muscle weakness. Mouse models exist for LGMD2D (Sgca-/-) and 2F (Sgcd-/-). In a previous natural history study, we described the pathology in these mice at 34 weeks of age. However, the development of muscle pathology at younger ages has not been fully characterised yet. We therefore performed a study into age-related changes in muscle function and pathology by examining mice at different ages. From 4 weeks of age onwards, male mice were subjected to functional tests and sacrificed at respectively 8, 16 or 24 weeks of age. Muscle histopathology and expression of genes involved in muscle pathology were analysed for several skeletal muscles, while miRNA levels were assessed in serum. In addition, for Sgcd-/- mice heart pathology was assessed. Muscle function showed a gradual decline in both Sgca-/- and Sgcd-/- mice. Respiratory function was also impaired at all examined timepoints. Already at 8 weeks of age, muscle pathology was prominent, and fibrotic, inflammatory and regenerative markers were elevated, which remained relatively constant with age. In addition, Sgcd-/- mice showed signs of cardiomyopathy from 16 weeks of age onwards. These results indicate that Sgca-/- and Sgcd-/- are relevant disease models for LGMD2D and 2F.

A modified diet does not ameliorate muscle pathology in a mouse model for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by a lack of dystrophin protein. Next to direct effects on the muscles, this has also metabolic consequences. The influence of nutrition on disease progression becomes more and more recognized. Protein intake by DMD patients may be insufficient to meet the increased demand of the constantly regenerating muscle fibers. This led to the hypothesis that improving protein uptake by the muscles could have therapeutic effects. The present study examined the effects of a modified diet, which composition might stimulate muscle growth, on disease pathology in the D2-mdx mouse model. D2-mdx males were fed with either a control diet or modified diet, containing high amounts of branched-chain amino acids, vitamin D3 and ursolic acid, for six weeks. Our study indicates that the modified diet could not ameliorate the muscle pathology. No effects on bodyweight or weight of individual muscles were observed. Neither did the diet affect severity of fibrosis or calcification of the muscles.

Muscle biopsies in clinical trials for Duchenne muscular dystrophy - Patients' and caregivers' perspective.

The number of clinical trials for Duchenne muscular dystrophy is increasing. Many trials require muscle biopsies, which involve an invasive surgical procedure. Little is known about short- and long-term impacts of muscle biopsies as perceived by patients and caregivers. Therefore a survey was held among patients and their caregivers who participated in trials involving muscle biopsies, in seven countries. Seventy-eight responses were received. Analysis revealed that many patients and parents had significant anxiety before the biopsy. The main concern of caregivers was the required general anaesthesia. In most cases biopsies caused pain and temporarily hampered daily activities. The main long-term impact was scarring, although large variation in size was reported. Seventy-nine percent of caregivers were little bothered and 21% were moderately or severely bothered by the scar. Willingness to consider another biopsy in future protocols was higher for open-label studies than for placebo-controlled trials. Caregivers stressed the importance of knowing the results of biopsy analyses; only a minority actually received this information. Recommendations are made on the informed consent procedure regarding risks and consequences of muscle biopsies, and communication of results. Furthermore, efforts should be made to minimise the impact of biopsies through pain management and by considering plastic surgery.

A multi-source approach to determine SMA incidence and research ready population.

In spinal muscular atrophy (SMA), degeneration of motor neurons causes progressive muscular weakness, which is caused by homozygous deletion of the SMN1 gene. Available epidemiological data on SMA are scarce, often outdated, and limited to relatively small regions or populations. Combining data from different sources including genetic laboratories and patient registries may provide better insight of the disease epidemiology. To investigate the incidence of genetically confirmed SMA, and the number of patients who are able and approachable to participate in new clinical trials and observational research, we used both genetic laboratories, the TREAT-NMD Global SMA Patient Registry and the Care and Trial Sites Registry (CTSR). In Europe, 4653 patients were genetically diagnosed by the genetic laboratories in the 5-year period 2011 to 2015, with 992 diagnosed in 2015 alone. The data provide an estimated incidence of SMA in Europe of 1 in 3900-16,000 live births. Patient numbers in the national patient registries and CTSR were considerably lower. By far, most patients registered in the national patient registries and the CTSR live in Europe and are reported to have SMA type II. Considerable differences between countries in patient participation in the registries were observed. Our findings indicate that not all patients with SMA are accessed by specialist healthcare services and these patients may not have access to research opportunities and optimal care.

Prevalence, incidence and carrier frequency of 5q-linked spinal muscular atrophy - a literature review.

Spinal muscular atrophy linked to chromosome 5q (SMA) is a recessive, progressive, neuromuscular disorder caused by bi-allelic mutations in the SMN1 gene, resulting in motor neuron degeneration and variable presentation in relation to onset and severity. A prevalence of approximately 1-2 per 100,000 persons and incidence around 1 in 10,000 live births have been estimated with SMA type I accounting for around 60% of all cases. Since SMA is a relatively rare condition, studies of its prevalence and incidence are challenging. Most published studies are outdated and therefore rely on clinical rather than genetic diagnosis. Furthermore they are performed in small cohorts in small geographical regions and only study European populations. In addition, the heterogeneity of the condition can lead to delays and difficulties in diagnosing the condition, especially outside of specialist clinics, and contributes to the challenges in understanding the epidemiology of the disease. The frequency of unaffected, heterozygous carriers of the SMN1 mutations appears to be higher among Caucasian and Asian populations compared to the Black (Sub-Saharan African ancestry) population. However, carrier frequencies cannot directly be translated into incidence and prevalence, as very severe (death in utero) and very mild (symptom free in adults) phenotypes carrying bi-allelic SMN1 mutations exist, and their frequency is unknown. More robust epidemiological data on SMA covering larger populations based on accurate genetic diagnosis or newborn screening would be helpful to support planning of clinical studies, provision of care and therapies and evaluation of outcomes.

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.

A 3-base pair deletion, c.9711_9713del, in DMD results in intellectual disability without muscular dystrophy.

We have identified a deletion of 3 base pairs in the dystrophin gene (DMD), c.9711_9713del, in a family with nonspecific X-linked intellectual disability (ID) by sequencing of the exons of 86 known X-linked ID genes. This in-frame deletion results in the deletion of a single-amino-acid residue, Leu3238, in the brain-specific isoform Dp71 of dystrophin. Linkage analysis supported causality as the mutation was present in the 7.6 cM linkage interval on Xp22.11-Xp21.1 with a maximum positive LOD score of 2.41 (MRX85 locus). Molecular modeling predicts that the p.(Leu3238del) deletion results in the destabilization of the C-terminal domain of dystrophin and hence reduces the ability to interact with ß-dystroglycan. Correspondingly, Dp71 protein levels in lymphoblastoid cells from the index patient are 6.7-fold lower than those in control cell lines (P=0.08). Subsequent determination of the creatine kinase levels in blood of the index patient showed a mild but significant elevation in serum creatine kinase, which is in line with impaired dystrophin function. In conclusion, we have identified the first DMD mutation in Dp71 that results in ID without 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.

The effect of 6-thioguanine on alternative splicing and antisense-mediated exon skipping treatment for duchenne muscular dystrophy.

The severe muscle wasting disorder Duchenne muscular dystrophy (DMD) is caused by genetic defects in the DMD gene, leading to a complete absence of dystrophin protein. Of the therapeutic approaches addressing the underlying genetic defect, exon skipping through antisense oligonucleotides (AONs) is the closest to clinical application. Several strategies to improve the efficiency of this approach are currently being investigated, such as the use of small chemical compounds that improve AONmediated exon skipping levels. Recently, enhanced exon skipping in combination with a guanine analogue, 6-thioguanine (6TG) was reported for phosphorodiamidate morpholino oligomers (PMO). Here the effect of 6TG on the exon skipping efficacy of 2'-O-methyl phosphorothioate RNA (2OMePS) and PMO AONs in vitro and in vivo was further evaluated, as well as the effect of 6TG by itself. Results confirm an increase of exon skipping levels in vitro, however, in contrast to the previous report, no effect was observed in vivo. Importantly, 6TG treatment in vitro resulted in numerous additional DMD exon skipping events. This, in combination with the known cytotoxic effects of 6TG after incorporation in DNA, warrants reconsidering of the use of 6TG as enhancer of AON efficiency in DMD, were chronic treatment will be required.

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.