Publicação
Insights into AAV promoter activity and helper virus components interactions
| datacite.subject.fos | Departamento de Biologia Vegetal | pt_PT |
| dc.contributor.advisor | Fernandes, Ana Sofia | |
| dc.contributor.advisor | Zilhão, Rita, 1959- | |
| dc.contributor.author | Guerra, Júlia Reis de Lidon Brandão | |
| dc.date.accessioned | 2024-04-15T14:03:39Z | |
| dc.date.embargo | 2026-11-18 | |
| dc.date.issued | 2024 | |
| dc.date.submitted | 2023 | |
| dc.description | Tese de mestrado, Biologia Molecular e Genética , 2023, Universidade de Lisboa, Faculdade de Ciências | pt_PT |
| dc.description.abstract | Gene therapies using viral vectors expanded in the last two decades, being Adeno-associated virus (AAV)-based methods one of the most used with six clinical products approved. Accordingly, the efforts are now focused on the improvement of recombinant AAV (rAAV) production based on stable producer cell lines, devoid of replication-competent virus. Several studies have been focusing on the mechanisms of transactivation of AAV promoters by viral Rep proteins. However, the ability of each promoter to mediate independent ectopic gene expression in mammalian cells is not so well known. In this study, we assessed the activity and strength of each AAV promoter and compared it with other viral and cellular promoters by transient transfection, in HEK 293, A549 and Vero cells. Additionally, the impact of AdV helper components of serotype 2 (E2A, E4 and VA RNA) in promoter activity was also evaluated. We showed that AAV promoters were active in all tested cell lines, being p5 the strongest. Moreover, in 293 cells, the presence of AdV helper components enhanced AAV promoter activity as well as that of other studied promoters. In Vero and A549 cells, this effect was less pronounced and, in some cases, absent. This work also focuses on the manipulation of AdV helper components, namely the E2A and E4, to evaluate their contribution during rAAV production. Using a pHelper plasmid as backbone containing the full AdV2 genes E2A, E4 and VA RNAs, we generated eight plasmids with different combinations of these genes. The impact of each construct in the productivity of serotype 5, 8 and 9 was evaluated by transient transfection and compared relative to control (pHelper). Through this approach, we identified which minimal components of the E2A and E4 significantly impacted the overall rAAV productivity and the quality of produced virus. | pt_PT |
| dc.identifier.tid | 203606299 | pt_PT |
| dc.identifier.uri | http://hdl.handle.net/10451/64259 | |
| dc.language.iso | eng | pt_PT |
| dc.subject | Vírus adeno-associados | pt_PT |
| dc.subject | Genes auxiliares AdV | pt_PT |
| dc.subject | Promotores de AAV | pt_PT |
| dc.subject | Produção de rAAV | pt_PT |
| dc.subject | Teses de mestrado - 2024 | pt_PT |
| dc.title | Insights into AAV promoter activity and helper virus components interactions | pt_PT |
| dc.type | master thesis | |
| dspace.entity.type | Publication | |
| rcaap.rights | embargoedAccess | pt_PT |
| rcaap.type | masterThesis | pt_PT |
| thesis.degree.name | Tese de mestrado em Biologia Molecular e Genética | pt_PT |