Browsing by Author "Coutinho, M.F."
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- An Antisense Oligonucletide based therapy for a rare disease: in vitro and in vivo studiesPublication . Gonçalves, M.; Matos, L.; Santos, J.I.; Coutinho, M.F.; Prata, M.J.; Pires, M.J.; Oliveira, P.A.; Alves, SandraMucolipidosis type II (ML II) is a Lysosomal Storage Disorder caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose-6-phosphate marker to lysosomal enzymes allowing their targeting to lysosomes. From the several ML II mutations, the deletion of two nucleotides from GNPTAB exon 19 (c.3503_3504del) is the most frequent, making it a good target for a mutation specific therapy. In this study, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) for ML II. In a previous study1 on fibroblasts from ML II patients, ASOs were used to skip exon 19 of the GNPTAB pre-mRNA, successfully resulting in the production of an in-frame mRNA. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice.
- Development of RNA based approaches to exploit alternative therapies for Lysosomal Storage DiseasesPublication . Matos, L.; Santos, J.I.; Rocha, M.; Coutinho, M.F.; Gaspar, P.; Voltolini Velho, R.; Braulke, T.; Prata, M.J.; Alves, S.Treatment strategies such enzyme-replacement therapy and substrate reduction, among others, are available for some Lysosomal Storage Diseases, yet still with some limitations. In recent years, the RNA molecule became one of the most promising targets for therapeutic intervention and currently, a large number of RNA-based therapies are being investigated at the basic research level and in late-stage clinical trials. Actually, some of them are already approved for medical use (e.g. Spinal muscular atrophy and Duchenne muscular dystrophy). RNA-based approaches can act at pre-mRNA level (by splicing modulation/correction using antisense oligonucleotides or U1snRNA vectors), at mRNA level (inhibiting gene expression by siRNAs and antisense oligonucleotides) or at DNA level (by editing mutated sequences through the use of CRISPR/Cas). Currently, we are developing some of these therapeutic approaches for LSDs. Two main research lines are ongoing: one involves the use of antisense U1 snRNAs to overcome the effect of a splice site mutation causing Mucopolysaccharidosis type IIIC and the other is based on the use of splice switching oligonucleotides to induce the skipping and consequently circumvent the effects of the most common causal mutation in Mucolipidosis type II.
- From bedside to cell biology: a century of history on lysosomal dysfunctionPublication . Coutinho, M.F.; Matos, L.S.; Alves, S.Lysosomal storage disorders (LSDs) are a group of rare genetic diseases, generally caused by a deficiency of specific lysosomal enzymes, which results in abnormal accumulation of undegraded substrates. The first clinical reports describing what were later shown to be LSDs were published more than a hundred years ago. In general, the history and pathophysiology of LSDs has impacted on our current knowledge of lysosomal biology. Classically, depending on the nature of the substrates, LSDs can be divided into different subgroups. The mucopolysaccharidoses (MPSs) are those caused by impaired degradation of glycosaminoglycans (GAGs). Amongst LSDs, the MPSs are a major group of pathologies with crucial historical relevance, since their study has revealed important biological pathways and highlighted interconnecting pathological cascades which are still being unveiled nowadays. Here we review the major historical discoveries in the field of LSDs and their impact on basic cellular knowledge and practical applications. Attention will be focused on the MPSs, with occasional references to other LSDs. We will show as studies on the metabolic basis of this group of diseases have increased our knowledge of the complex degradative pathways associated with the lysosome and established the basis to the development of specific therapeutic approaches aiming at correcting or, at least ameliorating their associated phenotypes.
- Molecular and computational analyses of genes involved in mannose 6-phosphate independent traffickingPublication . Coutinho, M.F.; Lacerda, L.; Pinto, E.; Ribeiro, H.; Macedo-Ribeiro, S.; Castro, L.; Prata, M.J.; Alves, S.The newly-synthesized lysosomal enzymes travel to the trans-Golgi network (TGN) and are then driven to the acidic organelle. While the best-known pathway for TGN-to-endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP-2, implicated in the delivery of β-glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P-independent receptors, we screened the genes that encode for LIMP-2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease.
- Mucolipidosis Type II and Type III: Nine cases from one Indian centrePublication . Bhat, M.; Sanjeeva, G.N.; Maganti, M.; Devaiah, S.; Silji, G.; Undamatla, J.; Coutinho, M.F.; Alves, S.
- Mucopolysaccharidoses type III: toward a siRNA-containing nanoparticle targeted to brain cellsPublication . Coutinho, M.F.; Santos, J.I.; Gaspar, P.; Alves, S.The classical therapeutic approach for LSD, enzyme replacement therapy, would hardly rise as a potentially successful tool to reduce the disease burden in MPS III patients, as it is long known to have no impact on neuropathology. A tempting alternative, however, would be to block substrate accumulation upstream, by decreasing its synthesis. That concept is known as substrate reduction therapy (SRT). Having this in mind, we designed an RNA-based strategy based upon the selective downregulation of one gene involved in the very early stages of the glycosaminoglycans’ (GAG) biosynthethic cascade. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPS’ symptoms. As tools to achieve substrate reduction, we are evaluating a specific type of antisense oligonucleotides, able to trigger a naturally-occurring post-transcriptional gene silencing process called RNA interference: the small interfering RNAs (siRNAs). So far, the obtained results are quite promising with marked decreases of the target mRNA levels in all tested cell lines (MPS IIIA, IIIC and IIID patients’ fibroblasts). Currently, we are evaluating the effect of that decrease on the overall storage of GAGs 7 days post-transfection, also with promising results. Here we present an overview on the current results of this project, while discussing its next steps, namely the development and evaluation of vectors for in vivo delivery. Our goal is to develop targeted stable nucleic acid lipid particles (t-SNALPs) coupled with different ligands, which promote cell uptake of the ‘anti-GAG’ siRNAs in a variety of cells, including neurons.
- Prenatal skeletal dysplasia phenotype in severe MLII alpha/beta with novel GNPTAB mutationPublication . Aggarwal, S.; Coutinho, M.F.; Dalal, A.; Jain, S.J.; Prata, M.J.; Alves, S.We report a neonate who was diagnosed as a case of skeletal dysplasia during pregnancy, and was subsequently diagnosed as a case of MLII alpha/beta on the basis of clinical and radiological findings and molecular testing of the parents. A novel GNPTAB mutation c.1701delC [p.F566LfsX5] was identified in the father. The case reiterates the severe prenatal phenotype of MLII alpha/beta which mimics skeletal dysplasia and illustrates the utility of molecular genetic analysis in confirmation of diagnosis and subsequent genetic counselling.
- SCARB2 mutations as modifiers in Gaucher disease: the wrong enzyme at the wrong place?Publication . Coutinho, M.F.; Lacerda, L.; Gaspar, A.; Pinto, E.; Ribeiro, I.; Laranjeira, F.; Ribeiro, H.; Silva, E.; Ferreira, C.; Prata, M.J.; Alves, S.Unlike most lysosomal proteins, β-glucocerebrosidase (GCase), the hydrolase defective in Gaucher disease (GD), is delivered to lysosomes through its interaction with the transmembrane protein LIMP2. A few years ago, mutations in its coding gene, SCARB2, were reported to modify the severity of GD phenotype. The existence of a great variety of GD phenotypes is well-known, with numerous patients who carry identical genotypes presenting remarkable phenotypic variability. Over the years, that variability has been attributed to other genetic, epigenetic and/or environmental factors. Still, there is still much to learn on this subject. Recently, an association between Parkinson's disease (PD) and the presence of mutations in the GBA gene has been demonstrated. Moreover, there are also studies suggesting that genetic variants in the SCARB2 gene may also be risk factors for PD. We analysed the SCARB2 gene in the Portuguese cohort of 91 GD patients, having identified 3 different SCARB2 coding variants. Of those, 2 were known polymorphisms with high prevalence in the normal population (p.M159V and p.V396I) and the third was a novel coding variant, p.T398M, present in heterozigousity in a single patient. Our study demonstrated that, at least for the Portuguese population, genetic variability at SCARB2 does not account much to the GD phenotypic spectrum. Nevertheless, in vitro analyses of the novel p.T398M are envisaged, in order to further characterize the effect of this variant on the levels and sub-cellular location of GCase. The clinical presentation of the patient harbouring this coding variant will also be discussed.
- The disease modelling value of a folklore FAIRYtale: SHEDing light over a special group of genetic disordersPublication . Carvalho, S.; Santos, J.I.; Moreira, L.; Gaspar, P.; Gonçalves, M.; Encarnação, M.; Ribeiro, D.; Duarte, A.; Prata, M.J.; Coutinho, M.F.; Alves, SandraThe problem we are addressing: Despite extensive research, the links between accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of Mucopolysaccharidoses (MPSs) have yet to be further elucidated. These Lysosomal Storage Diseases (LSDs) present symptoms, which may (or may not) include critical musculoskeletal and cardiovascular alterations, respiratory problems, and serious neurological dysfunctions. The skeletal and brain systems are the hardest ones to access and, consequently, those in greatest need of additional knowledge and novel therapeutic solutions.
- The use of a modified U1 snRNA as a therapeutic strategy to correct a 5’ splice-site mutation in Mucopolysaccharidosis IIIC: in vitro steps towards an in vivo approachPublication . Santos, J.I.; Matos, L.; Rocha, M.; Coutinho, M.F.; Prata, M.J.; Alves, S.Genetic therapy directed towards the correction of RNA missplicing is being investigated not only at basic research level but even in late-stage clinical trials. Many mutations that change the normal splicing pattern and lead to aberrant mRNA production have been identified in Lysosomal Storage Disorders (LSDs). The Mucopolysaccharidosis IIIC (MPS IIIC) is a LSD caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. Splicing mutations represent one of the most frequent (~20%) genetic defects in MPS IIIC. Approximately 55% corresponds to 5' splice-site mutations which thus constitute a good target for mutation specific therapeutic approaches. Recently, we demonstrated in fibroblast cells that a modified U1snRNA vector designed to improve the definition of exon 2 5’ss of the HGSNAT can restore splicing impaired by the mutation c.234+1G>A.(Matos et al., 2014). Presently our goal is to evaluate in vivo the therapeutic potential of the modified U1snRNA by testing it in mice expressing the human splicing defect. For this, in a first step we tried to generate full-length splicing competent constructs of wild-type (wt) and c.234+1G>A HGSNAT by cloning the wt or the mutated HGSNAT splicing-competent cassettes into the pcDNA 3.1 backbone. According to the protocol reported by other researchers (Pinotti et al., 2009), plasmid vectors will be used to promote transient expression of the human HGSNAT wt or mutant alleles in mice. Here, we describe the cloning process followed to obtain the aforementioned splicing constructs. During the cloning steps different difficulties were found as, for example, in fragments amplification, ligation, and obtainment of bacterial transformants. Even so, positive bacterial colonies were obtained, selected, and amplified by colony PCR. However, DNA sequencing data showed the presence of different nucleotide point alterations in the obtained clones, invalidating its use for further steps. Therefore, plasmid constructs were ordered commercially. Now we are performing its transfection in Hep3B/COS-7 cells to confirm that they recapitulate the splicing process observed in wt and patient cDNA being thus ready to be expressed in mice to test the therapeutic effect of the modified U1snRNA. This work shows the different steps and difficulties of the cloning process to obtain HGSNAT expression constructs towards testing of an in vivo U1snRNA therapeutic approach.