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Brief Report Unverricht-Lundborg disease (ULD) is a common form of progressive myoclonic epilepsy caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. Presently, only pharmacological treatment and psychosocial support are available for ULD patients. To overcome the pathogenic effect of the ULD splicing mutation c.66G>A (exon 1), we investigated whether an antisense oligonucleotide therapeutic strategy could correct the defect in patient cells. A specific locked nucleic acid (LNA) antisense oligonucleotide was designed to block a cryptic 5′ss in intron 1. Overall, this approach allowed the restoration of the normal splicing pattern. Furthermore, the recovery was both sequence and dose-specific. In general, this work provides a proof of principle on the correction of a CSTB gene defect causing ULD through a mutation-specific antisense therapy. It adds evidence to the feasibility of this approach, joining the many studies that are paving the way for translating antisense technology into the clinical practice. The insights detailed herein make mutation-based therapy a clear candidate for personalized treatment of ULD patients, encouraging similar investigations into other genetic diseases. This work was funded by FCT (Fundação para a Ciência e a Tecnologia—MCTES, Portugal) project PTDC/BBB-BMD/6301/2014. Liliana Matos was grant recipient from FCT under the same project info:eu-repo/semantics/publishedVersion

15th Annual WORLDSymposium 2019 Scientific Meeting - Lysosomal Disease Research, 4-8 Feb 2019, Orlando, Florida, USA. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were found as an immune adaptive mechanism in bacteria and quickly were applied to various fields as a promising tool for gene editing. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by defects in lysosomal proteins leading to accumulation of undigested macromolecules within the cells. The lack of good in vitro models hinders research of the pathophysiologic mechanisms and the development of new therapies. Induced pluripotent stem cells (iPSCs) are patient-specific and can be differentiated in any cell type. The advantage of iPSCs is to enable targeted studies in cells with the patient’s own background leading to more straightforward results than other models. Combining CRISPR and iPSCs is, therefore, a promising strategy. We aim to use CRISPR/Cas-mediated gene editing to provide more specific cellular models of disease, to correct causal mutations in LSDs and to create mutants for functional studies. In this work, we generated and characterized iPSCs from human fibroblasts obtained from Gaucher and Fabry patients (through Gaslini Institute) and will edit them with a CRISP/Cas9 approach. Because both gene editing and iPSCs generation require manipulating the cell’s genome, we envisage multiple check points along the workflow. It will be useful to compare the “native” mutated cells with the corrected cells that modulate the “disease in a dish”. Gene editing is still recent and the methods require improvement, namely increasing transfection rates and mutagenesis efficiency with less off-targets. Nevertheless, CRISPR/Cas is a promising alternative to other therapies, and every result contributes to the enhancement of this technology, broadening the validation of CRISPR application and making it an accessible option. FCT Funding: PTDC/BIM-MEC/4762/2014 info:eu-repo/semantics/publishedVersion

Publicado em: Molecular Genetics and Metabolism 105 (2012) S15–S69. Disponivel em: http://www.sciencedirect.com/science/article/pii/S1096719211004343