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Addressing a challenging enzyme in vitro: proof of principle on the therapeutic potential of an antisense oligonucleotide approach for ML II
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1. To confirm the specificity of selected ASOs to skip the GNPTAB exon 19, a scramble ASO (expected to have no effect on GNPTAB gene) was transfected in WT & ML II patient fibroblasts. RT-PCR results showed that this ASO did not changed exon 19 splicing pattern proving the specificity of GNPTAB ASOs (Fig1A). Also, the enzymatic activity of various hydrolases was analysed in the same transfected cells. Results showed that the scramble ASO seems to interfere, even if little, with hydrolases activities of treated WT & ML II fibroblasts as shown by the differences in activities observed (Fig1B). The experiments number need to be increased to confirm these results.
2. To analyse the effect of exon 19 skipping on the GlcNAc-PT, a WT construct with the full GNPTAB cDNA (pGNPTAB_WT) and a mutant without exon 19 (pGNPTAB_delEx19) were tested in HEK293T cells. RT-PCR results after transfection showed the expected transcript pattern in both constructs (Fig2A). Moreover, GNPTAB protein expression was analysed by Western Blot (WB) using an antibody (Ab) specific for a myc-His tag located in constructs downstream the GNPTAB insert. Both constructs expressed a band corresponding to the α/β precursor, with or without exon 19. However, regarding the cleaved β-subunit the results are not so clear since we observed 2 bands (~48KDa) for the WT construct and further analysis is needed to understand if any of them corresponds to the target (Fig2B).
Both constructs were also transfected in ML II fibroblasts and cDNA analysis showed the expected transcript pattern. The activity of hydrolases will also be assessed to check if it increases with the delEx19 construct expression, suggesting a potential therapeutic effect.
3. We generated a novel Ab for the GlcNAc-PT β-subunit (rabbits). We expected to detect the protein in WT but not in ML II fibroblasts. However, WB results showed a band with the expected protein size in both cases (Fig3A). So, to test the Ab specificity different assays were done. The pre-bleed serum of the immunized rabbits was tested by WB and no target band was detected confirming that animals did not have Abs against the human protein (Fig3B). Also, the 2 synthetic β-subunit peptides used to immunize the animals were detected by WB confirming the specificity of the produced Abs (Fig3C). Protein sequencing (mass spectrometry) was also performed in extracts of WT & ML II fibroblasts after SDS-Page to search for the GNPTAB protein. As our Ab target has 45KDa, bands within 37-50KDa were analysed but the protein was not detected not even in the WT, suggesting that the protein amount used was insufficient for target protein detection. Finally, the Ab was tested by WB in HEK293T cells transfected with both constructs and the expected protein pattern at least for the α/β precursor was observed (Fig3D). This last experiment needs to be repeated, however results obtained in the various assays suggest that our Ab is specific for GNPTAB protein detection.
Descrição
Associate Laboratory for Animal and Veterinary Science (AL4AnimalS) 2023 Project Report.
Palavras-chave
Mucolipidose tipo II Terapias de RNA Oligonucleótidos Antisense Doenças Lisossomais de Sobrecarga Genética Humana Doenças Genéticas Relatório de Projeto