Percorrer por autor "Bragança, José"
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- 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.
- 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.
- Gene editing as a tool for developing cell based models of a lysosomal storage disorder: preliminary resultsPublication . Duarte, Ana Joana; Moreira, Luciana; Gaspar, Paulo; Alves, Sandra; Bragança, José; Amaral, OlgaIn this work, we aimed to establish a Fabry Disease (FD, OMIM: #301500) disease model using the CRISPR/Cas 9 system by knocking out a HDFa iPSC line. We also aimed to correct a nonsense mutation (p. W 287 X) in the iPSCs derived from a patient with FD. The cell lines used were generated in our laboratory, and the FD iPSC line is registered in the Human Pluripotent Stem Cell Registry with identification "INSAi 002-A". To fully evaluate the molecular and cellular physiological changes, further studies are still required. The development of innovative cell models, particularly for rare diseases like Lysosomal Storage Disorders, is beneficial for studying the pathophysiology of the disease.
- Gene editing in Lysosomal DiseasesPublication . Duarte, Ana Joana; Bragança, José; Coutinho, Francisca; Amaral, OlgaClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were found as an immune adaptive mechanism in bacteria and quickly applied to various fields as a gene editing tool. Gene editing methods, as a research tool to attempt in vitro correction, have been carried out in several disorders. Induced pluripotent stem cells (iPSCs) from patients with several genetic diseases, including Lysosomal Storage Diseases (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. 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 but it is hardly effective in non-neurological disease forms. The CRISPR/Cas9 genome-editing system is most promising for the establishment of disease models and for the potential correction of causal. Gene editing technologies and iPSCs provide a unique system for data analysis and research for target therapy.
- Generation of Cellular Models for Fabry Disease: Unlocking the Potential of iPSCs and Gene EditingPublication . Duarte, Ana Joana; Moreira, Luciana; Ribeiro, Diogo; Alves, Sandra; Gaspar, Paulo; Bragança, José; Amaral, OlgaIntroduction: Fabry Disease (FD) is a lysosomal storage disorder caused by mutations in the GLA gene, resulting in a defective α-GAL A enzyme. This deficiency leads to the accumulation of Gb3 and lyso-Gb3 within lysosomes, resulting in a multisystem disease. Through reprogramming, we obtained induced pluripotent stem cells (iPSCs) derived from fibroblasts of a patient with FD2 and from a wild-type (WT) control. We used CRISPR/Cas9 to correct the c.860G>A mutation present in the patient’s cells, as well as to generate a WT GLA knockout (KO). The resulting cells were then differentiated into cardiomyocytes, a cell type affected by this disease. Methods: We reprogrammed the fibroblasts into iPSCs using episomal vectors or Sendai virus. For gene editing, single-guide RNAs (sgRNAs) and Cas9 were nucleofected, and the editing was confirmed by Sanger sequencing. Following colony selection, isogenic cell lines were established. The FD iPSCs, the corrected FD iPSCs, and the WT iPSCs were then differentiated into iPSC-derived cardiomyocytes (iPSC-CMs). Results: Seven new cell models were generated. Functional studies of the FD iPSCs showed the maintenance of the molecular and biochemical characteristics and a normal karyotype. The KO cell line recapitulated the biological features observed in FD patient cells, with reduced GLA expression, lower α-Galactosidase A (α-Gal A) activity (1.5 nmol/h/mg protein), and Gb3 accumulation. The corrected cell line was generated with 75.8% efficiency and 69.6% on-target efficacy. Enzyme activity increased to 579 nmol/h/mg protein (vs. 0.78 nmol/h/mg protein in FD iPSCs), accompanied by a marked reduction in Gb3 levels. We successfully generated iPSC-CM lines, which were validated by qRT-PCR and immunofluorescence. Discussion: Cell modelling is essential for studying the pathophysiology of disease mechanisms. By retaining the characteristics of the original cells, iPSCs are a valuable biological resource for generating specific differentiated cell types affected by the disease, which would otherwise be difficult to access. This study also explored the therapeutic potential of gene editing as a promising approach to altering the course of rare diseases.
- 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.
- Induced pluripotent stem cells as genetic disease modelsPublication . Duarte, Ana Joana; Bragança, José; Amaral, OlgaLysosomal storage disorders (LSDs) are a group of genetic diseases characterised by lysosomal dysfunction. Some of the commonest LSDs are currently treated by enzyme replacement therapy. However, particularly in cases of advanced disease or late onset, results are discouraging. The lack of good ex vivo models hinders R&D and delays the understanding of the human pathophysiologic mechanisms. Thus, using iPSCs methods to generate the cell-targets to reproduce the disease, might help create ideal models for studying pathogenic mechanisms and to find new or more effective therapeutic strategies. iPSCs generated from somatic cells from patients are a necessary source for patient-specific studies since they maintain the patient’s genetic background. Material and Methods: Using commercially obtained skin fibroblasts, as a control, guarantees better consistency in technical conditions. In this study we used two different methods to achieve forced expression of Oct4, Sox2, Klf4 and c-Myc: a non-integrative polycistronic plasmid vector and the Sendai virus method Transformation conditions with different vehicles of delivery were tested: different reagents, concentration ratios and timings were compared. Posterior validation of cells pluripotent state is currently underway. Results: Fibroblasts are very difficult to transform but colonies were observed at around three weeks post-transfection using plasmid DNA. The Sendai virus method proved to be easier and faster. Aims: Currently we are generating iPSCs from human skin fibroblasts and intend to obtain a good cellular model for LSDs.
- Induced pluripotent stem cells as genetic disease models.Publication . Duarte, Ana Joana; Amaral, Olga; Bragança, JoséRare diseases (together representing 1 in 8000) provide a valuable model to study cellular mechanisms involved in health and disease. However, a major obstacle in the study of pathogenic mechanisms and evaluation of new therapeutic approaches is the accessibility of the target cells. Rare diseases of lysosomal accumulation (Lysosomal storage Disorders, LSDs) are multisystemicand exhibit different degrees of involvement, often presenting target cells that can only be obtained through very invasive procedures. The loss of lysosomal integrity influences the entire cellular environment and leads to impairment of cellular function. The importance of iPSCs as experimental models is vast since they provide an optimal platform for investigation pathological mechanisms.
- inducing a new startPublication . Duarte, Ana Joana; Bragança, José; Amaral, OlgaThe 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.