Gene Editing in Fabry Disease: A Strategy Delineation

Duarte, Ana J.; Moreira, Luciana; Ribeiro, Diogo; Amaral, Olga

http://hdl.handle.net/10400.18/8302

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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.