WO2024213573A1

WO2024213573A1 - Inducible expression system

Info

Publication number: WO2024213573A1
Application number: PCT/EP2024/059697
Authority: WO
Inventors: Maysam MANSOURI; Matthias BOZZA
Original assignee: Vector Biopharma Ag
Priority date: 2023-04-13
Filing date: 2024-04-10
Publication date: 2024-10-17

Abstract

The present invention provides a versatile inducible eukaryotic expression system. The system enables the high-level expression of any gene of interest and is devoid of any leakiness. The gene expression of interest can be effectively turned on and shut off upon introducing small molecules. The system is controlled at the DNA, the RNA and at protein level.

Description

BHIP-C09-09 PCT Inducible expression system Field of the invention The present invention provides a versatile inducible eukaryotic expression system. The system enables the high-level expression of any gene of interest and is devoid of any leakiness. The gene expression of interest can be effectively turned on and shut off upon introducing small molecules. The system is controlled at the DNA, the RNA and at protein level. Background of the invention Numerous systems are known that can be exploited for the expression of genes in eukaryotic cells. Gene expression can be turned on at a specific time or in response to specific signals. In its simplest form such inducible system uses a gene promoter that is responsive to specific signals, such as the addition of certain chemicals or changes in temperature. The gene of interest is placed under the control of this promoter, so that its expression can be controlled by the induction signal, thereby allowing the expression of genes in a temporal and spatial manner. Inducible eukaryotic expression systems however still have several shortcomings. One of the main challenges is the difficulty in achieving consistent and reproducible induction. This can be due to variations in the response of the induction signal, or differences in the level of expression between cells or between experiments. In addition, the induction signal may not be specific enough, leading to unintended effects on other genes or pathways. Another problem associated with inducible expression systems is the leakiness of many systems, i.e. a certain degree of expression even in the absence of an inducer. Finally, the induction of gene expression in many systems is not reversible. WO2021/232632 tries to address the leakiness of expression systems by utilizing multiple copies of the respective operators, minimal promoter sequences and adjusting the positioning of the operator and the TATA box in the promoter. The problem is solved to some extent, but the system still exhibits some leakiness. Leakiness is also still observed in the hepato-specific bidirectional system utilized in US2012/0225933 and the expression system of WO2023/050644. Similarly, US2020/0085021 utilizes a bidirectional system for the expression of heterologous proteins in transgenic animals. Focus of this BHIP-C09-09 PCT study is expression of the gene of interest, not the avoidance of leakiness. Appl Microbiol Biotechnol (2018) 102, 6357-72 reviews expression systems in fungi. There it is described, that there is no expression of the gene of interest in the absence of the inducer. What is not described is the continued production of the proteins of interest after removal of the inducer via transcripts produced during the ON state. This problem is addressed by the present invention, in which hammerhead structures are utilized to completely switch off expression after removal of the induced, making expression of the gene of interest completely reversible. The present invention describes the iterative development of a highly versatile eukaryotic expression system that addresses all aforementioned shortcomings. The system shows no expression in the Off state and a high expression rate upon induction, and it was engineered to be reversible. It furthermore allows for the tissue-specific expression of genes. This system provides maximal spatiotemporal control of the gene expression by combining multiple levels of control; DNA, RNA, and protein. Summary of the invention The present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and BHIP-C09-09 PCT wherein said system leads to the expression of the gene of interest in the presence of an inducer. In certain embodiments, said first compound responsive domain comprises ABI or a functional fragment or derivative of ABI and said second compound responsive domain is PYL1 or a functional fragment or derivative of PYL1, or wherein said first compound responsive domain is PYL1 or a functional fragment or derivative of PYL1 and said second compound responsive domain is ABI or a functional fragment or derivative of ABI. In certain embodiments, said first compound responsive domain comprises a polypeptide of SEQ ID No.1 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.2 or a functional fragment or derivative thereof. In other embodiments, said first compound responsive comprises a polypeptide of SEQ ID No.2 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.1 or a functional fragment or derivative thereof. In certain embodiments, said inducer is abscisic acid or a functional fragment or derivative of abscisic acid. In certain embodiments, said first compound responsive domain comprises VanR or a functional fragment or derivative of VanR and said second compound responsive domain is VanR or a functional fragment or derivative of VanR. In certain embodiments, said first compound responsive comprises a polypeptide of SEQ ID No.11 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.11 or a functional fragment or derivative thereof. In certain embodiments, said inducer is vanillic acid or a functional fragment or derivative of vanillic acid. In certain embodiments, said first compound responsive domain comprises GAI or a functional fragment or derivative of GAI and said second compound responsive domain is GID or a functional fragment or derivative of GID. In other embodiments, said first compound responsive domain is GID or a functional fragment or derivative of GID and said second compound responsive domain is GAI or a functional fragment or derivative of GAI. In certain embodiments, said first compound responsive comprises a polypeptide of SEQ ID No.12 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No. 13 or a functional fragment or derivative thereof. In other embodiments, said first compound responsive comprises a polypeptide of SEQ ID No.13 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.12 or a functional fragment or derivative thereof. In certain embodiments, said inducer is gibberellic acid or a functional fragment or derivative of gibberellic acid. BHIP-C09-09 PCT In certain embodiments, said operator unit binding domain is TetR or a functional fragment or derivative of TetR. In certain embodiments, said operator unit binding domain comprises a polypeptide of SEQ ID No.3 or a functional fragment or derivative thereof. In certain embodiments, said transactivator is selected from VP16 (SEQ ID No.8), VP64 (SEQ ID No. 9) and VPR (SEQ ID No.10). In certain embodiments, said transactivator is VP16 (SEQ ID No.8). In certain embodiments, said transactivator is VP64 (SEQ ID No. 9). In certain embodiments, said transactivator is VPR (SEQ ID No.10). In certain embodiments, said promoter of said first nucleic acid sequence and the promoter of said second nucleic acid sequence are identical. In certain embodiments, said promoter is a Pol II promoter. In certain embodiments, said promter is a CMV promoter. In certain embodiments, said promoter comprises the nucleic acid sequence of SEQ ID No.4. In certain embodiments, said first nucleic acid of the controller unit comprises a nucleic acid sequence encoding a linker which is located between said operator unit binding domain and said first compound responsive domain. In certain embodiments, said linker is a G4S-NLS-G4S linker or a G4S linker. In certain embodiments, said linker is a G4S-NLS-G4S linker. In certain embodiments, said linker is a G4S linker. In certain embodiments, said linker consists of an amino acid sequence selected from SEQ ID No.5 or SEQ ID No.6. In certain embodiments, said linker consists of the amino acid sequence of SEQ ID No.5. In certain embodiments, said linker consists of the amino acid sequence of SEQ ID No. 6. In certain embodiments, said reporter unit comprises a 3’UTR hammerhead sequence. In certain embodiments, said hammerhead sequence comprises the nucleic acid sequence of SEQ ID No.7. In certain embodiments, the present disclosure relates to a method to express a gene of interest in a eukaryotic target cell, said method comprising a) culturing eukaryotic cells containing the aforementioned expression system, b) adding an inducer to the eukaryotic cells, and c) continue culturing said eukaryotic cells, thereby allowing said eukaryotic cells to express said gene of interest. BHIP-C09-09 PCT Figure legends Figure 1 shows the components of the inducible expression system of the present disclosure. The inducible system is composed of the controller and the reporter unit. The controller unit encodes for the operator unit binding domain fused with the inducer-responsive element monomer and the operator unit transcriptional activator, which is linked with another inducer-responsive element monomer. The reporter unit comprises the DNA binding site of the operator unit binding domain and the gene of interest, and optionally a hammerhead. Figure 2 depicts the functioning of the inducible expression system of the present disclosure. An inducer, such as a plant-derived hormone, triggers the dimerization of the controller monomers forming a functional complex that can bind the DNA binding site on the reporter unit, starting the expression of the gene of interest. Doxycycline prevents the controller's binding to the reporter unit, abolishing the expression of the gene of interest. Figure 3 compares the induction of the expression of a gene of interest triggered by three different plant-derived inducers (abscisic acid, vanillic acid, and gibberellic acid). The abscisic acid generates the most potent gene expression induction, leading to more than 150 fold upregulation over the second- best compound. ABA = abscisic acid, VAN = vanillic acid, GE = gibberellic acid. Figure 4 compares the upregulation of the gene expression from three different transactivators :VP16, VP64, and VPR. VP16 and VP64 lead to a strong gene expression upregulation upon induction with abscisic acid, and VP16 showed 7 times higher inducibility. Figure 5 compares the induction of the gene expression with three different linkers between the operator unit binding domain and the first inducer responsive domain. All linkers tested were proven to be functional, however, the highest signal was generated with the linker G4S-NLS-G4S, which showed a luciferase activity of 3.8-fold higher than the second best. (G= Glycine, S= Serine, NLS= nuclear localization signal) Figure 6 compares the expression systems with and without a hammerhead structure at the 3’ UTR region of the gene of interest. Both constructs provide a robust transgene expression; however, despite having a lower absolute switchability, the construct containing the hammerhead was characterized by essentially a complete absence of basal gene expression in the absence of the inducer. Figure 7 depicts the construct's evolution and the orientation of the expression cassettes. In 1, the operator and the responsive units were placed on three vectors. In 2, the operators were combined in BHIP-C09-09 PCT one vector and tested when launched from the same or two different promoters, while the responsive unit was placed on a separate vector. In 3, all the components were placed on the same vector where the operator units were launched from different promoters, and the responsive elements were tested on the positive and negative DNA strands. Figure 8 shows that all the expression systems tested are functional. The highest level of expression induction was observed in the system where the different components are present on different vectors. Figure 9 shows the inducer-dependent transgene expression. Cells continuously supplemented with the inducer showed a steadily increased transgene expression, while cells treated with abscisic acid (ABA) only at time point 0 showed luciferase activity for 24 hours, which returned to baseline levels within 72 hours. DMSO-treated control cells did not show any reporter gene activity. Figure 10 shows the on-demand reversibility of the expression system with the introduction of doxycycline (DOX). Cells continuously supplemented with abscisic acid showed a steady increase in luciferase activity, while cells treated with ABA at time point 0 and DOX 24h later showed a return of the luciferase activity to baseline levels within 24 hours. DMSO-treated control cells did not show any luciferase activity. Definitions The term "expression system" as used herein refers to a vector or a vector system comprising a gene of interest which is transcribed into mRNA and/or translated into a polypeptide, protein, structural RNA, or another cellular component under appropriate conditions. The expression system may be an in vivo expression system or an in vitro expression system. In its general meaning, the expression system can be a eukaryotic or a prokaryotic expression system. The expression system of the present disclosure is a eukaryotic expression system. The term "vector" as used herein in its most general refers to any intermediate vehicles for a nucleic acid which, for example, enables said nucleic acid to be introduced into prokaryotic and/or eukaryotic host cells and, where appropriate, to be integrated into a genome. Such vectors are preferably replicated and/or expressed in the cell. Vectors comprise plasmids, phagemids, free nucleic acid BHIP-C09-09 PCT molecules, virus-like particles (VLPs), enveloped delivery vehicles (EDVs), lipid nanoparticles (LNPs), adeno-associated viruses (AAVs) and adenoviruses. The terms "control” or “controlled” as used herein in the context of expression systems refers to the willful initiation of expression of a gene of interest. Initiation of expression of the gene of interest can be achieved via the addition of an inducer as described herein. However, alternative ways to initiate the expression of a gene exist, such as the shift to a certain temperature. The terms "expression" and “expressing” are used herein in their most general meaning and include the production of RNA and/or protein. Expression may be transient or stable. With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of messenger RNA directs the assembly of a sequence of amino acids to make a peptide or protein. The terms "transcription" and "transcribing" as used herein relate to a process during which a nucleic acid molecule with a particular nucleic acid sequence (the "nucleic acid template") is read by an RNA polymerase so that the RNA polymerase produces a single-stranded RNA molecule. During transcription, the genetic information in a nucleic acid template is transcribed. Subsequently, the transcribed RNA may be translated into protein. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term "vector" as defined above. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for transcription. The cDNA may be obtained by reverse transcription of RNA. The terms “gene of interest” and “protein of interest” as used herein refer to a gene or protein, as applicable, that is to be transcribed or expressed with the vectors, nucleic acid molecules and methods of the present disclosure. The term “peptide” as used herein refers to as used herein refers to a molecule consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds. A peptide typically consists of not more than twenty amino acids linked via peptide bonds. The term "polypeptide" as used herein refers to a molecule consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds. A polypeptide typically consists of more than twenty amino acids linked via peptide bonds. BHIP-C09-09 PCT The term "nucleic acid" as used herein is art recognized and includes deoxyribonucleic acids (DNA) and a ribonucleic acids (RNA). It also includes chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate, and nucleic acids containing non-natural nucleotides and nucleotide analogs. The terms "DNA" or "DNA molecule" as used herein refers to a molecule which comprises deoxyribonucleotide residues and is entirely or substantially composed of deoxyribonucleotide residues. "Deoxyribonucleotide" relates to a nucleotide which lacks a hydroxyl group at the 2'-position of a beta-D-ribofuranosyl group. The terms "DNA" and "DNA molecule" comprise isolated DNA such as partially or completely purified DNA, essentially pure DNA. Synthetic DNA, and recombinantly generated DNA and includes modified DNA which differs from naturally occurring DNA by addition, deletion, substitution and/or alteration of one or more nucleotides. The terms "RNA" or "RNA molecule" as used herein refers to a molecule which comprises ribonucleotide residues and which is preferably entirely or substantially composed of ribonucleotide residues. The term "ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a beta -D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly generated RNA such as modified RNA which differs from naturally occurring RNA by addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxy nucleotides. These altered RNAs can be referred to as analogs, particularly analogs of naturally occurring RNAs. "Fragment" or "fragment of a nucleic acid sequence" relates to a part of a nucleic acid sequence, i.e. a sequence which represents the nucleic acid sequence shortened at the 5'- and/or 3'-end(s). Preferably, a fragment of a nucleic acid sequence comprises at least 80 percent, preferably at least 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent of the nucleotide residues from said nucleic acid sequence. The term "variant" with respect to, for example, nucleic acid and amino acid sequences, refers to any variants, in particular mutants, viral strains, splice variants, conformations, isoforms, allelic variants, species variants and species homologs. Complete gene sequencing often identifies numerous allelic variants for a given gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, wherein a degenerate nucleic acid according to the invention is a BHIP-C09-09 PCT nucleic acid that differs from a reference nucleic acid in codon sequence due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence. A virus homolog is a nucleic acid or amino acid sequence with a different virus of origin from that of a given nucleic acid or amino acid sequence. According to the invention, nucleic acid variants include single or multiple nucleotide deletions, additions, mutations and/or insertions in comparison with the reference nucleic acid. Deletions include removal of one or more nucleotides from the reference nucleic acid. Addition variants comprise 5'- and/or 3'-terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. Mutations can include but are not limited to substitutions, wherein at least one nucleotide in the sequence is removed and another nucleotide is inserted in its place (such as transversions and transitions), abasic sites, crosslinked sites, and chemically altered or modified bases. Insertions include the addition of at least one nucleotide into the reference nucleic acid. Variants of specific nucleic acid sequences preferably have at least one functional property of said specific sequences and preferably are functionally equivalent to said specific sequences, e.g. nucleic acid sequences exhibiting properties identical or similar to those of the specific nucleic acid sequences. Preferably the degree of identity between a given nucleic acid sequence and a nucleic acid sequence which is a variant of said given nucleic acid sequence will be at least 70 percent, preferably at least 75 percent, preferably at least 80 percent, more preferably at least 85 percent, even more preferably at least 90 percent or most preferably at least 95 percent, 96 percent, 97 percent, 98 percent or 99 percent. The degree of identity is preferably given for a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In preferred embodiments, the degree of identity is given for the entire length of the reference nucleic acid sequence. The term "promoter" or "promoter region" as used herein refers to a nucleic acid sequence which controls synthesis of a transcript, e.g. a transcript comprising a coding sequence, by providing a recognition and binding site for RNA polymerase. The promoter region may include further recognition or binding sites for further factors involved in regulating transcription of said gene. A promoter may control transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible" and initiate transcription in response to an inducer, or may be "constitutive" if transcription is not controlled by an inducer. An inducible promoter is expressed only to a very small extent or not at all, if an inducer is absent. In the presence of the inducer, the gene is "switched on" or the level of transcription is increased. This is usually mediated by binding of a specific transcription factor. BHIP-C09-09 PCT The term "untranslated region" or "UTR" as used herein refers to a region which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. A 3'-UTR, if present, is located at the 3' end of a gene, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" or "3’ untranslated region" does preferably not include the poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) tail (if present), e.g. directly adjacent to the poly(A) tail. A “5'-UTR” or "3’ untranslated region", if present, is located at the 5' end of a gene, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g. directly adjacent to the 5'-cap.5'- and/or 3'-untranslated regions may be functionally linked to an open reading frame, so as for these regions to be associated with the open reading frame in such a way that the stability and/or translation efficiency of the RNA comprising said open reading frame are increased. UTRs are implicated in stability and translation efficiency of RNA. An "isolated molecule" as used herein, is intended to refer to a molecule which is substantially free of other molecules such as other cellular material. The term "recombinant" as used herein refers to an entity or molecules which is made through genetic engineering. Recombinant entities or moieties are not occurring naturally. The terms "transactivator" or “transcriptional activator” as used herein refer to transcriptional activation domain. Exemplary transactivators include VP16, VP64, VP48, VP160, the p65 subdomain (e.g., from NFkB), the activation domain of EDLL and/or the TAL activation domain, histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, and NSD1, histone lysine demethylases such as JHDM2a/b, UTX and JMJD3, histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160 and CLOCK, and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2 and ROS1. The term “ABI” refers to a protein phosphatase of Arabidopsis thaliana (UniProt: P49597) which is a key component and repressor of the abscisic acid (ABA) signaling pathway that regulates numerous ABA responses. ABI has the following amino acid sequence: MEEVSPAIAGPFRPFSETQMDFTGIRLGKGYCNNQYSNQDSENGDLMVSLPETSSCSVSG SHGSESRKVLISRINSPNLNMKESAAADIVVVDISAGDEINGSDITSEKKMISRTESRSL FEFKSVPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHG GSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETV GSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQW NGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACE BHIP-C09-09 PCT MARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVV DLKPRRKLKSKPLN (SEQ ID No. 1) The term “PYL1” refers to the abscisic acid receptor of Arabidopsis thaliana (UniProt: Q8VZS8) required for abscisic acid (ABA)-mediatted responses such as stomatal closure and germination inhibition. ABI has the following amino acid sequence: MANSESSSSPVNEEENSQRISTLHHQTMPSDLTQDEFTQLSQSIAEFHTYQLGNGRCSSL LAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSR ERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEG NSEEDTRLFADTVIRLNLQKLASITEAMNRNNNNNNSSQVR(SEQ ID No. 2) The term “VanR” refers to a negative transcriptional regulator that occurs in bacteria. The VanR utilized in the present disclosure is derived from [Enterococcus avium; UniProt: A0A286Q5Q1] and has the following amino acid sequence: MPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVR LGARGYAARGVSSDQIRDAIEVRGVLEGFAARRLAERGMTAETHARFVALIAEGEALFAA GRLNGEDLDRYAAYNQAFHDTLASAAGNGAVESALARNGFEPFAAAGALALDLMDLPAEY EHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNTKVFEAAASAGAPLGAAWSIR AD (SEQ ID No. 11) The term “GAI” refers to a GRAS family transcription factor family protein from Arabidopsis thaliana (NCBI reference sequence: NP_172945.1) and has the following amino acid sequence: MKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNV QEDDLSQLATETVHYNPAELYTWLDSMLTDLN (SEQ ID No. 12) The term “GID” refers to a gibberellin receptor of the from Arabidopsis thaliana (UniProt: Q9LYC1) and has the following amino acid sequence: MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDGVFSFD VLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIYDTLCRRLV GLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALR AGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGK SLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISA FVNAEC (SEQ ID No. 13) The terms “abscisic acid” and “ABA” refers to a plant hormone (CAS 21293-29-8) involved in many plant developmental processes. Abscisic acid has the following structure: BHIP-C09-09 PCT

The terms “vanillic acid” and “VA” refers a compound (CAS 121-34-6) which is an intermediate in the production of vanillin from ferulic. Vanillic acid has the following structure:

The terms “gibberellic acid” and “GE” refers to a hormone found in plants and fungi (CAS 77-05-5). Gibberellic acid has the following structure:

The terms “TetR”, “Tet repressor” and “Tetracycline repressor”” refers to a protein involved in antibiotic resistance (UniProt: P0ACT4). TetR has the following amino acid sequence: MARLNRESVIDAALELLNETGIDGLTTRKLAQKLGIEQPTLYWHVKNKRALLDALAVEIL ARHHDYSLPAAGESWQSFLRNNAMSFRRALLRYRDGAKVHLGTRPDEKQYDTVETQLRFM TENGFSLRDGLYAISAVSHFTLGAVLEQQEHTAALTDRPAAPDENLPPLLREALQIMDSD DGEQAFLHGLESLIRGFEVQLTALLQIVGGDKLIIPFC (SEQ ID No. 3) The term “linker” in its broadest sense refers to a molecule or moiety linking two adjacent molecules or moieties. Linker length, linker composition and linker orientation can have an influence on the functioning of the adjacent molecules or moieties. The term “hammerhead” or “hammerhead ribozyme” refers to a RNA molecule motif that catalyzes reversible cleavage and joining reactions at a specific site within an RNA molecule. For example, BHIP-C09-09 PCT hammerhead ribozymes are catalytic RNA molecules capable of inducing site-specific cleavage of a phosphodiester bond within an RNA molecule. Hammerhead ribozymes can be used to reduce intracellular level of a specific mRNA coding for a protein. The minimal hammerhead ribozyme is composed of three base paired helices, separated by short linkers of conserved sequence as shown in the crystal structure described in Scott (Cell 199581: 991-1002). The structure-function relationships in ribozymes have been extensively reviewed (see, e.g., Hammann et al, RNA 201218: 871-885). A hammerhead ribozyme can also contain one or more non-naturally occurring nucleotides. Embodiments of the invention The present disclosure provides a novel, versatile eukaryotic inducible expression system. Expression of the gene of interest can be switched on by the addition of an inducer and switched off by the addition of an inhibitor. The sophisticated design of the system ensures that essentially no expression occurs in the off-state, i.e., prior to the addition of the inducer. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and BHIP-C09-09 PCT wherein said system leads to the expression of the gene of interest in the presence of an inducer. The components of the expression system are depicted in Figure 1. The expression system consists of a controller unit and a reporter unit. The reporter unit contains an operator unit and the gene of interest. Optionally the reporter unit additionally comprises a 3’UTR hammerhead sequence. The operator unit is the region to which the operator unit binding domain (which is encoded on the controller unit) binds. The controller unit contains two nucleic acid sequences. The first nucleic acid of the controller unit comprises a promoter and a nucleic sequence encoding the operator unit binding domain and a first compound responsive domain. The second nucleic acid of the controller unit comprises a promoter and a nucleic sequence encoding a second compound responsive domain and a transactivator. The first compound responsive domain and the second compound responsive domain can be dimerized via an inducer. Upon dimerization the transactivator fused to the second compound responsive domain comes into proximity to the operator unit binding domain, thereby activating the transcription from the operator unit. Compound responsive domains The present disclosure makes use of a two compound response domains which dimerize in the present of an appropriate inducer. Three different systems were tested in the present disclosure: ^ a system in which one response domain comprises ABI and the other response domain comprises PYL1. A heterodimer is formed in the presence of the inducer abscisic acid, ^ a system in which both response domain comprise VanR. A homo dimer is formed in the presence of the inducer vanillic acid, ^ a system in which one response domain comprises GAI and the other response domain comprises GID. A heterodimer is formed in the presence of the inducer gibberellic acid. In certain embodiments, said first compound responsive domain comprises ABI or a functional fragment or derivative of ABI. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1. BHIP-C09-09 PCT In certain embodiments, said second compound responsive domain comprises PYL1 or a functional fragment or derivative of PYL1. In other embodiments said second compound responsive domain comprises a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2. In other embodiments, said first compound responsive domain comprises ABI or a functional fragment or derivative of ABI, and said second compound responsive domain comprises PYL1 or a functional fragment or derivative of PYL1. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No. 1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and said second compound responsive domain comprises a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2. In certain embodiments, said first compound responsive domain comprises PYL1 or a functional fragment or derivative of PYL1. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2. In certain embodiments, said second compound responsive domain comprises ABI or a functional fragment or derivative of ABI. In other embodiments said second compound responsive domain comprises a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1. In other embodiments, said first compound responsive domain comprises PYL1 or a functional fragment or derivative of PYL1, and , said second compound responsive domain comprises ABI or a functional fragment or derivative of ABI. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No. 2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2, and said second compound responsive domain comprises a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises BHIP-C09-09 PCT (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and polypeptide of SEQ ID No.1, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and polypeptide of SEQ ID No.2, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.1, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. BHIP-C09-09 PCT In certain embodiments, said first compound responsive domain and said second compound responsive domain comprise VanR or a functional fragment or derivative of VanR. In other embodiments said first compound responsive domain and said second compound responsive domain comprise a polypeptide of SEQ ID No.11 or a functional fragment or derivative of a polypeptide of SEQ ID No.11. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.11, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.11, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. In certain embodiments, said first compound responsive domain comprises GAI or a functional fragment or derivative of GAI. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No.12 or a functional fragment or derivative of a polypeptide of SEQ ID No.12. In certain embodiments, said second compound responsive domain comprises GID or a functional fragment or derivative of GID. In other embodiments said second compound responsive domain comprises a polypeptide of SEQ ID No.13 or a functional fragment or derivative of a polypeptide of SEQ ID No.13. BHIP-C09-09 PCT In other embodiments, said first compound responsive domain comprises GAI or a functional fragment or derivative of GAI, and said second compound responsive domain comprises GID or a functional fragment or derivative of GID. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No. 12 or a functional fragment or derivative of a polypeptide of SEQ ID No.12, and said second compound responsive domain comprises a polypeptide of SEQ ID No.13 or a functional fragment or derivative of a polypeptide of SEQ ID No.13. In certain embodiments, said first compound responsive domain comprises GID or a functional fragment or derivative of GID. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No.13 or a functional fragment or derivative of a polypeptide of SEQ ID No.13. In certain embodiments, said second compound responsive domain comprises GAI or a functional fragment or derivative of GAI. In other embodiments said second compound responsive domain comprises a polypeptide of SEQ ID No.12 or a functional fragment or derivative of a polypeptide of SEQ ID No.12. In other embodiments, said first compound responsive domain comprises GID or a functional fragment or derivative of GID, and , said second compound responsive domain comprises GAI or a functional fragment or derivative of GAI. In other embodiments said first compound responsive domain comprises a polypeptide of SEQ ID No. 13 or a functional fragment or derivative of a polypeptide of SEQ ID No.13, and said second compound responsive domain comprises a polypeptide of SEQ ID No.12 or a functional fragment or derivative of a polypeptide of SEQ ID No.12. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, BHIP-C09-09 PCT (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and polypeptide of SEQ ID No.12, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.23, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and polypeptide of SEQ ID No.13, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.12, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. BHIP-C09-09 PCT Inducer The expression system of the present disclosure can be induced by the addition of an inducer. The inducer leads to the dimerization of the first compound responsive domain and the second compound responsive domain. Since the first compound responsive domain is fused to the operator unit binding domain and the second compound responsive domain is fused to the transactivator, the transactivator comes into proximity of the operator unit binding domain, thereby inducing expression of the gene of interest. The inducers exemplified in present disclosure are abscisic acid (ABA), vanillic acid (VA) and gibberellic acid (GE). Abscisic acid leads to the dimerization of ABI and PYL1. Vanillic acid leads to the dimerization of VanR (homodimer). Gibberellic acid leads to the dimerization of GAI and GID. Therefore, in certain embodiments the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and ABI or a functional fragment or derivative of ABI, (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising PYL1 or a functional fragment or derivative of PYL1 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and BHIP-C09-09 PCT wherein said inducer is abscisic acid. In other embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid. In certain embodiments the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and PLY1 or a functional fragment or derivative of PLY1, BHIP-C09-09 PCT (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising ABI or a functional fragment or derivative of ABI and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid. In other embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2, (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid. BHIP-C09-09 PCT In certain embodiments the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and VanR or a functional fragment or derivative of VanR, (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising VanR or a functional fragment or derivative of VAnR and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is vanillic acid. In other embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.11 or a functional fragment or derivative of a polypeptide of SEQ ID No. 11, (b) a second nucleic acid sequence comprising BHIP-C09-09 PCT (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.11 or a functional fragment or derivative of a polypeptide of SEQ ID No.11, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is vanillic acid. In certain embodiments the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and GAI or a functional fragment or derivative of GAI, (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising GID or a functional fragment or derivative of GID and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is gibberellic acid. In other embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, BHIP-C09-09 PCT wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.12 or a functional fragment or derivative of a polypeptide of SEQ ID No. 12, (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.13 or a functional fragment or derivative of a polypeptide of SEQ ID No.13, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is gibberellic acid. In certain embodiments the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and GID or a functional fragment or derivative of GID, (b) a second nucleic acid sequence comprising (i) a promoter and, BHIP-C09-09 PCT (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising GAI or a functional fragment or derivative of GAI and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is gibberellic acid. In other embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.13 or a functional fragment or derivative of a polypeptide of SEQ ID No. 13, (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.12 or a functional fragment or derivative of a polypeptide of SEQ ID No.12, and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is gibberellic acid. BHIP-C09-09 PCT Operator unit binding domain The expression system of the present disclosure makes use of a operator unit binding domain. Said operator unit binding domain binds to the operator unit of the reporter unit. If the first compound responsive domain dimerized with the first compound responsive domain, i.e. if the inducer is present, the transactivator comes into proximity with the operator unit binding domain, thereby inducing the expression of the gene of interest. The operator unit binding domain exemplified in the present disclosure is TetR. Therefore, in certain embodiments the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said operator unit binding domain is TetR or a functional fragment or derivative of TetR. Preferably said first compound responsive domain is ABI, preferably a polypeptide comprising the amino acid sequence of SEQ ID No. 1, said second compound responsive domain is PYL1, preferably a polypeptide comprising the amino acid sequence of SEQ ID No.2, and said inducer is BHIP-C09-09 PCT abscisic acid. Alternatively, said first compound responsive domain is PYL1, preferably a polypeptide comprising the amino acid sequence of SEQ ID No. 2, said second compound responsive domain is ABI, preferably a polypeptide comprising the amino acid sequence of SEQ ID No.1, and said inducer is abscisic acid. In other embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said operator unit binding domain comprises a polypeptide of SEQ ID No.3 or a functional fragment or derivative of TetR. Preferably said first compound responsive domain is ABI, preferably a polypeptide comprising the amino acid sequence of SEQ ID No. 1, said second compound responsive domain is PYL1, preferably a polypeptide comprising the amino acid sequence of SEQ ID No.2, and said inducer is abscisic acid. Alternatively, said first compound responsive domain is PYL1, preferably a polypeptide comprising the amino acid sequence of SEQ ID No.2, said second compound responsive domain is ABI, preferably a polypeptide comprising the amino acid sequence of SEQ ID No.1, and said inducer is abscisic acid. BHIP-C09-09 PCT Transactivator In certain embodiments, the present disclosure also makes use of a transactivator. The transactivators exemplified in the present disclosure are VP16, VPR and VP64: ^ VP16: MDLLVDELFADMNADGASPPPPRPAGGPKNTPAAPPLYATGRLSQAQLMPSPPM PVPPAALFNRLLDDLGFSAGPALCTMLDTWNEDLFSALPTNADLYRECKFLSTL PSDVVEWGDAYVPERTQIDIRAHGDVAFPTLPATRDGLGLYYEALSRFFHAELR AREESYRTVLANFCSALYRYLRASVRQLHRQAHMRGRDRDLGEMLRATIADRYY RETARLARVLFLHLYLFLTREILWAAYAEQMMRPDLFDCLCCDLESWRQLAGLF QPFMFVNGALTVRGVPIEARRLRELNHIREHLNLPLVRSAATEEPGAPLTTPPT LHGNQARASGYFMVLIRAKLDSYSSFTTSPSEAVMREHAYSRARTKNNYGSTIE GLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVA MAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGID EYGG (SEQ ID No. 8) ^ VP64: DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML (SEQ ID No. 9) ^ VPR: MEQAPEDQGPQREPHNEWTLELLEELKNEAVRHFPRIWLHGLGQHIYETYGDTW AGVEAIIRILQQLLFIHFRIGCRHSRIGVTQQRRARNGASRS (SEQ ID No. 10) Therefore, in certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and BHIP-C09-09 PCT (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said transactivator is selected from VP16, VPR and VP64. Preferably said transducer is VP16. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and BHIP-C09-09 PCT wherein said transactivator is selected from a nucleic acid sequence selected from SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10. Preferably said transducer has the nucleic acid sequence of SEQ ID No.8. Promoter The expression system of the present disclosure makes use of a controller unit. The controller units comprises a first and a second nucleic acids sequence which both contain a Pol II promoter. The promoter of these two nucleic acid sequences can be identical or can be different. In certain embodiments said promoters are identical. In certain preferred embodiments said promoter is a CMV promoter. In other preferred embodiments said promoter comprises the nucleic acid sequence of SEQ ID No.4: GACATTGATTATTGAGTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGAC GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCT CCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT CGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA GCAGAGCT. Therefore, in certain embodiments the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, BHIP-C09-09 PCT (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said the promoter of said first nucleic acid sequence and the promoter of said second nucleic acid sequence are identical. Preferably, said promoter is a CMV promoter. More preferably, said promoter comprises the nucleic acid sequence of SEQ ID No.4. In other embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, BHIP-C09-09 PCT wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said the promoter of said first nucleic acid sequence and the promoter of said second nucleic acid sequence are different. Linker The first nucleic acid sequence of the controller units comprises a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain. Said operator unit and said first compound responsive domain may be separated by a linker. The linker exemplified in the present disclosure are a G4S-NLS-G4S linker and G4S linker. The G4S-NLS-G4S linker consists of the amino acid sequence of SEQ ID No.5. The G4S linker consists of the amino acid sequence of SEQ ID No.6. Therefore, in certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit, a linker and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit and a nucleic acid sequence encoding a gene of interest, and BHIP-C09-09 PCT wherein said system leads to the expression of the gene of interest in the presence of an inducer. Preferably said linker is selected from a G4S-NLS-G4S linker and G4S linker, most preferably said linker is a G4S-NLS-G4S linker. Alternatively, said linker is selected from an amino acid sequence of SEQ ID No.5 and SEQ ID No.6. Most preferably said linker consists of the amino acid sequence of SEQ ID No.5. Hammerhead The reporter unit of the expression system may further comprise a 3’UTR hammerhead sequence downstream of the gene of interest. The hammerhead structure destabilizes RNA, thereby being responsible for a quick recycling leading to a minimal leakiness of the expression system. Any known hammerhead structure may be employed in the expression system of the present disclosure. Exemplified herein is the hammerhead structure comprising the nucleic acid sequence of SEQ ID No. 7. Therefore, in certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, BHIP-C09-09 PCT wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. Certain preferred embodiments In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising BHIP-C09-09 PCT (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and ABI or a functional fragment or derivative of ABI, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising PYL1 or a functional fragment or derivative of PYL1 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. BHIP-C09-09 PCT In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and ABI or a functional fragment or derivative of ABI, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising PYL1 or a functional fragment or derivative of PYL1 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and (b) a second nucleic acid sequence comprising BHIP-C09-09 PCT (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding TetR or a functional fragment or derivative of TetR and ABI or a functional fragment or derivative of ABI, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising PYL1 or a functional fragment or derivative of PYL1 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid. Preferably said TetR comprises a polypeptide of SEQ ID No. 3 or a functional fragment or derivative of TetR. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. BHIP-C09-09 PCT In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding TetR or a functional fragment or derivative of TetR and a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid. Preferably said TetR comprises a polypeptide of SEQ ID No. 3 or a functional fragment or derivative of TetR. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding TetR or a functional fragment or derivative of TetR and a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and BHIP-C09-09 PCT (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid, and wherein said transactivator is selected from VP16 and VP64. Preferably said inducer is abscisic acid. Preferably said TetR comprises a polypeptide of SEQ ID No.3 or a functional fragment or derivative of TetR. Preferably said transactivator is selected from VP16. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding TetR or a functional fragment or derivative of TetR and a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2 and a transactivator, BHIP-C09-09 PCT wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid, wherein said transactivator is selected from VP16 and VP64, and wherein said the promoter of said first nucleic acid sequence and the promoter of said second nucleic acid sequence are identical. Preferably said TetR comprises a polypeptide of SEQ ID No.3 or a functional fragment or derivative of TetR. Preferably said transactivator is selected from VP16. Preferably said promoter is a CMV promoter. More preferably, said promoter comprises the nucleic acid sequence of SEQ ID No.4. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. In certain embodiments, the present disclosure relates to an expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding TetR or a functional fragment or derivative of TetR, a linker, and a polypeptide of SEQ ID No.1 or a functional fragment or derivative of a polypeptide of SEQ ID No.1, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a polypeptide of SEQ ID No.2 or a functional fragment or derivative of a polypeptide of SEQ ID No.2 and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and 3’UTR hammerhead sequence, BHIP-C09-09 PCT wherein said system leads to the expression of the gene of interest in the presence of an inducer, and wherein said inducer is abscisic acid, wherein said transactivator is selected from VP16 and VP64, and wherein said the promoter of said first nucleic acid sequence and the promoter of said second nucleic acid sequence are identical. Preferably said TetR comprises a polypeptide of SEQ ID No.3 or a functional fragment or derivative of TetR. Preferably said transactivator is selected from VP16. Preferably said promoter is a CMV promoter. More preferably, said promoter comprises the nucleic acid sequence of SEQ ID No.4. Preferably said linker is selected from a G4S-NLS-G4S linker and aG4S linker, more preferably a G4S-NLS-G4S linker. More preferably, said linker consists of the amino acid sequence od SEQ ID No.5 or the amino acid sequence of SEQ ID No.6, preferably the amino acid sequence od SEQ ID No.5. Preferably, said hammerhead structure comprises the nucleic acid sequence of SEQ ID No.7. Methods and uses The expression system disclosed herein has numerous uses. High level expression of any gene of interest can be induced. The system furthermore is substantially devoid of any expression in the absence of an inducer. Gene expression can also be effectively be shut off by way of an inhibitor. The inhibitor exemplified herein is doxycycline. The present disclosure provides a method to express a gene of interest in a eukaryotic target cell, said method comprising a) culturing eukaryotic cells containing the expression system disclosed herein, b) adding an inducer to the eukaryotic cells, c) continue culturing said eukaryotic cells, thereby allowing said eukaryotic cells to express said gene of interest. The present disclosure also provides a method to control the expression of a gene of interest in a eukaryotic target cell, said method comprising a) culturing eukaryotic cells containing the expression system disclosed herein, BHIP-C09-09 PCT b) adding an inducer to the eukaryotic cells, c) continue culturing said eukaryotic cells as long as the expression of the gen of interest is desired and d) shut down expression of the gene of interest by adding an inhibitor. Preferably said inhibitor is doxycycline. Examples Example 1: Components of the system The inducible vector system of the present disclosure was iteratively developed. It consists of various features which function together and in their entirety enable specific expression of a gene of interest in a target cell. The system is silent in the sense that essentially no expression of the gene of interest occurs in the Off state, i.e. without the addition of an inducer. Upon addition of the inducer expression of the gene of interest occurs. The system allows for the tissue-specific expression of the gene of interest by selecting an appropriate promoter. The system is also reversible and can furthermore be shut off via the addition of an inhibitor that shuts off gene expression. The tight regulation of the system is achieved via the control of three levels: DNA, RNA and protein. On the DNA level, a specific promoter is responsible for the induction of gene expression. On the RNA level, a hammerhead structure destabilizes RNA, thereby being responsible for quick recycling leading to a minimal leakiness of the system. Finally, protein dimerization triggered by an inducer initiates the gene expression. The components of the system are depicted in Figure 1. The inducible system is composed of the controller and the reporter unit. The controller unit encodes for the operator unit binding domain fused with the responsive element monomer, and the operator unit transcriptional activator is linked with another responsive element monomer. The reporter unit is composed of the DNA binding site of the operator unit binding domain and the gene of interest. The mechanism of the system is depicted in Figure 2. An inducer, such as plant-derived abscisic acid, triggers the dimerization of the controller monomers forming a functional complex that can bind the DNA binding site on the reporter unit, starting the expression of the gene of interest. Doxycycline prevents the controller's binding to the reporter unit, abolishing the expression of the gene of interest. BHIP-C09-09 PCT Example 2: General material and methods Materials: Abscisic acid (Sigma-Aldrich), vanillic acid (Sigma-Aldrich) and gibberellic acid (Merck) Experimental procedure to test gene expression: Cells were transfected and grown in mammalian cells culturing media supplemented with 10% Fetal Bovine Serum (FBS). To induce gene expression, the respective inducer was added at the indicated concentrations. Cells were further grown in the aforementioned culturing conditions, and the expression of the luciferase reporter gene was measured using a Tekan plate reader. Example 3: Exploration of various inducers In a first step various inducers were tested for their capability to induce the expression of the gene of interest. Three different inducer systems were tested Experimentally, the inducers ABA, VA, and GE were added to Hek293T cells, equipped with the respective compound responsive domains, and cultured in DMEM supplemented with 10% FBS at the indicated concentration. The cells were kept in culture in the presence of the inducer for 72h prior to reporter gene expression analysis. In this test, the controller and the operator units placed on different vectors were introduced into the recipient cells through lipofection following the manufacturer's guidelines. Inducers (abscisic acid, vanillic acid and gibberellic acid were added at a concentration of 100 uM]. The results are shown in Figure 3. All three inducers tested led to an induction of the gene of interest, as measured by luciferase activity. Abscisic acid proved to be the most potent inducer of gene expression, leading to an upregulation of more than 150 folds over the second-best compound. Example 4: Exploration of various transactivators BHIP-C09-09 PCT In the next step, various transactivators were tested for their capability to induce the expression of the gene of interest in the presence of ABA. The following transactivators were tested: ^ VP16 (SEQ ID No.8) ^ VP64 (SEQ ID No.9) ^ VPR (SEQ ID No.10) Results are shown in Figure 4. Transactivators VP16 and VP63 both led to a strong upregulation of expression of the gene of interest upon induction with abscisic acid. Upregulation with VP16 was 7-times higher than with VP64. Transactivator VPR showed expression of the gene of interest also without the addition of abscisic acid. Example 5: Optimization of the linker In the next step it was tested if different linkers between the operator unit binding domain and the first compound responsive domain impact the expression of the gene of interest. The testing was carried using the ABA as inducer and the VP16 as transactivator. The following linkers were tested: ^ a G4S-NLS-G4S linker of sequence GGGGS - PKKKRKV - GGGGS (SEQ ID No.5) ^ a G4S linker of sequence GGGGS (SEQ ID No.6) ^ no linker Results are shown in Figure 5. All linkers tested showed strong induction of the gene of interest. The highest induction was observed with linker G4S-NLS-G4S, which showed a luciferase activity 3.8- fold higher than the second-best tested linker (G4S). Example 6: Evaluation of a 3’ UTR hammerhead structure In the next step it was tested if the introduction of a hammerhead structure 3’ of the gene of interest has a beneficial impact on the expression system. The construct containing the best linker, the VP16 transactivator, and the ABA-responsive elements was considered for the testing. The hammerhead has the following nucleic acid sequence: CCTCCTCGTCCCCGGATGTGCTTTCCGGGCTGACGAGTCCGTGAGGACGAAACGAGGAGG (SEQ ID No.7). BHIP-C09-09 PCT Results are shown in Figure 6. A strong induction of expression of the gene of interest was observed with and without the hammerhead structure. The system with the hammerhead structure showed a complete absence of gene expression in the absence of the inducer. Example 7: Single vs. multiple vector system In this experiment, it was tested whether there is a difference in expression of the gene of interest if the various components of the system are encoded on the same or different vectors. To do so different construct combinations were tested, which are depicted in Figure 7: (1) the first nucleic acid of the controller unit (comprising ABI), the second nucleic acid of the controller unit (comprising PYL1) and the reporter unit (comprising the gene of interest) are encoded on different vectors, (2) the first nucleic acid of the controller unit (comprising ABI) and the second nucleic acid of the controller unit (comprising PYL1) are encoded on the same vector separated by the polypeptide self-cleavage sequence P2A, but the reporter unit (comprising the gene of interest) is encoded on a different vector, (3) the first nucleic acid of the controller unit (comprising ABI) and the second nucleic acid of the controller unit (comprising PYL1) are encoded on the same vector but the expression is driven by two different promoters and the reporter unit (comprising the gene of interest) is encoded on a different vector, (4) the first nucleic acid of the controller unit (comprising ABI), the second nucleic acid of the controller unit (comprising PYL1) and the reporter unit (comprising the gene of interest) are encoded on the same vector, and the gene of interest is transcribed in the same direction as ABI and PYL, and (5) the first nucleic acid of the controller unit (comprising ABI), the second nucleic acid of the controller unit (comprising PYL1) and the reporter unit (comprising the gene of interest) are encoded on the same vector, and the gene of interest is transcribed from the antisense DNA strand as ABI and PYL. The results are shown in Figure 8. The system is functional in all variations tested. The highest level of upregulation of the gene of interest was observed in the system in which the first nucleic acid of the controller unit, the second nucleic acid of the controller unit, and the reporter unit are encoded on different vectors. BHIP-C09-09 PCT Example 8: Reversibility of the system In this experiment it was tested if the expression of the gene of interest is reversible. At time point 0, gene expression was induced by adding abscisic acid at 50 µM. After 24 hours in one sample, cells were cultured in the presence of the inducer, while in another sample, the media containing ABA was replaced with fresh non-supplemented culturing media. Control cells were treated with DMSO only. Luciferase activity was measured for all samples every 24 hours. Results are shown in Figure 9. Cells cultured in the presence of the inducer showed a steady increase in luciferase activity, while cells, where the media was replaced showed a decrease in the reporter gene activity that returned to baseline within 72 hours. DMSO-treated control cells did not show any luciferase activity. Example 9: Possibility to witch the system off In this experiment it was tested if an appropriate inhibitor could switch off the expression of the gene of interest after induction. At time point 0, gene expression was induced via the addition of abscisic acid at 50 µM. After 24 hours in one sample cells were cultured in the presence of the inducer, while in another sample, doxycycline was added at a concentration of 0.75 µM. Control cells were treated with DMSO only. Luciferase activity was measured for all samples every 24 hours. The results are shown in Figure 10. As in Example 8, cells continuously supplemented with the inducer showed a continued increase in luciferase activity, while cells that were supplemented with abscisic acid at time point 0 and treated with doxycycline after 24 hours, showed a decrease in luciferase activity that returned to baseline within 24 hours. DMSO-treated control cells did not show any luciferase activity.

Claims

BHIP-C09-09 PCT Claims 1. An expression system for the controlled expression of a gene of interest in a eukaryotic cell comprising a controller unit and a reporter unit, wherein said controller unit comprises (a) a first nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit binding domain comprising a polypeptide binding to the operator unit of the reporter unit and a first compound responsive domain, and (b) a second nucleic acid sequence comprising (i) a promoter and, (ii) a nucleic sequence encoding an operator unit transcriptional activator domain comprising a second compound responsive domain and a transactivator, wherein said reporter unit comprises a nucleic acid sequence comprising an operator unit, a nucleic acid sequence encoding a gene of interest and a 3’UTR hammerhead sequence, and wherein said system leads to the expression of the gene of interest in the presence of an inducer. 2. The expression system according to claim 1, wherein said first compound responsive domain comprises ABI or a functional fragment or derivative of ABI and said second compound responsive domain is PYL1 or a functional fragment or derivative of PYL1, or wherein said first compound responsive domain is PYL1 or a functional fragment or derivative of PYL1 and said second compound responsive domain is ABI or a functional fragment or derivative of ABI, preferably wherein said first compound responsive domain comprises a polypeptide of SEQ ID No.1 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.2 or a functional fragment or derivative thereof, or wherein said first compound responsive comprises a polypeptide of SEQ ID No.2 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.1 or a functional fragment or derivative thereof. 3. The expression system according to claim 2, wherein said inducer is abscisic acid or a functional fragment or derivative of abscisic acid. BHIP-C09-09 PCT 4. The expression system according to claim 1, wherein said first compound responsive domain comprises VanR or a functional fragment or derivative of VanR and said second compound responsive domain is VanR or a functional fragment or derivative of VanR, preferably wherein said first compound responsive comprises a polypeptide of SEQ ID No.11 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.11 or a functional fragment or derivative thereof. 5. The expression system according to claim 4, wherein said inducer is vanillic acid or a functional fragment or derivative of vanillic acid. 6. The expression system according to claim 1, wherein said first compound responsive domain comprises GAI or a functional fragment or derivative of GAI and said second compound responsive domain is GID or a functional fragment or derivative of GID, or wherein said first compound responsive domain is GID or a functional fragment or derivative of GID and said second compound responsive domain is GAI or a functional fragment or derivative of GAI, preferably wherein said first compound responsive comprises a polypeptide of SEQ ID No.12 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No. 13 or a functional fragment or derivative thereof, or wherein said first compound responsive comprises a polypeptide of SEQ ID No.13 or a functional fragment or derivative thereof and said second compound responsive domain comprises a polypeptide of SEQ ID No.12 or a functional fragment or derivative thereof. 7. The expression system according to claim 6, wherein said inducer is gibberellic acid or a functional fragment or derivative of gibberellic acid. 8. The expression system according to any one of the preceding claims, wherein said operator unit binding domain is TetR or a functional fragment or derivative of TetR, wherein said operator unit binding domain comprises a polypeptide of SEQ ID No.3 or a functional fragment or derivative thereof. 9. The expression system according to any one of the preceding claims, wherein said transactivator is selected from VP16 (SEQ ID No.8), VP64 (SEQ ID No.9) and VPR (SEQ ID No.10). 10. The expression system according to any one of the preceding claims, wherein said promoter of said first nucleic acid sequence and the promoter of said second nucleic acid sequence are identical. 11. The expression system according to claim 10, wherein said promoter is a Pol II promoter, preferably a CMV promoter, more preferably wherein said promoter comprises the nucleic acid sequence of SEQ ID No.4. BHIP-C09-09 PCT 12. The expression system according to any one of the preceding claims, wherein said first nucleic acid of the controller unit comprises a nucleic acid sequence encoding a linker which is located between said operator unit binding domain and said first compound responsive domain. 13. The expression system according to claim 12, wherein said linker is a G4S-NLS-G4S linker or a G4S linker, preferably wherein said linker consists of an amino acid sequence selected from SEQ ID No. 5 or SEQ ID No.6. 14. The expression system according to any one of the preceding claims, wherein said hammerhead sequence comprises the nucleic acid sequence of SEQ ID No.7. 15. A method to express a gene of interest in a eukaryotic target cell, said method comprising a) culturing eukaryotic cells containing the expression system of any one of claims 1-14, b) adding an inducer to the eukaryotic cells, and c) continue culturing said eukaryotic cells, thereby allowing said eukaryotic cells to express said gene of interest.