Search Results (30)

RNA-based therapeutics are reshaping the treatment landscape for skeletal muscle disorders by enabling modulation of RNA processing or direct correction of disease-causing alleles. In Duchenne muscular dystrophy (DMD), four antisense oligonucleotides—eteplirsen, golodirsen, viltolarsen, and casimersen—have received FDA approval; these phosphorodiamidate morpholino oligomers (PMOs) induce exon skipping to restore the reading frame and enable expression of internally truncated dystrophin. Beyond splice switching, RNA therapeutics include RNase H-active gapmers and steric-blocking antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) that mediate post-transcriptional gene silencing, and RNA-guided gene-modifying technologies such as CRISPR systems that can reframe or repair endogenous alleles. Despite major progress in DMD, broader clinical impact remains constrained by inefficient delivery to skeletal and especially cardiac muscle, the need for repeat administration for most modalities, and safety considerations that limit dose escalation and durability. Next-generation approaches aim to overcome these barriers through peptide- or antibody-conjugated oligonucleotides that enhance cellular uptake and tissue distribution, alternative chemistries with improved stability and potency, and viral or non-viral platforms for durable splice modulation. In parallel, CRISPR-based strategies—including base and prime editing—offer the prospect of one-time correction, while raising important questions regarding delivery, immunogenicity, editing specificity, and long-term safety. This review synthesizes recent advances in antisense and gene-modifying strategies for skeletal muscle and highlights practical priorities for translation, including improved muscle/heart delivery, controllable safety mechanisms, scalable manufacturing, and standardized biomarker-to-clinical outcome relationships. Full article

Bacterial stem and root rot (BSRR), caused by Dickeya dadantii, poses a severe threat to global sweetpotato production, yet the genetic architecture underlying resistance remains elusive. To dissect these mechanisms, we conducted a high-resolution genome-wide association study (GWAS) on 135 diverse accessions, integrating two-year field phenotyping with best linear unbiased prediction (BLUP) and 6.8 million single-nucleotide polymorphism (SNP) markers. This approach mapped nine quantitative trait loci (QTLs) exhibiting significant allelic dosage-dependent effects, with the major locus, qBSRR.6.1 was the primary discriminator between resistant and susceptible genotypes. Crucially, transcriptomic profiling within these loci revealed distinct expression patterns: IbTCP5 and IbERF003 (located in qBSRR.5.1 and qBSRR.6.2) were highly expressed in the susceptible cultivar ‘Xinxiang’ but suppressed in the resistant ‘Guangshu87’. Furthermore, BSRR challenge identified IbPUB4, IbKCS5, and IbLig1 as priority candidate genes involved in defense, with expression patterns suggesting roles in ubiquitin-mediated protein turnover, cuticular wax biosynthesis, and DNA repair, respectively. In stark contrast, IbPUB25 was constitutively upregulated in ‘Xinxiang’, potentially acting as a negative regulator of immunity via degradation of target proteins. These findings elucidate the polygenic, dosage-sensitive nature of BSRR resistance and prioritize specific targets for future functional characterization. Pyramiding favorable alleles of positive candidates while silencing potential negative regulators like IbPUB25 offers a promising avenue for developing durable, high-resistance sweetpotato varieties. Full article

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease for which there is currently no cure. Dominant mutations in the TARDBP gene are causative of ALS. In particular, the p. G376D substitution in TDP-43 causes familial ALS and it is associated with TDP-43 mislocalization in the cytosol, increased presence of cytoplasmic aggregates, and lysosomal and mitochondrial dysfunction. We previously designed a small interfering RNA (siRNA) that specifically targets and silences the mutant allele and we demonstrated that, in patient-derived fibroblasts, it can reduce TDP-43 aggregation, decrease oxidative stress, and improve cell viability. Here, we investigated the ability of this siRNA to revert some ALS-associated pathological phenotypes in motor neurons derived from induced pluripotent stem cells (iPSCs), as motor neurons are the primary cells affected in ALS. siRNA treatment reduced TDP-43 mislocalization, enhanced lysosomal function and cell viability, and decreased oxidative stress. These findings indicate that this allele-specific siRNA effectively reverses key ALS-related cellular deficits in motor neurons, representing a promising candidate for targeted therapy in patients carrying the TDP-43 G376D mutation. Full article

Background/Objectives: Angelman syndrome is a neurodevelopmental disorder resulting from a deficiency of the maternally inherited UBE3A gene. In mature neurons, UBE3A expression is restricted to the maternal allele due to tissue-specific genomic imprinting, while the paternal allele is silenced in cis by the UBE3A antisense transcript (UBE3A-ATS). To date, numerous strategies have been employed to activate paternal UBE3A expression. In this study, we utilized RNA interference (RNAi) to investigate the downregulation of UBE3A-ATS in mouse primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons. Methods: To induce paternal UBE3A expression, we employed small interfering RNA (siRNA) oligonucleotides (20 mouse candidates and 47 human candidates) and lentiviral short hairpin RNA (LV-shRNA) targeting SNORD115 to suppress UBE3A-ATS expression in both mouse primary neurons and iPSCs. Subsequently, we assessed the expression levels of Angelman syndrome-related neighboring and target genes at the transcript and, where applicable, protein levels. Results: Following treatment with siSnord115 or LV-shSnord115, we observed a reduction in Ube3a-ATS and a corresponding activation of paternal Ube3a RNA and protein expression in both Ube3a^P-YFP/m+ and Ube3a^p+/m− mouse primary neurons. A similar effect was observed upon treatment with LV-shSNORD115s in human iPSC-derived neurons. Conclusions: shRNA-mediated inhibition of Ube3a-ATS by targeting Snord115 effectively restores Ube3a/UBE3A expression in both mouse neurons and human iPSCs. While promising, the mild reduction in Snord116 raises concerns about potential off-target effects. AAV-based delivery of shRNA shows potential, but its translational applicability remains to be evaluated in vivo. Full article

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. The efficacy and safety of lipoplexes was previously demonstrated in vitro in halting variant expression in the peripheral blood of patients. Preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before clinical investigations can be undertaken, according to regulatory authorities in Europe. In the light of the foregoing, this study aims to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed specific siRNA duplexes for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 h post-transfection, reaching higher significant knockdown after 72 h. Lipoplexes efficiently suppress pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre-clinical platform for testing of in vivo lipoplexes in CRISPR-engineered NOD/ShiLtJ mice carrying the R619W mutation. Full article

The BEST1 gene encodes a transmembrane protein in the retinal pigment epithelium (RPE) in the eye, that functions as a calcium-dependent chloride channel (CaCC). Pathogenic variants in BEST1 are the underlying cause for bestrophinopathies, a group of inherited retinal disorders that vary in their pattern of inheritance, clinical appearance, and underlying molecular disease mechanisms. Currently, there are no treatments available for any of the bestrophinopathies, and gene therapy represents an attractive strategy due to the accessibility of the eye and slow disease progression. While gene augmentation may be effective for a subset of bestrophinopathies, others require allele-specific silencing or correction of the disease-causing variant to reconstitute expression of the BEST1 protein. This review aims to give an overview of the clinical diversity of bestrophinopathies and proposes the molecular disease mechanism of the pathogenic BEST1 variant as an important parameter for the choice of treatment strategy. Furthermore, we discuss the potential of different mutation-specific and mutation-independent CRISPR/Cas9-based gene editing strategies as a future treatment approach for bestrophinopathies. Full article

X chromosome inactivation (XCI) is a fundamental epigenetic process that balances X-linked gene expression between females and males by silencing one X chromosome in female cells. Variability or skewing of XCI can influence the clinical presentation of X-linked disorders. Bain type X-linked intellectual disability syndrome (MRXSB), caused by mutations in the X-linked HNRNPH2 gene, is characterized by intellectual disability, developmental delay, and neurological abnormalities. In female patients, XCI heterogeneity complicates disease modeling and therapeutic development. Induced pluripotent stem cells (iPSCs) offer a unique platform to study patient-specific disease mechanisms, but the dynamics of XCI during iPSC reprogramming, maintenance, and differentiation are not fully understood. In this study, we generated 12 iPSC clones from fibroblasts of a female MRXSB patient heterozygous for the HNRNPH2 c.340C > T mutation. Four clones expressed the mutant HNRNPH2 allele and eight expressed the wild-type allele, indicating X chromosome reactivation (XCR) followed by random XCI during reprogramming. Importantly, these XCI patterns remained stable during long-term iPSC propagation and subsequent differentiation into the three germ layers and neural stem cells. Our findings provide new insights into XCI and XCR dynamics in the context of X-linked neurodevelopmental disorders and emphasize the importance of careful clone selection for accurate disease modeling using iPSC-based approaches. Full article

B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma (FL), evade CD4+ T-cell immunity through novel HLA class II-associated immunosuppressive mechanisms. Despite expressing surface HLA-DR, these tumors fail to activate antigen-specific CD4+ T cells, independent of co-stimulation or PD-L1 checkpoint inhibition. We identified lymphoma-secreted factors that broadly disrupt HLA class II-mediated antigen presentation in both malignant B cells and dendritic cells (DCs), silencing T-cell responses. This inhibition is allele-independent (affecting DR1, DR4, DR7) but spares HLA class I-mediated CD8+ T-cell recognition, indicating a targeted immune evasion strategy. Biochemical and mass spectrometry (MALDI-MS) analyses revealed unique low-molecular-weight peptides (693–790 Da) in BL cells, absent in normal B cells, which may mediate this suppression. Functional fractionation confirmed bioactive inhibitory fractions in lymphoma lysates, further implicating tumor-intrinsic molecules in immune escape. These findings highlight a previously unrecognized axis of B-cell lymphoma immune evasion, where secreted factors disable HLA class II function across antigen-presenting cells. Therapeutically, neutralizing these immunosuppressive molecules could restore CD4+ T-cell surveillance and enhance immunotherapies in B-cell malignancies. This work underscores the importance of HLA class II dysfunction in lymphoma progression and identifies candidate targets for reversing immune suppression. Full article

Cardiac arrhythmias remain a major source of morbidity and mortality, often stemming from molecular and structural abnormalities that are insufficiently addressed by current pharmacologic and interventional therapies. Gene therapy has emerged as a transformative approach, offering precise and durable interventions that directly target the arrhythmogenic substrate. Across the spectrum of inherited and acquired arrhythmias—including long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, atrial fibrillation, and post-infarction ventricular tachycardia—gene-based strategies such as allele-specific silencing, gene replacement, CRISPR-mediated editing, and suppression-and-replacement constructs are showing growing translational potential. Advances in delivery platforms, including cardiotropic viral vectors, lipid nanoparticle-encapsulated mRNA, and non-viral reprogramming tools, have further enhanced the specificity and safety of these approaches. Additionally, innovative applications such as biological pacemaker development and mutation-agnostic therapies underscore the versatility of genetic modulation. Nonetheless, significant challenges remain, including vector tropism, immune responses, payload limitations, and the translational gap between preclinical models and human electrophysiology. Integration of patient-derived cardiomyocytes, computational simulations, and large-animal studies is expected to accelerate clinical translation. This review provides a comprehensive synthesis of the mechanistic rationale, therapeutic strategies, delivery platforms, and translational frontiers of gene therapy for cardiac arrhythmias. Full article

Background/Objectives: Prader–Willi Syndrome (PWS) is a neurodevelopmental disease associated with multiple behavioral features, including a prevalence for psychosis. The genetic causes of PWS are well characterized and involve the silencing or deletion of the paternal copy of a region of chromosome 15q11–13. One gene within this region, Snord116, a non-coding RNA, has been determined to have a determinant role in the manifestation of PWS. However, it remains unclear as to how the deletion of this allele can affect activity in the brain and influence psychosis-like behaviors. Methods: In this study, we assessed the effects of the microdeletion of the paternal copy of Snord116 on regional neural activity in psychosis-associated brain regions and psychosis-like behaviors in mice. Results: The results suggest that Snord116 deletion causes increased c-Fos expression in the hippocampus and anterior cingulate cortex. Snord116 deletion also results in behavioral phenotypes consistent with psychosis, most notably in stressful paradigms, with deficits in sensorimotor gating and augmented contextual as well as cued fear conditioning. Conclusions: These results implicate the targets of Snord116 in the presentation of a psychosis-like state with regional specificity. Full article

Background/Objectives: MicroRNAs (miRNAs) are molecules involved in biological regulation processes, including type 2 diabetes and its complications development. Single nucleotide polymorphisms (SNPs) can alter miRNA mechanisms, resulting in loss or gain effects. VEGFA is recognized for its role in angiogenesis. However, its overexpression can lead to deleterious effects, such as disorganized and inefficient vasculature. Under hyperglycemic conditions, VEGFA expression seems to increase, which may contribute to the development of microvascular and macrovascular diabetic complications. Several miRNAs are associated with VEGFA regulation and seem to act in the prevention of dysregulated expression. This study aimed to investigate SNPs in miRNA regions related to the loss effect in VEGFA regulation, examining their frequency and potential physiological effects in the development of diabetic complications. Methods: VEGFA-targeting miRNAs were identified using the R package multimiR, with validated and predicted results. Tissue expression analysis and SNP search were data-mined with Python 3 for miRNASNP-v3 SNP raw databases. Allele frequencies were obtained from dbSNP. The miRNA–mRNA interaction comparison was obtained in the miRmap tool through Python 3. MalaCards were used to infer physiological disease association. Results: The variant rs371699284 was selected in hsa-miR-654-3p among 103 potential VEGFA-targeting miRNAs. This selected SNP demonstrated promising results in bioinformatics predictions, tissue-specific expression, and population frequency, highlighting its potential role in miRNA regulation and the resulting loss in VEGFA-silencing efficiency. Conclusions: Our findings suggest that carriers of rs1238947970 may increase susceptibility to diabetic microvascular and macrovascular complications. Furthermore, in vitro and in silico studies are necessary to better understand these processes. Full article

Piwi-interacting RNAs (piRNAs) play a crucial role in silencing transposable elements (TEs) in the germ cells of Metazoa by acting as sequence-specific guides. Originating from distinct genomic loci, called piRNA clusters, piRNA can trigger an epigenetic conversion of TE insertions into piRNA clusters by means of a paramutation-like process. However, the variability in piRNA clusters’ capacity to induce such conversions remains poorly understood. Here, we investigated two Drosophila virilis strains with differing capacities to produce piRNAs from the subtelomeric RhoGEF3 and Adar gene loci. We found that active piRNA generation correlates with high levels of the heterochromatic mark histone 3 lysine 9 trimethylation (H3K9me3) over genomic regions that give rise to piRNAs. Importantly, the maternal transmission of piRNAs drives their production in the progeny, even from homologous loci previously inactive in piRNA biogenesis. The RhoGEF3 locus, once epigenetically converted, maintained enhanced piRNA production in subsequent generations lacking the original allele carrying the active piRNA cluster. In contrast, piRNA expression from the converted Adar locus was lost in offspring lacking the inducer allele. The present findings suggest that the paramutation-like behavior of piRNA clusters may be influenced not only by piRNAs but also by structural features and the chromatin environment in the proximity to telomeres, providing new insights into the epigenetic regulation of the Drosophila genome. Full article

Prostate-specific antigen remains a cornerstone biomarker for prostate cancer diagnosis and management. However, the molecular mechanisms regulating its expression, particularly through DNA methylation, are not fully understood. Here, we report a comprehensive analysis of allele-specific CpG and CCWGG methylation in the proximal PSA promoter across aggressive (PC3), indolent (LNCaP), benign (BPH1), and normal (HPrEpiC) prostate cell lines and provide insights into the unique methylation patterns associated with these states. Our findings reveal that PC3 cells, representing an aggressive PCa phenotype, exhibit complete biallelic methylation of the PSA promoter, leading to PSA gene silencing. Conversely, LNCaP cells display a fully unmethylated promoter with biallelic PSA expression. Interestingly, BPH1 cells display a monoallelic CG/CCWGG methylation pattern, yet fail to express PSA, suggesting imprinting defects or RNA decay mechanisms. Notably, acquisition of biallelic PSA promoter methylation status in PC3 was accompanied by upregulation of DNMT1, whereas unmethylated PSA promoter state in LNCaP was associated with downregulation of DNMT1. These findings highlight distinct methylation patterns in the PSA promoter that differentiate between aggressive, indolent, and benign prostate states. Translating this epigenetic insight into clinical diagnostics could enhance the precision of PSA-based diagnostics, addressing limitations such as false negatives in PSA testing for aggressive PCa. Further exploration of CCWGG methylation’s role in imprinting and monoallelic expression is warranted, particularly in patient-derived samples. Full article

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing in B cells. To assess the functional impact of the proposed causal variant in C-type lectin domain family 16, member A (CLEC16A), we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), the CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus—particularly the risk allele—drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and could underlie the signal at 16p13. Full article

Paramutation, a specific epigenetic phenomenon first identified in Zea mays by Alexander Brink in the 1950s, has since been observed in different plant and animal species. What sets paramutation apart from other gene silencing processes is its ability for one silenced allele (referred to as paramutagenic) to silence another allele (paramutable) in trans. The resultant silenced allele (paramutated) remains stable across generations, even after separating from the paramutagenic allele, and acquires paramutagenic properties itself. This hereditary behavior facilitates the rapid dissemination of specific gene expression patterns or phenotypes within populations, disrupting the Hardy–Weinberg equilibrium even without other evolutionary pressures. Despite extensive research, the molecular mechanisms underlying paramutation are still not fully understood, although numerous studies suggest the involvement of RNA-mediated changes in DNA methylation and in the chromatin structure of silenced genes. In this paper, we report preliminary evidence regarding horizontal paramutation transfer at the pl1 (purple plant1) regulatory gene involved in the accumulation of anthocyanin in several plant tissues such as tassel, kernel, and cob. A paramutated pl1′ allele arose spontaneously in a pl1 population, and in this study, we found evidence of silencing events spatially associated in the field, suggesting a possible horizontal transfer of silencing among nearby plants. Full article