Browsing by Author "Gaspar, P."
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- 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.
- Genetically Modulated Substrate Reduction Therapy for Sanfilippo syndrome – proof of principlePublication . Santos, J.I.; Coutinho, Maria Francisca; Gaspar, P.; Alves, S.Introduction: Mucopolysaccharidosis type III (MPS III) refers to a group of five autosomal recessive neurodegenerative lysosomal storage disorders caused by the incomplete lysosomal degradation of the heparan sulphate (HS) that accumulates in patient cells and triggers disease. The main characteristic of this disease is the degeneration of the central nervous system, resulting in mental retardation and hyperactivity. Currently, there is no effective therapy available, with treatment limited to clinical management of neurological symptoms. Methods: Taking advantage of the RNA interference (RNAi) technology potential, we have designed and assayed a specific siRNA targeting an early stage of the HS biosynthetic cascade (XYLT1) in order to promote an effective reduction of the accumulating substrate. Fibroblasts from MPS III patients were transfected with the designed siRNA. Total RNA was extracted and target mRNA levels evaluated through real-time PCR. The effect on GAGs accumulation was quantified over time using a modified 1,9-dimethylmethylene blue assay. Results: Proof of principle on the effect of siRNA targeting XYLT1 was achieved for two independent control cell lines, with 8-12 fold decreases on the target mRNA levels, after 24h of incubation with concentrations of 20nM of each siRNA. Subsequent analysis on the effect of the same siRNA on MPS III cell lines resulted in significant lower expression of XYLT1 in types A, B and C, after 24-48h of siRNA incubation. Studies on type B are also ongoing. For types C and D, we have already assessed the treatment effect on storage and observed a significant reduction (50-70%) on the total GAGs levels. Conclusions: The effect of siRNA targeting XYLT1 was achieved, resulting in significant lower levels of XYLT1 mRNA. Studies on MPS IIIB are ongoing. Moreover, a significant reduction on GAGs’ accumulation was observed, and we are currently addressing this storage in the remaining MPS III cell lines.
- 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.
- Simultaneous quantitation of sphingoid bases by UPLC-ESI-MS/MS with identical13C-encoded internal standardsPublication . Mirzaian, M.; Wisse, P.; Ferraz, M.J.; Marques, A.R.A.; Gaspar, P.; Oussoren, S.V.; Kytidou, K.; Codée, J.D.C.; van der Marel, G.; Overkleeft, H.S.; Aerts, J.M.Free sphingoid bases (lysosphingolipids) of primary storage sphingolipids are increased in tissues and plasma of several sphingolipidoses. As shown earlier by us, sphingoid bases can be accurately quantified using UPLC-ESI-MS/MS, particularly in combination with identical13C-encoded internal standards. The feasibility of simultaneous quantitation of sphingoid bases in plasma specimens spiked with a mixture of such standards is here described. The sensitivity and linearity of detection is excellent for all examined sphingoid bases (sphingosine, sphinganine, hexosyl-sphingosine (glucosylsphingosine), hexosyl2-sphingosine (lactosylsphingosine), hexosyl3-sphingosine (globotriaosylsphingosine), phosphorylcholine-sphingosine) in the relevant concentration range and the measurements show very acceptable intra- and inter-assay variation (<10% average). Plasma samples of a series of male and female Gaucher Disease and Fabry Disease patients were analyzed with the multiplex assay. The obtained data compare well to those earlier determined for plasma globotriaosylsphingosine and glucosylsphingosine in GD and FD patients. The same approach can be also applied to measure sphingolipids in the same sample. Following extraction of sphingolipids from the same sample these can be converted to sphingoid bases by microwave exposure and subsequently quantified using13C-encoded internal standards.
- 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.