Spatial transcriptomics reveal markers of histopathological changes in Duchenne muscular dystrophy mouse models.

Duchenne muscular dystrophy is caused by mutations in the DMD gene, leading to lack of dystrophin. Chronic muscle damage eventually leads to histological alterations in skeletal muscles. The identification of genes and cell types driving tissue remodeling is a key step to developing effective therapies. Here we use spatial transcriptomics in two Duchenne muscular dystrophy mouse models differing in disease severity to identify gene expression signatures underlying skeletal muscle pathology and to directly link gene expression to muscle histology. We perform deconvolution analysis to identify cell types contributing to histological alterations. We show increased expression of specific genes in areas of muscle regeneration (Myl4, Sparc, Hspg2), fibrosis (Vim, Fn1, Thbs4) and calcification (Bgn, Ctsk, Spp1). These findings are confirmed by smFISH. Finally, we use differentiation dynamic analysis in the D2-mdx muscle to identify muscle fibers in the present state that are predicted to become affected in the future state.

Deacetylation Inhibition Reverses PABPN1-Dependent Muscle Wasting.

Reduced poly(A)-binding protein nuclear 1 (PABPN1) levels cause aging-associated muscle wasting. PABPN1 is a multifunctional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome with the proteome in mouse muscles expressing short hairpin RNA to PABPN1 (shPab). We found greater variations in the proteome than in mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be decreased in shPab versus control proteomes (63%). Acetylome profiling in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via alternative polyadenylation site utilization. Most importantly, SIRT1 deacetylase inhibition by sirtinol increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting. VIDEO ABSTRACT.

Natural disease history of the dy2J mouse model of laminin α2 (merosin)-deficient congenital muscular dystrophy.

Merosin deficient congenital muscular dystrophy 1A (MDC1A) is a very rare autosomal recessive disorder caused by mutations in the LAMA2 gene leading to severe and progressive muscle weakness and atrophy. Although over 350 causative mutations have been identified for MDC1A, no treatment is yet available. There are many therapeutic approaches in development, but the lack of natural history data of the mouse model and standardized outcome measures makes it difficult to transit these pre-clinical findings to clinical trials. Therefore, in the present study, we collected natural history data and assessed pre-clinical outcome measures for the dy2J/dy2J mouse model using standardized operating procedures available from the TREAT-NMD Alliance. Wild type and dy2J/dy2J mice were subjected to five different functional tests from the age of four to 32 weeks. Non-tested control groups were taken along to assess whether the functional test regime interfered with muscle pathology. Respiratory function, body weights and creatine kinase levels were recorded. Lastly, skeletal muscles were collected for further histopathological and gene expression analyses. Muscle function of dy2J/dy2J mice was severely impaired at four weeks of age and all mice lost the ability to use their hind limbs. Moreover, respiratory function was altered in dy2J/dy2J mice. Interestingly, the respiration rate was decreased and declined with age, whereas the respiration amplitude was increased in dy2J/dy2J mice when compared to wild type mice. Creatine kinase levels were comparable to wild type mice. Muscle histopathology and gene expression analysis revealed that there was a specific regional distribution pattern of muscle damage in dy2J/dy2J mice. Gastrocnemius appeared to be the most severely affected muscle with a high proportion of atrophic fibers, increased fibrosis and inflammation. By contrast, triceps was affected moderately and diaphragm only mildly. Our study presents a complete natural history dataset which can be used in setting up standardized studies in dy2J/dy2J mice.

Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne.

Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients.

Natural disease history of mouse models for limb girdle muscular dystrophy types 2D and 2F.

Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F mice.

Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.

Studying the role of dystrophin-associated proteins in influencing Becker muscular dystrophy disease severity.

Becker muscular dystrophy is characterized by a variable disease course. Many factors have been implicated to contribute to this diversity, among which the expression of several components of the dystrophin associated glycoprotein complex. Together with dystrophin, most of these proteins anchor the muscle fiber cytoskeleton to the extracellular matrix, thus protecting the muscle from contraction induced injury, while nNOS is primarily involved in inducing vasodilation during muscle contraction, enabling adequate muscle oxygenation. In the current study, we investigated the role of three components of the dystrophin associated glycoprotein complex (beta-dystroglycan, gamma-sarcoglycan and nNOS) and the dystrophin homologue utrophin on disease severity in Becker patients. Strength measurements, data about disease course and fresh muscle biopsies of the anterior tibial muscle were obtained from 24 Becker patients aged 19 to 66. The designation of Becker muscular dystrophy in this study was based on the mutation and not on the clinical severity. Contrary to previous studies, we were unable to find a relationship between expression of nNOS, beta-dystroglycan and gamma-sarcoglycan at the sarcolemma and disease severity, as measured by muscle strength in five muscle groups and age at reaching several disease milestones. Unexpectedly, we found an inverse correlation between utrophin expression at the sarcolemma and age at reaching disease milestones.

Quantitative MRI and strength measurements in the assessment of muscle quality in Duchenne muscular dystrophy.

The purpose of this study was to assess leg muscle quality and give a detailed description of leg muscle involvement in a series of Duchenne muscular dystrophy patients using quantitative MRI and strength measurements. Fatty infiltration, as well as total and contractile (not fatty infiltrated) cross sectional areas of various leg muscles were determined in 16 Duchenne patients and 11 controls (aged 8-15). To determine specific muscle strength, four leg muscle groups (quadriceps femoris, hamstrings, anterior tibialis and triceps surae) were measured and related to the amount of contractile tissue. In patients, the quadriceps femoris showed decreased total and contractile cross sectional area, attributable to muscle atrophy. The total, but not the contractile, cross sectional area of the triceps surae was increased in patients, corresponding to hypertrophy. Specific strength decreased in all four muscle groups of Duchenne patients, indicating reduced muscle quality. This suggests that muscle hypertrophy and fatty infiltration are two distinct pathological processes, differing between muscle groups. Additionally, the quality of remaining muscle fibers is severely reduced in the legs of Duchenne patients. The combination of quantitative MRI and quantitative muscle testing could be a valuable outcome parameter in longitudinal studies and in the follow-up of therapeutic effects.

Clinical characterisation of Becker muscular dystrophy patients predicts favourable outcome in exon-skipping therapy.

OBJECTIVE: Duchenne and Becker muscular dystrophy (DMD/BMD) are both caused by mutations in the DMD gene. Out-of-frame mutations in DMD lead to absence of the dystrophin protein, while in-frame BMD mutations cause production of internally deleted dystrophin. Clinically, patients with DMD loose ambulance around the age of 12, need ventilatory support at their late teens and die in their third or fourth decade due to pulmonary or cardiac failure. BMD has a more variable disease course. The disease course of patients with BMD with specific mutations could be very informative to predict the outcome of the exon-skipping therapy, aiming to restore the reading-frame in patients with DMD. METHODS: Patients with BMD with a mutation equalling a DMD mutation after successful exon skipping were selected from the Dutch Dystrophinopathy Database. Information about disease course was gathered through a standardised questionnaire. Cardiac data were collected from medical correspondence and a previous study on cardiac function in BMD. RESULTS: Forty-eight patients were included, representing 11 different mutations. Median age of patients was 43 years (range 6-67). Nine patients were wheelchair users (26-56 years). Dilated cardiomyopathy was present in 7/36 patients. Only one patient used ventilatory support. Three patients had died at the age of 45, 50 and 76 years, respectively. CONCLUSIONS: This study provides mutation specific data on the course of disease in patients with BMD. It shows that the disease course of patients with BMD, with a mutation equalling a 'skipped' DMD mutation is relatively mild. This finding strongly supports the potential benefit of exon skipping in patients with DMD.

Prolonged Ambulation in Duchenne Patients with a Mutation Amenable to Exon 44 Skipping.

Duchenne muscular dystrophy has a severe disease course, though variability exists. Case reports suggest a milder disease course of patients amenable to exon 44 skipping. In this study, we analyzed this and show that age at wheelchair dependence in patients with a dystrophin deletion requiring exon 44 skipping is postponed compared to patients with a deletion skippable by exon 45, 51 and 53 (10.8 versus 9.8 years; P 0.020). This may be explained by more frequent spontaneous exon 44 skipping in patients with a deletion flanking exon 44. This finding has important implications for the development of future Duchenne trials.

Dystrophin levels and clinical severity in Becker muscular dystrophy patients.

OBJECTIVE: Becker muscular dystrophy (BMD) is characterised by broad clinical variability. Ongoing studies exploring dystrophin restoration in Duchenne muscular dystrophy ask for better understanding of the relation between dystrophin levels and disease severity. We studied this relation in BMD patients with varying mutations, including a large subset with an exon 45-47 deletion. METHODS: Dystrophin was quantified by western blot analyses in a fresh muscle biopsy of the anterior tibial muscle. Disease severity was assessed using quantitative muscle strength measurements and functional disability scoring. MRI of the leg was performed in a subgroup to detect fatty infiltration. RESULTS: 33 BMD patients participated. No linear relation was found between dystrophin levels (range 3%-78%) and muscle strength or age at different disease milestones, in both the whole group and the subgroup of exon 45-47 deleted patients. However, patients with less than 10% dystrophin all showed a severe disease course. No relation was found between disease severity and age when analysing the whole group. By contrast, in the exon 45-47 deleted subgroup, muscle strength and levels of fatty infiltration were significantly correlated with patients' age. CONCLUSIONS: Our study shows that dystrophin levels appear not to be a major determinant of disease severity in BMD, as long as it is above approximately 10%. A significant relation between age and disease course was only found in the exon 45-47 deletion subgroup. This suggests that at higher dystrophin levels, the disease course depends more on the mutation site than on the amount of the dystrophin protein produced.

Serum matrix metalloproteinase-9 (MMP-9) as a biomarker for monitoring disease progression in Duchenne muscular dystrophy (DMD).

To identify serum biomarkers that allow monitoring of disease progression and treatment effects in Duchenne muscular dystrophy (DMD) patients, levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1) and osteopontin (OPN) were determined in 63 DMD patients on corticosteroid therapy. These proteins were selected for their role in the pathogenesis of muscular dystrophy. Levels of MMP-9 and TIMP-1 were significantly higher in sera of DMD patients compared to healthy controls, whereas the OPN levels showed no significant difference. MMP-9 levels were also observed to be significantly higher in older, nonambulant patients, compared to ambulant patients. Longitudinal data from a smaller cohort of DMD patients followed up for over 4years showed that MMP-9, but not TIMP-1 increased significantly with age. Hence, MMP-9 is a potential DMD biomarker for disease progression. Future studies have to confirm whether serum MMP-9 levels can be used to monitor therapeutic response.

Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients.

Theoretically, 13% of patients with Duchenne muscular dystrophy may benefit from antisense-mediated skipping of exon 51 to restore the reading frame, which results in the production of a shortened dystrophin protein. We give a detailed description with longitudinal follow up of three patients with Becker muscular dystrophy with in-frame deletions in the DMD gene encompassing exon 51. Their internally deleted, but essentially functional, dystrophins are identical to those that are expected as end products in DMD patients treated with the exon 51 skipping therapy. The mild phenotype encourages further development of exon 51 skipping therapy.

Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in the absence of functional protein. In the majority of cases these are out-of-frame deletions that disrupt the reading frame. Several attempts have been made to restore the dystrophin mRNA reading frame by modulation of pre-mRNA splicing with antisense oligonucleotides (AOs), demonstrating success in cultured cells, muscle explants, and animal models. We are preparing for a phase I/IIa clinical trial aimed at assessing the safety and effect of locally administered AOs designed to inhibit inclusion of exon 51 into the mature mRNA by the splicing machinery, a process known as exon skipping. Here, we describe a series of systematic experiments to validate the sequence and chemistry of the exon 51 AO reagent selected to go forward into the clinical trial planned in the United Kingdom. Eight specific AO sequences targeting exon 51 were tested in two different chemical forms and in three different preclinical models: cultured human muscle cells and explants (wild type and DMD), and local in vivo administration in transgenic mice harboring the entire human DMD locus. Data have been validated independently in the different model systems used, and the studies describe a rational collaborative path for the preclinical selection of AOs for evaluation in future clinical trials.

Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides.

Antisense oligonucleotides (AONs) can be used to correct the disrupted reading frame of Duchenne muscular dystophy patients (DMD). We have a collection of 121 AONs, of which 79 are effective in inducing the specific skipping of 38 out of the 79 different DMD exons. All AONs are located within exons and were hypothesized to act by steric hindrance of serine-arginine rich (SR) protein binding to exonic splicing enhancer (ESE) sites. Indeed, retrospective in silico analysis of effective versus ineffective AONs revealed that the efficacy of AONs is correlated to the presence of putative ESE sites (as predicted by the ESEfinder and RESCUE-ESE software). ESE predicting software programs are thus valuable tools for the optimization of exon-internal antisense target sequences.

Duplications in the DMD gene.

The detection of duplications in Duchenne (DMD)/Becker Muscular Dystrophy (BMD) has long been a neglected issue. However, recent technological advancements have significantly simplified screening for such rearrangements. We report here the detection and analysis of 118 duplications in the DMD gene of DMD/BMD patients. In an unselected patient series the duplication frequency was 7%. In patients already screened for deletions and point mutations, duplications were detected in 87% of cases. There were four complex, noncontiguous rearrangements, with two also involving a partial triplication. In one of the few cases where RNA was analyzed, a seemingly contiguous duplication turned out to be a duplication/deletion case generating a transcript with an unexpected single-exon deletion and an initially undetected duplication. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied. As with deletions, duplications occur nonrandomly but with a dramatically different distribution. Duplication frequency is highest near the 5' end of the gene, with a duplication of exon 2 being the single most common duplication identified. Analysis of the extent of 11 exon 2 duplications revealed two intron 2 recombination hotspots. Sequencing four of the breakpoints showed that they did not arise from unequal sister chromatid exchange, but more likely from synthesis-dependent nonhomologous end joining. There appear to be fundamental differences therefore in the origin of deletions and duplications in the DMD gene.

Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells.

As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional Becker muscular dystrophy (BMD)-like dystrophins and potential conversion of severe DMD into milder BMD phenotypes. Thus far we have used 2'-O-methyl phosphorothioate (2OMePS) AONs. Here, we assessed the skipping efficiencies of different AON analogs containing morpholino-phosphorodiamidate, locked nucleic acid (LNA) or peptide nucleic acid (PNA) backbones. In contrast to PNAs and morpholinos, LNAs have not yet been tested as splice modulators. Compared to the most effective 2OMePS AON directed at exon 46, the LNA induced higher skipping levels in myotubes from a human control (85 versus 20%) and an exon 45 deletion DMD patient (98 versus 75%). The morpholino-induced skipping levels were only 5-6%, whereas the PNA appeared to be ineffective. Further comparative analysis of LNA and 2OMePS AONs containing up to three mismatches revealed that LNAs, while inducing higher skipping efficiencies, show much less sequence specificity. This limitation increases the risk of adverse effects elsewhere in the human genome. Awaiting further improvements in oligochemistry, we thus consider 2OMePS AONs currently the most favorable compounds, at least for targeted DMD exon 46 skipping.