Browsing by Author "Gaspar, Paulo"
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- Assessing Niemann-Pick Type C (NP-C) through a multi-omics approach genomic and transcriptomic profile of challenging casesPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Ribeiro, Isaura; Santos, Juliana Inês; Gaspar, Paulo; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraNiemann-Pick type C (NP-C) is a neurodegenerative Inherited Metabolic Disease with a heterogeneous clinical presentation, due to mutations in either the NPC1 or NPC2 genes. We studied patients with clinical diagnosis of NP-C but presenting inconclusive results regarding biomarkers testing and molecular analysis. Using NGS- targeted DNA sequencing we have identified some novel putative mutations and subsequent cDNA analysis allowed us to stablish the functional effect of a silent mutation (previously reported as a polymorphism) in the NPC1 splicing process. We demonstrated that this mutation leads to exon skipping, frameshift and premature stop codon and identified it in two NP-C patients from two unrelated Portuguese families. This mutation most likelly leads to a very unstable transcript that was overlooked. Also, to better characterize the pathomechanisms related to specific disease-causing mutations in NP-C patients, we analysed gene expression profiles in cultured skin fibroblasts and compared them to control individuals using Massively Parallel RNA Single-Cell Sequencing (MARS-Seq). The most prominent hits from this transcriptomics analysis were validated by qRT-PCR. The MARS-Seq analysis showed that a number of genes were upregulated and a significant number of the highly enriched genes are related to the unfold protein response (UPR) and Endoplasmic Reticulum (ER) stress, in a specific patient, which deserves further studies. ER stress is a hallmark of many neurodegenerative diseases, including LSD and can be due to misfolded/unfolded proteins as result of a specific missense mutation. Our preliminary results suggest that UPR activation is variable among NP-C patients, and this is likely to depend on the mutation type. Several other factors may contribute to this though, which could explain the heterogeneous presentation of this pathology. Additionally, we have investigated the same patients studied in MARS-Seq at the protein and celular levels. Interestingly, and according to recently published work, we observed that, for the analyzed mutations a significant part of the mutated NPC1 protein was retained/ delayed in the ER.
- 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.
- Biomarcadores nas doenças lisossomais de sobrecarga: o que são e o que nos dizem?Publication . Gaspar, Paulo; Rocha, Hugo; Neiva, Raquel; Azevedo, Olga; Maia, Tabita; Aguiar, Patrício; Cardoso, Teresa; Chaves, Paulo; Alves, Sandra; Vilarinho, LauraAs Doenças Lisossomais de Sobrecarga (DLSs) são um conjunto de patologias raras, crónicas, multissistémicas com modo de apresentação e gravidade muito variáveis. No seu conjunto afetam aproximadamente 1:5000 nados vivos e são causadas pela acumulação de metabolitos não degradados no interior do lisossoma. As esfingolipidoses são o grupo de DLS com maior prevalência e incluem a doença de Gaucher, a doença de Fabry e a doença de Niemann-Pick. Por vezes, parte do produto primário de acumulação, através de uma reação de desacilação, é convertido na respetiva base esfingoide, que se encontra bastante elevada em casos de patologia. Em alguns doentes, estes compostos já se encontram aumentados, mesmo antes do aparecimento dos primeiros sintomas. Os tratamentos disponíveis para estas patologias (terapia de substituição enzimática, terapia de redução de substrato de chaperones farmacológicos) conduzem a um decréscimo da concentração destas bases esfingoides, tornando o doseamento destes compostos útil também para aferir a eficácia da terapêutica utilizada. Pela primeira vez em Portugal, é disponibilizado o estudo destes biomarcadores para as DLSs, através da tecnologia de espectrometria de massa em tandem (MS/MS). Os autores apresentam os resultados obtidos do doseamento destes lisolípidos em diferentes doentes de DLSs, com a clara demonstração da especificidade destes biomarcadores, permitindo um diagnóstico atempado, um melhor conhecimento da evolução da doença e monitorização da eficácia do respetivo tratamento.
- 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 an antisense oligonucleotide-mediated exon skipping therapeutic strategy for Mucolipidosis II: validation at RNA levelPublication . Matos, Liliana; Vilela, Regina; Rocha, Melissa; Santos, Juliana Inês; Coutinho, Maria Francisca; Gaspar, Paulo; Prata, Maria João; Alves, SandraLysosomal Storage Disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undegraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there is currently a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy.
- Development of an antisense-mediated exon skipping approach as a therapeutic option for the ML II-causing mutation c.3503_3504delTCPublication . Matos, Liliana; Vilela, Regina; Coutinho, Maria Francisca; Gaspar, Paulo; Alves, SandraLysosomal storage disorders (LSDs) are a group of a rare inherited metabolic disorders.
- Development of an antisense-mediated exon skipping therapeutic strategy to correct a frequent causing mutation in Mucolipidosis IIPublication . Matos, Liliana; Vilela, Regina; Coutinho, Maria Francisca; Gaspar, Paulo; Alves, SandraLysosomal Storage Disorders (LSDs) are a group of rare inherited diseases caused by the malfunction of the lysosomal system, resulting in the accumulation of undegraded substrates inside the lysosomes and leading to severe and progressive pathology. Among them is Mucolipidosis II (ML II), one of most severe LSDs, which is caused by the total or near total deficiency of the GlcNAc-phosphotransferase, a key enzyme for the correct trafficking of lysosomal hydrolases to the lysosome. GlcNAc-phosphotransferase is a multimeric enzyme and is encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutations is a dinucleotide deletion on exon 19 of the GNPTAB gene that disrupts the reading frame and prevents the production of an active GlcNAc-phosphotransferase, which in turn impairs the proper targeting of lysosomal enzymes. Despite broad understanding of the molecular causes behind this and other LSDs, the same progress has not been observed in the development of new therapies, with current treatments still mostly symptomatic and presenting several limitations. Therefore, alternative options should be investigated in order to provide patients and families with better healthcare and more promising therapies. One possibility is the modulation of splicing by antisense oligonucleotides (AOs) with the purpose of altering the splicing pattern, the mature mRNA and ultimately the final protein product. Acknowledging this, the present study intends to design and develop a RNA-based therapeutic agent through the use of AOs capable of inducing the skipping of exon 19 of the GNPTAB gene and consequently circumvent the effects of the most common ML II causal mutation. The approach is presently ongoing and different 2’O-Methyl AOs were designed and synthesized to target the GNPTAB exon 19 and promote its skipping. We have already succeeded in inducing the skipping of exon 19 in control and ML II patient fibroblasts. At biochemical level, 48 hours following transfection, enzyme activity suffered a small increase in patients fibroblasts for all enzymes tested, even if the results are still much lower than the observed for healthy controls. In conclusion, this work constitutes a proof of principle for correcting in vitro the defects caused by a frequent ML II mutation with antisense therapy. Acknowledgments: This work is supported by the project PTDC /BBB-BMD/6301/2014 (SPLICETHER) financed by Fundação para a Ciência e a Tecnologia – Portugal.
- Doenças lisossomais de sobrecarga em Portugal: aspetos bioquímicos, genéticos e epidemiológicosPublication . Gaspar, Paulo; Neiva, Raquel; Silva, Lisbeth; Vilarinho, Laura
- Doenças lisossomais de sobrecarga: da epidemiologia genética ao desenvolvimento de modelos de doençaPublication . Moreira, Luciana; Coutinho, Maria Francisca; Moutinho, Maria Eduarda; Almeida, Matilde Barbosa; Gonçalves, Francisca; Carvalho, Sofia; Amaral, Olga; Duarte, Ana Joana; Encarnação, Marisa; Gaspar, Paulo; Gonçalves, Mariana; Matos, Liliana; Ribeiro, Diogo; Rocha, Hugo; Santos, Juliana Inês; Alves, Sandra; .
- FIND: a importância de um diagnósticoPublication . Gaspar, Paulo; Alves, Sandra; Leão Teles, Elisa; Vilarinho, LauraO Projeto FIND é um estudo de colaboração entre a SDHM da SPP e o INSA e permite disponibilizar uma ferramenta de diagnóstico aos médicos que sigam um doente com suspeita clínica de mucopolissacaridose (MPS).