Browsing by Author "Duarte, A.J."
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- Foreseen accomplishments through precision cellular and molecular biologyPublication . Amaral, Olga; Duarte, A.J.LSDs therapies generate large expenses, having a major effect on health systems and family resources. It is, therefore inevitable to attempt to develop effective and economically viable models and therapeutic approaches. Through the development of specific cellular models, we hope to contribute to the increase of choices in terms of therapeutic interventions. Generation and application of iPS cells for the establishment of cellular models for lysosomal diseases has been applied to a few disorders. This approach can allow easier access to disease-specific cells, which will retain the original genotype. We expect that its application to LSDs will provide the means to study pathogenic mechanisms and therapeutic options.
- Rapid and cost-effective method for the detection of the c.533G>A mutation in the HEXA genePublication . Ribeiro, D.; Duarte, A.J.; Amaral, O.Tay-Sachs disease is a rare autosomal recessive neurodegenerative disorder that results from mutations in the HEXA gene, leading to β-hexosaminidase A (HexA) α subunit deficiency. An unusual variant of Tay-Sachs disease is known as the B1 variant. Previous studies indicated that, in northern Portugal, this is not only the most common variant but also one of the most prevalent lysosomal storage diseases. Additionally, this variant might also show a higher prevalence in populations of Portuguese and Spanish ancestry. A single mutation is invariably present in at least one of the alleles of B1 variant patients, HEXA mutation c.533G >A. To implement a method for c.533G >A testing in individuals and populations, we have optimized two distinct mutation analysis techniques, one based on restriction fragment length polymorphism analysis and the other based on allelic discrimination. We present the comparison of both methods and their advantages. Mutation screening by allelic discrimination proved to be particularly useful for the studying of large samples of individuals. It is time saving and highly reproducible, and under the conditions used, its cost is lower than the cost of polymerase chain reaction-based restriction fragment length polymorphism analysis.
- To CRISPR or not to CRISPRPublication . Duarte, A.J.; Amaral, Olga; Bragança, JoséThe CRISPR/Cas9 genome editing system is a most promising tool but the application of this method to Lysosomal Storage diseases (LSDs) still needs to be explored. 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 and it is effective in a few diseases. However, there are many limits to its cost-effective application. The lack of good in vitro models hinders R&D and the understanding of the human pathophysiologic mechanisms. Using CRISPR/Cas9-mediated gene editing, to either make a model and test therapeutic approaches or to correct a causal mutation and examine the potential effects on the cellular environment will likely provide important information for functional studies. Induced pluripotent stem cells (iPSCs) from patients, with several genetic diseases, including 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. Therefore, these iPSCs, differentiated into the disease specific target cell types, offer an ideal model for studying pathogenic mechanisms and therapies. Gene editing using CRISPR/Cas9 in combination with iPSCs seems like a perfect match. Currently, we are generating iPSCs from human fibroblasts and intend to proceed with gene editing. However, despite the apparently simple approach, there are still problems with CRISPR. Besides the ethical problems, there are practical problems such as diminishing off-targeting. Therefore, the question is, how feasible will CRISPR/Cas9 be? No doubt it is promising and valuable but will it be safe enough?
- Unverricht-Lundborg disease: development of splicing therapeutic approaches for a patient with an homozygous mutation in the cystatin B genePublication . Matos, L.; Duarte, A.J.; Ribeiro, D.; Jordan, P.; Prata, M.J.; Chaves, J.; Desviat, L.R.; Pérez, B.; Amaral, O.; Alves, S.Unverricht-Lundborg disease (ULD) is the most common form of progressive myoclonic epilepsy worldwide. It is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion, missense, nonsense, frameshift and mutations that may lead to alternative splicing have been described as causal of ULD. Recently, our group described an ULD patient who is homozygous for a new synonymous mutation (c.66G>A; p.Q22Q) located at the last nucleotide of exon 1. The transcriptional profile analysis allowed the identification of two CSTB splice variants, one of normal size with the G>A change and other with partial inclusion of intron 1 due to activation of a cryptic splice-site inside the intronic sequence. To correct the splice defect, here we developed antisense oligonucleotide and U1snRNA mediated therapeutic strategies. U1 is required for splice donor site (SDS) recognition of pre-mRNAs and initiates the splicing process. The mutation c.66G>A interferes with the recognition of the SDS by U1. In a first approach, to reduce missplicing we generated four U1 construct isoforms with increasing complementarity to the SDS. Transfection of patient-derived fibroblasts with different concentrations of the adapted U1 vectors did not allowed the correction of the aberrant transcript. In a second strategy, we have designed a specific lock nucleic-acid (LNA) oligonucleotide to block the activated cryptic splice-site in intron 1. Normal splicing pattern of a single transcript with the synonymous change G>A was successfully rescued after LNA transfection in patient cells. The therapeutic effect showed to be dose-dependent. These results suggest that antisense therapy might be a potential alternative or adjunct treatment strategy for patients holding splicing changes in CSTB gene. As far as we know this is the first report of a patient tailored therapy in cells of an ULD patient.
- Unverricht–lundborg disease: report of a new mutationPublication . Freitas, Joel; Pinto, Eugénia; Duarte, A.J.; Amaral, Olga; Chaves, Joao; Lopes-Lima, J.P301: Unverricht-Lundborg disease is the most frequent cause of progressive myoclonic epilepsy. CSTB mutations, with cystatin B loss of function, have been described as the major cause of this disease.