A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.     

Antisense Oligonucleotides Conjugated with Lipophilic Compounds: Synthesis and In Vitro Evaluation of Exon Skipping in Duchenne Muscular Dystrophy

Our groups previously reported that conjugation at 3′-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (T_m) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5′-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5′-UDC- and 5′,3′-bis-UDC-ASO 51.     

Serum Levels of MicroRNA-206 and Novel Mini-STR Assays for Carrier Detection in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disorder in which the detection of female carriers is of the utmost importance for genetic counseling. Haplotyping with polymorphic markers and quantitation of creatine kinase levels (CK) allow tracking of the at-risk haplotype and evidence muscle damage, respectively. Such approaches are useful for carrier detection in cases of unknown mutations. The lack of informative markers and the inaccuracy of CK affect carrier detection. Therefore, herein we designed novel mini-STR (Short Tandem Repeats) assays to amplify 10 loci within the DMD gene and estimated allele frequencies and the polymorphism information content among other parameters in 337 unrelated individuals from three Mexican populations. In addition, we tested the utility of the assays for carrier detection in three families. Moreover, given that serum levels of miR-206 discern between DMD patients and controls with a high area under the curve (AUC), the potential applicability for carrier detection was assessed. The serum levels of miR-206 of non-carriers (n = 24) and carriers (n = 23) were compared by relative quantitation using real-time PCR (p < 0.05), which resulted in an AUC = 0.80 in the Receiver Operating Characteristic curve analysis. In conclusion, miR-206 has potential as a “liquid biopsy” for carrier detection and genetic counseling in DMD.     

Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal–epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.     

Alternative Splicing Regulation of Low-Frequency Genetic Variants in Exon 2 of TREM2 in Alzheimer’s Disease by Splicing-Based Aggregation

TREM2 is among the most well-known Alzheimer’s disease (AD) risk genes; however, the functional roles of its AD-associated variants remain to be elucidated, and most known risk alleles are low-frequency variants whose investigation is challenging. Here, we utilized a splicing-guided aggregation method in which multiple low-frequency TREM2 variants were bundled together to investigate the functional impact of those variants on alternative splicing in AD. We analyzed whole genome sequencing (WGS) and RNA-seq data generated from cognitively normal elderly controls (CN) and AD patients in two independent cohorts, representing three regions in the frontal lobe of the human brain: the dorsolateral prefrontal cortex (CN = 213 and AD = 376), frontal pole (CN = 72 and AD = 175), and inferior frontal (CN = 63 and AD = 157). We observed an exon skipping event in the second exon of TREM2, with that exon tending to be more frequently skipped (p = 0.0012) in individuals having at least one low-frequency variant that caused loss-of-function for a splicing regulatory element. In addition, genes differentially expressed between AD patients with high vs. low skipping of the second exon (i.e., loss of a TREM2 functional domain) were significantly enriched in immune-related pathways. Our splicing-guided aggregation method thus provides new insight into the regulation of alternative splicing of the second exon of TREM2 by low-frequency variants and could be a useful tool for further exploring the potential molecular mechanisms of multiple, disease-associated, low-frequency variants.     

Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.     

Therapeutic Prospects of Exon Skipping for Epidermolysis Bullosa

Epidermolysis bullosa is a group of genetic skin conditions characterized by abnormal skin (and mucosal) fragility caused by pathogenic variants in various genes. The disease severity ranges from early childhood mortality in the most severe types to occasional acral blistering in the mildest types. The subtype and severity of EB is linked to the gene involved and the specific variants in that gene, which also determine its mode of inheritance. Current treatment is mainly focused on symptomatic relief such as wound care and blister prevention, because truly curative treatment options are still at the preclinical stage. Given the current level of understanding, the broad spectrum of genes and variants underlying EB makes it impossible to develop a single treatment strategy for all patients. It is likely that many different variant-specific treatment strategies will be needed to ultimately treat all patients. Antisense-oligonucleotide (ASO)-mediated exon skipping aims to counteract pathogenic sequence variants by restoring the open reading frame through the removal of the mutant exon from the pre-messenger RNA. This should lead to the restored production of the protein absent in the affected skin and, consequently, improvement of the phenotype. Several preclinical studies have demonstrated that exon skipping can restore protein production in vitro, in skin equivalents, and in skin grafts derived from EB-patient skin cells, indicating that ASO-mediated exon skipping could be a viable strategy as a topical or systemic treatment. The potential value of exon skipping for EB is supported by a study showing reduced phenotypic severity in patients who carry variants that result in natural exon skipping. In this article, we review the substantial progress made on exon skipping for EB in the past 15 years and highlight the opportunities and current challenges of this RNA-based therapy approach. In addition, we present a prioritization strategy for the development of exon skipping based on genomic information of all EB-involved genes.     

An Antisense Oligonucleotide against a Splicing Enhancer Sequence within Exon 1 of the MSTN Gene Inhibits Pre-mRNA Maturation to Act as a Novel Myostatin Inhibitor

Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in rhabdomyosarcoma cells. Remarkably, it did not decrease GDF11 mRNA levels, indicating myostatin-specific inhibition. As expected, KMM001 enhanced the proliferation of human myoblasts. We conclude that KMM001 is a novel myostatin inhibitor that inhibits pre-mRNA maturation. KMM001 has great promise for clinical applications and should be examined for its ability to treat various muscle-wasting conditions.     

Smad8 Is Increased in Duchenne Muscular Dystrophy and Suppresses miR-1, miR-133a,and miR-133b

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor β (TGFβ) signaling. In this report, we investigated the major transducers of TGFβ signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx^5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.     

Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.     

Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids,Amido-Bridged Nucleic Acids,and Guanidine-Bridged Nucleic Acids

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (T_m) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same T_m values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower T_m values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.     

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.     

Natural History of a Mouse Model Overexpressing the Dp71 Dystrophin Isoform

Dystrophin is a 427 kDa protein that stabilizes muscle cell membranes through interactions with the cytoskeleton and various membrane-associated proteins. Loss of dystrophin as in Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle weakness and cardiac dysfunction. Multiple promoters along the dystrophin gene (DMD) give rise to a number of shorter isoforms. Of interest is Dp71, a 71 kDa isoform implicated in DMD pathology by various animal and patient studies. Strong evidence supporting such a role for Dp71, however, is lacking. Here, we use del52;WT mice to understand how Dp71 overexpression affects skeletal and cardiac muscle phenotypes. Apart from the mouse Dmd gene, del52;WT mice are heterozygous for a full-length, exon 52-deleted human DMD transgene expected to only permit Dp71 expression in muscle. Thus, del52;WT mice overexpress Dp71 through both the human and murine dystrophin genes. We observed elevated Dp71 protein in del52;WT mice, significantly higher than wild-type in the heart but not the tibialis anterior. Moreover, del52;WT mice had generally normal skeletal muscle but impaired cardiac function, exhibiting significant systolic dysfunction as early as 3 months. No histological abnormalities were found in the tibialis anterior and heart. Our results suggest that Dp71 overexpression may have more detrimental effects on the heart than on skeletal muscles, providing insight into the role of Dp71 in DMD pathogenesis.     

Skeletal Ryanodine Receptors Are Involved in Impaired Myogenic Differentiation in Duchenne Muscular Dystrophy Patients

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting following repeated muscle damage and inadequate regeneration. Impaired myogenesis and differentiation play a major role in DMD as well as intracellular calcium (Ca²⁺) mishandling. Ca²⁺ release from the sarcoplasmic reticulum is mostly mediated by the type 1 ryanodine receptor (RYR1) that is required for skeletal muscle differentiation in animals. The study objective was to determine whether altered RYR1-mediated Ca²⁺ release contributes to myogenic differentiation impairment in DMD patients. The comparison of primary cultured myoblasts from six boys with DMD and five healthy controls highlighted delayed myoblast differentiation in DMD. Silencing RYR1 expression using specific si-RNA in a healthy control induced a similar delayed differentiation. In DMD myotubes, resting intracellular Ca²⁺ concentration was increased, but RYR1-mediated Ca²⁺ release was not changed compared with control myotubes. Incubation with the RYR-calstabin interaction stabilizer S107 decreased resting Ca²⁺ concentration in DMD myotubes to control values and improved calstabin1 binding to the RYR1 complex. S107 also improved myogenic differentiation in DMD. Furthermore, intracellular Ca²⁺ concentration was correlated with endomysial fibrosis, which is the only myopathologic parameter associated with poor motor outcome in patients with DMD. This suggested a potential relationship between RYR1 dysfunction and motor impairment. Our study highlights RYR1-mediated Ca²⁺ leakage in human DMD myotubes and its key role in myogenic differentiation impairment. RYR1 stabilization may be an interesting adjunctive therapeutic strategy in DMD.     

Effects of Low-Intensity and Long-Term Aerobic Exercise on the Psoas Muscle of mdx Mice: An Experimental Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a muscle disease characterized by the absence of the protein dystrophin, which causes a loss of sarcolemma integrity, determining recurrent muscle injuries, decrease in muscle function, and progressive degeneration. Currently, there is a need for therapeutic treatments to improve the quality of life of DMD patients. Here, we investigated the effects of a low-intensity aerobic training (37 sessions) on satellite cells, peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α protein (PGC-1α), and different types of fibers of the psoas muscle from mdx mice (DMD experimental model). Wildtype and mdx mice were randomly divided into sedentary and trained groups (n = 24). Trained animals were subjected to 37 sessions of low-intensity running on a motorized treadmill. Subsequently, the psoas muscle was excised and analyzed by immunofluorescence for dystrophin, satellite cells, myosin heavy chain (MHC), and PGC-1α content. The minimal Feret’s diameters of the fibers were measured, and light microscopy was applied to observe general morphological features of the muscles. The training (37 sessions) improved morphological features in muscles from mdx mice and caused an increase in the number of quiescent/activated satellite cells. It also increased the content of PGC-1α in the mdx group. We concluded that low-intensity aerobic exercise (37 sessions) was able to reverse deleterious changes determined by DMD.     

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.     

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-β and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.