Biology subjects

Goyenvalle, A.

Publications and source records attributed to Goyenvalle, A..

5 recordsLinked to original sources

bioRxiv2026

Neonatal Delivery of Exon Skipping Therapeutics Maximizes Brain Dystrophin Rescue in the Mdx52 Mouse Model of Duchenne Muscular Dystrophy

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.

neuroscience↗

bioRxiv2025

Evaluating exon-skipping therapies targeting the central nervous system in Duchenne muscular dystrophy using high-resolution spatial transcriptomics

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.

neuroscience↗

bioRxiv2025

Improving angiogenesis ameliorates the efficacy of ASO-based exon-skipping for the treatment of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe X-linked disease caused by mutations in the DMD gene, resulting in the absence of functional dystrophin. Antisense oligonucleotide (ASO)-based therapies aim to restore the open reading frame and produce a truncated but functional dystrophin protein. Although several ASOs are FDA-approved, dystrophin restoration in patient biopsies remains low, underlining the need to improve ASO efficacy. One major limitation is poor ASO biodistribution to skeletal muscle, influenced by both ASO chemistry and pathological features of dystrophic tissue. In DMD patients and mdx mice, microvascular abnormalities and impaired angiogenesis likely restrict ASO delivery. Here, we hypothesized that enhancing muscle vascularization could improve ASO biodistribution and therapeutic outcomes. Mdx mice were treated with a pro-angiogenic treatment prior to ASO administration targeting exon 23 of dystrophin pre-mRNA. Angiogenic stimulation increased capillary density and improved ASO delivery, exon skipping, and dystrophin expression compared to ASO alone. These molecular improvements were associated with increased myofiber size, larger mean cross-sectional area, and decreased serum myomesin levels, without signs of toxicity. This study provides proof-of-concept that promoting angiogenesis can enhance the efficacy of ASO-based treatments, offering a complementary strategy to improve therapeutic outcomes in DMD.

molecular biology↗

bioRxiv2024

A Genome-wide CRISPR screen unveils WDR91 protein as a promoter of productive ASO activity

Antisense oligonucleotides (ASOs) belong to promising therapeutics for the treatment of neurologic, muscular and metabolic disorders. Several ASOs have been approved so far and more than a hundred clinical trials are currently underway covering a dozen therapeutic areas. Yet, the mechanisms of internalization and cell trafficking of these molecules remain poorly understood. Moreover, with only a small fraction of ASOs reaching the correct cellular compartment following systemic delivery, the majority of targeted diseases requires recurrent injections of ASOs. A deeper understanding of these mechanisms would guide the improvement of their potency and thus, reduce the amount of delivered ASOs and their potential side-effects. Here, using a CRISPR screen, we investigated intracellular proteins involved in ASOs efficiency using a whole genome approach and identified several potential regulators which could significantly impact ASOs potency in melanoma cells. We validated WD Repeat Domain 91 (WDR91), a regulator of endosomal maturation, as a modulator whose depletion significantly inhibits ASO productive activity. This study provides the first list of ASO modulators using a biologically relevant assay to estimate the role of these proteins. In conclusion, these data could lead to a better understanding of the mechanisms favoring productive uptake or improved endosomal escape of ASOs.

molecular biology↗

bioRxiv2022

Novel uAUG creating variants in the 5UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare vascular disorder causing abnormal vessel formation and characterized by autosomal dominant transmission. The associated considerable variability in symptoms and clinical severity complicate the management of the disease. In clinical routine, 3 main genes, ACVRL1 (also known as ALK1), ENG and SMAD4 are screened for pathogenic variants at the origin of HHT. About 80% of HHT cases are caused by pathogenic coding variants in ACVRL1 and ENG. However, at least 15% remain with no molecular explanations in the 3 main genes. We here report the identification of 2 never reported variants, c.-79C>T and c.-68G>A, in the 5UTR of ENG in 2 unrelated HHT patients. These 2 variants are predicted to create upstream AUGs (uAUGs) in the 5UTR, which are in frame with a stop codon located within the CoDing Sequence (CDS), thus generating Overlapping upstream Open reading frames (uoORFs). In other cases, uAUGs can lead to fully upstream ORFs (uORFs) when associated with stop codons located within the 5UTR or to elongated CDS (eCDS) when in frame with the CDS and associated with the main stop codon. In order to assess the pathogenicity of these ENG variants, we performed in vitro functional assays based on the expression of wild-type and mutant constructs in human cells. We found that these 5UTR ENG variants were associated with a decrease of protein levels in HeLa and HUVEC cells. They were also associated with a decreased ability to activate BMP9-stimulated ALK1 receptor in a BMP-response element (BRE) assay. This assay is a mandatory element before providing a definitive molecular diagnosis and has been so far applied only on coding ENG variants. We applied the same experimental workflow on 3 additional uoORF-creating variants (c.-142A>T, c.-127C>T and c.-10C>T) and one eCDS-creating variant (c.-9G>A) in the 5UTR of ENG previously reported in HHT patients. We found that all the analysed uoORF-creating variants alter endoglin levels and function. A comparison of our experimental results with patients clinical characteristics suggests that uoORF-creating variants leading to ENG protein levels [≤] 40% in vitro would be associated with HHT. Additional experiments relying on an artificial deletion in our mutated constructs show that created uAUGs predicted to create uoORFs are able to initiate the translation indicating that the associated effect is likely caused by an alteration of the translation mechanism. Overall, we here identified two never reported 5UTR ENG variations in HHT patients and shed new lights on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.

molecular biology↗