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Projeto de investigação
Fabry disease: from iPS cells to Genomic Editing
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Gene Editing in Fabry Disease: A Strategy Delineation
Publication . Duarte, Ana J.; Moreira, Luciana; Ribeiro, Diogo; Amaral, Olga
The 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.
Gene editing as a tool for developing cell based models of a lysosomal storage disorder: preliminary results
Publication . Duarte, Ana Joana; Moreira, Luciana; Gaspar, Paulo; Alves, Sandra; Bragança, José; Amaral, Olga
In 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.
Generation of Cellular Models for Fabry Disease: Unlocking the Potential of iPSCs and Gene Editing
Publication . Duarte, Ana Joana; Moreira, Luciana; Ribeiro, Diogo; Alves, Sandra; Gaspar, Paulo; Bragança, José; Amaral, Olga
Introduction: 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.
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Fundação para a Ciência e a Tecnologia
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Número da atribuição
SFRH/BD/118009/2016