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Is Antisense Oligonucleotide-Mediated Exon Skipping a Potential Therapeutic Approach for Mucolipidosis II?

2025-11Documento de conferência Acesso aberto
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datacite.subject.fosCiências Médicas
datacite.subject.fosCiências Naturais
dc.contributor.authorGonçalves, Mariana
dc.contributor.authorMoreira, Luciana
dc.contributor.authorEncarnação, Marisa
dc.contributor.authorDuarte, Ana Joana
dc.contributor.authorGaspar, Paulo
dc.contributor.authorSantos, Juliana Inês
dc.contributor.authorCoutinho Maria Francisca
dc.contributor.authorPrata, Maria João
dc.contributor.authorOmidi, Maryam
dc.contributor.authorPohl, Sandra
dc.contributor.authorSilva, Frederico
dc.contributor.authorOliveira, Paula
dc.contributor.authorMatos, Liliana
dc.contributor.authorAlves, Sandra
dc.date.accessioned2026-02-11T12:02:15Z
dc.date.available2026-02-11T12:02:15Z
dc.date.issued2025-11
dc.description.abstractIntridution: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs lysosomal hydrolases trafficking. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) to promote targeted skipping of GNPTAB exon 19, which harbors c.3503_3504del, the most frequent disease-causing variant. Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping, successfully generating an in-frame mRNA1. Now, our aim is to determine if this in-frame transcript leads to increased GlcNAc-PT levels. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts, but activity levels were similar in ML II and control fibroblasts (treated/non-treated) showing that the assay is not proper to measure endogenous levels. To overcome this, we designed 3 constructs: a WT (full GNPTAB cDNA), a del_ex19 (without exon 19) and a mutant (with the mutation c.3503_3504del) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Also, we measured the activity of several hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment. To further validate this therapy we also generated a novel GlcNAc-PT antibody in rabbits. Results: Our results showed that HEK293T cells were able to express all the constructs. The WB of both WT and del_ex19 constructs showed bands corresponding to the α/β precursor. However, only the WT construct expressed the β-subunit, suggesting that there is no GlcNAc-PT activity in the absence of exon 19. As expected, in the delTC construct WB no α/β precursor band was detected. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts after treatment. However, only the α-Gal values were statistically significant, but the WB analysis for this enzyme did not reveal any band in ASO-treated ML II cells. We also developed a novel antibody for GlcNAc-PT. Preliminary results showed a β-subunit band both in control and patient fibroblasts (unexpected), but in overexpression both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess its specificity. Conclusion: Our ASO-based approach effectively promotes exon 19 skipping. However, this strategy, as far as we have been able to prove, is not able to restore any GlcNAc-PT enzymatic activity. Further validation, including co-localization studies are planned to clarify these findings.eng
dc.description.sponsorshipInstituto Nacional de Saúde Dr. Ricardo Jorge. Bolsa da Sociedade Portuguesa de Doenças Metabólicas (SPDM) de apoio à investigação Dr. Aguinaldo Cabral 2019” (2020DGH1834) Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), project AL4A-PROJ-LT3.5. PhD grant (2022.13676.BD) from the Portuguese Foundation for Science and Technology (FCT)
dc.identifier.urihttp://hdl.handle.net/10400.18/10896
dc.language.isoeng
dc.peerreviewedyes
dc.relationInnovative & personalized RNA-based therapies for rare diseases and development of models for their testing: application to Mucolipidosis II
dc.relation.hasversionhttps://spgh.net/wp-content/uploads/2014/04/liv_resumos_spgh25-1.pdf
dc.rights.uriN/A
dc.subjectMucolipidose tipo II
dc.subjectAntisense Oligonucleotide Therapy
dc.subjectExon-Skipping
dc.subjectMucolipidosis II
dc.subjectOligonucléotidos Antisense
dc.subjectGenética Humana
dc.subjectDoenças Genéticas
dc.subjectDoenças Lisossomais de Sobrecarga
dc.subjectTerapias de RNA
dc.titleIs Antisense Oligonucleotide-Mediated Exon Skipping a Potential Therapeutic Approach for Mucolipidosis II?eng
dc.typeconference object
dspace.entity.typePublication
oaire.awardTitleInnovative & personalized RNA-based therapies for rare diseases and development of models for their testing: application to Mucolipidosis II
oaire.awardURIhttp://hdl.handle.net/10400.18/10895
oaire.citation.conferenceDate2025-11
oaire.citation.conferencePlaceCoimbra, Portugal
oaire.citation.title29ª Reunião Anual da Sociedade Portuguesa de Genética Humana (SPGH), 20-22 novembro 2025
oaire.versionhttp://purl.org/coar/version/c_b1a7d7d4d402bcce
relation.isProjectOfPublication337d6c06-975b-4c8f-8a41-f47d51b7386f
relation.isProjectOfPublication.latestForDiscovery337d6c06-975b-4c8f-8a41-f47d51b7386f

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