WO2019010551A1 - Plateforme génétique pour la surexpression hétérologue associée à la sélection de cellules hautement productrices de protéines - Google Patents

Plateforme génétique pour la surexpression hétérologue associée à la sélection de cellules hautement productrices de protéines Download PDF

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WO2019010551A1
WO2019010551A1 PCT/BR2017/050184 BR2017050184W WO2019010551A1 WO 2019010551 A1 WO2019010551 A1 WO 2019010551A1 BR 2017050184 W BR2017050184 W BR 2017050184W WO 2019010551 A1 WO2019010551 A1 WO 2019010551A1
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expression
atf6
xbp1
proteins
cells
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Portuguese (pt)
Inventor
Marco Aurélio KRIEGER
Rafael Luis KESSLER
Luis Roberto Benghi SOARES
Henrique PRETI
Leon SU
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Instituto De Biologia Molecular Do Parana - Ibmp
SPL Evolution Ltd
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Instituto De Biologia Molecular Do Parana - Ibmp
SPL Evolution Ltd
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Priority to BR112020000406-5A priority patent/BR112020000406B1/pt
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biotechnology, more specifically in the field of mutation or genetic engineering, more specifically genetic manipulation of eukaryotic cells.
  • a genetic manipulation system of eukaryotic cells composed of lineages (i) integration system based on transposons, (ii) regulation system for epigenetic expression by insulators, (iii) co-expression of a reporter gene through a site internal ribosome binding (IRES, bi-cistronic mRNA) or two independent promoters.
  • IRS site internal ribosome binding
  • a platform for heterologous overexpression was created associated with the selection of highly protein-producing cells.
  • EP2316955A1 discloses systems and methods for producing proteins of interest. It employs genetically modified animal or plant cells that have increased ability to process proteins due to co-expression of Atf6 or Xbp1. Although the idea is similar to the genetic platform in question, the object of analysis is of no use since the proteins Atf6 and Xbp1 were expressed individually, transiently and without conditional control.
  • the present invention uses a synthetic gene to express a fusion of Atf6-Xbp1 by integration into the genome of the target cell and in a conditional (tetracycline-regulated) manner, which allows the production of highly secretory cell banks for production and impact.
  • the Atf6-Xbp1 fusion facilitates the positive impact of the activation of the UPR pathway on the productivity of recombinant proteins, since different proteins may have increase specifically mediated by Atf6 and / or Xbp1.
  • the invention NZ544335A discloses a method for increasing expression and protein secretion based on co-expression of Atf-6 and Xbp-1. There is no point in the subject of analysis by using different vectors to achieve higher levels of expression, including antibiotic selection and metatrexate-mediated gene amplification, which implies genetic instability.
  • the present invention makes use of FACS for selection of highly stable positive cells, in addition to expressing the Atf6-Xbp1 fusion.
  • the invention WO201099296 discloses the patent of the commercial transposition system of piggybac, the structure of the vectors being similar to that of the object of analysis. It is no use to invent the genetic platform because the transposon is distinct.
  • the present invention uses the Sleeping Beauty (SB) transposase, which is already in the public domain (WO1999025817A2).
  • SB Sleeping Beauty
  • the expression of recombinant proteins is through the cloning of heterologous genes into plasmids containing genetic elements necessary for expression in host cells, such as compatible promoters, transcription termination signals and selectable elements such as antibiotic resistance genes etc.
  • This allows the selection of the cells containing the heterologous gene from other negative cells in the population.
  • the genetic set containing the heterologous gene and the selection gene be transmissible through the cell generations and for this, integration of such a genetic pool into the genome of the host cell is necessary.
  • the present invention solves the problems cited above for production of recombinant proteins in mammalian cells in two complementary ways: i - development of integration vectors for stable expression of the transgene; ii - development of genetically modified CHO cell lines in order to control and increase expression of the protein of interest.
  • the integration vectors for stable expression of the transgene in eukaryotic cells consist of:
  • FACS fluorescence-activated cell sorter
  • the present invention consists of:
  • the innovation presented here deals with the solution of the difficulties of the state of the art for establishing and maintaining cell banks by introducing a genetic set incorporating elements known as "insulators", which guarantees the unaltered expression of expression cassettes included between two isolators, independent of the site of integration into the genome (Mol Cell, 2013 May 23; 50 (4): 461-74).
  • the expression cassette consists of a cytomegalovirus (CMV) or CAG viral promoter promoting the expression of the gene of interest and an IRES (Mountford, PS and Smith, AG Internai ribosome entry sites and dicistronic RNAs in mammalian transgenesis.
  • CMV cytomegalovirus
  • IRES Internai ribosome entry sites and dicistronic RNAs in mammalian transgenesis.
  • the entire cassette is flanked by two Sleeping Beauty (SB) transposase recombination sites that measure the transposition of the entire cassette to the target cell genome in the presence of the transposase.
  • Transposase SB is transiently expressed by the co-transfection of a second plasmid containing the transposase gene, but incapable of being integrated into the cell's genome.
  • SB Sleeping Beauty
  • an indeterminate number of independent integrations using the Sleeping Beauty-mediated recombination (Crit Rev Biochem Mol Biol. 2016 Oct 4: 1-27) is possible.
  • such a genetic platform can be used for heterologous expression in CHO cells of any protein, including complex proteins composed of various polypeptide chains and / or extensively modified post-translationally.
  • the heterologous overexpression gene platform system has been successfully used for recombinant expression of several proteins, including fibrinogen (3 chains), thrombin, erythropoietin, monoclonal antibodies (2 chains), among
  • a genetic manipulation system of eukaryotic cells composed of a a transposon-mediated gene transformation system, an epigenetic expression-regulating system and, in addition, linked to a reporter system (fluorescent protein).
  • a reporter system fluorescent protein
  • the co-expression of accessory proteins conferring increased productivity of the protein of interest can be regulated in an inducible TetON-like system in modified CHO cells.
  • Figure 1 is a schematic of the different types of vectors available in the Ingres System together with the epizomal vector used for transposal expression of the transposase after transfection.
  • Figure 2 shows the construction variants of the integration vector ("Expression vector") of the ingress system; in “A”, two independent promoters and in “B”, bicistrônico mRNA with IRES.
  • FIG 3 shows the two tetracycline-inducible systems (TetON).
  • TetON TetR
  • B a bidirectional promoter for rtTA (CHO rtTA Fat).
  • Figure 4 shows selection of highly secretory cells using endoplasmic reticulum labeling (Gordo system).
  • Figure 5 shows the direct correlation between expression level of the reporter gene and productivity of the protein of interest (in this case, for expression of a monoclonal antibody).
  • Figure 6 shows the expression of Thrombin using RFP as the reporter gene and the two steps typically used for selection of homogenous populations expressing high levels of the transgene.
  • transposon element is a DNA sequence capable of moving from one region to another in a cell genome. This phenomenon, called transposition, was discovered by Barbara McCinktock in the 1950s, which earned him the Nobel Prize in Medicine in 1983.
  • Deoxyribonucleic acid transposons act by excising the deoxyribonucleic acid sequence from its initial position and inserting it in a new location by the action of an enzyme called transposase. Because of these characteristics, DNA tranposons are widely used in the field of biotechnology for gene marking, especially for the insertion of new genes into a given genome.
  • insulators are elements present in the DNA sequence that prevent interactions between adjacent chromatin domains (Miklos Gaszner & Gary Felsenfeld Nature Reviews Genetics 7, Insulators: exploiting transcriptional and epigenetic mechanisms). These elements maintain an active chromatin configuration and, thus, the gene (s) regulated by it has high transcription rates.
  • the third component used in the developed vectors is the use of fluorescent reporters.
  • the main reporter used in this system are fluorescent proteins derived from GFP (green fluorescent protein).
  • the green fluorescent protein is a protein produced by the cnidarian Aequorea victoria, which emits fluorescence in the green zone of the visible spectrum. The gene encoding this protein has been isolated and is currently widely used in genetic engineering techniques.
  • the proteins (NM_021950.3), CD25 (NM_000417) and CD90 / Thy1.2 (NM_001311160.1) can be used as reporter associated with detection with specific antibodies conjugated to fluorophores, which provides for a greater comprehensiveness of the use of the technique in relation to the reporter gene and fluorescence detection spectra.
  • the presence of the reporter gene allows the selection of recombinant protein producing cells by separating cells with the highest fluorescence intensity in FACS type flow cytometry equipment.
  • Fig. 1 shows an overview of the operation of the platform of the ingress system and its different constructions.
  • This system basically has two different vectors: one containing the gene encoding the Sleeping Beauty transposase and one second containing the gene of interest to be expressed together with a label.
  • the first vector (“Vector transposase”) aims at the transient expression of the transposase, which will be responsible for integrating the target sequence, constructed in the second vector ("Expression vector”) into the genome through the recombination of the inverted terminal repeat sequences , sequences recognized by the transposase).
  • the expression vector is composed of 5 ' ITR and 3' ITR recombination sequences, insulators for epigenetic regulation, CAG promoter (CA 2733104C) or CMV, the gene of interest followed by the IRES sequence for internal binding to the ribosome.
  • the reporter gene may vary as can be seen in Fig. 1B. After transfection with both vectors, the cells are selected by FACS (Fig. 1A and Fig. 1B).
  • Fig. 2 Two forms of expression of the reporter gene are shown in the vectors of the genetic platform for heterologous overexpression (Fig. 2): I) gene expression system containing two independent, hybrid, non-proprietary (PF) promoters of high efficiency (Fig. 2A); (Fig. 2B).
  • PF non-proprietary promoters of high efficiency
  • FIG. 2B Two forms of expression of the reporter gene are shown in the vectors of the genetic platform for heterologous overexpression
  • PF hybrid, non-proprietary promoters of high efficiency
  • Fig. 2B Two forms of expression of the reporter gene are shown in the vectors of the genetic platform for heterologous overexpression (Fig. 2): I) gene expression system containing two independent, hybrid, non-proprietary (PF) promoters of high efficiency (Fig. 2A); (Fig. 2B).
  • PF hybrid, non-proprietary promoters of high efficiency
  • FIG. 2B Two forms of expression of the reporter gene are shown in the vectors of the genetic platform for heterologous overexpression (Fig. 2):
  • Such an element is a nucleotide sequence that allows translation initiation in the middle of a messenger RNA sequence (Jerry Pelletier & Nahum Sonenberg Nature 334, 320-325, 1988 Internai initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA ).
  • Fig. 2 there are two schematic representations of the developed genetic systems (two independent promoters or using IRES).
  • the genetic platform also enables an increase in the expression levels of the protein of interest by the use of genetically modified CHO cells. Since the synthesis, folding, and post-translational modifications of secreted proteins occurs in the endoplasmic reticulum (RE) and Golgi complex, there is a direct relationship between ER size, number of ribosomes and the cell's ability to synthesize. Highly secretory cells, such as plasmocytes, typically have a very prominent ER and a cellular and molecular phenotype directed to the expression and secretion of proteins. This highly secretory phenotype is here termed "Fat”.
  • the increase in protein production capacity by the RE-Golgi system depends on the cell's ability to cope with high protein processing demand. To do this, the cells must adapt to the stress state of RE-Golgi. Otherwise, they may enter the cell death pathway (apoptosis).
  • the stress adaptation of RE-Golgi triggers the unfolded protein response (UPR), which involves the activation of gene expression regulatory proteins (among them Atf6 and Xbp1), which in turn induce expression of a set of enzymes (chaperones, enzymes involved in glycosylation, formation of disulfide bridges and vesicle traffic) capable of dealing with the high demand for protein synthesis.
  • Atf6 and Xbp1 proteins are the major signal transducers known in the UPR pathway, and in vivo antibody secreting plasmocytes express high levels of these proteins (Nat Immunol 2016 Mar; 17 (3): 323-30).
  • Co-expression of such proteins in recombinant systems can significantly increase the level of expression of a transgene (Mol Immunol. 2004 Jul; 41 (9): 919-27; EP2316955A1, US2005106222 (A1)).
  • the central component of the genetic platform presented here is the ability to modify the eukaryotic cell line through the introduction of a modified synthetic gene containing the synthesis code of two fused proteins (Atf6 and Xbp1), which induce the stress-adaptive phenotype of the endoplasmic reticulum of the cell, what we call induction of the Fat phenotype.
  • Atf6-Xbp1 on the tetracycline control using a CHO cell line expressing the tatracyclin repressor (NC_00521 1 .1), TetR (Adv Drug Deliv Rev. 2009 Jul 2; 61 (7-8): 527-41).
  • the "Fat Tetro CHO" line (Fig. 3A) was produced using the Integration System integration vectors described above, but in this case using a CMV promoter (WO2012099540A1). After obtaining a stable line expressing TetR (CHO TetR strain), the Atf6-Xbp1 fusion was transfected.
  • the fusion was cloned into a modified version of the insert vector containing 8 replicates of the tetracycline operator (TetO, US5654168A) above the expression cassette promoter, generating the 8TetO expression cassette -Atf6-Xbp1, which was integrated into the CHO-TetR line, then producing the line referred to herein as CHO TetR Fat (Fig. 3A), in which the conditional expression of the Atf6-Xbp1 fusion is regulated by the addition of tetracycline (or the analogue doxycycline).
  • TetO tetracycline operator
  • Atf6-Xbp1 expression is induced.
  • the induction of a cellular metabolism focused on protein synthesis and traffic, or fat phenotype occurs after reaching a biomass of interest, which culminates in a 5 to 20-fold increase in productivity in relation to the use of the system vectors in unmodified CHO cells.
  • a second method of regulated expression based on a bidirectional promoter transcribed by rtTA (reversed tetracycline trans-activator) (Adv Drug Deliv Rev. 2009 Jul 2; 61 (7-8): 527-41).
  • the modified CHO line, CHO rtTA Fat was constructed using the scheme shown in Fig. 3B.
  • the line "CHO rtTA” was constructed after transfection of a vector of the system, which expresses the rtTA (reversed tetracycline trans-activator). The rtTA line was then used for transfection of the vector containing a bi-directional promoter for rtTA and reporter gene.
  • rtTA transcribes the bidirectional promoter, leading to expression of the Atf6-Xbp1 fusion and the reporter gene (Fig. 3B).
  • Fig. 3A and 3B the addition of tetracycline, or doxycycline, activates the expression of the Atf6-Xbp1 fusion and the reporter gene.
  • modified CHO cell lines described above (CHO TetR and CHO rtTA), together with the modified versions of the ingot system vectors containing tetracycline-specific inducible promoters can also be used as a basis for regulated expression of any transgene of interest .
  • Some sulfonated fluorescent ureas specifically label components of the endoplasmic reticulum (ER) and show fluorescence intensity directly proportional to ER size.
  • ER-specific fluorescent dye ER-Tracker Green or ER-Tracker Red
  • cells with large RE strong labeling for ER-Tracker
  • Productivity analyzes by ELISA evidenced an approximately three-fold increase in expression levels in ER-Tracker positive cells (Fig. 4, Gordo +).
  • the genetic platform (Ingresso System) consists of the use of a heterologous expression system to optimize the industrial process of production and quality control of recombinant proteins, from (a) the use of optimized cell lines, (b (c) favoring high levels of transcription due to the presence of insulators (independent of the integration site), (d) the possibility of real-time monitoring of expression levels (reporter protein), (e) the use of an efficient chromosomal integration system, ) ability to select highly productive cell populations and (f) ability to induce a high productivity secretory phenotype by the regulated expression of Atf6-Xbp1.
  • the use of these genetic systems allows for transposon-mediated transfection and insertion, which significantly increase the percentage of incorporation of the intact cassette into the genome of the target cell.
  • the Insulator gene system provides high levels of expression of the genes inserted in the system (gene of interest and reporter gene) and, in addition, the selection of cells by fluorescence intensity allows to select populations (highly homogeneous subpopulations or clones) in a significantly shorter time than traditional methodologies, in addition to considerably higher expression yields due to the use of Fat Tet-ON lines.
  • the functionality of the system is evident in the direct correlation of the expression level of the reporter gene with the productivity of the protein of interest. In Fig. 5 an example is shown using the production of a monoclonal antibody.
  • the cells were transformed using the RFP protein as the reporter gene. After eleven days of growth, the transformed culture underwent the first round of selection, where the cells that were detectable fluorescence were collected in relation to the non-transfected cells. At this time, less than 5% of the culture cells had this expression profile. However, after 18 days of transformation (7 days after the first cycle of selection by cell sorting), more than 50% of the cells present high production levels of the exogenous proteins, as measured by the reporter gene. These results can be evaluated in Fig.
  • III- Possibility of control of the levels of expression and monitoring of population dynamics of the culture in real time.

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Abstract

La présente invention concerne i) la construction de vecteurs d'intégration pour la surexpression hétérologue, associés à un système de sélection de cellules et de surveillance en temps réel de la productivité par co-expression de protéines rapporteuses, ii) la sélection de cellules hautement sécrétrices de la protéine d'intérêt par marquage avec des sondes fluorescentes spécifiques pour le réticulum endoplasmique (RE), iii) la construction d'une lignée génétiquement modifiée de cellules CHO pour l'expression conditionnelle de protéines d'intérêt sous le contrôle de tétracycline/doxycycline (système TetON), iv) développement d'un gène synthétique contenant les formes actives des protéines Atf6 et Xbp1 fusionnées en un unique polypeptide et de lignées de cellules CHO exprimant la fusion Atf6-Xbp1 sous le contrôle (ou non) de tétracycline, v) surexpression et haute productivité de protéines recombinantes par co-expression régulée d'Atf6-Xbp1.
PCT/BR2017/050184 2017-07-10 2017-07-10 Plateforme génétique pour la surexpression hétérologue associée à la sélection de cellules hautement productrices de protéines Ceased WO2019010551A1 (fr)

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BR112020000406-5A BR112020000406B1 (pt) 2017-07-10 Plataforma genética para superexpressão heteróloga associada à seleção de células altamente produtoras de proteínas

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Cited By (4)

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CN108728477A (zh) * 2017-04-24 2018-11-02 华东理工大学 一种高效的转座突变系统及构建方法
US11672874B2 (en) 2019-09-03 2023-06-13 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration
US12319925B2 (en) 2021-05-11 2025-06-03 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration
US12404502B2 (en) 2015-09-03 2025-09-02 CHO Plus, Inc. Hybrid yeast cell lines for high level production of recombinant protein

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12404502B2 (en) 2015-09-03 2025-09-02 CHO Plus, Inc. Hybrid yeast cell lines for high level production of recombinant protein
CN108728477A (zh) * 2017-04-24 2018-11-02 华东理工大学 一种高效的转座突变系统及构建方法
US11672874B2 (en) 2019-09-03 2023-06-13 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration
US12319925B2 (en) 2021-05-11 2025-06-03 Myeloid Therapeutics, Inc. Methods and compositions for genomic integration

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