title: Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.
abstract: Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: A F
authors.initials: AF
authors.lastname: Rodrigues
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: M R
authors.initials: MR
authors.lastname: Guerreiro
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: A S
authors.initials: AS
authors.lastname: Formas-Oliveira
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: P
authors.initials: P
authors.lastname: Fernandes
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering Group, Magdeburg, Germany.
authors.forename: A-K
authors.initials: AK
authors.lastname: Blechert
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering Group, Magdeburg, Germany.
authors.forename: Y
authors.initials: Y
authors.lastname: Genzel
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: P M
authors.initials: PM
authors.lastname: Alves
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, USA.
authors.forename: W S
authors.initials: WS
authors.lastname: Hu
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal. avalente@itqb.unl.pt.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. avalente@itqb.unl.pt.
authors.forename: A S
authors.initials: AS
authors.lastname: Coroadinha
mesh_terms: D002460:Cell Line; D015536:Down-Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D019344:Lactic Acid / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D012190:Retroviridae / Q000254:growth & development*; D019562:Viral Load; D014776:Virus Cultivation / Q000379:methods*
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D019344:Lactic Acid; D017346:Protein Serine-Threonine Kinases
keywords: functional genomics; glycolysis; hypoxia inducible factor 1; lactate; pyruvate dehydrogenase kinase; recombinant virus