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Applications of iPSCs in Gaucher Disease

Publication . Amaral, Olga; duarte, ana; Ribeiro, Diogo; Santos, Renato; Bragança, José

In recent years, human induced pluripotent cell (hiPSC) models have slowly become a trend in experimental modelling of disease, following and complementing animal based models. Human iPSCs provide an innovative manner for modelling Gaucher Disease (GD). Since 2008 several groups have created iPSCs models from GD patients, with various genotypes, and differentiated iPSCs to neural precursors and macrophages among many other types of cells. hiPSC models have been developed from multiple GD donors, recapitulating the disease phenotypic hallmarks. These models have provided a new platform for pathophysiology studies and for the testing of small molecules with therapeutic goals.

2020-05-25Conference object

Restricted access

Foreseen accomplishments through precision cellular and molecular biology

Publication . Amaral, Olga; Duarte, A.J.

LSDs therapies generate large expenses, having a major effect on health systems and family resources. It is, therefore inevitable to attempt to develop effective and economically viable models and therapeutic approaches. Through the development of specific cellular models, we hope to contribute to the increase of choices in terms of therapeutic interventions. Generation and application of iPS cells for the establishment of cellular models for lysosomal diseases has been applied to a few disorders. This approach can allow easier access to disease-specific cells, which will retain the original genotype. We expect that its application to LSDs will provide the means to study pathogenic mechanisms and therapeutic options.

2016-07Conference object

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To CRISPR or not to CRISPR

Publication . Duarte, A.J.; Amaral, Olga; Bragança, José

The CRISPR/Cas9 genome editing system is a most promising tool but the application of this method to Lysosomal Storage diseases (LSDs) still needs to be explored. In LSDs, enzyme replacement therapy (ERT, regular supplementation of the defective enzyme) is the most common treatment to clear the accumulated substrates in patient cells and it is effective in a few diseases. However, there are many limits to its cost-effective application. The lack of good in vitro models hinders R&D and the understanding of the human pathophysiologic mechanisms. Using CRISPR/Cas9-mediated gene editing, to either make a model and test therapeutic approaches or to correct a causal mutation and examine the potential effects on the cellular environment will likely provide important information for functional studies. Induced pluripotent stem cells (iPSCs) from patients, with several genetic diseases, including LSDs, have been successfully established. Patient-derived iPSCs present the advantage of having the patient’s genetic background with all corresponding influences on the disease’s mechanism. Therefore, these iPSCs, differentiated into the disease specific target cell types, offer an ideal model for studying pathogenic mechanisms and therapies. Gene editing using CRISPR/Cas9 in combination with iPSCs seems like a perfect match. Currently, we are generating iPSCs from human fibroblasts and intend to proceed with gene editing. However, despite the apparently simple approach, there are still problems with CRISPR. Besides the ethical problems, there are practical problems such as diminishing off-targeting. Therefore, the question is, how feasible will CRISPR/Cas9 be? No doubt it is promising and valuable but will it be safe enough?

2017-02-21Conference object

Restricted access

inducing a new start

Publication . Duarte, Ana Joana; Bragança, José; Amaral, Olga

The lack of good disease models limits the understanding of the human pathophysiologic mechanisms and hinders investigation research and development of new therapies. In 2006, Yamanaka’s group expressed four transcription factors (Oct4, Sox2, Klf4, and c-Myc) producing induced pluripotent stem cells (iPSCs), allowing the development of new strategies for pathogenesis modeling and drug testing. iPSCs generated from somatic cells from patients are a desirable source for patient-specific studies since they maintain the patient’s genetic background. In this work, we ultimately aim to develop induced pluripotent stem cells (iPSCs) from Lysosomal storage disorders (LSDs) patient’s fibroblasts and normal controls to produce disease models. Thus, using iPSCs methods to generate the cell-targets to reproduce the disease may create an ideal model for studying pathogenic mechanisms. The initial biological material being used consists of commercially obtained human control dermal fibroblasts. This type of material guarantees better consistency in technical conditions. We are testing two different non-integrative polycistronic plasmid vectors in order to achieve forced expression of the Yamanaka’s transcription factors. For this achievement, transformation conditions with different vehicles of delivery were tested: different transfection reagents, concentration ratios, and timings were compared. Since we are beginning this work from zero, only a few very preliminary results were obtained and will be presented.

2017-05Conference object

Open access

Tay Sachs disease variant B1: iPSC and NGS as the basis for a cellular model

Publication . Ribeiro, Diogo; Duarte, Ana J.; Moreira, Luciana; Santos, Renato; Encarnaçao, Marisa; Silva, Lisbeth; Coutinho, M. Francisca; Alves, Sandra; Amaral, Olga

Tay 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.

2022-09Conference object

Open access