Browsing by Author "Santos, Renato"
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- Advances in sphingolipidoses: CRISPR-Cas9 editing as an option for modelling and therapyPublication . Amaral, Olga; Santos, RenatoSphingolipidoses are inherited genetic diseases characterized by the accumulation of glycosphingolipids. Sphingolipidoses (SP), which usually involve the loss of sphingolipid hydrolase function, are of lysosomal origin, and represent an important group of rare diseases among lysosomal storage disorders. Initial treatments consisted of enzyme replacement therapy, but, in recent decades, various therapeutic approaches have been developed. However, these commonly used treatments for SP fail to be fully effective and do not penetrate the blood-brain barrier. New approaches, such as genome editing, have great potential for both the treatment and study of sphingolipidoses. Here, we review the most recent advances in the treatment and modelling of SP through the application of CRISPR-Cas9 genome editing. CRISPR-Cas9 is currently the most widely used method for genome editing. This technique is versatile; it can be used for altering the regulation of genes involved in sphingolipid degradation and synthesis pathways, interrogating gene function, generating knock out models, or knocking in mutations. CRISPR-Cas9 genome editing is being used as an approach to disease treatment, but more frequently it is utilized to create models of disease. New CRISPR-Cas9-based tools of gene editing with diminished off-targeting effects are evolving and seem to be more promising for the correction of individual mutations. Emerging Prime results and CRISPR-Cas9 difficulties are also discussed.
- Applications of iPSCs in Gaucher DiseasePublication . Amaral, Olga; duarte, ana; Ribeiro, Diogo; Santos, Renato; Bragança, JoséIn 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.
- Differential gene expression in cells from Fabry and Gaucher diseases: cell reprogramming and culture agingPublication . Amaral, Olga; Santos, Renato; Ribeiro, DiogoGene expression varies deeply, even in the same individual’s cells, depending on stress factors, cell aging, gene variants or gene and cell manipulation In this work, gene expression can be seen to shift in many genes, using fibroblasts from donors with Fabry and Gaucher type 3 genetic diseases Tests were also made to compare the impact of aging cultures Real time qRT PCR protocols for genes of interest were used for expression comparison after cell induction and reprogramming.
- A first step to open the neuronal box of Gaucher CellsPublication . Ribeiro, Diogo; Duarte, Ana; Santos, Renato; Amaral, OlgaThis work focuses on the differentiation and gene expression characterization of neural progenitor cells obtained from human induced pluripotent cells (hiPSCs) reprogrammed from type 3 GD (GD3) fibroblasts. GD3 patient fibroblasts (from an international cell bank) were cultured and reprogramed as previously described (https://doi.org/10.1016/j.scr.2019.101595). The resulting hiPSCs were differentiated into pre-neuronal cells and, at this stage, they were examined. The gene expression behavior of all neurogenesis genes (NES, MAP2 and OTX2) was similar but higher expression was observed in GD3 hiPSCs than in GD3 neural progenitor cells. With this work, we can conclude that, when working with hiPSCs in the process of creating disease-specific cell models it is most important to carry out a general gene expression characterization of the different cell lines involved in all stages.
- Gaucher Disease and iPSCs: what does the future hold?Publication . Amaral, Olga; Duarte, Ana; Ribeiro, Diogo; Santos, Renato; Bragança, JoséIntroduction: Currently we are working with induced pluripotent stem cells (iPSCs) in order to generate cells with the same genetic background as the donor patients and further differentiate them in cell types of interest for the study of specific diseases. We are manipulating human skin fibroblasts and have included multiple checkpoints along the procedures to avoid potential pitfalls.
- Induced pluripotent stem cell line (INSAi001-A) from a Gaucher disease type 3 patient compound heterozygote for mutations in the GBA1 genePublication . Duarte, Ana Joana; Ribeiro, Diogo; Santos, Renato; Moreira, Luciana; Bragança, José; Amaral, OlgaGaucher Disease (GD) type 3 is a neurological form of a multisystemic autosomal recessive disorder belonging to the group of lysosomal storage diseases. Causal mutations in the glucocerebrosidase 1 (GBA1) commonly lead to abnormal protein and GD, heterozygosity is a genetic risk factor for Parkinson's disease. This work describes the use of a non-integrative approach using Sendai Virus delivery to establish induced Pluripotent Stem Cells (iPSCs) from fibroblasts from a GD type 3 patient. Differentiation of iPSCs can be employed to generate a variety of complex cell types with a high degree of genetic complexity that would otherwise be unattainable.
- Induced pluripotent stem cell line (INSAi002-A) from a Fabry Disease patient hemizygote for the rare p.W287X mutationPublication . Duarte, Ana; Ribeiro, Diogo; Santos, Renato; Moreira, Luciana; Bragança, José; Amaral, OlgaFabry Disease (FD) is a multisystemic X-linked disorder that belongs to the group of lysosomal storage disorders (LSDs). Causal mutations on alpha-galactosidase A (α-Gal A) commonly lead to abnormal protein and consequently to FD. Since it is an X-linked disease, males are primarily affected. This work describes the generation of induced Pluripotent Stem Cells (iPSCs) from skin fibroblasts from a FD patient, using non-integrative episomal vectors. Differentiation of iPSCs can be applied to generate a variety of cell types with high degree of genetic complexity that would otherwise be difficult to obtain.
- 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.