Angela Johana Espejo-Mojica

Tay-Sachs disease is a rare genetic disorder characterized by the accumulation of GM2 ganglioside in neuronal lysosomes due to deficient β-hexosaminidase A (HexA) activity. Progressive GM2 storage leads to severe neurodegeneration, including developmental delay, motor weakness, seizures, ataxia, and early death, typically by five years of age. Previous studies have elucidated several neuronal mechanisms, including apoptosis, endoplasmic reticulum stress, neuroinflammation, and demyelination, these investigations have focused almost exclusively on neurons. However, other components of the central nervous system, particularly astroglia, may play a critical role in disease pathophysiology as suggested by studies in related lysosomal storage disorders. To address this gap, we generated an astrocyte-like model deficient in HexA by targeted knockdown of the HEXA gene in U87MG astrocytoma cells. The resulting cell line recapitulates key pathological features, including lysosomal accumulation, increased neutral lipid content, reduced mitochondrial mass, and elevated reactive oxygen species production. Transcriptomic analysis revealed significant alterations in pathways associated with neuronal degeneration, synaptic organization, mitochondrial dysfunction, and ganglioside metabolism. In summary, this model reproduces some classical cellular alterations reported in Tay-Sachs disease and could potentially provide novel insight into astrocyte involvement in its pathophysiology. These findings support the relevance of non-neuronal cells in disease pathophysiology and establish this system as a valuable platform for screening potential novel mechanisms and therapeutic approaches. Furthermore, this approach highlights the importance of integrating cell type specific models to better understand disease heterogeneity and providing insights into the progressive neurodegeneration of Tay-Sachs disease, positioning this model as a valuable tool for studying its underlying pathophysiology.

The interaction of a set of four N-acetyl-glucosamine (GlcNAc) glycomimetics with human N-acetyl-glucosaminidase (NAGLU), the genetically defective enzyme in patients suffering from mucopolysaccharidosis (MPS) IIIB, also known as Sanfilippo B syndrome, was investigated to identify potential pharmacological chaperones. Glycomimetic–NAGLU binding was initially studied by molecular docking simulations and a thermal shift assay. The effects of the glycomimetics on NAGLU activity enhancement were studied in fibroblast cells from seven MPS IIIB patients. A significant increase in NAGLU activity in four cell lines in the presence of glycomimetic MK 8719, a molecule tested in a Phase 1 study in healthy volunteers to treat Alzheimer’s disease, was demonstrated. Furthermore, MK 8719 prevented the increase in glycosaminoglycan (GAG) levels in four MPS IIIB fibroblast cells, suggesting that this molecule may be worth investigating further as a pharmacological chaperone for MPS IIIB. These results represent an important contribution towards the development of a specific therapy for MPS IIIB.

Background: Tay–Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the HEXA gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast Pichia pastoris, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. Methods: Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, GM2AP and HEXB expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. Results: Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the HEXB gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. Conclusions: Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells.

Background/Objectives: Tay–Sachs disease (TSD) is a neurodegenerative disorder caused by a deficiency in β-hexosaminidase A (HexA), which accumulates GM2 gangliosides, primarily in neurons. Currently, therapeutic options are limited, highlighting the need for new strategies such as gene therapy. Despite their effectiveness, viral vectors can elicit adverse immune responses; consequently, non-viral vectors are being explored as an alternative. We have previously investigated the use of CRISPR/Cas9 nickase (nCas9) as a potential tool for treating TSD. Here, we expanded our study by evaluating the PP6D5 polymer as a novel non-viral vector for delivering the CRISPR/nCas9 system to restore HexA activity. Methods: First, we evaluated the PP6D5-mediated CRISPR/nCas9 system’s transfection efficiency in NIH-3T3 fibroblasts, U87MG astrocytoma, SHSY5Y neuroblastoma, and TSD fibroblasts. We then evaluated the potential of PP6D5 to correct the gene defect in TSD fibroblasts. Results: The results showed that PP6D5 exhibited significantly higher transfection efficiency compared to lipofectamine 3000 in all tested cell models. In TSD fibroblasts, transfection with both HEXA and HEXB cDNAs increased the HexA activity levels by up to 7.4-fold, compared to a 3.2-fold increase in cells transfected only with HEXA cDNA after 15 days post-transfection. These levels were up to 4.5-fold higher than those observed in lipofectamine-mediated transfection. Additionally, PP6D5-mediated CRISPR/nCas9-based genome editing led to a significant reduction in the lysosomal mass of TSD fibroblasts. Conclusions: This study provides promising evidence for the use of the PP6D5 polymer as a non-viral vector for delivering CRISPR/nCas9-based gene therapy in TSD. The use of the PP6D5 polymer may offer some advantages that viral vectors cannot, such as a reduction in cytotoxicity and higher TE in difficult-to-transfect cell lines. Furthermore, this type of polymeric vector has not been extensively explored for gene therapy, making this study an important contribution to the development of non-viral delivery systems for the treatment of neurodegenerative diseases.

Background/Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, was declared a public health emergency in early 2020. The infection initiates when the receptor-binding domain (RBD) of the viral spike protein binds to human angiotensin-converting enzyme 2 (ACE2). Despite the success of vaccination efforts, the emergence of new variants highlights the ongoing need for treatments targeting these evolving strains. In silico methods previously identified peptides BP2, BP9, and BP11 as being capable of disrupting the RBD-ACE2 interaction, though their efficacy has not been experimentally validated until now. Methods: In this study, these peptides were recombinantly produced in the yeast Komagataella phaffii, and the activity was assessed in vitro using binding assays with multiple RBD variants and the inhibition of the RBD-ACE2 interaction. Results: The production yield for BP2, BP9, and BP11 was 14.34, 4.01, and 1.35 mg per culture liter, respectively. Noteworthy, the three BPs interacted with the RBD of SARS-CoV-2 variants of concern, with BP2 showing higher recognition. Finally, the BPs showed an RBD/hACE2 interaction blocking capacity with IC₅₀ values between 1.03 and 5.35 nM, with BP2 showing the lowest values among the evaluated peptides. Conclusions: These results demonstrate that BP2, specifically, is a promising candidate for the development of novel therapeutic interventions targeting SARS-CoV-2 and other coronaviruses that use hACE2 for cellular entry.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N−acetyl‐alpha‐glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood‐brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay–Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.