Engineered cell lines for increased protein production
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Publication Number
US-10577613-B1
Publication Date
2020-03-03
Expiration Date
Abstract
The present disclosure relates to engineered cells that include genetic alterations leading to up- or down-regulation of certain genes in the cells for improved production of a recombinant protein. Also provided are methods of preparing and using such cells.
Core Innovation
The invention provides an isolated engineered mammalian cell with one or more genetic alterations that increase expression or activity of hk1 (hexokinase 1), MAPK3 (mitogen-activated protein kinase 3), CAP (catabolite activator protein) and Nrf2 (nuclear factor (erythroid-derived 2)-like 2), as compared to a corresponding mammalian cell without the genetic alterations. The engineered cell lines are described in the context of recombinant protein production.
The engineered cells are configured to avoid the Warburg effect via coordinated gene modulation across categories that include catabolism and anabolic/secretion/growth/anti-apoptosis pathways. The framework includes gene categories associated with glycolysis and Krebs/energy metabolism, protein folding and secretion, and transcription initiation.
Engineered clones with different expression and growth characteristics are compared. Clone 76, having much higher HK1 expression, is associated with higher growth/confluency and higher titer than clone 54, and clone 73 with catabolic and survival enhancements shows high titer. The engineered cell and its genetic modulation are further described using example gene sets, polynucleotide types and cell hosts, including CHO cells.
Claims Coverage
The partial content provides two independent claim themes, covering genetic alterations that increase expression or activity of hk1, MAPK3, CAP, and Nrf2 versus a corresponding unaltered mammalian cell, and isolated mammalian cells containing exogenous polynucleotides encoding hk1, MAPK3, CAP, and Nrf2. Across these independent claim themes, the main inventive features are the defined set of four genes or protein activities and, depending on the claim, the nature of the genetic change.
Increased expression or activity of hk1, MAPK3, CAP and Nrf2
An isolated engineered mammalian cell comprising one or more genetic alterations resulting in increased expression or activity of hk1 (hexokinase 1), MAPK3 (mitogen-activated protein kinase 3), CAP (catabolite activator protein) and Nrf2 (nuclear factor (erythroid-derived 2)-like 2), as compared to a corresponding mammalian cell without the genetic alterations.
Exogenous polynucleotides encoding hk1, MAPK3, CAP and Nrf2
An isolated mammalian cell comprising one or more exogenous polynucleotides encoding hk1 (hexokinase 1), MAPK3 (mitogen-activated protein kinase 3), CAP (catabolite activator protein) and Nrf2 (nuclear factor (erythroid-derived 2)-like 2).
Overall, the independent claim coverage centers on engineered mammalian cells defined by modulation of hk1, MAPK3, CAP, and Nrf2, either by genetic alterations that increase expression or activity relative to an unaltered corresponding cell, or by inclusion of exogenous polynucleotides encoding those factors.
Stated Advantages
Improve recombinant protein production while avoiding the Warburg effect through balanced catabolic and anabolic/secretion/growth/anti-apoptosis pathway modulation.
Higher growth/confluency and higher titer are associated with increased HK1 expression in compared clones.
Provide engineered clones with high titer when catabolic and survival enhancements are present.
Documented Applications
Recombinant protein production in engineered mammalian/CHO cell lines configured for gene up- or down-regulation to improve output while avoiding the Warburg effect.
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