Percorrer por autor "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.
- Can an Antisense Oligonucleotide Exon Skipping Rewrite the Story of N-Acetylglucosamine-1-Phosphotransferase Deficiency?Publication . Gonçalves, M.; Moreira, L.; Encarnação, M.; Gaspar, P.; Duarte, A.J.; Santos, J.I.; Coutinho, M.F.; Prata, M.J.; Omidi, M.; Pohl, S.; Silva, F.; Oliveira, P.; Matos, L.; Alves, S.Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs the trafficking of lysosomal hydrolases. Of all ML II mutations, c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs). Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping in pre-mRNA, successfully generating an in-frame mRNA (Matos et al., 2020). Now, our aim is to determine whether this in-frame transcript leads to increased GlcNAc-PT levels improving ML II cellular phenotype.
- 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.
- An engineered U1 snRNA-based therapeutic approach can efficiently rescue a 5’ splice site mutation causing Mucolipidosis type IIIPublication . Peretto, L.; Gonçalves, M.; Santos, J.I.; Duarte, A.J.; Moreira, L.; Encarnação, M; Coutinho, M.F.; Pinotti, M.; Balestra, D.; Alves, S.; Matos. L.A significant number of splicing mutations have been identified in Lysosomal Storage Disorders (LSDs). Mucolipidosis III (ML III) is a LSD caused by GlcNAc-1-phosphotransferase deficiency, which impairs the trafficking of lysosomal hydrolases. 10% of the genetic defects in ML III are splicing mutations, and around 45% affect 5' splice-sites (ss) thus constituting a good target for mutation specific therapies. The use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s - ExSpeU1s) has been applied as a potential therapeutic strategy to correct 5’ss defects. Here we used engineered U1 snRNAs to correct the GNPTAB exon 17 skipping caused by the 5’ss mutation (c.3335+6T>G) found in a ML III patient. First, we performed transfection of exon-trapping minigenes expressing exon 17 surrounded by a portion of introns - pGNPTAB_WT and pGNPTAB_+6, in HEK293T cells to analyze if they reproduce the WT and mutant splicing patterns. Then, to evaluate the potential of 2 modified U1’s, 3 ExSpeU1s and 2 modified U6’s to restore mRNA splicing, these vectors were cotransfected into HEK293T cells along with the mutant +6 minigene as well as electroporated in patient’s fibroblasts. Then, cells were harvested, and RT-PCR analysis was performed. Both minigenes reproduced the control or ML III patient cDNA’s splicing patterns, thus, different concentrations of the modified U1’s and ExSpeU1s were tested together with the mutant minigene. The cDNA analysis showed almost 100% of exon 17 inclusion when one of the ExSpeU1s, was overexpressed in HEK293T cells. The combination of the 2 modified U6’s with the modified U1’s or the ExSpeU1s allowed exon 17 inclusion at some extent, but not as effectively as with the best ExSpeU1 alone. The electroporation of the 2 modified U1’s and of the 3 ExSpeU1s was done, and the cDNA analysis of patient’s fibroblasts treated with 2 ExSpeU1s (ExSpeU1 int17-1 or int17-2) showed around 35% and 15% of exon 17-including transcripts, respectively. To confirm these results, given that the lentiviral transduction is a more efficient delivery technique than electroporation, the gene cassettes of the 2 most promising ExSpeU1s were cloned in a lentivirus vector and after obtaining the viral mediums, their transduction in patient’s fibroblasts is being optimized. The cDNA analysis of preliminary experiments is still ongoing. In conclusion, we have developed an RNA therapy based on engineered U1 snRNAs for a ML III 5’ss mutation. We showed that an ExSpeU1 (binding downstream of the mutated 5´ss) can restore proper exon 17 definition in vitro, opening the opportunity for a personalized therapeutic intervention.
- An engineered U1 snRNA-based therapeutic approach can efficiently rescue a 5’ splice site mutation causing Mucolipidosis type IIIPublication . Peretto, L.; Gonçalves, M.; Santos, J.I.; Duarte, A.J.; Moreira, L.; Encarnação, M.; Coutinho, M.F.; Pinotti, M.; Balestra, D.; Alves, S.; Matos, L.A significant number of splicing mutations have been identified in Lysosomal Storage Disorders (LSDs). Mucolipidosis III (ML III) is a LSD caused by GlcNAc-1-phosphotransferase deficiency, which impairs the trafficking of lysosomal hydrolases. 10% of the genetic defects in ML III are splicing mutations, and around 45% affect 5' splice-sites (ss) thus constituting a good target for mutation specific therapies. The use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s - ExSpeU1s) has been applied as a potential therapeutic strategy to correct 5’ss defects. Here we used engineered U1 snRNAs to correct the GNPTAB exon 17 skipping caused by the 5’ss mutation (c.3335+6T>G) found in a ML III patient.
- 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.