Percorrer por autor "Duarte, Ana J."
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- Cell lines for the study of Lysosomal Storage Diseases: conservation and identityPublication . Correia, Maria I.; Duarte, Ana J.; Ribeiro, Diogo; Amaral, OlgaThe use of cell lines has revolutionized research in the area of human genetics. The possibility of allowing, at a low cost and relative ease, practical access to biological material bearing in mind the benefit to the patient/family by obtaining samples with adequate informed consent, is a great asset. The accessibility of cell lines from biobanks allows access to samples with all the ethical and feasibility concerns observed. Cell lines are usually cryopreserved in liquid nitrogen and can be maintained in viable conditions for long periods of time. Cell lines allow studies of the causes of the disease, namely: the establishment of cell models, for a better understanding of the pathophysiology; the study of gene interactions; toxicity assays and drug tests; gene editing studies and other types of research. Using fibroblasts from patients with Lysosomal Storage Diseases (LSDs), it was already possible in this laboratory to revert cells to the stem cell state by creating induced pluripotent stem cells (iPSCs) to serve as a model in future studies. This clearly demonstrates the potential of cell lines for research. As with other cell lines, iPSCs can be cryopreserved which increases their potential for use. In order to guarantee the integrity and viability of cryopreserved cell lines, in laboratories, not exclusively dedicated to cell culture (as would be the case of a biobank), it would be advisable to periodically perform random thawing of samples in order to guarantee the identity of the preserved cells, genetic stability and absence of contaminants. There are several ways to do this however, in this work, we present some of the techniques used based on minimal procedures to ensure the cellular integrity of cryopreserved lines. It would be desirable, even in small laboratories, that procedures like these were adopted in a standardized and routine way, to facilitate the success of the subsequent use of cells in research.
- 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.
- From fibroblasts to cardiomyocytes and beyondPublication . Duarte, Ana J.; Ribeiro, Diogo; Braganca, Jose; Amaral, OlgaFrom Fabry disease causing effect to personalised cell model of cardiomyocytes and future mutation correction applying a prime-editing approach.
- Gene Editing in Fabry Disease: A Strategy DelineationPublication . Duarte, Ana J.; Moreira, Luciana; Ribeiro, Diogo; Amaral, OlgaThe use of iPSCs, in the last years became wide spread, even in our group at INSA, the use of iPSCs to develop models of disease is now envisaged for various Lysosomal Storage Diseases. Such cell models are being used to experiment several types of therapeutic methodologies, as well as approaches that interfere with normal pathways to provide understanding about pathologic mechanisms, and gene editing is particularly interesting among the latter strategies. Recently, a new CRISPR-based method – Prime Editing (PE) – provides all-possible base-to-base conversions, “indels”, and combinations; the human genome can be edited without the need of double-strand breaks (DSBs) or donor DNA templates. This method proved its efficacy to correct a pathogenic insertion that causes Tay-Sachs disease (HEXA 1278+TATC; OMIM 606869). In this work, our aim is to correct one of the Fabry Disease (FD) causing mutations, the p.W287X, located on the GLA gene (OMIM 300644). For this purpose, our strategy is to use a construct that uses a one-step golden gate digestion-ligation cloning that is called Prime Editing All-in-One (PEA1) plasmid, consisting in a cassette for expression of all PE3 components and a selection marker. A few years ago we developed iPSCs from skin fibroblasts of patients. The present correction approach will be tested in our FD iPSC line. At this moment, we are initiating the work but we hope to achieve positive results soon. The use of new genetic engineering tools, like PE, and its use as possible therapeutic strategy should provide further comprehension of FD and act as a potential therapy.
- 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.
- Tay Sachs disease variant B1: iPSC and NGS as the basis for a cellular modelPublication . Ribeiro, Diogo; Duarte, Ana J.; Moreira, Luciana; Santos, Renato; Encarnaçao, Marisa; Silva, Lisbeth; Coutinho, M. Francisca; Alves, Sandra; Amaral, OlgaTay Sachs disease variant B1 (TSD B1; OMIM 272800) is a neurodegenerative lysosomal storage disease (LSD) which, although rare, is the most frequent form of TSD in Portugal. The mutation p.R178H (c.533G>A; rs28941770), associated with TSD B1, leads to a mutant HexA protein with altered kinetics and reduced residual activity. The availability of disease-relevant cell types derived from induced pluripotent stem cells (iPSCs) provides a model for studying the pathogenic mechanisms and, eventually, test therapeutic approaches for TSD B1 patients. The main objectives of this project is, by using iPSCs, to generate a neuronal TSD B1 specific cellular model and to implement the genetic profiling by Next Generation Sequencing (NGS) to examine potential changes in the manipulated cells. In the present work, the iPSC reprogramming and differentiation into neural progenitor cells (NPCs) is presented, as well as the NGS results from the donor cells. As a first step, iPSCs from a control fibroblast cell line and from TSD B1 fibroblasts were obtained by using a non-integrative approach with episomal vectors, the control was further differentiated into NPCs. When generating iPSCs it is important to have multiple characterisation steps. The iPSCs have to mimic the donor background at genetic and protein levels. Furthermore, iPSCs or iPSC-derived lines need to be free of the reprogramming and differentiation markers while presenting specific cell-type markers. The frailty of these cells and adverse conditions (first lockdowns and later accidental deprivation of liquid nitrogen) led to the interruption of the iPSC work. In the meantime, we did a lysosomal-related gene profiling of the DNA from the original cell lines. By using an in-house customized NGS panel, we obtained results of the cells in a “naïve” state to later compare with TSD B1 iPSCs and NPCs obtained from control iPSCs. The results presented consist of the preliminary work to obtain a cell model of TS-B1.