Browsing by Author "Matos, L."
Now showing 1 - 5 of 5
Results Per Page
Sort Options
- 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.
- 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.
- 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 . Matos, L.; Santos, J.I.; 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.
- 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.