Monitoring Epigenetic Traits in situ Cell Models of Mucopolysaccharidosis IVA (MPS IVA)

Lysosomal Storage Diseases (LSDs) represent a group of approximately 70 rare inherited metabolic disorders, each one resulting from a specific deficiency or dysfunction in lysosomal enzymes, transport proteins, or activator molecules(Platt et al., 2018). Lysosomes, essential cellular organelles, are responsible for the controlled degradation and processing of a wide range of intra- and extra- cellular macromolecules, including complex lipids, proteins, glycosaminoglycans, glycoproteins, and glycolipids(Lastoria et al., 2025). Disruption of this fundamental catabolic process due to an underlying genetic defect, undegraded substrates progressively accumulate within lysosomes, leading to aberrant intracellular buildup that manifests as widespread cellular dysfunction, chronic tissue damage, and a broad spectrum of clinical manifestations. These pathologies can affect virtually every organ and system in the human body, including the central nervous system (often with devastating neurodegeneration), cardiovascular system (cardiomyopathies), renal system (renal failure), hepatic system (hepatomegaly), splenic system (splenomegaly), and skeletal system (multiple dysostoses)(Lastoria et al., 2025; Parenti et al., 2015). The heterogeneity of clinical symptomatology in LSDs is a hallmark feature and a major challenge for diagnosis. Symptom onset can vary dramatically, ranging from the neonatal period in severe early-onset forms with poor prognosis to adulthood in milder, slowly progressive forms. This wide phenotypic variability not only hampers early patient identification but also contributes to delayed diagnosis which is a critical barrier that directly impacts the effectiveness of available therapeutic interventions. Available treatments such as enzyme replacement therapy (ERT), substrate reduction therapy (SRT), and more recently, gene therapies, have shown to be improve life quality with better outcomes when initiated in the earliest stages of the disease, ideally before irreversible tissue damage and show up of severe symptoms(Leal et al., 2020)(Lastoria et al., 2025; Leal et al., 2020; Parenti et al., 2015; Sun, 2018). However, the gap between symptom onset (often subtle and nonspecific at first) and diagnostic confirmation can span several years, during which the underlying pathology progresses. On the other hand, epigenetics has emerged as a crucial dimension in understanding the pathogenesis of human diseases, revealing an additional layer of biological complexity and regulation(Farsetti et al., 2023; Saul & Kosinsky, 2021). For instance, an important initial milestone in establishing a direct connection between epigenetic mechanisms and human diseases, is the research conducted in the early 80s on imprinting disorders and cancer(Feinberg, 2013). Despite that this correlation between aberrant epigenetics and disease instauration has been widely studied for more than three decades, LSDs has been attributed mainly to point mutations or genetic rearrangements that alter the DNA sequence encoding the affected lysosomal proteins, however, the relation between epigenetics and LSD onset has been barely addressed during the last decade(Hassan et al., 2017). Epigenetics studies heritable changes in gene expression that occur without alterations in the underlying DNA nucleotide sequence(Berger et al., 2009). These epigenetic mechanisms act as a dynamic bridge between the genetic information encoded in DNA and environmental influences, modulating gene activity in a reversible yet stable manner. Among epigenetic mechanisms, DNA methylation and histone posttranslational modifications have been widely studied in mammals. While DNA methylation process mainly involves the covalent addition of a methyl group to the C5 position of cytosines (5-methylcytosine or 5mC), predominantly within CpG sites(Mattei et al., 2022). Histone post-translational modifications are more diverse, including acetylation, methylation, phosphorylation, and ubiquitination, and are predominately located at the N terminus of histones(Millán-Zambrano et al., 2022; Musselman et al., 2012). These histone PTMs are crucial regulators of gene expression, acting by influencing chromatin structure and serving as docking sites for regulatory proteins. These epigenetic marks have a generally well recognized effect on gene expression, for instance most CpG sites in the genome are methylated, except in CpG-rich promoter regions, known as CpG islands, which are typically unmethylated to allow gene transcription(Skvortsova et al., 2019). On the other hand, histone PTMs can alters chromatin structure by changing the electrostatic charge of histones, thereby modulating their affinity for DNA and influencing the compaction level of chromatin; like lysine acetylation which results in a reduced electrostatic interaction between the negatively charged DNA and the histones, leading to a more "open" and relaxed chromatin structure (euchromatin), generally promoting gene activation(Kouzarides & Berger, 2007). Furthermore, these histone PTMs and their combinations create a “histone code” which is recognized by specific "reader" proteins that recruit other complexes and either activate or repress gene transcription(Wang & Patel, 2011). Thus, histone PTMs act as a dynamic language, dictating the accessibility of DNA and orchestrating the recruitment of regulatory complexes, thereby precisely controlling which genes are expressed. Among the different histone PTMs, H3K9me3 (trimethylation of lysine 9 on histone H3) is a well-established hallmark of heterochromatin and plays a crucial role in gene silencing, genome stability, and transposon repression. It is commonly found in transcriptionally inactive regions of the genome and is essential for maintaining epigenetic memory during cell division(Becker et al., 2017). H3K9me3 is recognized by heterochromatin protein 1 (HP1), which contributes to chromatin compaction and the propagation of the heterochromatic state. Its dysregulation is associated with various diseases, including cancer, neurodevelopmental disorders, and aging-related pathologies(Kumar & Kono, 2020; Nicetto & Zaret, 2019). Different biosensor models have been developed for monitoring epigenetic marks such as DNA methylation, histone modifications, and non-coding RNAs, including optical, electrochemical, and nano-biosensors(Li et al., 2019). Traditional approaches like immunosorbent assays and mass spectrometry offer high specificity but are often limited by complex protocols and lack of real-time capability(Singh, 2025). Recent advances have produced engineered protein-based and fluorescence biosensors, such as bimolecular fluorescence complementation (BiFC) probes, which enable live-cell, single-cell resolution tracking of epigenetic modifications—a significant advantage for studying dynamic processes and cellular heterogeneity(Mendonca et al., 2022). In situ biosensors, stand out for their ability to quantify and sort viable cells based on their epigenetic status in real time, preserving cell integrity and allowing subsequent functional analyses, provide rapid, non-destructive, and multiplexed detection of epigenetic marks, making them especially powerful for basic research and diagnostic applications(Wettschurack et al., 2020). Emerging research suggests that aberrant DNA methylation patterns may play a critical role in the pathogenesis and progression of LSDs(Kido et al., 2023; La Cognata et al., 2020). These epigenetic alterations can directly influence the expression of key genes involved in lysosomal biogenesis, lysosomal enzyme activity, autophagy (a vital cellular degradation process for lysosomal homeostasis), or metabolic signaling pathways (such as the mTOR pathway), all of which are intrinsically linked to lysosomal function and are disrupted in these diseases(La Cognata et al., 2020). For instance, studies have documented dysregulated methylation in the promoter region of the GLA gene (which encodes α-galactosidase A) in patients with Fabry disease, suggesting that aberrant methylation may contribute to reduced enzymatic activity independently of the primary genetic mutation or even exacerbate the phenotype(Shen et al., 2022). Similarly, changes in methylation patterns have been identified in genes related to autophagy and lysosomal function in other LSDs, indicating that these epigenetic events are not mere bystanders but may actively modulate disease severity(Mousavi et al., 2023; Smith et al., 2023). Similarly, aberrant histone PTMs have been also correlated to LSDs(Horsthemke, 2024). For example, Sanfilippo syndrome (Mucopolysaccharidosis Type IIIA, MPS-IIIA), a neurodegenerative LSD, showed that deficiency of the lysosomal enzyme SGSH led to increases in open chromatin and histone acetylation at thousands of putative microglia-specific enhancers. Being associated with upregulated genes involved in lysosomal function and immune signaling, suggesting a direct link between lysosomal stress and altered histone acetylation contributing to the neuroinflammatory phenotype(Balak et al., 2024). Similarly, in MPSIIB and MPSIVA changes in DNA methylation, H3K14acetylation, and H3K9me3 suggest individual-specific epigenetic responses that might contribute to phenotypic heterogeneity(Vargas-López et al., 2024). LSDs often exhibit significant clinical heterogeneity, even among patients with the same genetic mutation. Epigenetic mechanisms, including histone modifications and DNA methylation, are proposed to contribute to this variability. Environmental factors or modifier genes could induce different histone PTM patterns, influencing the severity, age of onset, and specific organ involvement in LSDs(Hassan et al., 2017). Thus, identifying and characterizing these specific epigenetic signatures not only deepens our understanding of the complex pathophysiology of LSDs, offering new insights into the underlying molecular mechanisms, but also opens an unprecedented avenue for the development of novel, non-invasive biomarkers that could complement or even surpass the limitations of current diagnostic methods. To address this knowledge gap, we propose the use of sensors able to monitor changes in situ of genomic DNA methylation and H3K9me3, for specific cell models of LSDs syndromes. Specifically, we will study the epigenetics changes in Mucopolysaccharidosis IVA (MPS IVA), one of the LSDs that we have extensively studied from the basic and therapeutic point of view(Espejo‐Mojica et al., 2020; Leal et al., 2023; Puentes-Tellez et al., 2020). These sensors will allow us to increase our understanding of the molecular basis of these group of disorders, as may serve as a platform for the study of other common or rare diseases.