Browsing by Author "Oliveira, Paula"
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- Antisense oligonucleotide exon-skipping as a therapeutic approach for a rare diseasePublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana I.; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Omidi, Maryam; Pohl, Sandra; Alves, SandraMucolipidosis II (MLII) is a Lysosomal Storage Disorder caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase, which is responsible for the Mannose- 6-Phosphate marker addition to lysosomal enzymes. Of all MLII mutations, the 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) for MLII. Previously, on MLII patients’ fibroblasts, ASOs were used to skip exon 19 of the GNPTAB pre-mRNA, successfully resulting in the production of an in-frame mRNA[1]. Now, our aim is to analyze if these results are translated to the enzymatic and cellular phenotype level.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studiesPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II alpha/beta (ML II alpha/beta) is one of the most severe Lysosomal Storage Disorders and is 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, which allow their targeting to lysosomes. Of the several mutations that occur in ML II alpha/beta, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II alpha/beta. In a previous in vitro study in ML II alpha/beta patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA1. 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. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg/kg and groups 3 and 6 were injected with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 days (groups 1, 2, 3) or 7 days (groups 4, 5, 6) post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24h or 48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done. 1. Matos L, Vilela R, Rocha M, et al. Development of an antisense oligonucleotide-mediated exon skipping therapeutic strategy for Mucolipidosis II: validation at RNA level. Hum Gene Ther, 2020, 31(13-14):775-783.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studiesPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II alpha/beta (ML II) is one of the most severe Lysosomal Storage Disorders and is 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, which allow their targeting to lysosomes. Of the several mutations that occur in ML II, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II. In a previous in vitro study in ML II patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully 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. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 with AO at 25 mg/kg and groups 3 and 6 with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 or 7 days post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24/48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II α/β: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraGenetic therapy directed towards the correction of RNA mis-splicing is being investigated at basic research and 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). Mucopolysaccharidosis IIIC (MPS IIIC) is one of those LSDs caused by mutations in the HGSNAT gene that encodes an enzyme involved in heparan sulphate degradation. Splicing mutations are one of the most frequent (~20%) genetic defects in MPS IIIC. Approximately 55% correspond to 5' splice-site (ss) mutations thus constituting a good target for mutation-specific therapeutic approaches. Recently, we have demonstrated in fibroblast cells that a modified U1 snRNA 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. Currently, our goal is to evaluate in vivo the therapeutic potential of that modified U1 snRNA by testing it in mice expressing the human splicing defect. For this purpose, two full-length constructs were generated by cloning the wild-type (wt) or the mutated HGSNAT splicing-competent cassettes in the pcDNA 3.1 vector. Then, in an in vitro assay, the wt or mutated construct was transfected in Hep3B and COS-7 cells. After molecular analysis it was observed that both minigenes reproduce the healthy control and patient cDNA’s splicing pattern. Therefore, both constructs were used to generate mice of the C57BL/6 strain expressing the human mutation c.234+1G>A in the liver and test its modified U1-mediated rescue in vivo. Wt or mutant minigenes were administrated in mice by hydrodynamic injection following a reported protocol(1). After 48 hours animals were sacrificed, the liver was collected and molecular analysis was performed. Preliminary results showed expression of the HGSNAT cDNA from the mutant construct in the liver of at least one animal. Thus, further tests will be carried out to optimize some limiting points, such as the administration of the minigenes (e.g. increase of injection volume from 7% to 8-9% of mice body weight; inclusion of an in vivo transfection reagent to enhance delivery efficiency) and the use of other mice strain. 1. Balestra D, et al. (2014) J Thromb Haemost 12(2):177–185.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II α/β: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II α/β (ML II α/β) is one of the most severe Lysosomal Storage Disorders and is 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, which allow their targeting to lysosomes. Of the several mutations that occur in ML II α/β, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II α/β. In a previous in vitro study in ML II α/β patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully 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. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg/kg and groups 3 and 6 were injected with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 days (groups 1, 2, 3) or 7 days (groups 4, 5, 6) post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24h or 48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done.
- Assessing the potential of RNA-based therapeutics for a group of Lysosomal Storage Diseases with neurological involvementPublication . Santos, Juliana Inês; Gonçalves, Mariana; Matos, Liliana; Gaspar, Paulo; Pires, Maria João; Oliveira, Paula; Prata, Maria JoãoDuring the first two decades of the 21st century, remarkable progresses have been achieved in the field of RNA-based therapeutics. From antisense RNA to RNA modification, the therapeutic potential of RNA-based technologies has nothing but increased. In our lab, we have been addressing the potential of different RNA-based drugs to either correct or ameliorate the sub-cellular phenotype of a number of severe, life-threatening diseases: the so-called Lysosomal Storage Disorders (LSDs). Among them, we are focusing our efforts on those which present with a predominant neurological phenotype, since there are virtually no approved treatments for any of them. Briefly, two major research lines are being pursued: the first relies on the design of mutation-specific approaches to correct abnormal splicing processes in LSD-related genes, whenever they underlie pathology. The second depends upon selective downregulation of genes involved in the biosynthethic cascades that give origin to the substrates that accumulate in each pathology. Here we present an overview on our results with both approaches on Sanfilippo syndrome, a sub-group of severe neurodegenerative LSDs. For the mutation-specific, splicing correction approach, we are using U1snRNA vectors to restore the splicing defect caused by the HGSNAT mutation c.234+1G>A, that leads to Sanfilippo C disease. We started by demonstrating in vitro that a modified U1snRNA vector designed to improve the definition of HGSNAT exon 2 could partially restore its normal splicing process. Now, we are evaluating its therapeutic potential in vivo, in mice expressing the human splicing defect. For the substrate reduction approach, we are using siRNAs. By acting over a specific biosynthethic cascade, siRNAs promote an overall decrease of the accumulating substrate. So far, we have already tested this approach in patients’ fibroblasts and observed a high inhibition of the target mRNAs and a decrease in storage. Overall, there are substantial differences between these two approaches but they also face common challenges and show equally promising results.
- Characterization of the Human Papillomavirus 16 Oncogenes in K14HPV16 Mice: Sublineage A1 Drives Multi-Organ CarcinogenesisPublication . Cochicho, Daniela; Nunes, Alexandra; Gomes, João Paulo; Martins, Luís; Cunha, Mário; Medeiros-Fonseca, Beatriz; Oliveira, Paula; Bastos, Margarida M.S.M.; Medeiros, Rui; Mendonça, Joana; Vieira, Luis; Gil da Costa, Rui M.; Felix, AnaThe study of ()-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.
- Development of an antisense oligonucleotides-based therapy for mucolipidosis type II α/β: in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraIntroduction: The RNA molecule has become an increasingly promising target for the therapy of various diseases. Mucolipidosis type II α/β (ML II α/β) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1- phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate (M6P) marker to lysosomal enzymes, which allow their targeting to lysosomes. GlcNAc-1- phosphotransferase is encoded by the GNPTAB and GNPTG genes. Of the several mutations that occur in ML II α/β, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, being a good target for a mutation specific therapy as there is no therapy for this disease 1,2. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II. In a previous in vitro study in ML II patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA 3. Currently, our objective is to evaluate the therapeutic potential of this approach in vivo in mice (Mus musculus) of the strain C57BL/6. Material & Methods: Eighteen animals with an average body mass of 25 g were used. During the study, individual body weight, food and water intake were recorded. The animals were divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg / kg and groups 3 and 6 were injected with AO at 50 mg / kg. All animals were injected intraperitoneally and sacrificed after 4 days (groups 1, 2 and 3) or after 7 days (groups 4, 5 and 6). At the end of the experiment, the organs were collected, weighted and frozen at -80ºC, for later RNA extraction, cDNA synthesis and PCR. Statistical analysis was performed using the GraphPad Prism® for Windows program. Statistical significance between groups was determined by analysis of variance (ANOVA), followed by a Bonferroni test. All ethical issues were followed by the guidelines of the Portuguese General Directorate of Food and Veterinary. Results: Our results show that mice from groups 5 and 6 have a lower liver mass compared to the other groups, with significant differences (p <0.05). Liver of animals from group 3 has a lower weight compared to the liver of animals from group 2 and group 4, with a statistically significant difference. However, exon 19 skipping was not observed in any organ sample using any of the tested doses or incubation periods. Conclusions: To explain these first in vivo results we can theorize that the doses administered were not sufficient to achieve a response or the AO may have had a high clearance rate or did not recognize the target RNA. Moreover, the collected organs should have been preserved in liquid nitrogen to maintain RNA integrity. Other experiments will be done in the near future to overcome these preliminary data. References: [1] R. V. Velho et al. Human mutation. [2] M. F. Coutinho, et al. Biochemistry research international [3] L. Matos, et al. Human Gene Therapy.
- E se a retirada de um exão pudesse ter um efeito terapêutico? Caminhos de uma abordagem terapêutica molecular para uma doença rara através de exon-skippingPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraAtualmente, as abordagens terapêuticas de RNA estão em grande desenvolvimento e algumas destas terapias foram já aprovadas para uso clínico. Uma das estratégias mais conhecidas tem como base a utilização de oligonucleótidos antisense (OA), pequenos oligonucleótidos sintéticos que se ligam aos RNA alvo por meio de emparelhamento de bases. Estas moléculas podem atuar de diferentes formas, sendo a diminuição da expressão génica devido à degradação pela RNase-H dos mRNA alvo e a modulação do processo de splicing para induzir a inclusão ou exclusão de um exão (exon skipping), as mais amplamente aplicadas. Particularmente para as doenças raras, diferentes aplicações de OA estão em desenvolvimento, com vários ensaios clínicos em curso e com 3 destas terapêuticas já em prática clínica. As Doenças Lisossomais de Sobrecarga (DLS) são um grupo de doenças hereditárias do metabolismo raras que podem originar uma patologia grave e progressiva devido a uma disfunção lisossomal específica. Atualmente, não existe cura para as DLS, embora para algumas destas doenças haja estratégias de tratamento. Assim, as abordagens terapêuticas de RNA podem representar uma importante estratégia terapêutica alternativa ou adjuvante. O grupo de investigação em DLS do Departamento de Genética Humana do INSA – Porto, tem contribuído para o desenvolvimento de novas abordagens terapêuticas de RNA tendo sido já obtidos resultados positivos para diferentes DLS. Para a validação pré-clínica de novas terapias, tanto os modelos in vitro como in vivo são necessários e largamente utilizados. Num estudo anterior efetuado in vitro, para a mutação c.3503_3504delTC no gene GNPTAB, que origina a Mucolipidose tipo II α/β, foi testada uma estratégia terapêutica de exon-skipping utilizando diferentes OA em fibroblastos de doentes, tendo resultado na produção de um mRNA in-frame1. Neste estudo, pretendeu-se avaliar o potencial terapêutico desta abordagem tanto in vitro (fibroblastos de murganho C57BL/6) como in vivo (murganhos C57BL/6) e os resultados preliminares obtidos serão apresentados e discutidos. 1. Matos L, Vilela R, Rocha M, et al. Development of an antisense oligonucleotide-mediated exon skipping therapeutic strategy for Mucolipidosis II: validation at RNA level. Hum Gene Ther, 2020, 31(13-14):775-783.
- Mutation-adapted U1snRNA as a therapeutic strategy for Mucopolysaccharidosis IIIC: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraA significant number of mutations that change the splicing process and lead to aberrant mRNA production have been identified in Lysosomal Storage Disorders (LSDs). Mucopolysaccharidosis type IIIC (MPS IIIC) is a very rare LSD caused by mutations in the HGSNAT gene which encodes an enzyme involved in heparan sulphate degradation. Splicing mutations are one of the most frequent (~20%) genetic defects in MPS IIIC. Around 55% correspond to 5' splice-site (ss) mutations thus constituting a good target for splicing therapeutics. Recently, we have demonstrated that a modified U1snRNA vector designed to improve the definition of the HGSNAT exon 2 5’ss can restore splicing impaired by the mutation c.234+1G>A. Currently, our goal is to evaluate in vivo the therapeutic potential of that modified U1 snRNA by testing it in mice expressing the human splicing defect. For this purpose, two full-length constructs were generated by cloning the wt or the mutated HGSNAT splicing-competent cassettes in the pcDNA 3.1 vector. Then, the wt and the mutant constructs were transfected in Hep3B and COS-7 cells. Both minigenes reproduce the healthy control and patient cDNA’s splicing pattern. Therefore, they were used to generate C57BL/6 mice expressing the wt (c.241+1G) or mutant (c.234+1A) alleles in the liver. These mice can be used for testing the modified U1 snRNA efficacy in vivo. Thus, wt or mutant minigenes were administrated in mice by hydrodynamic injection following a protocol described by Balestra et al. After 48h, animals were sacrificed, the liver was collected, and the molecular analysis was performed. Preliminary results showed the expression of the mutant construct in the liver of at least one animal. Thus, further tests will be carried out to optimize experimental conditions, by testing other forms of minigenes administration and using other mice strains.