GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the β-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay–Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood–brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.

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.

Lysosomal storage diseases (LSDs) are a group of about 50 inborn errors of metabolism characterized by the lysosomal accumulation of partially or non-degraded molecules due to mutations in proteins involved in the degradation of macromolecules, transport, lysosomal biogenesis or modulators of lysosomal environment. Significant advances have been achieved in the diagnosis, management, and treatment of LSDs patients. In terms of approved therapies, these include enzyme replacement therapy (ERT), substrate reduction therapy, hematopoietic stem cell transplantation, and pharmacological chaperone therapy. In this review, we summarize the Colombian experience in LSDs thorough the evidence published. We identified 113 articles published between 1995 and 2019 that included Colombian researchers or physicians, and which were mainly focused in Mucopolysaccharidoses, Pompe disease, Gaucher disease, Fabry disease, and Tay-Sachs and Sandhoff diseases. Most of these articles focused on basic research, clinical cases, and mutation reports. Noteworthy, implementation of the enzyme assay in dried blood samples, led to a 5-fold increase in the identification of LSD patients, suggesting that these disorders still remain undiagnosed in the country. We consider that the information presented in this review will contribute to the knowledge of a broad spectrum of LSDs in Colombia and will also contribute to the development of public policies and the identification of research opportunities.

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.