Insights into AAV promoter activity and helper virus components interactions

Bibliographic Details
Main Author: Guerra, Júlia Reis de Lidon Brandão
Publication Date: 2023
Format: Master thesis
Language: eng
Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
Download full: http://hdl.handle.net/10451/64259
Summary: Tese de mestrado, Biologia Molecular e Genética , 2023, Universidade de Lisboa, Faculdade de Ciências
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spelling Insights into AAV promoter activity and helper virus components interactionsVírus adeno-associadosGenes auxiliares AdVPromotores de AAVProdução de rAAVTeses de mestrado - 2024Departamento de Biologia VegetalTese de mestrado, Biologia Molecular e Genética , 2023, Universidade de Lisboa, Faculdade de CiênciasGene 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.Fernandes, Ana SofiaZilhão, Rita, 1959-Repositório da Universidade de LisboaGuerra, Júlia Reis de Lidon Brandão202420232026-11-18T00:00:00Z2024-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10451/64259TID:203606299enginfo:eu-repo/semantics/embargoedAccessreponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiainstacron:RCAAP2025-03-17T15:14:28Zoai:repositorio.ulisboa.pt:10451/64259Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireinfo@rcaap.ptopendoar:https://opendoar.ac.uk/repository/71602025-05-29T03:37:43.114738Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologiafalse
dc.title.none.fl_str_mv Insights into AAV promoter activity and helper virus components interactions
title Insights into AAV promoter activity and helper virus components interactions
spellingShingle Insights into AAV promoter activity and helper virus components interactions
Guerra, Júlia Reis de Lidon Brandão
Vírus adeno-associados
Genes auxiliares AdV
Promotores de AAV
Produção de rAAV
Teses de mestrado - 2024
Departamento de Biologia Vegetal
title_short Insights into AAV promoter activity and helper virus components interactions
title_full Insights into AAV promoter activity and helper virus components interactions
title_fullStr Insights into AAV promoter activity and helper virus components interactions
title_full_unstemmed Insights into AAV promoter activity and helper virus components interactions
title_sort Insights into AAV promoter activity and helper virus components interactions
author Guerra, Júlia Reis de Lidon Brandão
author_facet Guerra, Júlia Reis de Lidon Brandão
author_role author
dc.contributor.none.fl_str_mv Fernandes, Ana Sofia
Zilhão, Rita, 1959-
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Guerra, Júlia Reis de Lidon Brandão
dc.subject.por.fl_str_mv Vírus adeno-associados
Genes auxiliares AdV
Promotores de AAV
Produção de rAAV
Teses de mestrado - 2024
Departamento de Biologia Vegetal
topic Vírus adeno-associados
Genes auxiliares AdV
Promotores de AAV
Produção de rAAV
Teses de mestrado - 2024
Departamento de Biologia Vegetal
description Tese de mestrado, Biologia Molecular e Genética , 2023, Universidade de Lisboa, Faculdade de Ciências
publishDate 2023
dc.date.none.fl_str_mv 2023
2024
2024-01-01T00:00:00Z
2026-11-18T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/64259
TID:203606299
url http://hdl.handle.net/10451/64259
identifier_str_mv TID:203606299
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/embargoedAccess
eu_rights_str_mv embargoedAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
instname:FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron:RCAAP
instname_str FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
instacron_str RCAAP
institution RCAAP
reponame_str Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
collection Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
repository.name.fl_str_mv Repositórios Científicos de Acesso Aberto de Portugal (RCAAP) - FCCN, serviços digitais da FCT – Fundação para a Ciência e a Tecnologia
repository.mail.fl_str_mv info@rcaap.pt
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