Browsing by Author "Matos, Liliana"
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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.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studiesPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II alpha/beta (ML II alpha/beta) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II alpha/beta, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II alpha/beta. In a previous in vitro study in ML II alpha/beta patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA1. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg/kg and groups 3 and 6 were injected with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 days (groups 1, 2, 3) or 7 days (groups 4, 5, 6) post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24h or 48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done. 1. Matos L, Vilela R, Rocha M, et al. Development of an antisense oligonucleotide-mediated exon skipping therapeutic strategy for Mucolipidosis II: validation at RNA level. Hum Gene Ther, 2020, 31(13-14):775-783.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studiesPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II alpha/beta (ML II) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II. In a previous in vitro study in ML II patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 with AO at 25 mg/kg and groups 3 and 6 with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 or 7 days post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24/48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II α/β: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraGenetic therapy directed towards the correction of RNA mis-splicing is being investigated at basic research and in late-stage clinical trials. Many mutations that change the normal splicing pattern and lead to aberrant mRNA production have been identified in Lysosomal Storage Disorders (LSDs). Mucopolysaccharidosis IIIC (MPS IIIC) is one of those LSDs caused by mutations in the HGSNAT gene that encodes an enzyme involved in heparan sulphate degradation. Splicing mutations are one of the most frequent (~20%) genetic defects in MPS IIIC. Approximately 55% correspond to 5' splice-site (ss) mutations thus constituting a good target for mutation-specific therapeutic approaches. Recently, we have demonstrated in fibroblast cells that a modified U1 snRNA vector designed to improve the definition of exon 2 5’ss of the HGSNAT can restore splicing impaired by the mutation c.234+1G>A. Currently, our goal is to evaluate in vivo the therapeutic potential of that modified U1 snRNA by testing it in mice expressing the human splicing defect. For this purpose, two full-length constructs were generated by cloning the wild-type (wt) or the mutated HGSNAT splicing-competent cassettes in the pcDNA 3.1 vector. Then, in an in vitro assay, the wt or mutated construct was transfected in Hep3B and COS-7 cells. After molecular analysis it was observed that both minigenes reproduce the healthy control and patient cDNA’s splicing pattern. Therefore, both constructs were used to generate mice of the C57BL/6 strain expressing the human mutation c.234+1G>A in the liver and test its modified U1-mediated rescue in vivo. Wt or mutant minigenes were administrated in mice by hydrodynamic injection following a reported protocol(1). After 48 hours animals were sacrificed, the liver was collected and molecular analysis was performed. Preliminary results showed expression of the HGSNAT cDNA from the mutant construct in the liver of at least one animal. Thus, further tests will be carried out to optimize some limiting points, such as the administration of the minigenes (e.g. increase of injection volume from 7% to 8-9% of mice body weight; inclusion of an in vivo transfection reagent to enhance delivery efficiency) and the use of other mice strain. 1. Balestra D, et al. (2014) J Thromb Haemost 12(2):177–185.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II α/β: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II α/β (ML II α/β) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II α/β, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II α/β. In a previous in vitro study in ML II α/β patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg/kg and groups 3 and 6 were injected with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 days (groups 1, 2, 3) or 7 days (groups 4, 5, 6) post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24h or 48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done.
- Assessing the potential of RNA-based therapeutics for a group of Lysosomal Storage Diseases with neurological involvementPublication . Santos, Juliana Inês; Gonçalves, Mariana; Matos, Liliana; Gaspar, Paulo; Pires, Maria João; Oliveira, Paula; Prata, Maria JoãoDuring the first two decades of the 21st century, remarkable progresses have been achieved in the field of RNA-based therapeutics. From antisense RNA to RNA modification, the therapeutic potential of RNA-based technologies has nothing but increased. In our lab, we have been addressing the potential of different RNA-based drugs to either correct or ameliorate the sub-cellular phenotype of a number of severe, life-threatening diseases: the so-called Lysosomal Storage Disorders (LSDs). Among them, we are focusing our efforts on those which present with a predominant neurological phenotype, since there are virtually no approved treatments for any of them. Briefly, two major research lines are being pursued: the first relies on the design of mutation-specific approaches to correct abnormal splicing processes in LSD-related genes, whenever they underlie pathology. The second depends upon selective downregulation of genes involved in the biosynthethic cascades that give origin to the substrates that accumulate in each pathology. Here we present an overview on our results with both approaches on Sanfilippo syndrome, a sub-group of severe neurodegenerative LSDs. For the mutation-specific, splicing correction approach, we are using U1snRNA vectors to restore the splicing defect caused by the HGSNAT mutation c.234+1G>A, that leads to Sanfilippo C disease. We started by demonstrating in vitro that a modified U1snRNA vector designed to improve the definition of HGSNAT exon 2 could partially restore its normal splicing process. Now, we are evaluating its therapeutic potential in vivo, in mice expressing the human splicing defect. For the substrate reduction approach, we are using siRNAs. By acting over a specific biosynthethic cascade, siRNAs promote an overall decrease of the accumulating substrate. So far, we have already tested this approach in patients’ fibroblasts and observed a high inhibition of the target mRNAs and a decrease in storage. Overall, there are substantial differences between these two approaches but they also face common challenges and show equally promising results.
- Correction of a Splicing Mutation Affecting an Unverricht-Lundborg Disease Patient by Antisense TherapyPublication . Matos, Liliana; Duarte, Ana Joana; Ribeiro, Diogo; Chaves, João; Amaral, Olga; Alves, SandraUnverricht-Lundborg disease (ULD) is a common form of progressive myoclonic epilepsy caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. Presently, only pharmacological treatment and psychosocial support are available for ULD patients. To overcome the pathogenic effect of the ULD splicing mutation c.66G>A (exon 1), we investigated whether an antisense oligonucleotide therapeutic strategy could correct the defect in patient cells. A specific locked nucleic acid (LNA) antisense oligonucleotide was designed to block a cryptic 5′ss in intron 1. Overall, this approach allowed the restoration of the normal splicing pattern. Furthermore, the recovery was both sequence and dose-specific. In general, this work provides a proof of principle on the correction of a CSTB gene defect causing ULD through a mutation-specific antisense therapy. It adds evidence to the feasibility of this approach, joining the many studies that are paving the way for translating antisense technology into the clinical practice. The insights detailed herein make mutation-based therapy a clear candidate for personalized treatment of ULD patients, encouraging similar investigations into other genetic diseases.
- Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS IIPublication . Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R.; Pérez, Belén; Alves, SandraThis data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].
- Desenvolvimento de um ensaio de sequenciação de nova geração para acelerar o diagnóstico molecular das doenças lisossomais de sobrecargaPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Silva, Lisbeth; Matos, Liliana; Ribeiro, Diogo; Nogueira, Célia; Gaspar, Paulo; Vilarinho, Laura; Alves, SandraAs doenças lisossomais de sobrecarga (DLS) são um grupo de cerca de 70 doenças hereditárias do metabolismo. A sua apresentação clínica é muito heterogénea, variando desde formas pré-natais, até apresentações infantis ou na idade adulta, sendo frequente a presença de atraso psicomotor e neurodegeneração progressiva. Nas DLS, um diagnóstico molecular preciso é muito importante dado que novas terapias têm sido desenvolvidas e se encontram disponíveis. Para a maioria destas doenças o diagnóstico é difícil devido à considerável heterogeneidade clínica e à sobreposição de sintomas com outras doenças, podendo os doentes permanecer sem diagnóstico durante décadas. A sequenciação de nova geração (NGS), sendo a tecnologia de sequenciação mais avançada no momento, torna-se uma metodologia essencial num laboratório dedicado ao diagnóstico de doenças metabólicas, incluindo as DLS. Desde o início de 2017, foram incluídos neste projeto 18 doentes com suspeita clínica de DLS, tendo sido esclarecida a etiologia molecular em 39% (7/18). Este estudo contribuiu assim para alargar o espectro mutacional das DLS, permitindo o aconselhamento genético aos familiares, oferecer diagnóstico pré-natal molecular e selecionar a abordagem terapêutica mais adequada.
- Development of a U1 snRNA-adapted gene therapeutic strategy to correct 5’ splicing defects in lysosomal storage disordersPublication . Alves, Sandra; Pérez, Belén; Desviat, Lourdes; Matos, LilianaStudy aims: To overcome the pathogenic effect of two 5’ splice donor site (SDS) mutations diagnosed in LSDs patients, we have exploited the use of the antisense-U1 snRNA mediated therapeutic strategy to correct both 5’ SDS mutations that were reported in patients with Mucopolysaccharidosis type I (MPS I) (3) and Mucolipidosis III alpha/beta (ML III alpha/beta).