DGH - Apresentações orais em encontros nacionais
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Browsing DGH - Apresentações orais em encontros nacionais by Author "Alves, Sandra"
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- Abordagens terapêuticas de exon-skipping em doenças raras – aplicação à Neurofibromatose tipo 1Publication . Alves, SandraA Neurofibromatose tipo 1 (NF1) é uma das doenças neurocutâneas mais frequentes.
- 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.
- Development of a U1 snRNA-adapted gene therapeutic strategy to correct 5’ splicing defects in lysosomal storage disordersPublication . Matos, Liliana; Alves, Sandra
- Development of an antisense oligonucleotides-based therapy for mucolipidosis type II α/β: in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraIntroduction: The RNA molecule has become an increasingly promising target for the therapy of various diseases. Mucolipidosis 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 (M6P) marker to lysosomal enzymes, which allow their targeting to lysosomes. GlcNAc-1- phosphotransferase is encoded by the GNPTAB and GNPTG genes. 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, being a good target for a mutation specific therapy as there is no therapy for this disease 1,2. 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 3. Currently, our objective is to evaluate the therapeutic potential of this approach in vivo in mice (Mus musculus) of the strain C57BL/6. Material & Methods: Eighteen animals with an average body mass of 25 g were used. During the study, individual body weight, food and water intake were recorded. The animals were 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 intraperitoneally and sacrificed after 4 days (groups 1, 2 and 3) or after 7 days (groups 4, 5 and 6). At the end of the experiment, the organs were collected, weighted and frozen at -80ºC, for later RNA extraction, cDNA synthesis and PCR. Statistical analysis was performed using the GraphPad Prism® for Windows program. Statistical significance between groups was determined by analysis of variance (ANOVA), followed by a Bonferroni test. All ethical issues were followed by the guidelines of the Portuguese General Directorate of Food and Veterinary. Results: Our results show that mice from groups 5 and 6 have a lower liver mass compared to the other groups, with significant differences (p <0.05). Liver of animals from group 3 has a lower weight compared to the liver of animals from group 2 and group 4, with a statistically significant difference. However, exon 19 skipping was not observed in any organ sample using any of the tested doses or incubation periods. Conclusions: To explain these first in vivo results we can theorize that the doses administered were not sufficient to achieve a response or the AO may have had a high clearance rate or did not recognize the target RNA. Moreover, the collected organs should have been preserved in liquid nitrogen to maintain RNA integrity. Other experiments will be done in the near future to overcome these preliminary data. References: [1] R. V. Velho et al. Human mutation. [2] M. F. Coutinho, et al. Biochemistry research international [3] L. Matos, et al. Human Gene Therapy.
- Doenças Lisossomais de Sobrecarga: Perspetiva Geral e Novas Abordagens de Diagnóstico Molecular por Next Generation SequecingPublication . Alves, SandraRare genetic diseases, the diagnosis is difficult due to considerable clinical overlap and clinical variability. Usually multiple samples and tests are often required before a diagnosis is reached.
- E se a retirada de um exão pudesse ter um efeito terapêutico? Caminhos de uma abordagem terapêutica molecular para uma doença rara através de exon-skippingPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraAtualmente, as abordagens terapêuticas de RNA estão em grande desenvolvimento e algumas destas terapias foram já aprovadas para uso clínico. Uma das estratégias mais conhecidas tem como base a utilização de oligonucleótidos antisense (OA), pequenos oligonucleótidos sintéticos que se ligam aos RNA alvo por meio de emparelhamento de bases. Estas moléculas podem atuar de diferentes formas, sendo a diminuição da expressão génica devido à degradação pela RNase-H dos mRNA alvo e a modulação do processo de splicing para induzir a inclusão ou exclusão de um exão (exon skipping), as mais amplamente aplicadas. Particularmente para as doenças raras, diferentes aplicações de OA estão em desenvolvimento, com vários ensaios clínicos em curso e com 3 destas terapêuticas já em prática clínica. As Doenças Lisossomais de Sobrecarga (DLS) são um grupo de doenças hereditárias do metabolismo raras que podem originar uma patologia grave e progressiva devido a uma disfunção lisossomal específica. Atualmente, não existe cura para as DLS, embora para algumas destas doenças haja estratégias de tratamento. Assim, as abordagens terapêuticas de RNA podem representar uma importante estratégia terapêutica alternativa ou adjuvante. O grupo de investigação em DLS do Departamento de Genética Humana do INSA – Porto, tem contribuído para o desenvolvimento de novas abordagens terapêuticas de RNA tendo sido já obtidos resultados positivos para diferentes DLS. Para a validação pré-clínica de novas terapias, tanto os modelos in vitro como in vivo são necessários e largamente utilizados. Num estudo anterior efetuado in vitro, para a mutação c.3503_3504delTC no gene GNPTAB, que origina a Mucolipidose tipo II α/β, foi testada uma estratégia terapêutica de exon-skipping utilizando diferentes OA em fibroblastos de doentes, tendo resultado na produção de um mRNA in-frame1. Neste estudo, pretendeu-se avaliar o potencial terapêutico desta abordagem tanto in vitro (fibroblastos de murganho C57BL/6) como in vivo (murganhos C57BL/6) e os resultados preliminares obtidos serão apresentados e discutidos. 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.
- A investigação em Sanfilippo: abordagens terapêuticas e Projeto Fada dos dentesPublication . Alves, Sandra; Coutinho, Maria FranciscaSobre a investigação em Sanfilippo (abordagens terapêuticas e Projeto Fada dos dentes).
- Lysosome revisited – Connecting Rare and Common IllnessesPublication . Coutinho, Maria Francisca; Alves, SandraOur perception of the lysosome and its role in the cell has suffered a tremendous change since its discovery by Christian de Duve, 60 years ago. Traditionally depicted as cell's waste bin, this small organelle used to be regarded as just an end-point degradative compartment. Nevertheless, the early discovery of a group of rare life-threatening diseases which resulted from failures in that degradation process, the so-called Lysosomal Storage Disorders (LSDs), prompted the search for the underlying causes of such abnormalities and led to a greater understanding of lysosomal function and physiology. Nowadays, the lysosome is no longer viewed as a terminal organelle, but rather as the central regulator of cellular metabolic homeostasis, coordinating a very complex and interactive set of intracellular organelles that have a wide array of specialized functions: the endosomal-lysosomal system. During this same period, lysosomal dysfunction was also extensively documented in other diseases that afflict a much higher number of individuals than all LSDs together. The first clues on this association came from the recognition that Gaucher disease (GD) patients had a higher risk of developing multiple myeloma and/or PD. Numerous reports over the last decades have focused the comorbidity between this GD and either of those common conditions. But GD patients are far from being the only individuals afflicted by an LSD, who present a higher risk of developing a second disorder. There are several case reports of both patients and carriers of specific LSDs presenting with neuropathological findings suggestive of PD (-synuclein accumulation and inclusions, as well as loss of neurons in the substantia nigra), for example. Recently, that same 'risk association' was also documented for patient cohorts of Niemann-Pick type C, Mucopolysaccharidosis type III C and Fabry disease and the idea that a more general mechanism relating lysosomal dysfunction and the development of -synuclein aggregation disorders exists, is gaining supporters. Additional striking connections between common diseases and rare LSD are being unveiled nowadays. Mutations in one of the genes which is long-known to cause frontotemporal dementia (the most common cause of dementia in people under age 65) when mutated in one of its alleles, were recently shown to cause a novel LSD, neuronal ceroid lipofuscinosis 11, when affecting both its copies. In summary, we have come a long way since the first descriptions of LSDs were published and their underlying biochemical basis unveiled. Parallels across groups of traditionally unrelated neurological disorders have emerged, reshaping the way we think about the role of the lysosome and the whole system it controls. Pathophysiological boundaries have blurred between LSDs and other disorders including both early-onset neurogenetic conditions and late-onset neurodegenerative diseases. Ultimately, a better understanding of lysosomal dysfunction in all these conditions may provide valuable insights towards the development of therapeutic strategies for a multitude of presently untreatable disorders. So, let us wait for the next 60 years, and hope they will be as groundbreaking as the last ones.
- Molecular characterization of Portuguese patients with pathologies related to the lysosomal multienzymatic complex: Sialidosis and Galactosialidosis.Publication . Coutinho, Maria Francisca; Lacerda, Lúcia; Prata, Maria João; Ribeiro, Helena; Alves, Sandra
- National Mirror Group Portugal - European Joint Programme on Rare Diseases, EJP RDPublication . Alves, SandraO National Mirror Group (NMG-P) do Programa Europeu Conjunto para as Doenças Raras é coordenado pelo Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) em colaboração com a Fundação para a Ciência e Tecnologia (FCT) e a Direção-Geral da Saúde e pretende criar um ecossistema sustentável e permitir um círculo virtuoso entre investigação, cuidados de saúde e inovação médica.