WO2017017131A1 - Protéines répresseurs tet à codons optimisés - Google Patents

Protéines répresseurs tet à codons optimisés Download PDF

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WO2017017131A1
WO2017017131A1 PCT/EP2016/067883 EP2016067883W WO2017017131A1 WO 2017017131 A1 WO2017017131 A1 WO 2017017131A1 EP 2016067883 W EP2016067883 W EP 2016067883W WO 2017017131 A1 WO2017017131 A1 WO 2017017131A1
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vector
cell
tetr
cells
expression
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Tejinder Kaur Bhinder
Alan Peter Lewis
Celeste PALLANT
Conrad VINK
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Glaxosmithkline Intellectual Property Development Limited
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Definitions

  • the present invention relates to improved tet repressor proteins, nucleotide sequences encoding such repressor proteins, and vectors containing such sequences, for use in inducible gene expression systems in human cells.
  • Tetracycline resistance genes may be located on mobile genetic elements such as plasmids.
  • the expression of certain tet resistance genes is controlled by transcriptional regulators known as tetracycline Repressors (Tet repressors or TetR proteins).
  • Tet repressors transcriptional regulators known as tetracycline Repressors (Tet repressors or TetR proteins).
  • Tet repressors transcriptional regulators known as tetracycline Repressors (Tet repressors or TetR proteins).
  • Tet repressors transcriptional regulators known as tetracycline Repressors (Tet repressors or TetR proteins).
  • Tet repressors transcriptional regulators known as tetracycline Repressors (Tet repressors or TetR proteins).
  • Tet repressors transcriptional regulators known as tetracycline Repressors (T
  • tetracycline (tet) resistance system from Escherischia coli has been utilized to provide inducible gene expression constructs suitable for use in eukaryotic cells (see, e.g., Gossen and Bujard, PNAS USA 89(12):5547-51 (1992)).
  • a target gene of interest is operably linked to a tet-inducible promoter. Binding of tetracycline to tetR homodimers results in derepression of the promoter (Yao et al., Hum. Gen. Ther. 9, 1939- 1950 (1998)).
  • One aspect of the present invention are codon optimised nucleotide sequences encoding a tetracycline repressor protein, and vectors comprising such sequences.
  • a further aspect of the present invention is host cells comprising an expression vector comprising a codon optimised nucleotide sequence encoding a tetR protein.
  • a further aspect of the present invention is a method of transfecting a cell with an expression vector comprising a codon optimised nucleotide sequence encoding a tetR protein.
  • a further aspect of the present invention is a method of producing a lentiviral vector packaging cell comprising transfecting a host cell with (a) an expression vector comprising a codon optimised nucleotide sequence encoding a tetR protein, and (b) expression vectors comprising a lentiviral GagPol sequence, a lentiviral Rev sequence, and a VSVg sequence, where one or more of said lentiviral sequences is tet-inducible.
  • a further aspect of the present invention is a method of producing a lentiviral vector, the method comprising (1) transfecting a host cell with (a) an expression vector comprising a codon optimised nucleotide sequence encoding a tetR protein, and (b) expression vectors comprising a lentiviral GagPol sequence, a lentiviral Rev sequence, and a VSVg sequence, where one or more of said lentiviral sequences is tet-inducible; (2) culturing said transfected host cell, and (3) inducing expression of the tet-inducible gene(s) under conditions suitable for the production of lentiviral vectors.
  • Figure 1 provides a generalized schematic of lentiviral vector production and host cell transduction.
  • a packaging host cell is transfected with plasmid expression vectors encoding lentiviral packaging genes and a transgene of interest (Green Fluorescent Protein or GFP in this schematic).
  • Viral protein expression results in the assembly of proteins at the host membrane, and generation of a viral vector.
  • Figure 2A is a graph of TetR protein expression in cells transfected with different vector constructs containing TetR. Cells were transfected with 1.25ug of plasmid construct per 10 6 cells. Untransfected cells were used as controls.
  • Figure 2B is a graph of TetR protein expression in cells transfected with one of four different vector constructs containing TetR. Cells were transfected with 2.5ug of plasmid construct per 10 6 cells. Untransfected cells were used as controls.
  • Figure 3 illustrates viral titre produced in cells transfected with BACmod-WT lenti packaging constructs and TetR vectors containing either WT (SEQ ID NO:l), hCO (SEQ ID NO:3) or GSKCO (SEQ ID NO:4) TetR. Cells that did not contain the TetR vector (No TetR) and untransfected cells (UT) were used as controls The Y-axis is viral titre produced (TU/mL).
  • Figure 4 graphs viral titer averages from Figure 3 on a linear scale.
  • Figure 5A - 5D provides schematics of the pcDNA6/TR plasmid ( Figure 5A), pMA- BACmod-WT-TR-lr-ZeoR plasmid ( Figure 5B), pMA-BACmod-humTR-lr-ZeoR plasmid ( Figure 5C), and pMA-BACmod-GSKTR-lr-ZeoR plasmid ( Figure 5D).
  • Tet-inducible gene expression systems Tet-inducible gene expression systems.
  • the tetracycline (tet) resistance system from Escherischio coli has been utilized to provide inducible gene expression constructs suitable for use in eukaryotic cells (see, e.g., Gossen and Bujard, PNAS USA 89(12):5547-51 (1992)).
  • the TetR protein is highly specific for its tetO DNA binding sites, and can recognize such binding sequences even in more complex eukaryotic genomes.
  • a host cell is designed to stably express the Tet repressor, such as by containing a plasmid encoding the TetR protein.
  • the host cell also contains plasmids or other genetic vectors that carry a gene of interest under the control of a promoter, where the promoter contains one or more tetracycline operator (tetO) sites. Due to the presence of TetR protein, expression of the gene of interest is repressed in the absence of tetracycline (though a basal level of transcription may occur) and induced in the presence of tetracycline.
  • the system may comprise a control expression plasmid, such as one that expresses B-galactosidase upon induction with tetracycline.
  • Rex(TM) system (I nvitrogen by Life Technologies), which utilizes a regulatory vector (pcDNA 6/TR) which encodes a tet repressor under the control of the human CMV promoter.
  • induced expression means the expression of an identified gene of interest in response to an inducer, where the induced level of expression is greater than the basal (un-induced) level of expression of that gene.
  • inducers are known in the art; the appropriate inducer depends on the genetic expression system, as would be known by one of skill in the art. In tet-inducible systems, the inducer is a tetracycline antibiotic.
  • Tetracyclines are a group of antibiotics having a common molecular structure containing four hydrocarbon rings, and include naturally-occuring compounds as well as synthetic and semi-synthetic compounds.
  • tetracyclines or “tetracycline compound” comprises, but is not limited to: tetracycline, oxytetracycline, doxycycline, minocycline, chlortetracycline, tigecycline.
  • Codon bias refers to differences in the frequency of occurrence of synonymous nucleotide triplets in the coding DNA.
  • the degeneracy found in the genetic code allows each amino acid to be encoded by between one and six synonymous codons allowing many alternative nucleic acid sequences to encode the same protein (Gustafsson, Govindarajan, and Minshull, Trends Biotechnol. 22(7): 346-53 (2004)).
  • the frequencies with which different codons are used within genes is referred to codon usage and can vary significantly between two genera or species of organisms, or among multiple genera or species of organisms. Different species often show particular preferences for one of the several codons that encode the same amino acid.
  • Codon optimisation is a technique used in to modify genetic sequences with the intent of increasing the rate of expression of a gene in a heterologous expression system; typically the nucleotide sequence encoding a protein of interest is codon optimized such that the codon usage more closely resembles the codon bias of the host cell, while still coding for the same amino acid sequence.
  • the nucleotide sequence encoding a protein of interest is codon optimized such that the codon usage more closely resembles the codon bias of the host cell, while still coding for the same amino acid sequence.
  • packaging cell it is therefore of interest to develop a highly efficient vector production process in order to achieve a suitable viral titre.
  • Codon optimisation tailored to an expression system increases the speed and efficiency of translation.
  • increasing the rate of translation by codon optimisation can lead to misfolding of the resultant protein, negatively impacting its stability or substrate specificity (Zhou et al, Nature 495(7439): 111-115 (2013); Kimchi-Sarfaty, Science 315(5811):525-8 (2007)).
  • codon optimization of the heterologous gene sequence to suit the host cell may increase the rate of gene expression.
  • vectors providing efficient expression of functional Tet repressor proteins in eukaryotic host cells (including human cells) that contain tet-inducible genes of interest are desirable.
  • Viral Vectors/ packaging cell lines Viral Vectors/ packaging cell lines. Viral vectors designed to modify a subject's stem cells by the incorporation of a therapeutic gene are based on members of the retrovirus family due to the ability of retrovirus to integrate their genetic payload into the host's genome (provirus). Retroviral vectors are designed to keep the essential proteins required for packaging and delivery of the viral genome, but any non-essential accessory proteins including those responsible for the viral disease profile are removed. Retroviral vectors are thus Retroviruses modified to carry a therapeutic gene for delivery into target cells. Highly efficient vector production processes are desirable in order to achieve a suitable viral titre.
  • Lentiviral vectors produced according to the present invention can be used to transfect target cells and induce the expression of the gene of potential therapeutic interest.
  • Lentiviral vectors such as those based upon Human Immunodeficiency Virus Type 1 (HIV-1) are widely used as they are able to integrate into non-proliferating cells. Both coding and cis-acting sequences are required for viral vector construction, however, these sequences are separated to prevent the generation of replication-competent retroviruses.
  • dsDNA double- stranded DNA
  • the lentiviral vector has been made replication defective by splitting the genome into separate parts (Dull et al. 8463-71), which can then be individually expressed by plasmid vectors (transient transfection), or expressed in specifically engineered packaging cell lines.
  • Producer cell lines can be generated by transfecting a cell line capable of packaging viral vectors, such as the 293T cell, with the lentiviral packaging genes on individual plasmids which also carry unique eukaryotic selection markers. These producer cells are then kept in constant selection and are described as being stably transfected.
  • the packaging genes can be integrated into the packaging cell line's genome.
  • the viral proteins generated can package the separate transfer vector, including an LTR flanked therapeutic gene, but renders the resulting particle unable to generate further virions.
  • This multipart genome also known as the 3rd generation lentiviral vector packaging system (Dull et al. 8463-71) also avoids the risk of recombining with other wild type retroviruses that may be present in the target cell.
  • Figure 1 provides a generalized schematic of lentiviral vector production and host cell transduction.
  • a packaging host cell is transfected with plasmid expression vectors encoding lentiviral packaging genes and a transgene of interest (Green Fluorescent Protein or GFP in Figure 1).
  • Viral protein expression results in the assembly of proteins at the host membrane, and generation of a viral vector
  • lentiviral production is normally carried out in mammalian cells.
  • Cells for use in the invention include, but are not limited to, HEK 293, 293T, and T_REXTM (Life Technologies) cell lines.
  • Tet system in Lentiviral packaging cells Commercial-scale production of lentiviral vectors comprising therapeutic genes requires packaging cell lines capable of generating large quantities of viral vector. Inducible expression of viral packaging proteins is desirable in order to minimize metabolic burden on the host cell and/or where the expressed viral protein is cytotoxic to the cell. According to an aspect of the present invention, this can be achieved by placing expression of one or more viral genes under the control of a tet- inducible promoter, and introducing a vector encoding the TetR protein into the packaging host cell, where the tetR nucleotide sequence has been codon-optimized for that host cell.
  • the codon optimized tet-repressor sequence of the present invention can be carried by a vector and operationally linked to a promoter for expression in mammalian cells.
  • a promoter for expression in mammalian cells.
  • One such promoter is the human Cytomegalovirus (CMV) promoter.
  • CMV Cytomegalovirus
  • the vector may further comprise a selection marker to aid in generating stable cell lines transfected with the vector.
  • a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed.
  • the four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
  • the present invention includes all vectors as covered by this definition.
  • the vector carrying the codon optimized TetR sequence is a plasmid; in another embodiment, the vector is a Bacterial Artificial Chromosome (BAC).
  • the vectors of the present invention may include a promoter such as the human cytomegalovirus (CMV) immediate early promoter, spleen focus-forming virus (SFFV) promoter, Rous sarcoma virus (RSV) promoter, or human elongation factor 1-alpha (EFla) promoter.
  • CMV human cytomegalovirus
  • SFFV spleen focus-forming virus
  • RSV Rous sarcoma virus
  • EFla human elongation factor 1-alpha
  • the vectors of the present invention may also include a selection marker such as enzymes encoding resistance to an antibiotic, e.g., neomycin, puromycin, hygromycin blasticidin, or zeocin.
  • Artificial chromosomes have the capacity to accommodate and express heterologous genes inserted therein. Furthermore, large segments of DNA, multiple copies of the heterologous gene, and linked promoter element(s) can be retained in the artificial chromosomes, thereby providing a high-level of expression of the retroviralprotein(s).
  • Artificial Chromosome Expression refers to a fully functional mammalian cells
  • the ACE provides an extra genomic locus for targeted integration of heterologous DNA into a host cell.
  • a Bacterial Artificial Chromosome is a functional bacterial chromosomal structure, capable of accommodating up to 350kb of heterologous DNA that can stably replicate within a bacterial cell alongside the bacterial genome. Therefore, the BAC can be used to amplify large segments of DNA in a bacterial system and capable to shuttle this DNA to mammalian cells for either transient transfection, stable transfection or targetted integration into the host cell chromosomes, including the ACE.
  • the present inventors constructed a codon-optimized (CO) Tet repressor suitable for use in viral packaging cell lines where at least one viral gene is under the control of a tet- inducible promoter.
  • CO codon-optimized
  • the invention provides an isolated nucleotide comprising a codon optimised (CO) Tet repressor sequence of SEQ ID NO:3 or SEO ID NO: 4.
  • the invention provides a vector comprising a codon optimised (CO) Tet repressor sequence of SEQ ID NO:3 or SEQ ID NO: 4.
  • the vector comprises a promoter operably linked to the CO tet repressor sequence.
  • a further aspect of the present invention is a host cell comprising such an expression vector.
  • the invention provides a eukaryotic host cell comprising at least one heterologous target gene of interest, where: (a) the host cell comprises a vector comprising a codon optimised (CO) Tet repressor sequence of the present invention; and (b) the target gene is under the control of a promoter that includes at least one Tet Operon, such that the expression of the target gene is tet-inducible.
  • the host cell may be a human cell.
  • the host cell is a Human Embryonic Kidney 293 cell (HEK-293) or a HEK 293T cell.
  • the promoter that is operably linked to a target gene is a Cytomegalovirus promoter that additionally contains one or more Tet Operon sequences; such a promoter may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7-8.
  • SEQ ID NO:7 ( ⁇ :MV-T02(IH); ( MV promoter ;equence modified to contain tet operons)
  • the codon optimised sequence(s) or vector(s) are present episomally. In another embodiment of the invention, the codon optimised sequence(s) or vector(s) are integrated into the cell's genome.
  • a further embodiment of the invention provides a method of transfecting a cell with one or more of the expression vectors of the invention, wherein the transfection method comprises: (i) contacting the cell with a plasmid expression vector solution containing one or more plasmid expression vectors according to the invention; and (ii) growing the cells in a growth media.
  • a further aspect of the present invention is a method of producing a lentiviral vector packaging cell comprising transfecting a host cell with (a) an expression vector according to the present invention comprising a codon optimised nucleotide sequence encoding a tetR protein, and (b) expression vectors comprising a lentiviral GagPol sequence, a lentiviral Rev sequence, and a VSVg sequence, where one or more of said lentiviral sequences is tet- inducible.
  • a further aspect of the present invention is a method of producing a lentiviral vector, the method comprising (1) transfecting a host cell with (a) an expression vector according to the present invention comprising a codon optimised nucleotide sequence encoding a tetR protein, and (b) expression vectors comprising a lentiviral GagPol sequence, a lentiviral Rev sequence, and a VSVg sequence, where one or more of said lentiviral sequences is tet- inducible; (2) culturing said transfected host cell, and (3) inducing expression of the tet- inducible gene(s) under conditions suitable for the production of lentiviral vectors.
  • Insertion of a vector into the target cell is usually called transformation for bacterial cells and transfection for eukaryotic cells, although insertion of a viral vector may also be called transduction.
  • the skilled person will be aware of the different viral transfection methods commonly used, which include, but are not limited to, the use of physical methods, chemical reagents or cationic lipids. Many transfection methods require the contact of solutions of plasmid DNA to the cells, which are then grown and selected for a marker gene expression. The viral supernatants may then be harvested, purified and titred for further use.
  • compositions “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
  • a wild-type (WT) Tet Repressor nucleotide sequence was selected for codon optimization (SEQ ID NO:l, NCBI Gene ID: 4924774).
  • the WT TetR gene encodes the following a mino acid sequence (SEQ ID NO:2):
  • the WT seq uence was altered to provide two codon optimised sequences based on codon usage.
  • the sequences were further optimised for ⁇ 3750 iterations using a l l of the fol lowing pa ra meters: codon tandem repeats (repetition of identical codons); seconda ry structu re (hairpin loop formation); GC distribution; long ra nge repeats; DNA motifs (ARE motifs); Open Reading Fra me (ORF) optimisation; restriction sites - AsiSI, Ba m H I, BsiWI, EcoRI, Mlul, N hel, N hel, Pad, Pmel, Pvul, Sa il, Xba l, Xhol; cryptic splice sites - huma n.
  • huma n codon optimized (humCO) sequence was generated (SEQ I D NO:3) where a 70% Leto frequency threshold for ra re codons was set, discarding any codons below 70% of the theoretica l ratio (al l codons in eq ual a mounts).
  • GSK Codon Optimized (GSKCO) sequence was generated (SEQ I D NO:4) where a 90% Leto frequency threshold for ra re codons was set, disca rding a ny codons below 70% of the theoretica l ratio (al l codons in equal a mounts). Seq uence differences in SEQ I D NO:4 (compa red to WT TetR SEQ I D NO:l) a re indicated by underlini ng:
  • the pcDNATM6/TR vector is commercial ly availa ble from I nvitrogen.
  • pcDNATM6/TR vector contains the TetR gene under the control of the Huma n CMV i m mediate early promoter, a nd the blasticidin (bsd) resista nce gene under the control of the SV40 early promoter (for expression of BSD in mam ma lian cells to a llow for selection of stably tra nsfected mamma lia n cells).
  • the mu ltiple cloning site (MCS) seq uence is at residues 621-653, and the stop codon at residues 654-657, of SEQ I D NO:5 (referred to below as the WT-l nvitrogen TetR sequence).
  • MCS mu ltiple cloning site
  • Human 293T cells were transfected with four different plasmid constructs (see Figures 5A-%D), in two doses, as shown in Table 2. Each transfection (plasmid + dose) was run in triplicate. Un-transfected 293T cells were used as controls.
  • Hek 293T cells were plated at 10 6 cells per well in a six-well plate, cultured overnight at 37°C and 5% C0 2 , and transfected with 1.2ug or 2.5ug of DNA per construct.
  • Transfections were set up in triplicate and performed using the transfection reagent lipofectamine 2000 according to manufacturer's instruction.
  • the transfected and control cells were cultured for a further 48 hours and subsequently harvested for western blot analysis. Protein was isolated from the transfected and control cells, seperated by SDS-PAGE and the TetR protein band detected with a TetR specific antibody and visualised with a secondary antibody conjugated to HRP and HRP substrate. Protein loading was verified by a GAPDH control.
  • Total TetR protein band intensity was quantified by the programme I mageJ and the values plotted in figure 2.
  • WT is TetR of SEQ ID NO:l
  • hCO is TetR of SEQ ID NO:3
  • GSKCO is TetR of SEQ ID NO:4.
  • Example 3 Control of tet-inducible lentiviral packaging genes
  • Viral vector was made using lenti packaging constructs. 293T cells were transiently transfected with plasmid vectors carrying lentiviral packaging genes including GagPol, VSVg and Rev, each individually driven by a Tet02 controlled CMV promoter. Also included in the transfection were a GFP expressing transfer vector and TetR vectors containing either WT (SEQ ID NO:l), hCO (SEQ ID NO:3) or GSKCO (SEQ ID NO:4) TetR all utilising a non-Tet operon controlled CMV promoter. Cells that did not contain the TetR vector (No TetR) and untransfected cells (UT) were used as positive and negative viral vector controls
  • the presence of TetR protein is expected to decrease viral production levels.
  • 48 hours post transfection the viral vector supernatants were harvested. Viral titre of the supernatants was measured by the ability of the viral vector to deliver the GFP expression cassette into 293T cell chromatin. 293T cells were transduced with viral vector supernatant, cultured for 72hours and the percentage GFP positive cells analysed by FACS.
  • Results are shown in Figure 3, where the Y-axis is viral titre produced (TU/mL).
  • Figure 4 graphs viral titer averages from Figure 3 on a linear scale.
  • TetR sequences useful in tet-inducible gene expression systems in mammalian, specifically human, cells are provided herein.

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Abstract

L'invention concerne une séquence répresseur de tétracycline à codons optimisés (TetR) comprenant la séquence d'acide nucléique SEQ ID NO : 4 ou SEQ ID NO : 5, L'invention concerne également une cellule comprenant le vecteur et un procédé de transfection d'une cellule avec le vecteur.
PCT/EP2016/067883 2015-07-28 2016-07-27 Protéines répresseurs tet à codons optimisés WO2017017131A1 (fr)

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GB1513242.6A GB2540786A (en) 2015-07-28 2015-07-28 Codon optimised tet repressor proteins
GB1513242.6 2015-07-28

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WO2017017131A1 true WO2017017131A1 (fr) 2017-02-02

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JP2022505095A (ja) * 2018-10-17 2022-01-14 グラクソスミスクライン、インテレクチュアル、プロパティー、ディベロップメント、リミテッド アデノ随伴ウイルスベクタープロデューサー細胞株

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CN111386126A (zh) * 2017-10-25 2020-07-07 Nouscom股份公司 真核细胞系
CN111386126B (zh) * 2017-10-25 2024-01-30 Nouscom股份公司 真核细胞系
JP2022505095A (ja) * 2018-10-17 2022-01-14 グラクソスミスクライン、インテレクチュアル、プロパティー、ディベロップメント、リミテッド アデノ随伴ウイルスベクタープロデューサー細胞株
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GB201513242D0 (en) 2015-09-09

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