Percorrer por autor "Santos, Juliana I."
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- Antisense oligonucleotide exon-skipping as a therapeutic approach for a rare diseasePublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana I.; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Omidi, Maryam; Pohl, Sandra; Alves, SandraMucolipidosis II (MLII) is a Lysosomal Storage Disorder caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase, which is responsible for the Mannose- 6-Phosphate marker addition to lysosomal enzymes. Of all MLII mutations, the c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) for MLII. Previously, on MLII patients’ fibroblasts, ASOs were used to skip exon 19 of the GNPTAB pre-mRNA, successfully resulting in the production of an in-frame mRNA[1]. Now, our aim is to analyze if these results are translated to the enzymatic and cellular phenotype level.
- Establishment of a Human iPSC Line from Mucolipidosis Type II That Expresses the Key Markers of the DiseasePublication . Moutinho, Maria Eduarda; Gonçalves, Mariana; Duarte, Ana J.; Encarnação, Marisa; Coutinho, Maria Francisca; Matos, Liliana; Santos, Juliana I.; Ribeiro, Diogo; Amaral, Olga; Gaspar, Paulo; Alves, Sandra; Moreira, Luciana V.Mucolipidosis type II (ML II) is a rare and fatal disease of acid hydrolase trafficking. It is caused by pathogenic variants in the GNPTAB gene, leading to the absence of GlcNAc-1-phosphotransferase activity, an enzyme that catalyzes the first step in the formation of the mannose 6-phosphate (M6P) tag, essential for the trafficking of most lysosomal hydrolases. Without M6P, these do not reach the lysosome, which accumulates undegraded substrates. The lack of samples and adequate disease models limits the investigation into the pathophysiological mechanisms of the disease and potential therapies. Here, we report the generation and characterization of an ML II induced pluripotent stem cell (iPSC) line carrying the most frequent ML II pathogenic variant [NM_024312.5(GNPTAB):c.3503_3504del (p.Leu1168fs)]. Skin fibroblasts were successfully reprogrammed into iPSCs that express pluripotency markers, maintain a normal karyotype, and can differentiate into the three germ layers. Furthermore, ML II iPSCs showed a phenotype comparable to that of the somatic cells that originated them in terms of key ML II hallmarks: lower enzymatic activity of M6P-dependent hydrolases inside the cells but higher in conditioned media, and no differences in an M6P-independent hydrolase and accumulation of free cholesterol. Thus, ML II iPSCs constitute a novel model for ML II disease, with the inherent iPSC potential to become a valuable model for future studies on the pathogenic mechanisms and testing potential therapeutic approaches.
- Gapmer Antisense Oligonucleotides as a New Class of Genetic Substrate Reduction Agents in Mucopolysaccharidosis IIICPublication . Santos, Juliana I.; Gonçalves, Mariana; Almeida, Matilde B.; Rocha, Hugo; Duarte, Ana J.; Matos, Liliana; Moreira, Luciana V.; Encarnação, Marisa; Gaspar, Paulo; Prata, Maria J.; Coutinho, Maria F.; Alves, SandraBackground: Lysosomal storage disorders (LSDs) include over 70 rare inherited metabolic diseases caused by defective lysosomal enzymes or associated proteins, leading to the accumulation of undegraded substrates and progressive cellular dysfunction. Among these, Mucopolysaccharidoses (MPS) are a group characterized by storage of undegraded glycosaminoglycans (GAGs), including heparan, dermatan, keratan, and chondroitin sulfates. MPS type III (as known as Sanfilippo syndrome) predominantly affects the central nervous system (CNS) and manifests as severe neurodegeneration, behavioral abnormalities and cognitive decline. The subtype IIIC results from deficient activity of acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) enzyme. Currently, treatment options for MPS III are limited, increasing the need to find alternative therapies. RNA-based therapies have recently gained attention as powerful alternatives to traditional treatments, with several already approved for clinical use in other diseases. Among these, antisense oligonucleotides (ASOs) stand out as a highly promising class of molecules for personalized medicine. ASOs are short, chemically synthesized strands of nucleic acids designed to bind specifically to target RNA sequences, thereby influencing gene expression. In this work, we explored the application of gapmer ASOs as a genetic substrate reduction therapy (gSRT) for MPS IIIC, with the goal of decreasing the expression of a gene, XYLT1, involved in the synthesis of the accumulated substrate, heparan sulfate (HS).
- mRNA Degradation as a Therapeutic Solution for Mucopolysaccharidosis Type IIIC: Use of Antisense Oligonucleotides to Promote Downregulation of Heparan Sulfate SynthesisPublication . Santos, Juliana I.; Gonçalves, Mariana; Almeida, Matilde B.; Rocha, Hugo; Duarte, Ana J.; Matos, Liliana; Moreira, Luciana V.; Encarnação, Marisa; Gaspar, Paulo; Prata, Maria J.; Coutinho, Maria F.; Alves, SandraMucopolysaccharidosis type IIIC is a neurodegenerative lysosomal storage disorder (LSD) characterized by the accumulation of undegraded heparan sulfate (HS) due to the lack of an enzyme responsible for its degradation: acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT). Classical treatments are ineffective. Here, we attempt a new approach in genetic medicine, genetic substrate reduction therapy (gSRT), to counteract this neurological disorder. Briefly, we used synthetic oligonucleotides, particularly gapmer antisense oligonucleotides (ASOs), to target the synthesis of the accumulated compounds at the molecular level, downregulating a specific gene involved in the first step of HS biosynthesis, XYLT1. Our goal was to reduce HS production and, consequently, its accumulation. Initially, five gapmer ASOs were designed and their potential to decrease XYLT1 mRNA levels were tested in patient-derived fibroblasts. Subsequent analyses focused on the two best performing molecules alone. The results showed a high inhibition of the XYLT1 gene mRNA (around 90%), a decrease in xylosyltransferase I (XT-I) protein levels and a reduction in HS storage 6 and 10 days after transfection (up to 21% and 32%, respectively). Overall, our results are highly promising and may represent the initial step towards the development of a potential therapeutic option not only for MPS IIIC, but virtually for every other MPS III form. Ultimately, the same principle may also apply to other neuropathic MPS.
- Skipping the Pathology in Rare Diseases: Antisense exon-skipping therapy for Mucolipidosis type IIPublication . Matos, Liliana; Coutinho, Maria Francisca; Santos, Juliana I.; Gonçalves, Mariana; Gaspar, Paulo; Alves, SandraAbout Skipping the Pathology in Rare Diseases: Antisense exon-skipping therapy for Mucolipidosis type II