Probes for INS

Antisense oligonucleotides (AONs) are short, synthetic nucleic acid sequences that work by modulating exon incorporation at the level of pre-mRNA. In Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disorder caused by mutations in the DMD gene, AONs skip specific exons to correct the reading frame, producing an internally shortened but partly functional dystrophin protein. Golodirsen is an approved AON phosphorodiamidate morpholino oligomer (PMO) that specifically targets DMD exon 53. In the clinical study 4053-101, we demonstrated that intravenous golodirsen administration induces an unequivocal exon skipping and protein restoration in all the treated patients, but with inter-patient variability. We used fibroblasts isolated from the patients in this clinical trial, that were induced to undergo myogenic differentiation in vitro by expression of MyoD, to better understand the reasons behind the observed variability. We evaluated the amount and the molecular weight of dystrophin protein in treated and non-treated patient cells, by an automated capillary-based immunoassay (WES) system. In these in-vitro studies we demonstrated that the amount of protein was comparable to the previous in-vivo study and that the size of the restored protein was compatible with the different genomic deletions carried by patients. Next, we used an in-situ RNA hybridization technique, BaseScope, to investigate the sub-cellular localization of the DMD transcript in treated and non-treated differentiated patient-derived myogenic cells in vitro, which allowed us to assess the ratio of skipped and unskipped products. Our study provides additional information on the dynamics of DMD mRNA in patients and may help to better understand the biological reasons underpinning variability in dystrophin restoration that can be seen in AON clinical trials.

SELENON-related congenital myopathy is characterized by proximal weakness starting in infancy, early respiratory insufficiency, and early development of severe scoliosis. While changes in the SELENON gene, which encodes the protein SelN, are known to cause this disease the mechanisms through which loss of SelN lead to myopathy are not well understood. Previous studies suggest that SelN may have multiple roles in muscle, including regulating development of Type II muscle fibers, modulating excitation-contraction coupling through interactions with RYR1 and other muscle calcium channels, and possibly supporting satellite cell activation and proliferation following muscle injury. One particular challenge to understanding the role of SelN in skeletal muscle has been the inability to directly visualize SelN expression within muscle fibers and supporting cells due to a lack of robust antibodies for immunohistochemistry. Studies of mRNA expression and Western blot analysis of protein expression suggest significant post-transcriptional regulation of protein expression with an overall pattern of high expression in developing muscle and other developing tissues and low-level ubitquitous expression in mature tissues but evaluation of SelN expression in more limited sub-populations of cells has not been possible. Experiments in mouse suggest that loss of SelN expression results in decreased satellite cell proliferation following muscle injury. Here, I use a newly developed zebrafish model with mNeonGreen-tagged SelN to directly visualize SelN expression in satellite cells following muscle injury and show that SelN expression increases in activated satellite cells following mechanical muscle injury. This provides support SelN playing a role in satellite cell activation and proliferation during muscle repair following injury.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent muscular dystrophies, ranging from 1 in 8,333 to 1 in 20,000. Currently no treatment exists that alters the course of FSHD, and therapy development remains an unmet need in the field. Abnormal reactivation of the DUX4 gene in skeletal muscle has emerged as an underlying cause of muscle weakness and wasting in FSHD. We propose that DUX4 silencing is the most direct route to FSHD therapy. Toward this goal, we developed an AAV6-CRISPR-Cas13 strategy to silence DUX4 mRNA. Cas13 targets and cleaves RNA instead of DNA, and avoids potential risks of permanent off-target genome editing that could arise with DNA-targeting systems. Intramuscular delivery of an AAV6 vector encoding a PspCas13b enzyme and DUX4-targeting guide RNAs reduced DUX4 mRNA by >50% and improved histopathological outcomes in FSHD mice. To investigate possible off-target effects, we performed RNA-seq of treated versus control or untreated human myoblasts and also examined potential collateral RNA cleavage activity using a dual reporter system. Although we did not detect collateral cleavage, our RNA-sequencing results suggested some guide RNAs could induce potential off-target gene expression changes. We are currently exploring mechanisms to explain these differential off-target effects. To address whether PspCas13b can activate a mammalian host immune response, we injected wild-type mice with AAV-Cas13b and investigated immune cell infiltration and pro-inflammatory cytokine profiles. We find evidence of an immune response against PspCas13b in injected mouse muscles. Importantly, transient immunosuppression reduced immune responses to Cas13b in treated animals. In conclusion, our data support that Cas13b can target and reduce DUX4 expression in FSHD muscles, but minimizing cellular immune response may be necessary to translate AAV-Cas13b therapy.

The transient receptor potential cation channel subfamily V member 2 (TRPV2) is a stretch-sensitive calcium channel. Myocytes' damage induces TRPV2 expression on the sarcolemma, which causes calcium influx into the cytoplasm, and triggers degeneration. TRPV2 inhibition was effective in animal models of cardiomyopathy and muscular dystrophy (MD). Our pilot study showed that tranilast, a TRPV2 inhibitor, reduced brain natriuretic peptide (BNP) levels in two MD patients with advanced heart failure. Then, we planned a study to evaluate the safety and efficacy of tranilast for heart failure of MD patients. Subjects were MD patients whose serum BNP levels exceeded 100 pg/mL despite receiving standard therapy. Tranilast was administered orally at 100 mg thrice daily. The primary endpoint was the change in log (BNP) (⊿log [BNP]) at 6 months from baseline. The null hypothesis was determined based on a previous carvedilol study that resulted in a mean population ⊿log [BNP] of 0.18. TRPV2 expression on the mononuclear cell (MNC) surface, cardiac events, left ventricular fractional shortening (FS), human atrial natriuretic peptide (hANP), creatine kinase, and pinch strength were also assessed. Because of the poor general condition of many patients, among 18 patients included, 13 patients could be treated according to the protocol throughout the 6-month period. There were no serious adverse events related to tranilast except diarrhea, a known adverse effect. TRPV2 expression on the MNC surface was elevated at baseline and reduced after treatment. BNP, hANP, and FS remained stable. In the per-protocol set group, ⊿log [BNP] was -0.2 and significantly lower than that in the null hypothesis. In conclusion, tranilast is safe and effectively inhibits TRPV2 expression, even in MD patients with advanced heart failure. To evaluate the efficacy of tranilast in preventing heart failure, motor impairment, and respiratory failure, we are planning a study for mild MD patients.

Collagen VI (COLVI) is a critical myomatrix protein for skeletal muscle health and maintenance. There are 6 COL6 genes (COL6A1-COL6A6). Pathogenic variants in COL6A1, COL6A2, or COL6A3 cause COLVI-related dystrophies (COL6-RDs) with early-onset muscle weakness and loss of ambulation. Identifying novel therapeutic targets is critical for developing COL6-RDs therapies. Here, using in situ hybridization, we aim to identify, quantify, and locate all cell types that express the wild-type (WT) COL6 genes and a common, recurrent pseudoexon (PE)-inserting COL6A1 mutation in limb skeletal muscles and diaphragms of WT and COL6-RDs male mice at 10-day-old, and 6- and 20-month-old. We first analyzed published mouse skeletal muscle RNA-seq datasets to identify potential sensitive and specific mRNA cell markers (3/cell type). Then, we conducted a pilot marker validation experiment with RNAscope. We quantified and assessed the expression of the selected 33 markers’ transcripts in 3 non-overlapping fields of a quadriceps section from 2-month-old and 6-month-old mice (n=1). The specificity was reported as % of cells that had a marker for one cell type and lacked the markers of all other cell types. Our preliminary data indicated markers that were specific (Pdgfra: Fibro-adipogenic progenitors (FAPs) (98-100% specific), Myod1: satellite cells (SCs) (88-96%), and Pecam1: endothelial cells (endo) (92-100%)), non-specific (Esam: endo (57-77%), and Asb5: SCs (55-81%)), or specific in only one age group (Dpt: FAPs (2-month: 73%; 6-month: 89%), and Cdh5: endo (2-month: 80%; 6-month: 100%). We are currently validating each marker's specificity and sensitivity. We will identify and locate the cells that express the Col6 genes and the PE and quantify their transcripts expression levels in the various models and age groups. The findings of this project will provide additional insights into the roles of COLVI-producing cells in the pathogenesis of COL6-RDs and help direct therapeutic approaches.

Progressive respiratory muscle weakness and ineffective cough contributes to morbidity and mortality in children with neuromuscular diseases (NMD). Inspiratory muscle training (IMT) aims to preserve or improve respiratory muscle strength and reduce respiratory morbidity. This study aimed to determine the safety and efficacy of IMT in children with NMD. A randomised cross-over study compared three-month intervention (IMT) with control periods. During the intervention, children with NMD (5-18 years) from two provinces in South Africa performed 30 breaths (at 30% of inspiratory muscle strength (Pimax)) with an electronic threshold device, twice daily. During the control period participants did not perform any IMT. Twenty-three participants (median (IQR) age of 12.33 (10.03-14.17) years), mostly male (n=20) and non-ambulant (n=14) were included. No adverse events related to IMT were reported. There was no evidence of a difference in median patient hospitalisation and respiratory tract infection rates between control and intervention periods (p=0.60; p=0.21). During IMT, Pimax and peak cough flow improved with a mean (SD) of 14.57 (±15.67) cmH2O and 32.27 (±36.60) L/min, compared to a change of 3.04(±11.93)cmH2O (p=0.01) and -16.59 (±48.29) L/min (p=0.0005) during the control period. There was no evidence of change in spirometry, functional ability and total health-related quality of life scores following intervention. Patient satisfaction with IMT was high (median 8/10 (IQR 5-10)) and adherence was good. A three-month IMT programme in children with NMD is well tolerated, appears to be safe and is associated with a significant improvement in respiratory muscle strength and cough efficacy.

Congenital disorders of glycosylation (CDG) are a group of clinically and genetically heterogeneous diseases caused by disorders of glycoproteins synthesis. Patients manifest a wide range of symptoms, phenotypes, and severity, usually with neurological compromise. The conserved oligomeric Golgi (COG) complex plays an important role in vesicular tethering in retrograde Golgi transport. Mutation in this complex is considered a multiple-pathway CDG. Only 6 cases of pathogenic variants of COG1 have been reported in the literature. We present a 10 year-old-female born at term to healthy non-consanguineous Chilean parents. At birth, the main findings were weak suction, hypotonia, and high creatine kinase (CK). Due to development delay, hypotonia, and persistently elevated CK levels, sometimes over 10 times normal values, electromyography was performed, suggestive of a predominantly proximal myopathic compromise. Muscle biopsy revealed dystrophic changes and abnormal alpha-dystroglycan immunohistochemistry. The patient's symptoms progressed, and she currently continues with motor difficulties, muscle weakness, joint hypermobility, recurrent patellar dislocation, and severe progressive kyphoscoliosis. A lower limb muscular magnetic resonance image revealed mild fat replacement mainly on soleus and gastrocnemius muscles. No cognitive impairment or additional neurological symptoms have appeared, but persistent thrombocytopenia and intermittent leukopenia appeared after age 6 years. A neuromuscular NGS panel was negative, and exome sequencing revealed a homozygous frameshift mutation in COG1 gene (c.2665dupC, p.Arg889Profs*12). This mutation has been previously reported and is considered pathogenic. However, this is the first report of a COG1 mutation manifesting mainly as congenital muscular dystrophy with a musculoskeletal phenotype and without the intellectual phenotype expected due to the COG1 mutation. This communication expands the COG1 clinical spectrum, including muscle compromise and COG1 mutations as a potential gene candidate in the differential diagnosis of congenital muscular dystrophies.

SELENON-Related Myopathy (SELENON-RM) is a rare genetic disease caused by recessive mutations of the SELENON gene. It is characterized by the development of rigid spine, axial muscle weakness, and respiratory insufficiency. The most common histopathological feature in SELENON-RM patients is the presence of minicores in skeletal muscle biopsies, which are concentrated areas of mitochondrial depletion within fibers. Natural history data suggest that insulin-resistance as well as altered body mass index (BMI) are correlated with SELENON-RM prognosis. There is no cure or effective treatment for SELENON-RM. The SELENON gene encodes selenoprotein-N, an endoplasmic reticulum (ER) membrane glycoprotein with reducing catalytic activity. Selenoprotein-N has been shown to activate SERCA channels by reducing it at low Ca2+ concentrations in the ER. Additionally, it has also been shown to colocalize with Mitochondrial Associated Membranes (MAM), which are vital for mitochondrial function. However, the molecular mechanism(s) by which selenoprotein-N deficiency causes SELENON-RM is still unclear. A particular challenge has been the lack of cellular or animal models that exhibit readily assayable phenotypes. In this project, we aim to identify cellular and animal models suited for high throughput drug screening while elucidating the molecular mechanism of the disease. To do so, we tested selenoprotein-N deficient cells and zebrafish selenon-null models. Using different measures of metabolism, we found that Selenon-KO C2C12 cells and primary myoblasts isolated from Selenon-KO mice were metabolically impaired. We also assessed several phenotyping outcomes in zebrafish models, from embryonic development to adulthood. Our results showed muscle weakness during early development as well as reduced growth during the larval stage. Together, these data establish the potential for selenoprotein-N deficient cells and zebrafish as models for the discovery of therapeutic targets for SELENON-RM.

Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy developed for targeted skeletal and cardiac muscle expression of micro-dystrophin (a shortened, functional dystrophin protein). The objective of this phase 1/2a, single-dose, open-label clinical trial (NCT03375164) is to evaluate the safety of systemic delivery of delandistrogene moxeparvovec in patients with Duchenne muscular dystrophy (DMD). Four ambulatory patients with DMD (≥4 to ≤8 years old) were enrolled. Patients were given an intravenous infusion of delandistrogene moxeparvovec at a dose of 2.0x1014 vg/kg (supercoiled qPCR, linear plasmid standard equivalent of 1.33x1014 vg/kg) and prednisone (1 mg/kg/day) 1 day pre- to 30 days post-gene delivery. The primary outcome measure is safety. The secondary outcome measures include micro-dystrophin expression in pre- and post-muscle biopsies (Week 12 post-infusion). Key efficacy outcome measures include North Star Ambulatory Assessment (NSAA) and timed function tests. Previously, data from 3 years post-treatment were presented. Treatment-related adverse events (TRAEs) were mild to moderate, occurred mostly in the first 90 days of treatment, and all resolved. No serious adverse events (AEs), study discontinuations, or AEs associated with clinically relevant complement activation were reported. All patients demonstrated a clinically meaningful improvement on NSAA. Patients treated with delandistrogene moxeparvovec generally maintained muscle strength (Time to Rise and 4-stair Climb) and showed improvement in ambulation ability (10-metre and 100-metre Walk/Run) from baseline to Year 3. The observed safety profile and the enduring response following treatment provide proof of concept for continuation of clinical trials assessing delandistrogene moxeparvovec using single-dose gene therapy in patients with DMD. We present the latest long-term (4-year) safety and functional data from this study.

Hypokalemic periodic paralysis is a rare neuromuscular genetic disorder due to defect of ion channels and subsequent function impairment. It belongs to a periodic paralyses group including hyperkalemic periodic paralysis (HEKPP), hypokalemic periodic paralysis (HOKPP) and Andersen-Tawil syndrome (ATS). Clinical presentations are mostly characterized by episodes of flaccid generalized weakness with transient hypo- or hyperkalemia.A teenage boy presented to Emergency Department (ED) for acute weakness and no story of neurological disease, during the anamnestic interview he revealed that he had a carbohydrates-rich meal the previous evening. Through a focused diagnostic work-up the most frequent and dangerous causes of paralysis were excluded, but low serum potassium concentration and positive family history for periodic paralyses raised the diagnostic suspicion of HOKPP. After the acute management in ED, he was admitted to Pediatric Department where a potassium integration was started and the patient was counselled about avoiding daily life triggers. He was discharged in few days. Unfortunately, he presented again because of a new paralytic attack due to a sugar-rich food binge the previous evening. Again, he was admitted and treated by potassium integration. This time he was strongly made aware of the risks he may face in case of poor adherence to therapy or behavioral rules. Currently, after 15 months, the boy is fine and no new flare-ups are reported.HOKPP is a rare disease but symptoms can have a remarkable impact on patients' quality of life and can interfere with employment and educational opportunities. The treatment aims to minimize the paralysis attacks by restoring normal potassium level in order to reduce muscle excitability but it seems clear that a strong education of the patient about identification and avoidance triggering factors is essential to guarantee a benign clinical course. In our work we discuss the typical clinical presentation of these patients focusing on the key points of the diagnosis and on the challenges of therapeutic management especially in adolescence. A brief discussion of the most recent knowledge regarding this clinical condition follows.