Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2−/− mice (MDXxLcn2−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

Validation of DE50-MD dogs as a model for the brain phenotype of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disorder, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterization of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs including reduced attention, problem-solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, qPCR and RNAScope in situ hybridization we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally-relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.

TRF2 rescues pathogenic phenotypes in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart; however, these treatments do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that longer telomeres are protective against dilated cardiomyopathy. Here we investigated the role of telomeres as a target for therapy in DMD cardiomyocytes using human induced pluripotent stem cells (iPSCs) to model the disease. Compared to healthy controls, DMD cardiomyocytes exhibited reduced telomere lengths, cell size, nuclear size, and sarcomere density. The telomere-binding protein, TRF2, is a core component of the shelterin complex, which protects chromosome ends. TRF2 levels are reduced relative to healthy controls in DMD cardiomyocytes. We hypothesized that decreased TRF2 drives telomere attrition and subsequent cardiomyocyte death in the progression of dilated cardiomyopathy. Our data show that TRF2 overexpression prevented telomere attrition and also rescued deficits in cell size, nuclear size, sarcomere density, and calcium handling. These data highlight the benefits of TRF2 upregulation as a potential gene therapy to delay the onset of dilated cardiomyopathy.

Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence

Duchenne muscular dystrophy (DMD) is an X-linked recessive, infancy-onset neuromuscular disorder characterized by progressive muscle weakness and atrophy, leading to delay of motor milestones, loss of autonomous ambulation, respiratory failure, cardiomyopathy, and premature death. DMD originates from mutations in the DMD gene that result in a complete absence of dystrophin. Dystrophin is a cytoskeletal protein which belongs to the dystrophin-associated protein complex, involved in cellular signaling and myofiber membrane stabilization. To date, the few available therapeutic options are aimed at lessening disease progression, but persistent loss of muscle tissue and function and premature death are unavoidable. In this scenario, one of the most promising therapeutic strategies for DMD is represented by adeno-associated virus (AAV)-mediated gene therapy. DMD gene therapy relies on the administration of exogenous micro-dystrophin, a miniature version of the dystrophin gene lacking unnecessary domains and encoding a truncated, but functional, dystrophin protein. Limited transgene persistence represents one of the most significant issues that jeopardize the translatability of DMD gene replacement strategies from the bench to the bedside. Here, we critically review preclinical and clinical studies of AAV-mediated gene therapy in DMD, focusing on long-term transgene persistence in transduced tissues, which can deeply affect effectiveness and sustainability of gene replacement in DMD. We also discuss the role played by the overactivation of the immune host system in limiting long-term expression of genetic material. In this perspective, further studies aimed at better elucidating the need for immune suppression in AAV-treated subjects are warranted in order to allow for life-long therapy in DMD patients.

Influence of A Public Health Emergency On Care For Patients With Duchenne Muscular Dystrophy: An Online, Cross-Sectional Survey

Background: The public health emergency has created challenges for the care of patients, particularly those with chronic diseases such as Duchenne muscular dystrophy. To elucidate the challenges faced by Chinese patients with Duchenne muscular dystrophy during the public health emergency coronavirus disease 2019 pandemic, we conducted an online cross-sectional survey, the responses of which were collected between March 27 and June 30, 2021. Results: In total, valid questionnaire responses were obtained from 2,105 patients, of whom 49 lived in pandemic lockdown areas. Of the 2,056 responders from non-lockdown areas, 42.8% reduced their outside daily activities, 49.4% reduced their use of rehabilitation services, 39.7% postponed regular follow-up appointments, and 40.8% complained of accelerated declines in motor function over the previous year. The corresponding figures for the 49 participants from lockdown areas were almost all higher, with 67.3% reducing outside daily activities, 44.9% reducing their use of rehabilitation services, 79.6% postponing regular follow-up appointments, and 55.1% complaining of accelerated declines in motor function. When asked whether they expected more assistance from society than they had received before the pandemic, 60.8% of patients in non-lockdown areas and 87.8% of those in lockdown areas responded affirmatively. When asked whether they felt more anxious than they had before the pandemic and needed psychological counseling, 11.5% of respondents in non-lockdown areas and 18.4% of respondents in lockdown areas responded affirmatively. In non-lockdown areas, 76% of respondents had at least one telemedicine visit, and 71% of them thought that telemedicine was helpful. In lockdown areas, 91.8% had used telemedicine at least once, and 66.7% of them found it helpful.Conclusions: These public health emergency control measures have affected the care of patients with chronic diseases worldwide, particularly pronounced in lockdown areas. It is imperative that healthcare workers assist patients and establish more robust chronic disease management systems. Telemedicine is an effective model for providing healthcare to such patients.