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Genetic engineering of human cell lines for the improvement of viral vector production for gene therapy
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A terapia génica baseada em vetores virais toma partido dos mecanismos biológicos naturais para entregar genes terapêuticos e controlar a sua expressão nas células alvo do paciente. Vários produtos de terapia génica viral estão já no mercado, com cerca de metade destes utilizando vírus recombinantes da família Retroviridae. Estes são uma opção frequente devido à sua elevada capacidade de transporte de carga genética, elevada eficiência de transdução, integração estável de genoma em loci transcricionalmente ativos, quer as células estejam em divisão ou não e devido à expressão a longo termo do gene entregue. As previsões de receita no mercado de terapia génica viral até 2020 ultrapassam os 200 milhões de dólares, sem perspetivas de declínio pelo menos até 2026. No entanto, a produção de retrovírus recombinantes depara-se com vários desafios. A elevada produção de partículas não-infecciosas – aproximadamente 1 em cada 1000 partículas produzidas são infecciosas – e baixo rendimento das plataformas de produção, dois fatores que impõem custos de produção altos, são as barreiras mais difíceis de ultrapassar na transição clinico-mercado.
Vias metabólicas recrutadas em produção de retrovírus recombinantes foram previamente identificadas. Neste trabalho, cinco genes-alvo foram alvo de estudo numa linha celular produtora de retrovírus recombinantes. A sua sobre-expressão foi realizada através de transdução com vetores lentivirais. Os cinco genes considerados – BCL2, GSR, HSPA5, PDIA e XBP1 – pertencem a vias envolvidas em anti-apoptose, metabolismo de glutationa e síntese proteica no retículo endoplasmático. As populações resultantes foram caracterizadas quanto a crescimento celular, produtividade viral, expressão dos componentes virais e dos genes metabólicos.
Verificou-se um aumento de produtividade associada à sobre-expressão dos genes intervenientes na síntese proteica no retículo endoplasmático. Aumentos de 140% foram obtidos com o gene XBP1 – que está associado à resposta de proteínas mal conformacionadas. Aumentos mais modestos, na ordem dos 63% foram obtidos com o gene PDIA2 – que está associado à catálise de pontes dissulfureto. Por último, aumentos de 76% foram observados com HSPA5 – que está associado a um grande leque de processos envolvidos em síntese proteica no retículo endoplasmático. Não foram observados aumentos de produção com genes associados à anti-apoptose nem ao metabolismo de glutationa, nomeadamente BCL2 e GSR.
Os resultados aqui obtidos demonstram que engenharia metabólica é uma estratégia valiosa para o melhoramento de produção de retrovírus recombinantes. Três dos cinco genes abordados levaram ao aumento de produção de retrovírus recombinantes, o que apoia a síntese proteica como um alvo valioso na resolução dos constrangimentos encontrados na produção de retrovírus recombinantes. Este trabalho contribui para o campo da terapia génica baseada em vetores virais. O conhecimento gerado neste trabalho é relevante para outros vetores virais e para engenharia metabólica de linhas celulares humanas.
Gene therapy using viral vectors harnesses naturally occurring viral biological mechanisms to deliver therapeutic genes and control their expression in patient target cells. Several viral gene therapy products have already reached the market, with nearly half being based on recombinant viruses belonging to the Retroviridae family. These are a frequent option since they have large genetic payload capacity, high transduction efficiency, stable genome integration in transcriptionally active loci of dividing and non-dividing cells and sustained long-term expression of the delivered gene. Revenue predictions for viral gene therapy market in 2020 surpass 200 million US dollars, with no decline perspective until at least 2026. Yet, recombinant retroviral synthesis faces several challenges. High non-infective particle concentration – roughly 1 in every 1000 produced particles are infective – and low yields of current production platforms, both of which impose high production costs, present the hardest barriers to overcome in clinical to market transition. Previously, metabolic pathways recruited in recombinant retroviral production were identified. In this work, five target genes were overexpressed in a stable recombinant retrovirus producer cell line through lentiviral vector transduction with incremental target gene expression. The five considered genes – BCL2, GSR, HSPA5, PDIA and XBP1 – belong to pathways involved in anti-apoptosis, glutathione metabolism and endoplasmic reticulum protein synthesis. Resulting populations were characterized for cell growth, recombinant retrovirus productivity, viral components and metabolic gene expression. Increase in productivity was associated to overexpression of genes intervenient in endoplasmic reticulum protein synthesis. Increases of 140% were obtained with XBP1 gene – which is associated to unfolded protein response and thus, correct protein folding. More modest increases of 63% were attributed to PDIA2 gene – which is associated to disulfide bond catalysis. Lastly, increases of 73% can be observed with HSPA5 – which is associated to a wide range of protein synthesis processes within the endoplasmic reticulum. Improvements in productivity were not observed with anti-apoptotic nor glutathione associated genes, namely BCL2 and GSR. The results herein obtained demonstrate cell metabolic engineering as a valuable strategy to improve recombinant retroviral production. Three of the five targeted genes resulted in higher recombinant retroviral production supporting protein synthesis as powerful targets for debottlenecking recombinant retroviral production. This work contributes for the viral gene therapy field. The knowledge generated in this work is relevant to other viral vectors and for metabolic engineering of human derived cell lines.
Gene therapy using viral vectors harnesses naturally occurring viral biological mechanisms to deliver therapeutic genes and control their expression in patient target cells. Several viral gene therapy products have already reached the market, with nearly half being based on recombinant viruses belonging to the Retroviridae family. These are a frequent option since they have large genetic payload capacity, high transduction efficiency, stable genome integration in transcriptionally active loci of dividing and non-dividing cells and sustained long-term expression of the delivered gene. Revenue predictions for viral gene therapy market in 2020 surpass 200 million US dollars, with no decline perspective until at least 2026. Yet, recombinant retroviral synthesis faces several challenges. High non-infective particle concentration – roughly 1 in every 1000 produced particles are infective – and low yields of current production platforms, both of which impose high production costs, present the hardest barriers to overcome in clinical to market transition. Previously, metabolic pathways recruited in recombinant retroviral production were identified. In this work, five target genes were overexpressed in a stable recombinant retrovirus producer cell line through lentiviral vector transduction with incremental target gene expression. The five considered genes – BCL2, GSR, HSPA5, PDIA and XBP1 – belong to pathways involved in anti-apoptosis, glutathione metabolism and endoplasmic reticulum protein synthesis. Resulting populations were characterized for cell growth, recombinant retrovirus productivity, viral components and metabolic gene expression. Increase in productivity was associated to overexpression of genes intervenient in endoplasmic reticulum protein synthesis. Increases of 140% were obtained with XBP1 gene – which is associated to unfolded protein response and thus, correct protein folding. More modest increases of 63% were attributed to PDIA2 gene – which is associated to disulfide bond catalysis. Lastly, increases of 73% can be observed with HSPA5 – which is associated to a wide range of protein synthesis processes within the endoplasmic reticulum. Improvements in productivity were not observed with anti-apoptotic nor glutathione associated genes, namely BCL2 and GSR. The results herein obtained demonstrate cell metabolic engineering as a valuable strategy to improve recombinant retroviral production. Three of the five targeted genes resulted in higher recombinant retroviral production supporting protein synthesis as powerful targets for debottlenecking recombinant retroviral production. This work contributes for the viral gene therapy field. The knowledge generated in this work is relevant to other viral vectors and for metabolic engineering of human derived cell lines.
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Ciências biomédicas Biologia molecular Genética Terapêutica Vetores virais Retrovírus recombinantes Engenharia metabólica
Contexto Educativo
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Instituto de Higiene e Medicina Tropical