Browsing by Issue Date, starting with "2022-07-22"
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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.
- 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.