Ophélie Vacca

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, resulting in the loss of dystrophin expression. Beyond progressive muscle degeneration, nearly half of patients exhibit cognitive and behavioral impairments, highlighting the need for effective central nervous system (CNS) therapies. In this study, we investigated whether neonatal delivery could maximize brain dystrophin rescue in mdx52 mice using exon 51 skipping strategies. We compared systemic and intracerebroventricular (ICV) delivery of an antisense oligonucleotide (ASO-51) and an AAV9-mediated U7snRNA approach (scAAV9-U7-Ex51), administered neonatally. Neonatal intravenous ASO-51 induced widespread but transient exon skipping, resulting in limited brain dystrophin restoration. ICV administration markedly increased CNS exon skipping but did not overcome the limited persistence of ASO, leading to progressive loss of exon skipping and dystrophin rescue. In contrast, neonatal ICV delivery of scAAV9-U7-Ex51 achieved robust, widespread, and sustained exon 51 skipping throughout the brain, including the hippocampus, cortex, and cerebellum, resulting in 20–50% restoration of Dp427 expression. These findings demonstrate that early CNS-directed delivery enhances brain dystrophin restoration and establish AAV-U7-mediated exon skipping as a promising strategy for addressing CNS comorbidities in DMD.

The dystrophin gene encodes multiple dystrophin isoforms with tissue-specific functions, including several shorter isoforms expressed in the central nervous system and retina. While Duchenne muscular dystrophy (DMD) has historically been characterized as a primary myopathy resulting from loss of the full-length dystrophin Dp427, increasing clinical evidence indicates that dysfunction of shorter dystrophin isoforms contributes to significant extramuscular pathology, including retinal disease. In particular, loss of the Dp71 isoform has been implicated in retinal inflammation, blood–retinal barrier breakdown, and pathological angiogenesis. In this study, we investigated whether low-level residual expression of Dp71 is sufficient to mitigate retinal inflammation in the mdx3Cv mouse model, which displays reduced—but not absent—expression of multiple dystrophin isoforms. Western blot analysis revealed that mdx3Cv retinas express approximately 4% of wild-type Dp71 protein levels. Despite this marked reduction, mdx3Cv mice did not exhibit the inflammatory phenotype previously observed in Dp71-null mice. Retinal VEGF protein levels and VEGF receptor (FLT-1 and KDR) mRNA expression were preserved, while VEGF mRNA levels were modestly reduced. Furthermore, expression of inflammatory markers ICAM-1 and ALOX5AP, leukocyte adhesion to retinal vasculature, Aquaporin-4 expression, and BRB permeability to albumin were all comparable to wild-type littermates. Together, these findings demonstrate that minimal residual expression of Dp71 is sufficient to preserve retinal vascular homeostasis and prevent inflammatory and permeability defects in the mdx3Cv retina. These results further suggest that partial dystrophin restoration—at levels achievable with current exon-skipping or gene-based therapies—may be adequate to prevent or attenuate retinal pathology in DMD, providing a realistic and clinically relevant therapeutic target.

La formation et l’intégrité de la barrière hémato-rétinienne (BHR) sont nécessaires au maintien d’une bonne vision et la violation de cette barrière contribue à l’apparition d’un grand nombre de pathologies rétiniennes tel que la rétinopathie diabétique (RD) ou l’occlusion de la veine centrale de la rétine (OVCR). La dystrophine Dp71 est une protéine du cytosquelette associée à la membrane qui s’exprime majoritairement dans les cellules gliales de Müller. Son absence a été associée à une augmentation de la perméabilité vasculaire liée à la délocalisation et à la diminution de l’expression des canaux AQP4 et Kir4.1. La souris Dp71-null est donc un excellent modèle d’étude des pathologies rétiniennes présentant une rupture de la BHR. L’ensemble de nos résultats démontrent que chez la souris déficiente en Dp71 ayant une rupture de la BHR, il est possible de restaurer une perméabilité et une homéostasie rétinienne normale grâce à la surexpression de la Dp71 via les virus adéno-associés. Cette étude est à la base du développement de nouvelles stratégies thérapeutiques dans le traitement de pathologies associées à une rupture de la BHR et à un œdème maculaire, comme la RD ou l’OVCR.

The dystrophin gene encodes multiple dystrophin isoforms with tissue-specific functions, including several shorter isoforms expressed in the central nervous system and retina. While Duchenne muscular dystrophy (DMD) has historically been charac-terized as a primary myopathy resulting from loss of the full-length dystrophin Dp427, increasing clinical evidence indicates that dysfunction of shorter dystrophin isoforms contributes to significant extramuscular pathology, including retinal disease. In par-ticular, loss of the Dp71 isoform has been implicated in retinal inflammation, blood–retinal barrier breakdown, and pathological angiogenesis. In this study, we investigated whether low-level residual expression of Dp71 is sufficient to mitigate retinal inflammation in the mdx3Cv mouse model, which displays reduced—but not absent—expression of multiple dystrophin isoforms. Western blot analysis revealed that mdx3Cv retinas express approximately 4% of wild-type Dp71 protein levels. Despite this marked reduction, mdx3Cv mice did not exhibit the in-flammatory phenotype previously observed in Dp71-null mice. Retinal VEGF protein levels and VEGF receptor (FLT-1 and KDR) mRNA expression were preserved, while VEGF mRNA levels were modestly reduced. Furthermore, expression of inflammatory markers ICAM-1 and ALOX5AP, leukocyte adhesion to retinal vasculature, Aqua-porin-4 expression, and BRB permeability to albumin were all comparable to wild-type littermates. Together, these findings demonstrate that minimal residual expression of Dp71 is sufficient to preserve retinal vascular homeostasis and prevent inflammatory and permeability defects in the mdx3Cv retina. These results further suggest that partial dystrophin restoration—at levels achievable with current exon-skipping or gene-based therapies—may be adequate to prevent or attenuate retinal pathology in DMD, providing a realistic and clinically relevant therapeutic target.

Duchenne muscular dystrophy (DMD) is marked by progressive muscle degeneration due to dystrophin deficiency. Despite cognitive impairments in up to one–third of patients, central nervous system (CNS)–targeted dystrophin restoration remains relatively underexplored compared to muscle-focused therapies. Since dystrophin is expressed in the brain during development and postnatally, it is important to assess which aspects of CNS pathology could be rescued. However, studying Dmd is difficult due to its size, low–abundance transcripts, and with multiple isoforms, limiting the sensitivity and isoform resolution of standard sequencing methods. To overcome these limitations, we applied Xenium spatial transcriptomics to target splice junctions, enabling isoform-specific and exon 51 skipping events detection across brain regions and cell types. Using this approach, we analyzed mdx52 mice lacking the full–length and Dp140 isoforms, treated with two exon 51 skipping therapies (antisense oligonucleotides or AAV–U7ex51). We observed distinct spatial expression patterns in the wild type brain between the full-length isoforms (Dp427c/m/p1) and shorter isoforms (Dp71 and Dp40). The full-length isoforms were predominantly expressed in cortex layers 2/3–6b and the CA1 region, while the shorter isoforms were localized to cortex layer 1 and the dentate gyrus. Among the exon skipping therapies tested, U7ex51 delivered neonatally induced broad exon skipping in neurons and effectively restored full-length isoforms in the targeted cells. This study introduces the first subcellular-resolution spatial transcriptomic atlas of dystrophin presence and rescue in the CNS and demonstrates a framework for evaluating gene therapies in spatially and transcriptionally complex tissues such as the brain.

The mdx52 mouse model exhibits a common mutation profile associated with brain involvement in Duchenne muscular dystrophy (DMD), characterized by heightened anxiety, fearfulness, and impaired associative fear learning. Deletion of exon 52 disrupts the expression of two dystrophins found in the brain (Dp427 and Dp140), and is eligible for therapeutic exon-skipping strategies. We previously demonstrated that a single intracerebroventricular administration of an antisense oligonucleotide (ASO) targeting exon 51 of the Dmd gene could restore 5% to 15% of Dp427 expression. This treatment reduced anxiety and unconditioned fear in mdx52 mice, improved fear conditioning acquisition, and partially improved fear memory tested 24 hours later. To improve the restoration of Dp427 and induce a long-lasting therapeutic effect, we employed a vectorized approach using an AAV-U7snRNA vector to deliver antisense sequences to the brains of mdx52 mice. We evaluated two AAV serotypes known for their brain transduction efficiency (AAV9 and RH10) and two delivery routes, intracisterna magna and intracerebroventricular (ICV) injections, to maximize brain targeting. Based on GFP expression data, we selected the AAV9 capsid and a bilateral ICV delivery route. Using this approach, we demonstrated that ICV administration of AAV9-U7-Ex51M induced exon 51 skipping and restored Dp427 expression in the brains of adult mdx52 mice, though with significant variability among individuals. While a few mice showed high Dp427 expression levels, the average restoration was limited to approximately 6% to 12%. In conclusion, inducing exon skipping in the brains of adult mdx52 mice using the vectorized AAV9-U7 approach was less effective than synthetic ASO treatment and did not improve the emotional behavior of mdx52 mice.

Duchenne Muscular Dystrophy (DMD) is a severe genetic disorder characterized by progressive muscle degeneration due to mutations in the dystrophin gene. In addition to the well-known musculoskeletal, cardiac, and respiratory symptoms, DMD also involves significant central nervous system (CNS) manifestations, including cognitive, behavioral, and emotional deficits that profoundly affect patient quality of life. Despite advances in RNA-based therapies targeting muscle symptoms, CNS manifestations remain largely untreated. Preclinical studies in animal models have shown promising results in addressing these deficits through CNS-targeted delivery of antisense oligonucleotides, highlighting the potential of intrathecal and next-generation systemic delivery methods. This review explores the latest advancements in RNA therapeutics for DMD, focusing on overcoming the challenges of CNS delivery to address both muscular and neurological symptoms.

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood–brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the open reading frame and thus prevent production of functional dystrophin proteins. Recent advances in DMD treatment, notably exon skipping and AAV gene therapy, have achieved some success aimed at alleviating the symptoms related to progressive muscle damage. However, they do not address the brain comorbidities associated with DMD, which remains a critical aspect of the disease. The mdx52 mouse model recapitulates one of the most frequent genetic pathogenic variants associated with brain involvement in DMD. Deletion of exon 52 impedes expression of two brain dystrophins, Dp427 and Dp140, expressed from distinct promoters. Interestingly, this mutation is eligible for exon skipping strategies aimed at excluding exon 51 or 53 from dystrophin mRNA. We previously showed that exon 51 skipping can restore partial expression of internally deleted yet functional Dp427 in the brain following intracerebroventricular (ICV) injection of antisense oligonucleotides (ASO). This was associated with a partial improvement of anxiety traits, unconditioned fear response, and Pavlovian fear learning and memory in the mdx52 mouse model. In the present study, we investigated in the same mouse model the skipping of exon 53 in order to restore expression of both Dp427 and Dp140. However, in contrast to exon 51, we found that exon 53 skipping was particularly difficult in mdx52 mice and a combination of multiple ASOs had to be used simultaneously to reach substantial levels of exon 53 skipping, regardless of their chemistry (tcDNA, PMO, or 2′MOE). Following ICV injection of a combination of ASO sequences, we measured up to 25% of exon 53 skipping in the hippocampus of treated mdx52 mice, but this did not elicit significant protein restoration. These findings indicate that skipping mouse dystrophin exon 53 is challenging. As such, it has not yet been possible to answer the pertinent question whether rescuing both Dp427 and Dp140 in the brain is imperative to more optimal treatment of neurological aspects of dystrophinopathy.