GB2540786A - Codon optimised tet repressor proteins - Google Patents

Abstract

The invention provides a codon optimised tetracycline repressor (Tet R) sequence comprising the nucleic acid sequence of SEQ ID NO:4 or SEQ ID NO: 5, the sequence being optimised for expression in human cells. The sequence is preferably administered in a vector, which can be a plasmid vector. The vector may also include CMV promoter sequences and lentiviral genes. The invention also provides a cell comprising the vector and a method of transfecting a cell with the vector.

Description

DRAFT

Codon Optimised Tet Repressor Proteins

TECHNICAL FIELD

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.

BACKGROUND

The antibiotic effect of tetracycline antibiotics is due to their ability to inhibit protein synthesis in bacteria, mainly by binding to the mRNA translation complex. However, bacterial resistance to such antibiotics has evolved. 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). In the absence of a tetracycline antibiotic, the TetR protein forms a homodimer that binds with high affinity to DNA sequences (tet operators; tetO) in the promoter region of the resistance gene and represses transcription of the gene. However, if present, the antibiotic binds to TetR and reduces the affinity of TetR to the tetO binding site; the TetR:tetracycline complex dissociates from the TetO and allows transcription of the bacteria's resistance gene.

The 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)). In one such system, 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 ai, Hum. Gen. Ther. 9, 1939-1950 (1998)).

Improvements in tet-inducible systems that increase the level of expression of the tetR protein in the eukaryotic cell, or that increase the level of induced expression of a target gene of interest, or that decrease the basal expression of the target gene of interest, are desirable.

SUMMARY OF THE INVENTION

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 (S) inducing expression of the tet-inducible gene(s) under conditions suitable for the production of lentiviral vectors.

BRIEF DESCRIPTION OF THE FIGURES

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 106 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 106 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).

DETAILED DESCRIPTION

Tet-inducible gene expression systems. The 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)). The TetR protein is highly specific for its tetO DNA binding sites, and can recognize such binding sequences even in more complex eukaryotic genomes.

In one such system, 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. Additionally, the system may comprise a control expression plasmid, such as one that expresses B-galactosidase upon induction with tetracycline.

One such tetracycline-regulated expression system for mammalian cells is the T-Rex(TM) system (Invitrogen by Life Technologies), which utilizes a regulatory vector (pcDNA™6/TR) which encodes a tet repressor under the control of the human CMV promoter.

As used herein, "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. Various 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. As used herein, "tetracyclines" or "tetracycline compound" comprises, but is not limited to: tetracycline, oxytetracycline, doxycycline, minocycline, chlortetracycline, tigecycline.

Codon usage and Codon Optimization: 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. In the context of viral vector production in a eukaryotic host cell (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. However, it has been reported that 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 a I, Nature 495(7439): 111-115 (2013); Kimchi-Sarfaty, Science 315(5811):525-8 (2007)).

In host cells carrying heterologous genes, codon optimization of the heterologous gene sequence to suit the host cell (e.g., to correspond to the codon usage bias of the host cell's species or genus) may increase the rate of gene expression. In the context of tet-inducible systems, 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 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.

The use of lentiviral vectors in gene therapy of diseases like Diabetes mellitus or Murine haemophilia A has been described. The aim of gene therapy is to modify the genetic material of living cells for therapeutic purposes, and it involves the insertion of a functional gene into a cell to achieve a therapeutic effect. 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. Once the viral genome has been reverse transcribed from the transfer vector into double-stranded DNA (dsDNA) and has entered the nucleus of the cell, this nucleic acid is integrated into the host genome aided by the viral enzyme, integrase.

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. Alternatively, the packaging genes can be integrated into the packaging cell line's genome.

By isolating the lentiviral gag, pol, env and rev genes and by permitting their expression with accompanying (operationally linked) promoters, 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.

The various lentivirus generations are described in the following references. First generation: Naldini et al., Science, 272(5259):263-7 (1996). Second generation: Zufferey et al., Nat Biotechnol. 15(9):871-5 (1997). Third generation: Dull et al., J Virol. 72(11):8463-7 (1998). A review on the development of lentiviral vectors can be found in Sakuma et al., Biochem J. 443(3):603-18 (2012); Picango-Castro et al., Lentiviral-mediated gene transfer—a patent review. Exp Opin Therap Patents 18(5):525-539 (2008).

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

The skilled person will appreciate that 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_REX™ (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. One such promoter is the human Cytomegalovirus (CMV) promoter. The vector may further comprise a selection marker to aid in generating stable cell lines transfected with the vector.

The skilled person will appreciate that 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. In one embodiment of the present invention, the vector carrying the codon optimized TetR sequence is a plasmid; in another embodiment, the vector is a Bacterial Artificial Chromosome (BAC).

One aspect of the present invention is plasmid expression vectors, whose specific function is to express TetR in a mammalian host cell. Expression vectors generally have a promoter sequence that drives expression of the transgene, and a marker gene that will help select cells actively expressing the transgene. Accordingly, 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. Furthermore, 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. ACE and BAC system

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 (ACE) refers to a fully functional mammalian chromosomal structure that can stably replicate and segregate alongside endogenous mammalian chromosomes so that it is transmittable to host cell progeny. Therefore, the ACE provides an extra genomic locus for targeted integration of heterologous DNA into a host cell. A Bacterial Artificial Chromosome (BAC) 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.

Codon Optimisation of T repressor

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.

Accordingly, in a first embodiment, the invention provides an isolated nucleotide comprising a codon optimised (CO) Tet repressor sequence of SEQ ID NO:3 or SEQ ID NO: 4.

In another aspect, 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.

In another aspect, 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. In one embodiment the host cell is a Human Embryonic Kidney 293 cell (HEK-293) or a HEK 293T cell. In one aspect of the invention, 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:6 (CMV promoter sequence)

TGGCCATTGC ATACGTTGTA TCCATATCAT AATATGTACA TTTATATTGG CTCATGTCCA ACATTACCGC CATGTTGACA TTGATTATTG ACTAGTTATT AATAGTAATC AATTACGGGG TCATTAGTTC ATAGCCCATA TATGGAGTTC CGCGTTACAT AACTTACGGT AAATGGCCCG CCTGGCTGAC CGCCCAACGA CCCCCGCCCA TTGACGTCAA TAATGACGTA TGTTCCCATA GTAACGCCAA TAGGGACTTT CCATTGACGT CAATGGGTGG AGTATTTACG GTAAACTGCC CACTTGGCAG TACATCAAGT GTATCATATG CCAAGTACGC CCCCTATTGA CGTCAATGAC GGTAAATGGC CCGCCTGGCA TTATGCCCAG TACATGACCT TATGGGACTT TCCTACTTGG CAGTACATCT ACGTATTAGT CATCGCTATT ACCATGGTGA TGCGGTTTTG GCAGTACATC AATGGGCGTG GATAGCGGTT TGACTCACGG GGATTTCCAA GTCTCCACCC CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT CGTTTAGTGA ACCG SEQ ID N0:7 (CMV-T02(IH); CMV promoter sequence modified to contain tet operons)

GTTGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGAACCA AAATCAACGG GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTCTC CCTATCAGTG ATAGAGATCT CCCTATCAGT GATAGAGATC GTCGACGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCG SEQ ID N0:8 (CMV-T02(LT); CMV promoter sequence modified to contain tet operons)

GTTGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC ΓΓΆΆΓ(ΖΆΓΓΓ ΓΓΓ,ΓΓΓΰΦΦΟ ΆΓΠΨΓΆ ΆΨΆΆ ψπΆΓΠ.ΨΆΨΓ.Ψ ΨΓΓΓΆΨΆΓΖΨΆ ΆΓΓΖΓΓΆΆΨΆΠ.

GGACTTTCCA TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGAACCA AAATCAACGG GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTCTC CCTATCAGTG AT AGAGAT C T CCCTATCAGT GAT AGAGAT C GTCGACGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCG

In one embodiment of the invention, 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 (S) 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.

As used herein, the term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

References cited herein are incorporated by reference herein in their entirety.

The term "about" in relation to a numerical value x means, for example, X plus or minus 10%.

The invention will be further described by reference to the following, non-limiting, figures and examples.

EXAMPLES

Example 1: Codon optimisation of TetR A wild-type (WT) Tet Repressor nucleotide sequence was selected for codon optimization (SEQ ID NO:l, NCBI Gene ID: 4924774).

ATGTCTAGAT TAGATAAAAG TAAAGTGATT AACAGCGCAT TAGAGCTGCT

TAATGAGGTC GGAATCGAAG GTTTAACAAC CCGTAAACTC GCCCAGAAGC

TAGGTGTAGA GCAGCCTACA TTGTATTGGC ATGTAAAAAA TAAGCGGGCT

TTGCTCGACG CCTTAGCCAT TGAGATGTTA GATAGGCACC ATACTCACTT

TTGCCCTTTA GAAGGGGAAA GCTGGCAAGA TTTTTTACGT AATAACGCTA

AAAGTTTTAG ATGTGCTTTA CTAAGTCATC GCGATGGAGC AAAAGTACAT

TTAGGTACAC GGCCTACAGA AAAACAGTAT GAAACTCTCG AAAATCAATT

AGCCTTTTTA TGCCAACAAG GTTTTTCACT AGAGAATGCA TTATATGCAC

TCAGCGCTGT GGGGCATTTT ACTTTAGGTT GCGTATTGGA AGATCAAGAG

CATCAAGTCG CTAAAGAAGA AAGGGAAACA CCTACTACTG ATAGTATGCC

GCCATTATTA CGACAAGCTA TCGAATTATT TGATCACCAA GGTGCAGAGC

CAGCCTTCTT ATTCGGCCTT GAATTGATCA TATGCGGATT AGAAAAACAA CTTAAATGTG AAAGTGGGTC TTAA (624)

The WT TetR gene encodes the following amino acid sequence (SEQ ID NO:2): MSRLDKSKVI NSALELLNEV GIEGLTTRKL AQKLGVEQPT LYWHVKNKRA LLDALAIEML DRHHTHFCPL EGESWQDFLR NNAKSFRCAL LSHRDGAKVH LGTRPTEKQY ETLENQLAFL CQQGFSLENA LYALSAVGHF TLGCVLEDQE HQVAKEERET PTTDSMPPLL RQAIELFDHQ GAEPAFLFGL ELIICGLEKQ LKCESGS (207)

The WT sequence was altered to provide two codon optimised sequences based on codon usage. The sequences were further optimised for ~3750 iterations using all of the following parameters: codon tandem repeats (repetition of identical codons); secondary structure (hairpin loop formation); GC distribution; long range repeats; DNA motifs (ARE motifs); Open Reading Frame (ORF) optimisation; restriction sites - AsiSI, BamHI, BsiWI, EcoRI, Mlul, Nhel, Nhel, Pad, Pmel, Pvul, Sail, Xbal, Xhol; cryptic splice sites - human. A human codon optimized (humCO) sequence was generated (SEQ ID NO:3) where a 70% Leto frequency threshold for rare codons was set, discarding any codons below 70% of the theoretical ratio (all codons in equal amounts). ATGTCTAGGC TGGACAAGTC CAAGGTGATC AACTCCGCCC TGGAGCTCCT TAACGAGGTG GGGATTGAAG GGTTGACTAC CCGGAAACTG GCTCAGAAAC TGGGCGTCGA ACAGCCAACC CTGTACTGGC ACGTGAAGAA CAAGCGCGCT TTGCTGGATG CACTTGCCAT CGAGATGCTG GACAGGCACC ACAC AC AC T T TTGCCCCTTG GAGGGCGAAT CCTGGCAGGA CTTCCTGAGG AACAACGCCA AGAGTTTCCG CTGTGCCCTG TTGAGTCACA GGGACGGAGC AAAGGTTCAC CTTGGAACCA GACCCACCGA GAAGCAGTAC GAGACTCTGG AGAACCAGCT GGCCTTCCTG TGTCAGCAGG GCTTTAGCCT GGAGAACGCT CTGTACGCAC TGTCTGCCGT TGGCCACTTT ACACTCGGAT GCGTGCTGGA AGACCAGGAG CATCAGGTTG CCAAGGAAGA GAGGGAGACC CCTACCACTG ACAGCATGCC TCCACTTCTG AGACAGGCCA TCGAGCTCTT TGACCACCAG GGAGCAGAAC CCGCTTTCCT CTTCGGGCTG GAACTGATCA TCTGCGGCCT CGAAAAGCAG CTCAAGTGCG AAAGCGGCAG CTGA (624) A GSK Codon Optimized (GSKCO) sequence was generated (SEQ ID NO:4) where a 90% Leto frequency threshold for rare codons was set, discarding any codons below 70% of the theoretical ratio (all codons in equal amounts). Sequence differences in SEQ ID NO:4 (compared to WT TetR SEQ ID NO:l) are indicated by underlining:

ATGAG CAGGC TGGAC AAGAG CAAGG TGATC AACAG CGCCC TGGAG CTGCT

GAACG AGGTC GGCAT CGAGG GCCTG ACAAC CAGGA AGCTG GCCCA GAAAC

TCGGA GTGGA GCAGC CCACC CTGTA CTGGC ACGTG AAGAA CAAGC GCGCC

CTCCT GGACG CCCTG GCTAT TGAGA TGCTG GACAG GCACC ACACC CACTT

CTGCC CACTG GAGGG CGAGA GCTGG CAGGA CTTCC TGAGG AACAA CGCCA

AGAGC TTCAG GTGTG CCCTG CTGAG CCATA GGGAC GGCGC CAAGG TGCAC

CTGGG CACCA GGCCC ACCGA GAAGC AGTAC GAGAC CCTGG AGAAC CAGCT

GGCCT TCCTC TGCCA GCAGG GCTTC AGCCT GGAGA ATGCA CTGTA CGCCC

TGAGC GCCGT GGGCC ACTTC ACCCT GGGGT GCGTG CTGGA AGACC AGGAA

CACCA GGTGG CCAAG GAGGA GAGGG AGACC CCCAC CACCG ACTCT ATGCC

CCCCC TGCTG AGGCA GGCCA TCGAA CTGTT CGATC ACCAG GGCGC CGAGC

CCGCC TTTCT GTTCG GCCTG GAGCT GATCA TCTGC GGCCT GGAGA AGCAG CTGAA GTGCG AGAGC GGCTC CTGA (624)

The codon optimisation of both SEQ ID N0:3 and SEQ ID N0:4 preserves the amino acid sequence for the Tet R protein (SEQ ID N0:2)

The pcDNA™6/TR vector is commercially available from Invitrogen. The pcDNA™6/TR vector contains the TetR gene under the control of the Human CMV immediate early promoter, and the blasticidin (bsd) resistance gene under the control of the SV40 early promoter (for expression of BSD in mammalian cells to allow for selection of stably transfected mammalian cells).

Examination of the TetR sequence contained in the pcDNA™6/TR vector revealed that it includes a sequence comprising multiple cloning sites immediately upstream of the TetR stop codon (SEQ ID NO:5):

ATGTCTAGAT TAGATAAAAG TAAAGTGATT AACAGCGCAT TAGAGCTGCT TAATGAGGTC GGAATCGAAG GTTTAACAAC CCGTAAACTC GCCCAGAAGC TAGGTGTAGA GCAGCCTACA TTGTATTGGC ATGTAAAAAA TAAGCGGGCT TTGCTCGACG CCTTAGCCAT TGAGATGTTA GATAGGCACC ATACTCACTT TTGCCCTTTA GAAGGGGAAA GCTGGCAAGA TTTTTTACGT AATAACGCTA AAAGTTTTAG ATGTGCTTTA CTAAGTCATC GCGATGGAGC AAAAGTACAT TTAGGTACAC GGCCTACAGA AAAAC AG TAT GAAACTCTCG AAAATCAATT AGCCTTTTTA TGCCAACAAG GTTTTTCACT AGAGAATGCA TTATATGCAC TCAGCGCTGT GGGGCATTTT ACTTTAGGTT GCGTATTGGA AGATCAAGAG CATCAAGTCG CTAAAGAAGA AAGGGAAACA CCTACTACTG ATAGTATGCC GCCATTATTA CGACAAGCTA TCGAATTATT TGATCACCAA GGTGCAGAGC CAGCCTTCTT ATTCGGCCTT GAATTGATCA TATGCGGATT AGAAAAACAA CTTAAATGTG AAAGTGGGTC CGCGTACAGC GGATCCCGGG AATTCAGATC TTATTAA

The multiple cloning site (MCS) sequence is at residues 621-653, and the stop codon at residues 654-657, of SEQ ID N0:5 (referred to below as the WT-Invitrogen TetR sequence).

Example 2: Comparing TetR protein expression with WT-TetR and CO-TetR

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.

Table 2

Hek 293T cells were plated at 106 cells per well in a six-well plate, cultured overnight at 37°C and 5% C02, 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 ImageJ and the values plotted in figure 2.

Where the individual DNA constructs were transfected at a quantity of 1.25ug/transfection, little distinction could be made between the individual constructs and there was no difference in the background as compared to the UT control. However, when 2.5ug DNA was used per transfection, there appearred to be a greater amount of TetR protein in the humCO-TetR and GSKCO-TetR samples compared to the WT-TetR and pcDNA6/TR. Furthermore, the codon optimisation of GSKTetR appeared to be capable of increasing protein expression 2 fold.

TetR protein expression was compared using Western blot band density. Band density was quantified and compared using ImageJ, a Java-based image analysis software package available for download from the National Institutes of Health at http : // rsbweb (dot) nih (dot) gov /ij / download (dot) html. Results are shown in Figure 2A and 2B, where UT = untransfected control; pcDNA™6/TR is the regulatory vector pcDNA™6/TR (Invitrogen); WT is TetR of SEQ ID NO:l; hCO is TetR of SEQ ID NO:3; and GSKCO is TetR of SEQ ID NO:4. Each experimental condition was run in either duplicate or triplicate; each bar represents one experiment.

These results indicate that cells transfected with vectors containing the GSKCO optimized TetR sequence reliably provided the greatest TetR protein expression, compared to WT and hCO optimized TetR sequences.

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 respectively. In this example, where cells are not exposed to Doxycycline, 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). Background flourescence is established by the Untransfected (UT) cells. It is seen that both the hCO and GSKCO TetR vectors are capable of reducing gene expression to below that obtained with WT TetR and, in some cases, below that of UT cells.

Figure 4 graphs viral titer averages from Figure 3 on a linear scale.

Conclusion

Codon optimised TetR sequences useful in tet-inducible gene expression systems in mammalian, specifically human, cells are provided herein.

Claims (13)

We claim:

1. A codon optimised tetracycline repressor (TetR) sequence comprising the nucleic acid sequence of SEQ ID NO:4 or SEQ ID NO: 5.

2. A vector comprising a codon optimised TetR sequence according to claim 1.

3. A vector according to claim 2, wherein the vector comprises a human cytomegalovirus (CMV) promoter.

4. A vector according to claim 4, wherein the CMV promoter sequence is selected from SEQ ID NO:7 and SEQ ID NO:8.

5. A vector according to any one of claims 2-4, further comprising a selection marker gene.

6. A vector according to any one of claims 2-5, where said vector is a plasmid expression vector.

7. A vector according to claim 6, further comprising a selection marker gene.

8. A cell comprising a vector according to any one of claims 2-7.

9. A cell according to claim 8, wherein the cell is selected from the group consisting of HEK 293 and 293T cells.

10. The cell according to claim 8 or claim 9, wherein the vector is present episomally.

11. A method of transfecting a cell with a plasmid expression vector according to claim 7, wherein the transfection method comprises: (i) contacting a cell with a plasmid expression vector solution containing one or more plasmid expression vectors according to claim 6; and (ii) growing the cells in a growth media under conditions which allow for selection of cells which express the selection marker gene.

12. A method of transfection according to claim 11 wherein the plasmid expression vector solution used for the transfection further comprises (i) a plasmid containing a lentiviral gene.

13. A method of transfection according to claim 11 wherein the cell comprises a plasmid containing a lentiviral gene.