DGH - Apresentações orais em encontros internacionais
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- 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.
- Surveillance of invasive meningococcal disease in Portugal, from 2020 to 2024Publication . Bettencourt, Célia; Nunes, A.; VigLab-DM – Network for the Laboratory Surveillance of Meningococcal Disease; Bajanca-Lavado, M.P.; .Introduction: Since 2002, laboratory surveillance of Invasive Meningococcal Disease (IMD) has been carried out by the National Reference Laboratory for Neisseria meningitidis, at the National Institute of Health Doutor Ricardo Jorge, Portugal. This study aims to analyse the epidemiology of IMD and the genetic diversity of Neisseria meningitidis strains from 2020 to 2024. Material and Methods: Suspected IMD cases and N. meningitidis isolates were sent to the reference laboratory for confirmation and strain characterization. Invasive isolates were characterized by WGS (Illumina) and sequences were submitted to the PubMLST/Neisseria database. Results: Between 2020 to 2024, 125 IMD cases were confirmed. Annual incidence rate ranged from 0.36 cases/100,000 inhabitants in 2020 to 0.32 in 2023 [1, 2]. Serogroup B was the most prevalent (49.6%), followed by serogroups Y (14.4%), W (13.6%) and C (5.6%). Serogroup W mainly affected those over 45 years old (58.8%). In silico analysis of 89 (71.2%) isolates identified major clonal complexes (cc): B-cc213 (22%) and cc41/44 (18%), Y-cc23 (80%), W-cc11 (66.7%), and C-cc11/cc103 (33.3% each). Conclusions: Compared to previous studies (2003-2020), the incidence of IMD in Portugal has decreased [1-3]. However, serogroup B remains the leading cause of IMD, raising concerns, particularly due to cases in children and emerging clusters with low vaccination coverage (e.g. serogroup B cc213) [4]. In contrast, serogroup W cases have increased, especially among adults [2, 3]. This study highlights the importance of laboratory surveillance for understanding IMD epidemiology and monitoring long-term trends.