Overcoming the Challenges of Recombinant Adeno-Associated Virus Production
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/138711 |
Resumo: | Gene therapy is considered one of the most encouraging areas for the development of more ef- fective long-term therapeutics. These therapies rely on the delivery of genetic material to specific cells using a vector. Among the several vectors available, recombinant Adeno-Associated Viruses (rAAV) are regarded as one of the most promising viral vectors, due to the low immunogenic response, wide cellular tropism, and potential for stable expression. Despite the variety of manufacturing processes available, most of the rAAV produced in clinical trial settings are derived from transient transfection mechanisms. These systems have low scalability and high variability, which translates into high production costs. Stable producer cell line mechanisms offer an alternative to the transient transfection mechanisms by improving scalability. However, the time needed to obtain a high titer producing clone remains one of the major drawbacks. This thesis aimed to overcome these issues by using CRISPR/Cas9 genetic screens technology. To carry out the screen, two rAAV2 stable producer cell lines were developed in parallel. A cell line based on HeLaS3 was generated (clone H32D2) by transfecting a plasmid encoding all genetic material to generate rAAV2. After adaptation to suspension culture, the H32D2 clone achieved productivities levels similar to rAAV titers reported by Pharmaceutical Industry (10 11 vg/mL). In addition to the clone H32D2, a clone based on a packaging cell line (HeLaRC32) was generated. Production conditions were optimized identifying the temperature as a critical process parameter for the clone produced. The H32D2 clone was used to optimize and perform the genetic screen. Cells were infected with Lentiviral particles encoding a genome-wide CRISPR/Cas9 knockout library. rAAV2 high producing cells and low producing cells were sorted by Flow cytometry and will be used to analyze genes and pathways that have an impact on productivity and can be considered as future targets for cell engineer- ing. |
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Overcoming the Challenges of Recombinant Adeno-Associated Virus ProductionGene therapyAdeno-Associated VirusStable Producer cell lineCRISPR/Cas9Genetic ScreenDomínio/Área Científica::Engenharia e Tecnologia::Engenharia QuímicaGene therapy is considered one of the most encouraging areas for the development of more ef- fective long-term therapeutics. These therapies rely on the delivery of genetic material to specific cells using a vector. Among the several vectors available, recombinant Adeno-Associated Viruses (rAAV) are regarded as one of the most promising viral vectors, due to the low immunogenic response, wide cellular tropism, and potential for stable expression. Despite the variety of manufacturing processes available, most of the rAAV produced in clinical trial settings are derived from transient transfection mechanisms. These systems have low scalability and high variability, which translates into high production costs. Stable producer cell line mechanisms offer an alternative to the transient transfection mechanisms by improving scalability. However, the time needed to obtain a high titer producing clone remains one of the major drawbacks. This thesis aimed to overcome these issues by using CRISPR/Cas9 genetic screens technology. To carry out the screen, two rAAV2 stable producer cell lines were developed in parallel. A cell line based on HeLaS3 was generated (clone H32D2) by transfecting a plasmid encoding all genetic material to generate rAAV2. After adaptation to suspension culture, the H32D2 clone achieved productivities levels similar to rAAV titers reported by Pharmaceutical Industry (10 11 vg/mL). In addition to the clone H32D2, a clone based on a packaging cell line (HeLaRC32) was generated. Production conditions were optimized identifying the temperature as a critical process parameter for the clone produced. The H32D2 clone was used to optimize and perform the genetic screen. Cells were infected with Lentiviral particles encoding a genome-wide CRISPR/Cas9 knockout library. rAAV2 high producing cells and low producing cells were sorted by Flow cytometry and will be used to analyze genes and pathways that have an impact on productivity and can be considered as future targets for cell engineer- ing.A terapia génica é uma das áreas mais promissoras no desenvolvimento de bioterapêuticos efi- cazes. Na sua base encontram-se os vetores, essenciais para a entrega do material genético às células. Os vírus adeno-associados (AAV) são um dos vetores mais promissores, por não provocarem uma resposta imunitária acentuada, possuirem um alargado tropismo celular e poderem ser usados em siste- mas de expressão estável. Grande parte dos AAV recombinantes usados em ensaios clínicos são produzidos através de me- canismos de transfeção transiente. Embora estes sistemas sejam simples de implementar, a sua variabi- lidade e baixa adaptabilidade a escalas de produção maiores são desvantajosas, aumentando os custos de produção. As linhas celulares produtoras estáveis são uma alternativa, sendo facilmente adaptáveis a escalas de produção maiores. No entanto, a sua grande desvantagem prende-se com o tempo necessá- rio para o seu desenvolvimento e obtenção de títulos virais elevados. Esta tese teve como objetivo contribuir para a superação dos problemas acima descritos, reali- zando um screen genético usando a tecnologia CRISPR/Cas9. Duas linhas celulares produtoras de AAV foram desenvolvidas: 1) uma usando células HeLaS3 (H32D2), através da transfeção de um plasmídeo com todos os elementos necessários à produção de AAV. O clone foi adaptado a suspensão, obtendo- se produtividades semelhantes ao reportado em contexto industrial (10 11 vg/mL); 2) em paralelo, gerou- se um clone a partir de uma linha celular contendo os genes rep e cap (HeLaRC32). Neste caso, iden- tificou-se a temperatura como parâmetro crítico na produção de AAV. O clone H32D2 foi usado na otimização e realização do screen genético. As células foram infe- tadas com lentivírus contendo uma biblioteca genómica knockout de CRISPR/Cas9. Utilizou-se cito- metria de Fluxo para separar duas populações celulares: altamente produtoras e pouco produtoras. Ex- traindo o ADN destas populações será possível identificar genes que tenham impacto na produtividade, e considerar alvos para engenharia de futuras linhas celulares.Planells, JoseAlves, PaulaRUNMoura, Filipa Alves2022-05-26T17:42:25Z2022-022022-02-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/138711enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:16:03Zoai:run.unl.pt:10362/138711Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:49:11.595099Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production |
title |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production |
spellingShingle |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production Moura, Filipa Alves Gene therapy Adeno-Associated Virus Stable Producer cell line CRISPR/Cas9 Genetic Screen Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química |
title_short |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production |
title_full |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production |
title_fullStr |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production |
title_full_unstemmed |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production |
title_sort |
Overcoming the Challenges of Recombinant Adeno-Associated Virus Production |
author |
Moura, Filipa Alves |
author_facet |
Moura, Filipa Alves |
author_role |
author |
dc.contributor.none.fl_str_mv |
Planells, Jose Alves, Paula RUN |
dc.contributor.author.fl_str_mv |
Moura, Filipa Alves |
dc.subject.por.fl_str_mv |
Gene therapy Adeno-Associated Virus Stable Producer cell line CRISPR/Cas9 Genetic Screen Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química |
topic |
Gene therapy Adeno-Associated Virus Stable Producer cell line CRISPR/Cas9 Genetic Screen Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química |
description |
Gene therapy is considered one of the most encouraging areas for the development of more ef- fective long-term therapeutics. These therapies rely on the delivery of genetic material to specific cells using a vector. Among the several vectors available, recombinant Adeno-Associated Viruses (rAAV) are regarded as one of the most promising viral vectors, due to the low immunogenic response, wide cellular tropism, and potential for stable expression. Despite the variety of manufacturing processes available, most of the rAAV produced in clinical trial settings are derived from transient transfection mechanisms. These systems have low scalability and high variability, which translates into high production costs. Stable producer cell line mechanisms offer an alternative to the transient transfection mechanisms by improving scalability. However, the time needed to obtain a high titer producing clone remains one of the major drawbacks. This thesis aimed to overcome these issues by using CRISPR/Cas9 genetic screens technology. To carry out the screen, two rAAV2 stable producer cell lines were developed in parallel. A cell line based on HeLaS3 was generated (clone H32D2) by transfecting a plasmid encoding all genetic material to generate rAAV2. After adaptation to suspension culture, the H32D2 clone achieved productivities levels similar to rAAV titers reported by Pharmaceutical Industry (10 11 vg/mL). In addition to the clone H32D2, a clone based on a packaging cell line (HeLaRC32) was generated. Production conditions were optimized identifying the temperature as a critical process parameter for the clone produced. The H32D2 clone was used to optimize and perform the genetic screen. Cells were infected with Lentiviral particles encoding a genome-wide CRISPR/Cas9 knockout library. rAAV2 high producing cells and low producing cells were sorted by Flow cytometry and will be used to analyze genes and pathways that have an impact on productivity and can be considered as future targets for cell engineer- ing. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-05-26T17:42:25Z 2022-02 2022-02-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
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http://hdl.handle.net/10362/138711 |
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http://hdl.handle.net/10362/138711 |
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eng |
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eng |
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info:eu-repo/semantics/openAccess |
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openAccess |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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