Givinostat significantly improved exon skipping and dystrophin restoration in human DMD myoblasts carrying an exon 52 deletion, and combining givinostat with ASOs to enhance dystrophin restoration and muscle preservation in DMD.
Abstract
Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder caused by mutations in the DMD gene, with deletions within the exon 45–55 hotspot being the most common. Despite advances in antisense oligonucleotide (ASO)-mediated exon skipping, therapeutic efficacy remains limited by suboptimal transcript availability and delivery barriers. We investigated whether histone deacetylase inhibitors (HDACis), particularly givinostat, can enhance the efficacy of an exon 51-targeting ASO in the clinically relevant mdx52 mouse model. For the first time, we identified a pronounced 5′–3′ DMD transcript imbalance associated with exon 52 deletion, contrasting with the classical mdx model and potentially compromising ASO effectiveness. In human DMD myoblasts carrying an exon 52 deletion, givinostat significantly improved exon skipping and dystrophin restoration. In vivo, givinostat and ASO co-treatment resulted in a modest but significant increase in dystrophin levels compared to ASO alone (1.3-fold), along with reduced muscle fibrosis, improved fiber morphology, and better extracellular matrix organization. Notably, treated mice showed improved muscle function, as evidenced by a significant reduction in force loss after eccentric contractions, a clinically meaningful outcome. Although the precise molecular mechanisms underlying these effects remain to be fully elucidated, these findings support the therapeutic potential of combining givinostat with ASOs to enhance dystrophin restoration and muscle preservation in DMD.
Over the last decades, antisense oligonucleotides (AONs) have emerged as powerful tools to modulate alternative splicing, allowing exclusion of a specific exon from a target mRNA to bypass mutations or restore the reading frame, enabling production of a functional protein. In Duchenne muscular dystrophy (DMD), a lethal X-linked disorder affecting 1:5,000 males, four AON therapies have received accelerated approval. However, these treatments are applicable to only 27% of patients, leaving most individuals with rare mutations without therapeutic options. Here, we outline a methodology to identify relevant AONs for such populations. This approach aims to prequalify candidates and eliminate unsuitable leads before animal testing, reducing development time and costs. Following high-throughput screening, selected AONs are assessed for intravenous delivery compatibility and serum protein binding to support model selection and forecast human pharmacokinetics. As proof of concept, we applied this strategy to tricyclo-DNA AONs targeting an ultra-rare DMD exon 16 mutation. From a 59-member library, we defined a lead compound with efficient exon 16 skipping, favorable pharmacokinetic and safety profiles, able to rescue dystrophic transcriptomic and proteomic signatures. This study demonstrates the feasibility of developing a safe and effective lead compound within a reasonable time frame and cost, supporting compassionate use and future n = 1 trials.
Marine Geoffroy-Guiraud, C. Beley, Stephany Campuzano et al.· Molecular Therapy: Nucleic A...· 0 citations
Summary Duchenne muscular dystrophy (DMD) causes progressive muscle degeneration due to dystrophin deficiency. Dystrophin is also expressed in the brain during development and postnatally, yet a characterization of dystrophin isoform expression across brain cells and regions is lacking, limiting our understanding of the cognitive impairment affecting one-third of the patients and hampering the development of dystrophin-restoring drugs in the central nervous system (CNS). Here, we applied spatial transcriptomics to map Dmd isoforms across mouse brain regions and cell types. Mdx52 mice received exon 51-skipping therapies restoring the Dp427-sized isoform at the transcript and protein levels. We observed distinct spatial patterns: full-length isoforms localized to deeper cortical layers and CA1, while shorter isoforms were enriched in cortical layer 1 and dentate gyrus. We present evidence of isoform restoration, immune activation following treatment, and a framework to evaluate exon-skipping therapies in the CNS using spatial transcriptomics.
Qirong Mao, A. Ahmadi, Sharon I. de Vries et al.· iScience· 0 citations
Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle disease. DMD results from mutations preventing the production of the subsarcolemmal protein dystrophin, a protein critical for muscle stability. In the absence of dystrophin, muscle fibers are damaged and degenerate, leading to an inflamed and fibrotic muscle phenotype. DMD patients have a life expectancy of 20 to 40 years old and succumb to cardiac and respiratory failure. Adeno-associated virus (AAV) is a promising vector to deliver gene therapies (such as truncated dystrophin proteins, i.e., microdystrophin) to the muscle of DMD patients, due to its safety profile and broad tissue tropism. Currently, naturally occurring AAV variants including serotypes 8, 9, and rh74 are used in clinical trials, and an AAVrh74-based microdystrophin therapy (Elevidys) was approved in 2023 to treat DMD. Despite regulatory success, limited results are available from patients treated with Elevidys and in other clinical trials, and many questions remain about the biology of AAV vectors in muscle. Individual muscle cells, or myofibers, have different phenotypes (myofiber type), closely linked to their genetically regulated expression of different isoforms of the contractile protein myosin heavy chain. Based on previous literature, we hypothesized that AAV serotypes 8 and 9 may have a selective tropism for certain myofiber types, or restricted tropism in others. Fast-twitch myofiber types are susceptible to greater injury in DMD, and thus, benefit greater from microdystrophin gene transfer. To determine the myofiber type preference AAV serotypes 8 and 9 we performed histological analysis of reporter gene and microdystrophin expression in archival tissues from preclinical studies in dystrophic canines (cDMD model). We did not detect a myofiber type preference. However, the results from our histological analysis indicated that microdystrophin expression is maintained long-term (8-40 months following systemic delivery); and that micro-dystrophin expressing fibers were protected from atrophy. These results offer important insight for clinical development of microdystrophin gene therapies. Selecting the best AAV variants for muscle-directed gene therapy is a critical step in developing effective treatments. Currently, there is no way to evaluate systemic delivery of AAV vectors to human muscle. To overcome this barrier, we adapted a human skeletal muscle xenograft model, to use as a platform for AAV delivery. We characterized this model using 222 transplanted muscles from human donors, and developed rigorous methods to evaluate muscle transplant quality, and AAV transduction of the muscles. We showed for the first time, that human muscles transplanted in immune-deficient mice are efficiently transduced by AAV vectors at following intravenous injection of the host mice. We first used this platform to compare the three AAV serotypes in clinical trials (8, 9, and rh74). We found that all three have similar delivery efficiency (i.e., infection) in human muscles, but only AAV9 led to high-level mRNA production. These results not only present a novel finding on the superior efficiency of AAV9 but also highlight a new way to evaluate AAV vectors in a preclinical context. Other human transplantation models have been used to discover next-generation AAV serotypes for liver-directed gene therapy. We expect that the human muscle xenograft model has similar potential for discovery of serotypes with enhanced efficiency in human muscle.
Spinal Muscular Atrophy (SMA) is a neuromuscular genetic disorder resulting from the mutation or deletion of the Survival Motor Neuron 1 (SMN1) gene and the reduction of the Survival Motor Neuron (SMN) protein. As a result, SMN level in SMA depends on the almost identical copy gene SMN2, which produces a small amount of functional SMN due to a silent mutation in exon 7. SMN deficiency critically impairs spinal cord motoneuron (MN) function, causing progressive degeneration. Advances in SMA therapeutics have significantly improved clinical management and prognosis. However, therapeutic outcomes vary among SMA patients, resulting in broad heterogeneity in phenotypes and clinical trajectories; consequently, more focused investigation on the underlying disease mechanisms is essential. One of the FDA-approved treatments is nusinersen, an antisense oligonucleotide (ASO) designed to enhance SMN2 exon 7 splicing and increase SMN protein. The present study used non-SMA and SMA MNs differentiated from human induced Pluripotent Stem Cells (hiPSCs) to analyze the effect of a nusinersen-like ASO treatment on intracellular pathways altered in SMA MNs. ASO treatment efficiently increased SMN, prevented MN degeneration, and decreased apoptotic markers in SMA MNs. Furthermore, treatment increased Gemin3 protein and the NF-κB members, IKKβ and RelA. Nevertheless, ASO did not revert alterations of the autophagy markers LC3-II and p62/SQSTM1, and the calpain activation product α-fodrin 145/150 kDa. Our observations indicate that nusinersen-like ASO treatment might be insufficient to counteract the full spectrum of intracellular alterations occurring in SMN-reduced MNs. Therefore, supplementary compounds targeting these unrecovered pathways might supply additional protective effects on degenerating MNs.
María P. Miralles, Isabel Gimenez-Fernandez, María Beltran et al.· Cell Death and Disease· 0 citations
This study evaluates the therapeutic potential of the lipid-conjugated antimiR-23b, X82108, designed to promote MBNL1/2 upregulation through inhibition of miR-23b, and highlights X82108 as a promising systemic therapy for DM1.
D. Piqueras-Losilla, Andrea García-Rey, Aline Huguet-Lachon et al.· Cell Reports Medicine· 0 citations
Introduction: Givinostat, a histone deacetylase inhibitor, has been investigated for the treatment of Duchenne Muscular Dystrophy (DMD) and slow muscle degradation. DMD is a genetic neuromuscular degenerative disease that begins in early childhood. It is an X-linked recessive disorder characterized by mutations in the dystrophin gene and progressive and severe muscle weakness and atrophy are the main characteristics of this disease. DMD predominantly affects boys, while most females are asymptomatic carriers, with a global prevalence estimated at 19.8 cases per 100,000 live male births.
Objective: To evaluate the efficacy and safety of Givinostat for individuals with DMD based on evidence from randomized controlled trials (RCTs).
Methodology: We conducted a systematic review following PRISMA guidance, focusing on RCTs assessing Givinostat versus placebo, standard care, no treatment, or any active pharmacological intervention in DMD. CENTRAL, MEDLINE, Embase, PEDro and LILACS were searched (August 2025) without restrictions. Two reviewers independently selected studies, extracted data, and appraised risk of bias using the original Cochrane tool. Certainty of evidence was assessed using GRADE. The protocol was prospectively registered in the PROSPERO database (CRD420251107551).
Results: Two RCTs were included, although one is still ongoing. EPIDYS (n=179) compared Givinostat (13 mg to 47 mg twice daily) with placebo for 72 weeks. There is uncertainty regarding the effects of Givinostat on global symptom assessment (mean difference [MD] 1.91; 95% CI 0.30 to 3.53; n=120; very low certainty), serious adverse events (risk ratio [RR] 2.07; 95% CI 0.45 to 9.44; n=179; very low certainty), functional capacity assessed by two different instruments (n=120; very low certainty), and quality of life (MD 3.70; 95% CI −2.30 to 9.70; n=120; very low certainty). Givinostat may have little or no effect on the risk of any adverse events (RR 1.02; 95% CI 0.94 to 1.10; n=179; low certainty).
Conclusions: Current evidence on the efficacy of Givinostat remains uncertain, while it may have little or no effect on the risk of any adverse events. Further well-designed RCTs with larger sample sizes and varying disease stages are needed to reduce these uncertainties.
C. Latorraca, Bruna Carolina de Araújo, C. Cruz et al.· HTA Journal· 0 citations
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