In vitro and in silico characterization of competitive inhibition and repression of DUX4 target gene activation as a therapeutic approach for facioscapulohumeral muscular dystrophy (FSHD)
It is shown that DBD alone produces dose-dependent repression of DUX4-FL transcriptional activity in HEK293T cells, while a constitutively expressed DBD-KRAB fusion produces significantly greater repression than DBD alone, with a similar trend observed in C2C12 myoblasts.
Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is a rare neuromuscular disease caused by aberrant re-expression of the embryonic transcription factor DUX4 in skeletal muscle, which activates a toxic transcriptional program that drives progressive muscle wasting. No approved disease-modifying therapies currently exist. Prior work in mammalian and zebrafish models has shown that a truncated form of DUX4 retaining only its DNA-binding domain (DBD) lacks transactivation capacity and can suppress DUX4-FL-driven pathology; separately, dCas9/KRAB-based epigenetic repressors have demonstrated efficacy in silencing DUX4 expression, though CRISPR-based strategies face challenges from the repetitive nature of the D4Z4 locus, the immunogenicity associated with bacterial Cas proteins, and the payload limitations of gene delivery vehicles. Building on these findings, we corroborate that the DUX4 DBD, comprising both homeodomains, acts as a non-toxic competitive inhibitor of full-length DUX4 (DUX4-FL) at its genomic target sites, and extend this strategy by fusing the DBD to a human KRAB(ZNF10) domain, converting DUX4 from a transcriptional activator into a fully humanized epigenetic silencer of its own targets. Using a fluorescent DUX4-responsive reporter, we show that DBD alone produces dose-dependent repression of DUX4-FL transcriptional activity in HEK293T cells (200-fold at the highest inducible dose tested), while a constitutively expressed DBD-KRAB fusion produces significantly greater repression than DBD alone (949-fold versus 17-fold at a 25x molar ratio), with a similar trend observed in C2C12 myoblasts (47-fold versus 3.3-fold knockdown). To contextualize these findings and explore dosing considerations, we developed three complementary computational models – a transcription factor competitive binding model, a myotube diffusion model, and an ordinary differential equation (ODE) compartmental model – that illustrate how DBD concentration, intracellular diffusion, and population-level cell state transitions may relate to therapeutic efficacy. Together, these results corroborate and extend existing approaches into a single, fully humanized construct that may help circumvent the immunogenicity and delivery limitations of Cas-based systems.
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common dominant muscular dystrophies and remains without an approved disease modifying therapy. Caused by the aberrant expression of the cytotoxic gene DUX4, FSHD is typically diagnosed in adulthood, however clinical onset in children (<18 years of age) is often associated with a more severe and rapid disease. While clinical trials are underway, a lack of human-specific pre-clinical models limit effective testing of potential therapies, particularly in children. To fill this gap, we describe here the development of induced pluripotent stem cell-derived 2-and 3-dimensional skeletal muscle models of children with clinically defined mild, moderate, and severe FSHD. These iPSC-derived muscle models replicate key features of FSHD, including aberrant DUX4 mRNA expression, muscle atrophy, and weakness, which correlate with the individuals’ specific disease severity. Next, we assessed the efficacy of adenine base editing (ABE) as a potential gene therapy approach to treat FSHD. DUX4-targeted ABE reduced DUX4 mRNA expression, improved muscle area and force generation in the most severe individual. Together this work supports the use of iPSC-derived skeletal muscle models as a less invasive method to study childhood-onset FSHD and establishes targeted DUX4 gene editing therapies as a potential treatment approach.
P. Houweling, Vanessa G. Crossman, L. Kiriaev et al.· bioRxiv· 0 citations
Alpha-synuclein (SNCA) overexpression is implicated in Parkinson's disease (PD) pathogenesis, making SNCA downregulation a promising therapeutic strategy. We developed a SNCA-targeted transcriptional repression therapy using an all-in-one lentiviral vector (LV) carrying deactivated CRISPR/(d)Cas9, gRNA targeted at SNCA-intron1, and either the catalytic domain of DNA-methyltransferase3A (DNMT3A), or an engineered repressor molecule, a fusion of MeCP2's transcription repression domain (TRD) and KRAB. Therapeutic efficacy was evaluated following co-administration of the therapeutic and model vectors in a new PD mouse model, generated with an adeno-associated viral vector carrying an engineered minigene comprised of the human (h)A53T-SNCA expressed via the human native regulatory region. Both therapeutic vectors reduced expression of α-synuclein in the substantia nigra (SN), with LV/dSaCas9- KRAB-MeCP2(TRD) demonstrating greater repression. LV/dSaCas9- KRAB-MeCP2(TRD) also significantly reduced pathological α-synuclein aggregation and phosphorylation (Ser 129), and preserved tyrosine hydroxylase expression in the SN and the striatum. Behavioral analysis following LV/dSaCas9-KRAB-MeCP2(TRD) injection, showed significant improvement in motor deficits characteristic of our PD-mouse model. Preliminary safety assessments found normal blood counts, serum chemistry, and weights. Collectively, these findings provide in vivo proof-of-concept for SNCA-targeted transcriptional repression therapy in a PD-mouse model and support its further preclinical development toward investigational new drug enablement.
Bernadette O’Donovan, Joseph E. Rittiner, Suraj Upadhya et al.· Neurotherapeutics· 0 citations
Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder caused by CTG repeat expansions in the DMPK gene, leading to the formation of toxic RNA foci that sequester essential splicing regulators MBNL1/2. Beyond muscle impairment, DM1 affects also the brain, leading to significant cognitive deficits, behavioral abnormalities, and intellectual disabilities. This study evaluates the therapeutic potential of the lipid-conjugated antimiR-23b, X82108, designed to promote MBNL1/2 upregulation through inhibition of miR-23b. Systemic administration of X82108 in mice and non-human primates efficiently crosses the blood-brain barrier, increasing MBNL1 in the brain. In DMSXL transgenic mice, treatment increases Mbnl1/2, reduces toxic DMPK, and restores normal splicing patterns across all brain regions. These molecular improvements correlate with improved behavioral outcomes, including reduced impulsivity and normalized exploratory activity. Collectively, the findings highlight X82108 as a promising systemic therapy for DM1, targeting not only muscular features as we have previously shown but also DM1-related CNS alterations.
D. Piqueras-Losilla, Andrea García-Rey, Aline Huguet-Lachon et al.· Cell Reports Medicine· 0 citations
Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is caused in most cases by pathogenic variants in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Despite substantial progress in the development of gene therapy, restoring MECP2 expression remains challenging because MeCP2 is highly dosage-sensitive. Both deficiency and excessive expression of this protein are associated with severe neurological abnormalities. This makes simple viral vector-mediated replacement of MECP2 potentially unsafe and underscores the need for multilayered systems that control transgene expression. This review discusses current and emerging strategies for regulating MeCP2 expression in RTT, with an emphasis on non-coding RNA-based and epigenetic mechanisms. Particular attention is given to the limitations of conventional AAV-mediated gene therapy, the use of cell-specific and endogenous promoters, miRNA-regulated elements, autoregulatory systems, and post-transcriptional control of MECP2 expression. Strategies for reactivating the inactive X chromosome are also discussed, including XIST-dependent regulation and epigenome editing. In addition, the review considers CRISPR-mediated regulation, selective epigenetic activation, and combined therapeutic platforms that integrate viral delivery, RNA-dependent post-transcriptional control, and endogenous gene regulation. Overall, clinically applicable gene therapy for RTT will likely need to move beyond simple MECP2 replacement and instead rely on precise cell- and dose-dependent regulation of its expression. Non-coding RNA and epigenetic mechanisms represent important layers of such control and may contribute to the development of safer gene therapy strategies for RTT.
I. Kabdesh, A. Rizvanov, Y. Mukhamedshina· Non-Coding RNA· 0 citations
ASXL3 patient truncations in neurodevelopmental condition Bainbridge-Ropers syndrome are shown to mediates gain-of-function (GOF) by escaping nonsense-mediated decay and Cullin 4-dependent degradation, resulting in aberrant protein accumulation, widespread transcriptional dysregulation, and altered chromatin accessibility.
Y. Nakamura, T. Nguyen, N. Mor et al.· medRxiv· 0 citations
Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting and persistent chronic inflammation, yet the multi-lineage cellular drivers of its pathogenesis remain incomplete. In this study, single-cell RNA sequencing (scRNA-seq) identified Atp6ap2 as a profoundly upregulated gene across multiple skeletal muscle cell types-particularly endothelial cells, fibroblasts, and myoblasts-in both mdx and severe mdx mice. Weighted gene co-expression network analysis (WGCNA) linked Atp6ap2 expression to DMD progression, while enrichment analyses revealed that its dysregulation severely impairs vascular homeostasis and extracellular matrix integrity via the PI3K-Akt, focal adhesion, and cell cycle pathways. Utilizing Connectivity Map (CMap) analysis, we identified temozolomide (TMZ) as a top pharmacological candidate capable of reversing the ATP6AP2-associated gene signature. In vivo validation demonstrated that TMZ administration significantly enhanced motor coordination, balance, and grip strength in mdx mice, while markedly preserving dystrophic muscle architecture, reducing myofiber necrosis, and alleviating interstitial fibrosis. Mechanistically, integrated transcriptomic and metabolomic profiling revealed that TMZ induced profound metabolic and signaling shifts, modulating the Notch, MAPK, and Ras pathways, as well as autophagy and glycerophospholipid metabolism. Furthermore, scRNA-seq and cell-cell communication analyses indicated that TMZ dynamically reorganized multicellular networks, decreasing aberrant fibroblast and endothelial interactions. Crucially, immunofluorescence and Western blot validations confirmed that TMZ drastically attenuated the infiltration of F4/80-positive macrophages and suppressed their pro-inflammatory M1 polarization (indicated by reduced co-localization with iNOS and ATP6AP2), while successfully reversing the dysregulation of the ATP6AP2 axis and restoring its downstream targets MAP4K2, DGKE, and EFNA1. Collectively, our findings demonstrate that TMZ mitigates dystrophic pathology by targeting the ATP6AP2 signaling axis and dampening macrophage-mediated inflammatory responses, highlighting its potential as a novel immunopharmacological therapeutic strategy for DMD.
Lin Zhou, Yu Zhang, Xinxin Tan et al.· International Immunopharmaco...· 0 citations