Repressors for inducible expression in animals

Abstract

Novel gene repressor systems are provided that are useful in animal cells that include engineered repressors with altered ligand-binding domains (LBDs) and DNA-binding domains (DBDs), as well as mutated operator sequences. In one aspect, the systems enable independent, drug-inducible control of multiple genes within a single animal or animal cell line. In one aspect, the disclosure provides both tetracycline-responsive repressors and sulfonylurea (SU)-responsive repressors — e.g., variants that respond specifically to ethametsulfuron-methyl (Es) or chlorsulfuron (Cs). Mutations are introduced to either the LBD (to alter ligand specificity), DBD (to alter operator binding), or the operator sequence itself, resulting in orthogonal systems.

Description

Atty Dkt.: 114198-3110 REPRESSORS FOR INDUCIBLE EXPRESSION IN ANIMALS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Nos.63 / 684,784 and 63 / 701,458, filed on August 19, 2024 and September 30, 2024 respectively, the contents of each of which are incorporated herein by reference in their entireties. BACKGROUND

[0002] It is highly desirable in biology to be able to control expression of target genes, either endogenous or exogenous ones (transgenes). This allows the experimenter to express the target gene at a given time (perhaps after development or cell fate determination), to potentially activate the gene in a specific set of cells, and to turn off gene expression as well. Inducible gene expression systems are not well established in animals. Light penetrates quite poorly into intact animals, and optogenetic gene expression system have quite high background expression level (“leakiness”) and low levels of induction upon stimulation (“activation”). Small molecules, by contrast, can be easily delivered to all animal tissues, and ligand-gated gene expression systems are better studied. The only ligand-gated gene expression system robust enough to use in intact animals is the tetracycline repressor (TetR) / tetracycline (or similar antibiotics like doxycycline). TetR / Tet systems are ubiquitous in established animal systems, particularly mouse,1although they have been demonstrated in Drosophila,2C. elegans,3zebrafish,4and non-human primate,5as well.

[0003] Despite the success of the TetR / Tet systems for inducible gene expression in animals, this only allows a single gene to be regulated. Disease studies, drug screening, and other experiments would be greatly facilitated by the ability to independently regulate multiple genes. However, other ligand-inducible expression systems are quite poorly developed in animals6. To the best of Applicant’s knowledge, no ligand-inducible system besides TetR / Tet is available for critical studies in rodents and other animals. This disclosure provides repressor systems for expression in animal systems and provides related advantages as well. -1- 4871-7792-0235.1Atty Dkt.: 114198-3110 SUMMARY OF THE DISCLOSURE

[0004] The present disclosure introduces a significant advancement in multiplex gene regulation by enabling two or more independent genes to be precisely controlled within the same animal or mammalian cell. This is accomplished through the use of orthogonal combinations of repressors, ligands, and operators, thereby facilitating robust and selective gene expression patterns. Prior methodologies were limited in their ability to achieve such sophisticated levels of multiplexing; previous disclosures generally addressed gene regulation in eukaryotic cells without specificity toward animal or mammalian cell contexts.

[0005] A key innovation of this disclosure lies in the use of engineered DNA-binding domains (DBDs) that can recognize and bind to modified operator sequences, such as the TetO-4C5G operator. This, combined with carefully crafted mutations in both the DBD and ligand-binding domain (LBD), creates new, orthogonal repressor systems that do not cross- react, thus allowing for truly independent regulation of multiple genes in the same cell. The compositions, systems, and methods described herein are explicitly tailored for animal (mammalian) cells, overcoming the limitations of the prior art that were not directed to these specific cellular environments. This targeted approach opens new possibilities for complex gene circuit design and precise gene control in therapeutic, research, and biotechnological applications.

[0006] For example, Applicant provides herein a novel solution addressing the lack of ligand-inducible systems for investigating multiple independently regulated genes in animal systems. The TetR protein is composed of two domains: a ligand-binding domain (LBD) that binds tetracycline (Tet) and other antibiotics, and a DNA-binding domain (DBD) that binds to the TetO DNA operator sequence. In wild-type TetR, in the absence of Tet, TetR binds to TetO and represses transcription; in the presence of Tet, TetR changes conformation and dissociates from the DNA, allowing RNA polymerase and transcription factors to bind and transcribe the target gene.

[0007] In one embodiment, the new gene repressor systems, when combined with a repressor agent such as Tet or a sulfonylurea (SU) molecule, e.g. including ethametsulfuron-methyl (Es), a SU herbicide, and chlorsulfuron (Cs), constitutes a powerful inducible expression -2- 4871-7792-0235.1Atty Dkt.: 114198-3110 system for animals and animal cells. These were characterized in diverse mammalian cell culture experiments for demonstrated utility.

[0008] Applicant further demonstrates that mutants of the tetracycline repressor (TetR) designed to bind other molecules, for example, a sulfonylurea (SU) molecule, e.g., including an SU herbicide such as ethametsulfuron-methyl (Es) or chlorsulfuron (Cs), function quite well in mammalian cells. Applicant shows that these novel repressor systems are orthogonal with wild-type Tet and facilitate independent control of two genes with two small molecule ligands. Applicant grafted mutations that change the DNA-binding specificity for the repressors and show that they now function to induce expression at loci controlled by a mutant TetO operator. Finally, Applicant shows that the herbicide-responsive inducers can be used alongside the antibiotic-responsive ones to independently modulate distinct genes controlled separately by the wild-type and mutant TetO operators. These molecules are safe in mammals and are drug-like small molecules that will likely penetrate well in the tissue of mice and other animals. Such reagents can be used for basic science studies benefiting from the regulatable expression of two different genes, and potentially in disease studies.

[0009] The compositions and systems as described herein provides a means to control when various traits (e.g., fluorescence cell death, recombination, gene therapy payload, etc.) are turned on and off in animals or animal cells. Continuous expression of a gene with this trait is often problematic. "Controllable expression" systems let the user decide when the trait is on or off by which drug is added or removed at each time. This disclosure greatly expands the options for controllable expression, which has many commercial and academic applications.

[0010] In some aspects, the disclosure provides a gene repressor system including: a. a ligand-binding domain (LBD); b. a mutated DNA-binding domain (DBD); and c. an operator nucleotide that controls the expression of a target polynucleotide, and further wherein one or more of the LBD or the operator nucleotide include one or more mutations that change the DNA-binding specificity of the gene repressor system.

[0011] Isolated polynucleotides encoding the elements of the gene expression systems, are further provided herein. -3- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0012] In some aspects, the disclosure provides a system, further comprising a polynucleotide including a target gene sequence operably linked to a promoter including at least one operator sequence.

[0013] In some regards, the disclosure provides a gene expression system, wherein the gene repressor system is a tetracycline repressor system (TetR) and the ligand-binding domain binds to tetracycline (Tet) repressor agent.

[0014] In some embodiments, the disclosure provides a gene expression system, wherein the operator nucleotide includes wild-type TetO (SEQ ID NO: 7) or an operator variant, optionally the TetO-4C5G (SEQ ID NO: 8) variant.

[0015] In other aspects, the disclosure provides a gene expression system, wherein the LBD includes one or more mutations that alter the ligand binding activity of the LBD to bind to other molecules other than the LBD wild-type binding molecule.

[0016] In some regards, the disclosure provides a gene expression system, wherein the LBD binds to a repressor agent selected from tetracycline, a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

[0017] In some aspects, the disclosure provides a fusion protein, comprising a mutated LBD and a mutated DBD, wherein the fusion protein comprises a polypeptide comprising: L13-1-9 (SEQ ID NO: 4); L15-20 (SEQ ID NO: 3); CsL4.2-15 (SEQ ID NO: 5) and CsL4.2-20 (SEQ ID NO:6). Also provided are polynucleotides encoding the fusion proteins. Gene expression systems and host animal cells comprising these are further provided herein.

[0018] In some aspects, the disclosure provides a gene expression system, wherein the DBD includes a mutated tetracycline repressor (mTetR) selected from a mTetR having the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to wild-type TetR shown in SEQ ID NO: 1.

[0019] In some aspects, the disclosure provides a gene expression system, wherein the DBD includes, or consists essentially of, or consists of the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as shown in SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity thereto wherein the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation is retained. -4- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0020] In one embodiment, the disclosure provides a gene expression system, wherein one or more, two or more or all three of the ligand-binding domain (LBD); the DNA-binding domain (DBD); or the operator nucleotide is detectably labeled.

[0021] Also provided are multiple gene repressor systems, wherein the gene repressor system includes two or more gene repressor systems, wherein each of the two or more gene repressor systems work concurrently to modulate distinct target gene sequences controlled by a wild- type operator nucleotide or a mutated operator nucleotide. In one aspect, of the multiple gene expression system, at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) and the ligand-binding domain binds to tetracycline (Tet). In one aspect the multiple gene repressor system comprises two or more gene repressor systems as described above. In a further aspect, the two or more systems each comprise: a ligand- binding domain (LBD); b. a DNA-binding domain (DBD); and c. an operator nucleotide, wherein one or more of the LBD, DBD or the operator nucleotide include one or more mutations that change the DNA-binding specificity of the gene repressor system, such that the systems in combination work concurrently to modulate distinct target gene sequences. Examples of independent gene repressor systems are provided herein but in the multiple gene repressor system, it is not necessary that the DBD is a mutated polypeptide.

[0022] In some aspects, the disclosure provides a multiple gene expression system, wherein each of the two or more gene expression systems are on a single contiguous polypeptide.

[0023] In some aspects, the disclosure provides an isolated polynucleotide encoding a fusion protein selected from SEQ ID NO: 3 to 6, and equivalents thereof wherein in one aspect, the equivalents encode fusion proteins having at least 90% sequence identity to the reference fusion protein when evaluated under BLAST, wherein the reference the fusion proteins are independently selected from any one of SEQ ID NO: 3 to 6, with the proviso that the equivalent excludes the wild-type nucleic acid sequence individually for the respective polynucleotides encoding SEQ ID NO: 3 to 6.

[0024] Further provided are isolated polynucleotides encoding the gene expression systems or their individual elements, as disclosed herein.

[0025] In some aspects, the disclosure provides a vector including an isolated polynucleotide. The vector is for example, a viral vector or a plasmid vector. -5- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0026] In other aspects, the disclosure provides an isolated host cell including one or more gene expression system as described herein, or an isolated polynucleotide or a vector encoding same. The cell can be a procaryotic or a eukaryotic cell, preferably an animal cell.

[0027] In some aspects, the disclosure provides a method for controlling gene expression of a host cell by inserting into the host cell one or more gene expression system as described herein, and optionally contacting the cell with a repressor agent. The host cell is cultured for expression of the gene expression system. BRIEF DESCRIPTION OF THE FIGURES

[0028] FIG.1 (18 Panels): Three independent experiments (columns 1-3) are low cytometry histogram plots showing the GFP signal intensity of the cells transiently co-transfected with the TetO-sfGFP reporter plasmid and one of the repressor-expressing plasmids or an empty vector 24 hours after the addition of 1 µg / ml Tet, Es, Cs, or DMSO (0.1%, carrier solution,). Numbers represent the median intensity.

[0029] FIGS.2A – 2E: (FIG.2A, 8 Panels) Wild-type TetR mutated in its DBD is able to recognize the TetO-4C5G operator. All 3 mutation sets induce well with Tet (left column bright) and have low background (right column dim), essentially like wild-type (top row). (FIG.2B, 32 Panels) EsR L13-1-9 mutated in its DBD to recognize the TetO-4C5G operator. Two mutation sets produce high leakiness (bottom 2 rows, right columns bright). E37AP39K produced similar leakiness to wild-type (top 2 rows, right columns similarly dim), but its Es induction is slightly decreased relative to wild-type (2ndrow, left column dimmer than 1strow, left column). (FIG.2C, 32 Panels) EsR L15-20 mutated in its DBD to recognize the TetO-4C5G operator. One mutation set (V36AE37AP39K) produced extremely strong Es induction (3rdrow, left column), even higher than wild-type (1strow, left column). This mutation set produced slightly higher leakiness than wild-type (3rdrow, right column brighter than 1strow, right column). The other 2 mutation sets decreased Es activation (2ndand 4throws, left columns). (FIG.2D, 32 Panels) CsR CsL4.2-15 mutated in its DBD to recognize the TetO-4C5G operator. Two mutation sets produce high leakiness (bottom 2 rows, right columns bright). E37AP39K produced similar leakiness to wild-type (top 2 rows, right columns similarly dim), but its Cs induction is decreased relative to wild-type (2ndrow, left column dimmer than 1strow, left column). (FIG.2E, 32 Panels) CsR CsL4.2-20 mutated in -6- 4871-7792-0235.1Atty Dkt.: 114198-3110 its DBD to recognize the TetO-4C5G operator. Two mutation sets produce high leakiness (bottom 2 rows, right columns bright). E37AP39K produced similar leakiness to wild-type (top 2 rows, right columns similarly dim), but its Cs induction is decreased relative to wild- type (2ndrow, left column dimmer than 1strow, left column). Note that the leakiness of the wild-type CsR CsL4.2-20 is higher than the other SURs.

[0030] FIG.3: Left Panel: overlay of the signal from the mixture of the TetR- V36FE37AP39K-expressing cells transiently co-transfected with the TetO-4C5G-sfGFP reporter plasmid that were induced with Tet and the L13-1-9-expressing cells transiently co- transfected with the TetO-mScarlet-I3 reporter plasmid that were incubated with negative- control solvent DMSO (black dotted region), and the mixture of these cells that was incubated with Tet for 24 hours (white space bounded by black dots). Note strong green fluorescence, representing TetR-V36FE37AP39K-Tet induction, and weak red fluorescence, representing background leakiness of L13-1-9. Right Panel: overlay of the signal from the mixture of the TetR-V36FE37AP39K-expressing cells transiently co-transfected with the TetO-4C5G-sfGFP reporter plasmid that were incubated with DMSO and the L13-1-9- expressing cells transiently co-transfected with the TetO-mScarlet-I3 reporter plasmid that were induced with Es (black dotted region), and the mixture of these cells that was incubated with Es for 24 hours (white space bounded by black dots). Note strong red fluorescence, representing EsR L13-1-9-Es induction, and weak green fluorescence, representing background leakiness of TetR. Note that black and orange overlay in both panels, showing that the activity of the two repressors is unaffected by the presence of the other inducer.

[0031] FIGS.4A – 4B: (FIG.4A, 4 Panels) Raw FACS data from the HEK293 cells co- transfected with a TetR-V36FE37AP39K repressor, a Tet-responsive sfGFP reporter construct, a L13-1-9 repressor, and an Es-responsive mScarlet-I3 reporter construct. Tet- responsive sfGFP fluorescence brightness is shown in log scale along the x-axis; Es- responsive mScarlet-I3 fluorescence brightness is shown in log scale along the y-axis. DMSO: control shows negligible fluorescence. Es only: mScarlet-I3 fluorescence increases. Tet only: sfGFP fluorescence increases. Es+Tet: mScarlet-I3 and sfGFP fluorescence increases. (FIG.4B, 2 Panels) Quantification of raw data in double-positive Q2 quadrant in A. Left: red fluorescence signal increases upon treatment of cells with Es or Es+Tet, but is -7- 4871-7792-0235.1Atty Dkt.: 114198-3110 not affected by Tet treatment alone. Right: green fluorescence signal increases upon treatment of cells with Tet or Es+Tet, but is not affected by Es treatment alone.

[0032] FIG.5 graphically shows a representative DBD / LBD system. DETAILED DESCRIPTION

[0033] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation, the full bibliographic citations for some of which are found immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this disclosure pertains.

[0034] As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a single cell as well as a plurality of cells, including mixtures thereof.

[0035] As used herein, the term “comprising” is intended to mean that the methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define methods, shall mean excluding other elements of any essential significance to the method. In the context of a composition or combination, “consisting essentially of” a listing of active ingredients or agents that the recited elements are the only active agents in the composition or combination. “Consisting of” shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods can include additional steps and components (comprising) or alternatively including steps of no significance (consisting essentially of) or alternatively, intending only the stated method steps (consisting of).

[0036] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. The term “about” also includes the exact value “X” in addition to minor increments of “X” such as “X + 0.1” or “X – 0.1.” It also is to -8- 4871-7792-0235.1Atty Dkt.: 114198-3110 be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0037] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0038] “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient. A therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0039] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of -9- 4871-7792-0235.1Atty Dkt.: 114198-3110 one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. In one aspect, treatment excludes prophylaxis.

[0040] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human.

[0041] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0042] As used herein, the term “administration” and “administering” are used to mean introducing an agent into a subject. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracameral, intratumoral, epidural and intrathecal.

[0043] As used herein, a “vector” refers to a construct which is capable of delivering, and, in some embodiments expressing, a polynucleotide into a cell. Non-limiting examples of delivery vectors include viral vectors, nucleic acid expression vectors (such as a plasmid), naked DNA, and certain eukaryotic cells (e.g., producer cells). In some embodiments, -10- 4871-7792-0235.1Atty Dkt.: 114198-3110 nucleic acids described by the disclosure are delivered via a viral vector. Examples of viral vectors include retroviral vectors (e.g., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD 100), lentiviral vectors (e.g., HIV and FIV-based vectors), and herpesvirus vectors (e.g., HSV, HSV-1, HSV-2), as described by Chira et al. (Oncotarget, 2015, 6(31); 30673-30703). In some embodiments, nucleic acids described by the disclosure are delivered by an adeno-associated virus (AAV) vector (e.g., a recombinant AAV (rAAV) vector).

[0044] A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for. Plasmids are commercially available and known in the art.

[0045] “Repressors” are molecules that bind to an operator either in the absence or presence of a tetracycline ligand (tetracycline (Tet), doxycycline, anhydrotetracycline, or other tetracyclines) or a sulfonylurea ligand (e.g., ethametsulfuron-methyl (Es), chlorsulfuron (Cs), or other sulfonylurea herbicides). Those that bind in the presence of the ligand are known as reverse repressors. Some compositions feature sulfonylurea repressor (SUR) polypeptides that specifically bind to a tetracycline operator, with binding regulated by sulfonylurea compounds. In certain cases, these compositions include an isolated SUR polypeptide with at least one amino acid substitution in the ligand-binding domain, enabling regulated binding to a polynucleotide containing an operator sequence, where binding is dependent on the -11- 4871-7792-0235.1Atty Dkt.: 114198-3110 presence or absence of sulfonylurea. “A gene repressor system” intends a combination of elements when working in combination allow for the controlled expression of one or more target genes or polynucleotide. A well-known example is the Tet operator / repressor systems.

[0046] As used herein, the term ligand-binding domain “LBD” intends a molecule (i.e., polypeptide or protein) that contains a pocket that binds to a ligand, such as tetracycline (Tet), ethametsulfuron-methyl (Es), chlorsulfuron (Cs), or another sulfonylurea (SU) herbicide. For example, it is the region on a repressor protein (see e.g., FIG.5) that is responsible for interacting with and binding to the small molecules or ligands, also referred to herein as a repressor agent.

[0047] A “mutated” LBD is a mutated polypeptide that has altered binding activity as compared to the wild-type LBD.

[0048] As used herein the term DNA-binding domain “DBD” intends a molecule (i.e., a polypeptide or protein) that can recognize and bind to a specific DNA sequence – an operator. For example, it is the region on a repressor protein (see e.g., FIG.4) that allows it to recognize and bind to a particular DNA sequence, typically in a promoter or operator region of a gene.

[0049] A mutated DBD is a DBD that has a mutation that alters its ability to bind a promoter or operator. Non-limiting examples include a mutated tetracycline repressor (mTetR) selected from a mTetR having the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to wild-type TetR shown in SEQ ID NO: 1 or a DBD that comprises, or consists essentially of, or consists of the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as shown in SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity thereto wherein the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation is retained.

[0050] A “promoter” is a sequence of DNA located near the beginning of the gene. It acts as a binding site for RNA polymerase and other transcription factors, initiating the process where DNA is copied into RNA.

[0051] An “operator” in the context of gene expression is a genetic sequence that allows proteins responsible for transcription to attach to the DNA sequence. Repressor proteins can block transcription factors from binding to the operator. For example, it is a specific DNA -12- 4871-7792-0235.1Atty Dkt.: 114198-3110 sequence that acts as a binding site for a repressor protein. When the repressor protein binds to the operator, it physically blocks RNA polymerase from transcribing the downstream genes or polynucleotides, effectively preventing their expression. An operator variant is a DNA sequence that has been mutated to modify the binding of the repressor protein. An example of such is the TetO variant TetO-4C5G (SEQ ID NO: 8), and equivalents thereof that retain the mutated nucleotides as compared to wild-type TetO.

[0052] A “Tet operator (tetO)” intends the tet operator sequence (tetO) which is a 19- nucleotide sequence that is part of the tetracycline responsive element (TRE). The tetO sequence is TCC CTA TCA GTG ATA GAG A (SEQ ID NO: 7).

[0053] A “TetO-4C5G” operator intends the tet operator variant tetO-4C5G. See, e.g., Krueger et al. (2007) Gene Dec 1;404(1-2):93-100. The tetO-4C5G sequence is TCC CCG TCA GTG ACG GAG A (SEQ ID NO: 8).

[0054] A “molecule” intends a protein, polypeptide, small molecule, polypeptide or nucleic acid. In some aspects, as with the LBD, the molecule intends a polypeptide or protein.

[0055] A “ligand inducer” or “repressor agent” intends a molecule, such as tetracycline, ethametsulfuron-methyl (Es), chlorsulfuron (Cs), or another sulfonylurea (SU) herbicide, that binds to the ligand-binding domain (LBD) and influences the DNA-binding domain’s (DBD) ability to bind the operator DNA (enables binding to the operator DNA if it was not possible in the absence of the ligand inducer or promotes dissociation from the operator DNA if the protein was bound to the operator DNA in the absence of the ligand inducer). Non-limiting examples of register SU compounds (e.g., SU herbicides) include chlorsulfuron (W4189), thifensulfuron-methyl (M6316), tribenuron-methyl (L5300), metsulfuron- methyl (T6376), ethametsulfuron-methyl (A7881), chlorimuron-ethyl (F6025), rimsulfuron (E9636), nicosulfuron (V9630), and sulfometuron-methyl (T5648).

[0056] As used herein, the term “multiple gene regulation” (also referred to herein as a “collection”) means the regulation of two non-identical genes using two or more or more gene repressor systems, each with non-identical DBD, LBD, or operator allowing the simultaneous regulation of multiple genes in the same animal cell or organism.

[0057] Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations -13- 4871-7792-0235.1Atty Dkt.: 114198-3110 due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The host cell can be a prokaryotic or a eukaryotic cell. In some embodiments, the host cell is an animal cell, a mammalian cell, or a human cell or cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293T cell, or an a549 cell.

[0058] “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, mammals, humans, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, animal, mammal and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, canine, bovine, porcine, murine, rat, avian, reptilian and human.

[0059] “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2µm in diameter and 10 µm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0060] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.

[0061] Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, lipid nanoparticle and the like, HK polymers, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein -14- 4871-7792-0235.1Atty Dkt.: 114198-3110 excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0062] A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0063] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include lipid nanoparticles, HK polymers, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0064] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long- -15- 4871-7792-0235.1Atty Dkt.: 114198-3110 term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0065] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0066] A combination as used herein intends that the individual active ingredients of the compositions are separately formulated for use in combination and can be separately packaged with or without specific dosages. The active ingredients of the combination can be administered concurrently or sequentially.

[0067] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals such as non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, bat, rat, rabbit, guinea pig).

[0068] In one aspect, the term “equivalent” or “biological equivalent” of an antibody means the ability of the antibody to selectively bind its epitope protein or fragment thereof as measured by ELISA or other suitable methods. Biologically equivalent antibodies include, -16- 4871-7792-0235.1Atty Dkt.: 114198-3110 but are not limited to, those antibodies, peptides, antibody fragments, antibody variant, antibody derivative and antibody mimetics that bind to the same epitope as the reference antibody.

[0069] In one aspect, the term “equivalent” of “chemical equivalent” of a chemical means the ability of the chemical to selectively interact with its target protein, DNA, RNA or fragment thereof as measured by the inactivation of the target protein, incorporation of the chemical into the DNA or RNA or other suitable methods. Chemical equivalents include, but are not limited to, those agents with the same or similar biological activity and include, without limitation a pharmaceutically acceptable salt or mixtures thereof that interact with and / or inactivate the same target protein, DNA, or RNA as the reference chemical.

[0070] The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0071] The term “isolated” as used herein refers to molecules or biological or cellular materials being substantially free from other materials. In one aspect, the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues. -17- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0072] “Detecting” as used herein refers to determining the presence of a nucleic acid or marker of interest in a sample or the presence of a protein or marker of interest in a sample. Detection does not require the method to provide 100% sensitivity and / or 100% specificity.

[0073] “Detectable label” as used herein refers to a molecule or a compound or a group of molecules or a group of compounds used to identify a nucleic acid or protein of interest. In some cases, the detectable label can be detected directly. In other cases, the detectable label can be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label can be detected by various means and will depend on the nature of the detectable label. Detectable labels can be isotopes, fluorescent moieties, colored substances, and the like. Examples of means to detect detectable label include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means.

[0074] “TaqMan® PCR detection system” as used herein refers to a method for real time PCR. In this method, a TaqMan® probe which hybridizes to the nucleic acid segment amplified is included in the PCR reaction mix. The TaqMan® probe comprises a donor and a quencher fluorophore on either end of the probe and in close enough proximity to each other so that the fluorescence of the donor is taken up by the quencher. However, when the probe hybridizes to the amplified segment, the 5'-exonuclease activity of the Taq polymerase cleaves the probe thereby allowing the donor fluorophore to emit fluorescence which can be detected.

[0075] As used herein, the term “sample” or “test sample” refers to any liquid or solid material containing nucleic acids. In suitable embodiments, a test sample is obtained from a biological source (i.e., a "biological sample"), such as cells in culture or a tissue sample from an animal, preferably, a human. In an exemplary embodiment, the sample is a biopsy sample.

[0076] “Target nucleic acid” as used herein refers to segments of a chromosome, a complete gene with or without intergenic sequence, segments or portions a gene with or without intergenic sequence, or sequence of nucleic acids to which probes or primers are designed. Target nucleic acids can include wild type sequences, nucleic acid sequences containing mutations, deletions or duplications, tandem repeat regions, a gene of interest, a region of a -18- 4871-7792-0235.1Atty Dkt.: 114198-3110 gene of interest or any upstream or downstream region thereof. Target nucleic acids can represent alternative sequences or alleles of a particular gene. Target nucleic acids can be derived from genomic DNA, cDNA, or RNA. As used herein, target nucleic acid can be native DNA or a PCR-amplified product. Non-limiting examples include siRNA, mRNA, as well as DNA encoding a polypeptide, an antibody, an antibody fragment or a therapeutic polypeptide.

[0077] As used herein the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With high stringency conditions, nucleic acid base pairing will occur only between nucleic acids that have sufficiently long segments with a high frequency of complementary base sequences. Exemplary hybridization conditions are as follows. High stringency generally refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSC (saline sodium citrate) 0.2% SDS (sodium dodecyl sulfate) at 42°C., followed by washing in 0.1×SSC, and 0.1% SDS at 65°C. Moderate stringency refers to conditions equivalent to hybridization in 50% formamide, 5×Denhardt's solution, 5×SSC, 0.2% SDS at 42°C, followed by washing in 0.2×SSC, 0.2% SDS, at 65°C. Low stringency refers to conditions equivalent to hybridization in 10% formamide, 5×Denhardt's solution, 6×SSC, 0.2% SDS, followed by washing in 1×SSC, 0.2% SDS, at 50°C.

[0078] As used herein the term “substantially identical” refers to a polypeptide or nucleic acid exhibiting at least 50%, 75%, 85%, 90%, 95%, or even 99% identity to a reference amino acid or nucleic acid sequence over the region of comparison. For polypeptides, the length of comparison sequences will generally be at least 20, 30, 40, or 50 amino acids or more, or the full length of the polypeptide. For nucleic acids, the length of comparison sequences will generally be at least 10, 15, 20, 25, 30, 40, 50, 75, or 100 nucleotides or more, or the full length of the nucleic acid. Modes for Carrying Out the Disclosure

[0079] Applicant provides herein methods and compositions to control expression of multiple transgenes simultaneously in animals or their cells or tissues. It accomplishes -19- 4871-7792-0235.1Atty Dkt.: 114198-3110 this by combining many different mutations to the tetracycline repressor (TetR) to: 1. change ligand‐binding specificity, 2. change DNA‐binding specificity, and 3. convert repressors to activators, and vice versa. The result is the ability to control expression of multiple traits in animals using drugs. Repressor Systems and Collections Single Gene Repressor Systems

[0080] In one aspect, a gene repressor system is provided that comprises: a. a ligand- binding domain (LBD); b. a mutated DNA-binding domain (DBD); and c. an operator nucleotide that controls expression of a target gene or polynucleotide sequence, wherein one or more of the LBD or the operator nucleotide comprise one or more mutations such that the one or more mutations change the DNA-binding specificity of the gene repressor system. In a further aspect, the gene repressor system further comprises a polynucleotide encoding an expression product or a polynucleotide such as a therapeutic polynucleotide, e.g., siRNA, operably linked to a promoter comprising at least one operator sequence. In one aspect, the gene repressor system is a TetR system and the LBD binds to a Tet repressor agent. In a further aspect, the operator nucleotide is wild-type TetO or an operator variant, e.g., TetO-4C5G variant.

[0081] In a further aspect, the DBD of the system comprises one or more mutations that alter the DNA-binding activity of the DBD to bind to other molecules such as for example, operator nucleotide variants other than those bound by the tetracycline repressor system (TetR) DBD molecules, e.g., proteins, and specifically for example a wild-type operator, for example the wild-type TetO (SEQ ID NO: 7). In a further aspect, the DBD comprises the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to SEQ ID NO: 1 (wild-type TetR). Equivalents of the mutated DBDs are also provided by this disclosure, wherein the equivalent retains the one or more amino acids as shown in the DBD region of SEQ ID NOs: 2-6 or 9-22.

[0082] In one example, the LBD is a mutated tetracycline repressor LBD that binds to a molecule other than tetracycline. In yet a further aspect, the LBD binds to a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs). In one aspect, the LBD is selected from LBD is selected from the LBD region identified in SEQ ID NOs: 2- -20- 4871-7792-0235.1Atty Dkt.: 114198-3110 6 or 9-22, independently, or in one aspect, the LDB region shown in: L13-1-9 (SEQ ID NO: 4); L15-20 (SEQ ID NO: 3); CsL4.2-15 (SEQ ID NO: 5) and CsL4.2-20 (SEQ ID NO:6). Equivalents of the mutated LBDs are also provided by this disclosure, wherein the equivalent retains the one or more amino acids as shown in the LBD region of SEQ ID NOs: 2-6 or 9-22.

[0083] In a further aspect, the DBD comprises one or more mutations that alter the DNA- binding activity of the DBD to bind to other molecules such as for example, operator nucleotide variants other than those bound by the tetracycline repressor system (TetR) DBD molecules, e.g., proteins, and specifically for example a wild-type operator, for example the wild-type TetO (SEQ ID NO: 7). In a further aspect, the DBD comprises the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to SEQ ID NO: 1 (wild-type TetR).

[0084] In one example, the LBD is a mutated tetracycline repressor LBD that binds to a molecule other than tetracycline. In yet a further aspect, the LBD binds to a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs). In one aspect, the LBD is selected from LBD is selected from the LBD region identified in SEQ ID NOs: 2- 6 or 9-22, independently, or in one aspect, the LDB region shown in: L13-1-9 (SEQ ID NO: 4); L15-20 (SEQ ID NO: 3); CsL4.2-15 (SEQ ID NO: 5) and CsL4.2-20 (SEQ ID NO:6). Equivalents of the mutated LBDs are also provided by this disclosure, wherein the equivalent retains the one or more amino acids as shown in the LBD region of SEQ ID NOs: 2-6 or 9-22.

[0085] In a further aspect, the fusion polypeptide comprises one or more mutations in the DBD and / or LBD region, as compared to wild-type TetR, wherein the one or more mutations alter the DNA-binding activity of the repressor to bind to other molecules such as for example, operator nucleotide variants other than those bound by the tetracycline repressor system (TetR) and specifically for example a wild-type operator, for example the wild-type TetO (SEQ ID NO: 7). Examples of the fusion proteins are shown in SEQ ID NOs: 2-6 and 9-22, and equivalents thereof.

[0086] In one aspect, the LBD and the DBD are provided on the same protein or polypeptide. SEQ ID NOs: 2-6 and 9-22 and equivalents thereof are examples of such. -21- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0087] In one aspect, the operator is a Tet operator (tetO) e.g., TCC CTA TCA GTG ATA GAG A (SEQ ID NO: 7). In another aspect, the Tet operator is a tet operator variant identified herein as tetO-4C5G. The tetO-4C5G sequence is TCC CCG TCA GTG ACG GAG A (SEQ ID NO: 8).

[0088] In a further aspect, the system further comprises a ligand inducer or repressor agent such as tetracycline, ethametsulfuron-methyl (Es), chlorsulfuron (Cs), or another sulfonylurea (SU) herbicide, that binds to the ligand-binding domain (LBD) and influences the DNA- binding domain’s (DBD) ability to bind the operator DNA (enables binding to the operator DNA if it was not possible in the absence of the ligand inducer or promotes dissociation from the operator DNA if the protein was bound to the operator DNA in the absence of the ligand inducer). Non-limiting examples of register SU compounds (e.g., SU herbicides) include chlorsulfuron (W4189), thifensulfuron-methyl (M6316), tribenuron-methyl (L5300), metsulfuron- methyl (T6376), ethametsulfuron-methyl (A7881), chlorimuron-ethyl (F6025), rimsulfuron (E9636), nicosulfuron (V9630), and sulfometuron-methyl (T5648). Multiple Gene Repressor Systems

[0089] Further provided are multiple gene expressor systems comprising a collection of two or more gene repressor systems as described herein, wherein each of the two or more gene repressor systems work concurrently to modulate distinct target gene sequences controlled by a wild-type operator nucleotide or a mutated operator nucleotide. In one aspect, the LBD and the DBD of the gene repressor systems are provided on the same protein or polypeptide.

[0090] In a further aspect, the two or more systems of the multiple gene expressor system each comprise: a ligand-binding domain (LBD); b. a DNA-binding domain (DBD); and c. an operator nucleotide, wherein one or more of the LBD, DBD or the operator nucleotide include one or more mutations that change the DNA-binding specificity of the gene repressor system, such that the systems in combination work concurrently to modulate distinct target gene sequences. Examples of independent gene repressor systems are provided herein but in the multiple gene repressor system, it is not necessary that the DBD is a mutated polypeptide. -22- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0091] In one aspect, the gene repressor system or multiple gene expressor system is a tetracycline repressor system (TetR) or a mutated tetracycline repressor system (mTetR). In another aspect, at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) or a mutated tetracycline repressor system (mTetR).

[0092] In a further aspect, the DBD of the multiple system comprises one or more mutations that alter the DNA-binding activity of the DBD to bind to other molecules such as for example, operator nucleotide variants other than those bound by the tetracycline repressor system (TetR) DBD molecules, e.g., proteins, and specifically for example a wild-type operator, for example the wild-type TetO (SEQ ID NO: 7). In a further aspect, the DBD comprises the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to SEQ ID NO: 1 (wild-type TetR). Equivalents of the mutated DBDs are also provided by this disclosure, wherein the equivalent retains the one or more amino acids as shown in the DBD region of SEQ ID NOs: 2-6 or 9-22.

[0093] In one example, the LBD of the multiple system is a mutated tetracycline repressor LBD that binds to a molecule other than tetracycline. In yet a further aspect, the LBD binds to a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs). In one aspect, the LBD is selected from LBD is selected from the LBD region identified in SEQ ID NOs: 2-6 or 9-22, independently, or in one aspect, the LDB region shown in: L13-1-9 (SEQ ID NO: 4); L15-20 (SEQ ID NO: 3); CsL4.2-15 (SEQ ID NO: 5) and CsL4.2-20 (SEQ ID NO:6). Equivalents of the mutated LBDs are also provided by this disclosure, wherein the equivalent retains the one or more amino acids as shown in the LBD region of SEQ ID NOs: 2-6 or 9-22.

[0094] Further provided is a multiple gene expressor system or collection of two or more gene repressor systems as described herein, wherein each of the two or more gene repressor systems that work concurrently to modulate distinct target gene sequences controlled by a wild-type operator nucleotide or a mutated operator nucleotide. In one aspect, the LBD and the DBD of the gene repressor systems are provided on the same protein or polypeptide. In one aspect, at least one of the collections is a tetracycline repressor system (TetR) or a mutated tetracycline repressor system (mTetR). In another aspect, at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) or a mutated tetracycline repressor system (mTetR). In a further aspect, at -23- 4871-7792-0235.1Atty Dkt.: 114198-3110 least one of the collections of the DBD comprises one or more mutations that alter the DNA-binding activity of the DBD to bind to other molecules other than the DBD cognate molecule such as an operator variant. In one the LBD is a mutated tetracycline repressor that binds to a molecule other than tetracycline. In yet a further aspect, the LBD binds to a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs). In a further aspect, at least one of the DBD of the collection comprises the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to SEQ ID NO: 1 (wild-type TetR). In a further aspect, at least one of the repressor systems or the collection is a tetracycline repressor system (TetR) and the operator nucleotide comprises wild-type TetO (SEQ ID NO: 7) or the TetO-4C5G (SEQ ID NO: 8) variant. In a further aspect, at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) or a mTetR.

[0095] In alternative aspects of the system or the collection, the LBD comprises one or more mutations that alter the binding activity of the LBD to bind to other molecules other than the LBD cognate molecule. Non-limiting examples of such include an LBD that binds to a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

[0096] In one aspect, the operator is a Tet operator (tetO) e.g., TCC CTA TCA GTG ATA GAG A (SEQ ID NO: 7). In another aspect, the Tet operator is a tet operator variant identified herein as tetO-4C5G. The tetO-4C5G sequence is TCC CCG TCA GTG ACG GAG A (SEQ ID NO: 8).

[0097] In a further aspect, the system further comprises a ligand inducer or repressor agent such as tetracycline, ethametsulfuron-methyl (Es), chlorsulfuron (Cs), or another sulfonylurea (SU) herbicide, that binds to the ligand-binding domain (LBD) and influences the DNA- binding domain’s (DBD) ability to bind the operator DNA (enables binding to the operator DNA if it was not possible in the absence of the ligand inducer or promotes dissociation from the operator DNA if the protein was bound to the operator DNA in the absence of the ligand inducer). Non-limiting examples of register SU compounds (e.g., SU herbicides) include chlorsulfuron (W4189), thifensulfuron-methyl (M6316), tribenuron-methyl (L5300), metsulfuron- methyl (T6376), ethametsulfuron-methyl (A7881), chlorimuron-ethyl (F6025), rimsulfuron (E9636), nicosulfuron (V9630), and sulfometuron-methyl (T5648). -24- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0098] Further provided are multiple gene expressor systems comprising a collection of two or more gene repressor systems as described herein, wherein each of the two or more gene repressor systems work concurrently to modulate distinct target gene sequences controlled by a wild-type operator nucleotide or a mutated operator nucleotide. In one aspect, the LBD and the DBD of the gene repressor systems are provided on the same protein or polypeptide. Fusion Polypeptides

[0099] In a further aspect, the fusion polypeptide comprises one or more mutations in the DBD and / or LBD region, as compared to wild-type TetR, wherein the one or more mutations alter the DNA-binding activity of the repressor to bind to other molecules such as for example, operator nucleotide variants other than those bound by the tetracycline repressor system (TetR) and specifically for example a wild-type operator, for example the wild-type TetO (SEQ ID NO: 7). Examples of the fusion proteins are shown in SEQ ID NOs: 2-6 and 9-22, and equivalents thereof.

[0100] Also provided are fusion proteins is selected from: L13-1-9 (SEQ ID NO: 4); L15- 20 (SEQ ID NO: 3); CsL4.2-15 (SEQ ID NO: 5) and CsL4.2-20 (SEQ ID NO:6), TetR- V36FE37AP39K, and those presented as SEQ ID NOs.: 9 to 22, and equivalents of each thereof.

[0101] In a further aspect as described herein, the DBD of the system or collection comprises a mutated tetracycline repressor (mTetR) selected from the DBD region provided in a polypeptide selected from SEQ ID NOs: 2-6 or 9-22, and equivalents of that region. In a further aspect, the DBD is the DBD region of the mTetR having the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to wild-type TetR shown in SEQ ID NO: 1. Non-limiting examples of the DBD comprises, or consists essentially of, or consists of the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation as compared to the wild- type TetR shown in SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity thereto wherein the E37AP39K mutation, the V36AE37AP39K mutation, or the V36FE37AP39K mutation is retained. -25- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0102] Further provided are systems or collections wherein one or more, two or more or all three of the ligand-binding domain (LBD); the DNA-binding domain (DBD); and the operator nucleotide is detectably labeled.

[0103] Further provided are compositions comprising one or more gene expression system as described herein and a carrier. Polynucleotides, Vectors and Host Cells

[0104] This disclosure also provides isolated polynucleotides encoding the gene expression systems as described herein, wherein the polynucleotides are DNA or RNA. In one aspect, the polynucleotides comprise, or consist of, or consist essentially of a nucleic acid sequence encoding a polypeptides selected from SEQ ID NO: 2, 3-6, or 9-22, and equivalents of each thereof, wherein the polynucleotides encode polypeptides having at least 90% sequence identity to the reference polypeptide, when evaluated under BLAST run under default conditions, or to a nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2, 3-6, or 9-22 respectively, with the proviso that the equivalent excludes the wild-type nucleic acid sequence encoding polypeptides individually for the respective SEQ ID NO: 2, 3-6, or 9-22 polypeptides. The isolated equivalent polynucleotide can be DNA or RNA or hybrids thereof.

[0105] Further provided are vectors comprising an isolated polynucleotide comprising, or consisting of, or consisting essentially of a nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2, 3-6, or 9-22 and equivalents of each thereof having at least 90% sequence identity when evaluated under BLAST run under default conditions, to a nucleic acid sequence selected from SEQ ID NO: 2, 3-6, or 9-22 respectively, with the proviso that the equivalent excludes the wild-type nucleic acid sequence encoding individually for the respective polypeptides of SEQ ID NO: 2, 3-6, or 9-22. The isolated polynucleotide can be DNA or RNA or hybrids thereof.

[0106] Also provided is an isolated polynucleotide comprising, or consisting of, or consisting of SEQ ID NO: 8, and equivalents thereof.

[0107] Also provided are vectors comprising the polynucleotides described herein. Non- limiting examples of vectors include viral vectors, adeno-associated viral vectors, -26- 4871-7792-0235.1Atty Dkt.: 114198-3110 plasmids and baculovirus. In a specific embodiment, the vector is a viral vector or a plasmid vector.

[0108] Further provided is an isolated host cell comprising one or more of the gene expression system or collections as described herein, and / or an isolated polynucleotide an isolated polynucleotide comprising, or consisting of, or consisting essentially of a nucleic acid sequence encoding a polypeptide selected from SEQ ID NOs: 2, 3-6, or 9-22, and equivalents of each thereof having at least 90% sequence identity when evaluated under BLAST, to a nucleic acid sequence encoding a polypeptide selected from SEQ ID NOs: 2, 3-6, or 9-22, respectively, with the proviso that the equivalent excludes the wild-type nucleic acid sequence individually for the respective SEQ ID NOs: 2, 3-6, or 9-22. The isolated polynucleotide can be DNA or RNA or hybrids thereof. The isolated host cell can be a procaryotic or a eukaryotic cell, e.g., a mammalian, an animal or a plant cell.

[0109] Further provided are one or more gene expression system, fusion proteins, polynucleotides, vectors, or host cells and a carrier. The carrier can be a pharmaceutically acceptable carrier. Methods

[0110] Further provided are methods for controlling expression of a target gene linked to an operator nucleotide that controls expression of a target polynucleotide, the method comprising, or consisting essentially of, or yet further consisting of expressing a polynucleotide encoding the gene expression system or collection as described herein in a host cell and optionally contacting the cell with a repressor agent. The contacting can be in vitro or in vivo. In one aspect, the repressor agent is selected from tetracycline, a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

[0111] Also provided are methods of regulating transcription of a polynucleotide of interest in an animal host cell comprising providing a repressor agent selected from a sulfonylurea herbicide compound, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs) to a host cell such that the repressor agent binds to form a complex that modifies the binding properties to the operator. The host cell can be an animal cell, optionally a mammalian cell. -27- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0112] Kits are further provided herein, wherein the kit comprises one or more agents of the system or collection and instructions for use. Modes For Carrying Out the Disclosure

[0113] There are multiple unique aspects of this disclosure. Applicant is the first to combine these mutations in the binding pocket with published mutations that change DNA‐binding specificity and published mutations that convert repressors to activators, and vice versa. Applicant is the first to deploy such repressors and activators in animals and their cells and tissues.

[0114] This disclosure also brings together multiple sets of mutations to the microbial tetracycline repressor (TetR), which remains one of the dominant ways to control expression of transgenes in cells and organisms ‐ microbes, plants, animals, etc. Applicant combined different sets of mutations that regulate 3 different functions of TetR: 1. ligand (inducer) binding specificity, 2. DNA (operator) binding specificity, and 3. conformational coupling of inducer binding to DNA-binding. To the best of Applicant’s knowledge, Applicant is the first to combine these sets of mutations, and the first to use the resulting proteins in animal cells, where they work quite well. To the best of Applicant’s knowledge, Applicant is the first to use the designed herbicide‐responsive repressors in animal cells and demonstrate that the herbicides are well tolerated by animal cells, penetrate well, and are potent inducers. This system will open up new avenues for expressing transgenes in animal systems.

[0115] To the best of Applicant’s knowledge, Applicant also is the first to demonstrate that: the TetRs with redesigned binding pockets work as expected in animal cells; that different repressors can be driven with one of 2 different herbicides: ethametsulfuron‐ methyl (Es) and chlorsulfuron (Cs), in addition to the existing systems using tetracycline (Tet) or related antibiotics; that mutants specific for Tet, Es, and Cs can be mutually orthogonal and used simultaneously in animal cells; that these repressors can be combined with mutations changing the operator (TetO) sequence specificity and used orthogonally; that these repressors can be turned into activators by grafting known mutations.

[0116] The commercial applications of this disclosure include systems for controlling transgene expression in animal cells and animals. The TetR expression system is the basis -28- 4871-7792-0235.1Atty Dkt.: 114198-3110 for many commercial products in this space. This disclosure provides an alternative way to construct such products, as well as facilitate the development of revolutionary products allowing simultaneous multiple transgene control ‐ which do not exist currently. Non- limiting examples of applications include: 1. services to construct mice with inducible control, 2. a kit comprising the repressor system DNA, 3. bacteria or other cells expressing the system, allowing selection of new repressors and activators. Materials and Methods Cloning

[0117] Human codon-optimized sequences of SUR LBDs were synthesized by Twist and inserted in the place of the wild-type TetR LBD in the pcDNA6 / TR (“Tet Repressor”) plasmid (T-REx kit, Thermo-Fisher Scientific) via Gibson assembly. Reporter sfGFP and mScarlet-I3 genes were inserted in the pcDNA4 / TO (“Tet Operator”) vector (T-REx kit, Thermo-Fisher Scientific) via restriction cloning. pcDNA4 / TO-sfGFP reporter plasmid was further converted into pcDNA4 / TO-4C5G-sfGFP reporter vector and DBD mutations E37AP39K, V36AE37AP39K, and V36FE37AP39K were incorporated into all 5 repressor- encoding vectors via side-directed mutagenesis. Transient Transfections

[0118] HEK293 cells were plated in 24-well plates (30,000 cells per well). Next day the cells were transfected with 0.5 (co-transfection of 2 plasmids) or 1 μg (co-transfection of 4 plasmids) of DNA mixture and 2 μl of TurboFect (Thermo-Fisher Scientific) according to the manufacturer protocol. The DNA mixtures consisted of ~14:1 (w / w) pcDNA6 / TR:pcDNA4 / TO-reporter plasmid DNA ratio for experimental conditions and ~6:1 pcDNA4 / TR:pcDNA4 / TO-reporter plasmid DNA ratio for single stain controls. Unstained control cells were treated similarly, but no DNA was added in the reaction mixture.24 hours after transfection, the medium was replaced with fresh medium supplemented with 1 μg / mL Tet, Es, Cs, and / or the appropriate amount of DMSO (carrier solvent, max 0.2%).24 hours after induction, the cells were harvested, resuspended in serum-free Opti-MEM (with or without propidium iodide depending on the experiment), and analyzed by flow cytometry using BD FACSymphony A1 cell Analyzer. Fluorescence microscopy -29- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0119] Fluorescence images of the plates were taken using a Keyence BZ-X810 with 10x objective and the Chroma 49002 EGFP fluorescence filter set (excitation: 470 nm / 40 nm bandwidth; dichroic mirror 495 nm; emission: 525 nm / 50 nm bandwidth). Results

[0120] Applicant first sought to demonstrate that the new repressors expressed well and were well-tolerated in mammalian cells; that the small molecule SU inducers were not toxic and were delivered well to cells; and that the SUR-SU system can drive strong gene expression in mammalian cells with minimal background expression. HEK293 cells were transiently co- transfected with the TetO-sfGFP reporter plasmid and one of the repressor-expressing plasmids (wild-type TetR; 2 EsR constructs: L13-1-9 and L15-20; 2 CsR constructs: CsL4.2- 15 and CsL4.2-20) – or an empty vector control. Applicant then added Tet, Es, Cs, or DMSO (negative control), waited 24 hours for the reporter “target” genes to express, and measured the samples on fluorescence activated cell sorting (FACS) (FIG.1).

[0121] As seen in FIG.1, TetR responds strongly to Tet and neither SU ligand; EsR L13-1-9 and L15-20 responded strongly to Es but neither Tet nor Cs; CsR CsL4.2-15 responded strongly to Cs and weakly to Es; CsR CsL4.2-20 responded strongly to both Es and Cs; and with no transfected repressor, the GFP signal was equivalent in all cells.

[0122] Having shown that the new SUR repressors work well in mammalian cells, and that the SU ligands Es and Cs are delivered well, Applicant next incorporated published mutations to the DBD that alter DNA-binding specificity of the wild-type TetR, as preparation for creating orthogonal gene expression systems that can be used to independently modulate two different genes.

[0123] Three (3) published sets of mutations that allow the DBD of wild-type TetR to bind to a modified DNA operator sequence: “TetO-4C5G.”11were tested. The 3 mutation sets are E37AP39K, V36AE37AP39K, and V36FE37AP39K11. Applicant first made and tested these mutations being grafted onto a number of different repressors, including wild-type TetR, 2 ethametsulfuron-methyl (Es)-responsive repressors (EsR-L13-1-9 and EsR-L15-20), and 2 chlorsulfuron (Cs)-responsive repressors (CsR-4.2.15 and CsR-4.2.20). Applicant then tested them in transiently co-transfected cultured HEK293 cells, where they each drive expression of a superfolder GFP (sfGFP) reporter gene, under control of either the wild-type TetO -30- 4871-7792-0235.1Atty Dkt.: 114198-3110 operator (top row) with wild-type DBD-containing repressor, or the mutated TetO-4C5G operator and a repressor with one of the 3 combinations of DBD mutations (FIG.2). Each condition was also tested with a control DMSO (carrier solvent) addition, to test for leakiness of condition. (Note that camera exposure was 5 times longer for the DMSO control to better detect low-level leakiness.) For FIGS.2B-2E, 4 representative fields-of-view are shown for each condition.

[0124] These experiments show that the 3 mutation sets all work well with wild-type TetR to make Tet-responsive repressors recognizing the TetO-4C5G operator. Of the SURs, the Es- responsive EsR-L15-20 works quite well with the V36AE37AP39K mutation set, producing Es activation similar to wild-type and only slightly elevated leakiness (FIG.2C). This clone can be used in applications alongside TetR-TetO, and the 3 DBD mutants of TetR can be used alongside any repressor with TetO. The other repressors should be further mutated in the DBD and nearby regions to rescue high inducibility and low leakiness.

[0125] Applicant next investigated whether it would be possible to simultaneously use the TetR-Tet and SUR-SU systems in different cell populations. This could enable technologies such as expressing Gene1 in neurons under the control of Molecule1, and Gene2 in astrocytes under the control of Molecule2. The molecules can be added at different times or together, would be delivered to the cells expressing the appropriate switch, and modulate target gene expression. For these experiments, Applicant used two cell populations: wild-type TetR with the mutated DBD regulating expression of the TetO-4C5G-controlled sfGFP and a SUR with the wild-type DBD regulating expression of the TetO-controlled red fluorescent protein mScarlet-I3. To demonstrate the orthogonality of the systems, Applicant compared the mixtures of independently activated and non-activated cell populations with the mixtures of the two populations each incubated with the appropriate inducer for 24 hours (FIG.3).

[0126] These experiments showed that the mutated TetR and EsR-L13-1-9 can be used simultaneously, being induced by Tet and Es, respectively, with essentially zero cross-talk between the inducers and ligand-binding domains. That is, Tet doesn’t affect EsR in any way, and Es doesn’t affect TetR in any way. Thus, Applicant has shown simultaneous deployment of these two gene switches in animals to control 2 different target genes in different cell types is possible. -31- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0127] In a third set of experiments, Applicant determined if the two gene switches could be simultaneously used in the same cells. This is a more discriminating test, as it is now susceptible to the ligands binding to the wrong LBDs (with or without inducing dissociation from the DNA) and also the DBDs binding to the wrong operator elements, thus creating cross-talk between the two expression systems. Applicant thus tested simultaneous usage of the DBD-TetO and mutated DBD-TetO-4C5G repressor-operator pairs in the same cells. Applicant used the wild-type TetR with the mutated DBD, and the SUR with the wild-type DBD. Thus, Tet will regulate a transgene under the TetO-4C5G operator, and SU will regulate a transgene under the wild-type TetO operator. In this experiment, Tet drives sfGFP (green) fluorescence, and Es drives mScarlet-I3 (red) fluorescence (FIG.4). This configuration can be switched, i.e., wild-type TetR with wild-type TetO and mutated SUR with TetO-4C5G. This would allow a plug and play system because there are many existing reagents (e.g., mouse lines) with the TetR-TetO setup, and Applicant would want to create things that could plug seamlessly into those.

[0128] These experiments showed that the TetO-4C5G operator – TetR-V36FE37AP39K can be used in conjunction with the wild-type TetO operator – EsR independently inside the same cells, thus paving the way to independent control of expression of two genes within the same cell type – for instance, both presenilin and Aβ peptide in Alzheimer’s mouse models. Discussion

[0129] Together, Applicant have demonstrated that: 1. the SUR repressors function well in animal cells, with low background, high activation, and no obvious cellular toxicity, 2. the SU molecules are well delivered to cells, also producing no obvious toxicity, 3. the SUR-SU switches function well along TetR-Tet to drive reporter genes independently in different cell populations, and 4. the SUR-SU and TetR-Tet gene switches are orthogonal in ligand input and DNA-binding output in single cells, thus creating opportunities for expressing multiple genes in the same cell, either simultaneously or at different times, and to different extents, based on concentration of inducer.

[0130] The SU inducers that Applicant demonstrate here are non-toxic to animals and can be seamlessly used in mice and other model organisms. Given their small molecular weight, amphipathic nature, and presence of functional groups, these inducers appear “drug-like” and -32- 4871-7792-0235.1Atty Dkt.: 114198-3110 it is likely that they will be well tolerated in animals and even cross the blood-brain barrier (BBB). Chemically related molecules such as glimepiride readily cross the BBB12, where they are efficacious in treating and preventing ischemic stroke. Thus, Applicant’s repressor- inducer systems will be broadly useful in diverse cell types across the body, including in the brain behind the BBB.

[0131] US Patent No.8,777,503 provided systems for exclusive use in plants while the current system and methods are designed for use in higher order organisms, e.g., for use in animal, mammal and human cells and organisms. Moreover, the ‘503 Patent did not disclose how to incorporate mutations in the DNA-binding region of the repressor protein. Here, utility is established for mutations of the repressors at modified tetracycline operators, and provides mutations in both the DNA-binding region and the ligand binding regions to enable interaction of the proteins with the new operator. This allows for the use of multiple gene induction pathways in the same cell. The systems of the ‘503 are not applicable to simultaneous usage of two or more (e.g., three) operators and two or possibly three proteins to independently and inducibly regulate the expression of multiple genes in the same animal cell or organism.

[0132] Applicant has shown that the SUR-SU switches can be used in conjunction with TetR-Tet, either in different cell populations or the same cells. This creates numerous opportunities for beneficial reagents and commercial products, by facilitating the first-in- class possibility of controlling the expression of two genes independently in animals. If the switches were put upstream of activity-dependent reporters, this could create a system for labeling cellular activity in different cellular populations during different user-defined periods (by ligand delivery). If the switches are coupled to therapeutic target proteins, 2 different (potentially interacting) target molecules could be up- or down-regulated at will by driving expression of the target molecule, an inhibitory RNA against the mRNA of that target molecule, etc.

[0133] Many mouse experiments use engineered versions of the TetR repressor, coupling it to viral transactivator domains such as VP16, thus creating “Tet transactivators” (tTA).13Mutation of the repressor changes the ligand-dependent allosteric modulation of the protein such that it now binds DNA in the presence of ligand and dissociates in its absence, thus creating “reverse TetR” (rTetR). Fusion of the transactivator to this reverse repressor -33- 4871-7792-0235.1Atty Dkt.: 114198-3110 produces rtTA. These “Tet-OFF” (tTA) and “Tet-ON” (rtTA) systems have higher levels of induction and lower background levels than the isolated TetR and rTetR. It is likely that the SURs will work with the reverse mutations, producing rSURs, and the fusion with the VP16 transactivator, creating tSUA (“SU-OFF”) and rtSUA (“SU-ON”). These can be used in mammalian cell culture and in vivo in mice. The simultaneous use of Tet-ON / OFF and SU- ON / OFF in different cell populations and in the same cells also is provided herein. These reagents can dramatically expand options for inducible expression in mammalian cell culture and other animal systems. Clauses

[0134] Clause 1. A multiple gene repressor system comprising two or more gene repressor systems, wherein each of the gene repressor systems comprises: a ligand-binding domain (LBD); b. a DNA-binding domain (DBD); and c. an operator nucleotide that controls expression of a target polynucleotide, and optionally wherein LBD, DBD, or the operator nucleotide comprise one or more mutations that change the DNA-binding specificity of the gene repressor system, and further optionally wherein each of the two or more gene repressor systems work concurrently to modulate distinct target gene sequences controlled by a wild- type operator nucleotide or a mutated operator nucleotide.

[0135] Clause 2. The multiple gene expression system of clause 1, wherein at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) and the ligand- binding domain binds to tetracycline (Tet).

[0136] Clause 3. The multiple gene expression system of clause 1 or 2, wherein each of the two or more gene expression systems are on a single contiguous polynucleotide.

[0137] Clause 4. The multiple gene expression system of any one of clauses 1-3, further comprising a polynucleotide comprising a target gene sequence operably linked to a promoter comprising at least one operator sequence.

[0138] Clause 5. The multiple gene expression system of any one of clauses 1-4, wherein the operator nucleotide comprises wild-type TetO (SEQ ID NO: 7) or an operator variant, optionally the TetO-4C5G (SEQ ID NO: 8) variant. -34- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0139] Clause 6. The multiple gene expression system of any one of clauses 1-5, wherein the LBD comprises one or more mutations that alter the ligand binding activity of the LBD to bind to other molecules other than the LBD wild-type binding molecule.

[0140] Clause 7. The multiple gene expression system of clause 6, wherein the LBD binds to a repressor agent selected from tetracycline (Tet), a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

[0141] Clause 8. The multiple gene expression system of any one of clauses 1-7, wherein the mutated DBD comprises a mutated polypeptide as compared to amino acids 1-49 of SEQ ID NO: 1 and / or the mutated LBD polypeptide comprises a mutated polypeptide as compared to amino acids 50-207 of SEQ ID NO: 1.

[0142] Clause 9. The multiple gene expression system of any one of clauses 1-8, wherein the mutated DNA-binding domain (DBD) and / or the mutated LBD comprise a fusion protein with the individual LBD and DBD selected from those DBD and / or LBD domains identified in any polypeptides of SEQ ID NOs: 2-6 or 9-22, and equivalents of each thereof.

[0143] Clause 10. The multiple gene expression system of any one of clauses 1-9, comprising TetO-4C5G operator and TetR-V36FE37AP39K repressor; or TetO operator and L13-1-9 repressor.

[0144] Clause 11. The multiple gene expression system of any one of clauses 1-9, comprising TetO operator and wtTetR repressor; or TetO-4C5G operator and L15-20-V36AE37AP39K repressor.

[0145] Clause 12. The multiple gene expression system of any one of clauses 1-11, wherein the DBD and LBDs are selected from any one of SEQ ID NOs: 2-6 or 9-22, and equivalents of each thereof.

[0146] Clause 13. The multiple gene expression system of any one of clauses 1-11, wherein one or more, two or more or all three of the ligand-binding domain (LBD); the DNA-binding domain (DBD); or the operator nucleotide is detectably labeled.

[0147] Clause 14. A gene repressor system comprising: a. a ligand-binding domain (LBD); b. a mutated DNA-binding domain (DBD); and c. an operator nucleotide that controls expression of a target polynucleotide, and optionally wherein LBD or the operator nucleotide -35- 4871-7792-0235.1Atty Dkt.: 114198-3110 comprise one or more mutations that change the DNA-binding specificity of the gene repressor system.

[0148] Clause 15. The system of clause 14, further comprising a polynucleotide comprising a target gene sequence operably linked to a promoter comprising at least one operator sequence.

[0149] Clause 16. The system of clause 14 or 15, wherein the LBD comprises one or more mutation that changes the DNA-binding specificity of the gene repressor system.

[0150] Clause 17. The gene expression system of any one of clauses 14-16, wherein the gene repressor system is a tetracycline repressor system (TetR) and the ligand-binding domain binds to tetracycline (Tet) repressor agent.

[0151] Clause 18. The gene expression system of any one of clauses 14-17, wherein the operator nucleotide comprises wild-type TetO (SEQ ID NO: 7) or an operator variant, optionally the TetO-4C5G (SEQ ID NO: 8) variant.

[0152] Clause 19. The gene expression system of any one of clauses 14-18, wherein the LBD comprises one or more mutations that alter the ligand binding activity of the LBD to bind to other molecules other than the LBD wild-type binding molecule.

[0153] Clause 20. The gene expression system of clause 19, wherein the LBD binds to a repressor agent selected from tetracycline (Tet), a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

[0154] Clause 21. The gene expression system of any one of clauses 14-20, wherein the mutated DBD comprises a mutated polypeptide as compared to amino acids 1-49 of SEQ ID NO: 1 and / or the mutated LBD polypeptide comprises a mutated polypeptide as compared to amino acids 50-207 of SEQ ID NO: 1.

[0155] Clause 22. The gene expression system of any one of clauses 14-21, wherein the mutated DNA-binding domain (DBD) and / or the mutated LBD comprise a fusion protein with the individual LBD and DBD domains identified in any one of the polypeptides of SEQ ID NOs: 2-6 or 9-22, and equivalents of each thereof.

[0156] Clause 23. The gene expression system of any one of clauses 14-22, wherein the LBD and the DBD are fusion polypeptides selected from any one of SEQ ID NOs: 2-6 or 9-22. -36- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0157] Clause 24. The gene expression system of any one of clauses 14-23, comprising TetO-4C5G operator and TetR-V36FE37AP39K repressor; or TetO operator and L13-1-9 repressor.

[0158] Clause 25. The gene expression system of any one of clauses 14-24, comprising TetO operator and wtTetR repressor; or TetO-4C5G operator and L15-20-V36AE37AP39K repressor.

[0159] Clause 26. The gene expression system of any one of clauses 14-25, wherein one or more, two or more or all three of the ligand-binding domain (LBD); the DNA-binding domain (DBD); or the operator nucleotide is detectably labeled.

[0160] Clause 27. A multiple gene repressor system comprising two or more gene repressor systems, wherein each of the gene repressor systems comprises: a ligand-binding domain (LBD); b. a DNA-binding domain (DBD); and c. an operator nucleotide that controls expression of a target polynucleotide, and optionally wherein LBD, DBD, or the operator nucleotide comprise one or more mutations that change the DNA-binding specificity of the gene repressor system, or alternatively, wherein the two or more gene repressor systems are defined by any one of clauses 14-26, and further optionally wherein each of the two or more gene repressor systems work concurrently to modulate distinct target gene sequences controlled by a wild-type operator nucleotide or a mutated operator nucleotide.

[0161] Clause 28. The multiple gene expression system of clause 27, wherein at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) and the ligand-binding domain binds to tetracycline (Tet).

[0162] Clause 29. The multiple gene expression system of clause 27 or 28, wherein each of the two or more gene expression systems are on a single contiguous polynucleotide.

[0163] Clause 30. An isolated fusion polypeptide selected from any one of SEQ ID NOs: 2-6 or 9-22, and equivalents of each thereof.

[0164] Clause 31. An isolated polynucleotide encoding the isolated fusion polypeptide of clause 30, or an equivalent polynucleotide.

[0165] Clause 32. An isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2 to 6 or 9-22, and polynucleotides encoding -37- 4871-7792-0235.1Atty Dkt.: 114198-3110 equivalents having at least 90% sequence identity to any one of SEQ ID NO: 2 to 6 or 9 to 22 when evaluated under BLAST, or having at least 90% sequence identity to a nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2 to 6 or 9 to 22, with the proviso that the equivalent excludes the wild-type nucleic acid sequence individually for the respective nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2, 3-6, or 9-22.

[0166] Clause 33. An isolated polynucleotide encoding one or more of elements a through c of the gene expression system of any one of clauses 1-29.

[0167] Clause 34. A vector comprising an isolated polynucleotide of any one of clauses 31- 33.

[0168] Clause 35. The vector of clause 34, wherein the vector is a viral vector or a plasmid vector.

[0169] Clause 36. An isolated host cell comprising one or more of the gene expression system of any of clauses 1-29, the fusion polypeptide of claim 30, the isolated polynucleotide of any one of claims 31-33, or the vector of claim 34 or 35.

[0170] Clause 37. The isolated host cell of clause 36, wherein the cell is a procaryotic or a eukaryotic cell.

[0171] Clause 38. A method for controlling expression of a target gene linked to an operator nucleotide that controls expression of a target polynucleotide, comprising expressing a polynucleotide encoding the gene expression system of any of clauses 1-29 in a host cell, and optionally contacting the cell with a repressor agent.

[0172] Clause 39. The method of clause 38, further comprising a target polynucleotide.

[0173] Clause 40. The method of clause 38 or 39, wherein the host cell is an animal cell, optionally a mammalian cell.

[0174] Clause 41. The method of claim any one of clauses 38-40, wherein the repressor agent is selected from tetracycline (Tet), a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

[0175] Clause 42. A method of regulating transcription of a polynucleotide of interest in an animal host cell comprising: a) providing a repressor agent selected from a sulfonylurea -38- 4871-7792-0235.1Atty Dkt.: 114198-3110 herbicide compound, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs) to a host cell of any one of clause 36 or 37such that the repressor agent binds to form a complex that modifies the binding properties to the operator.

[0176] Clause 43. The method of clause 42, wherein the host cell is an animal cell, optionally a mammalian cell. Equivalents

[0177] Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosure embodied therein herein disclosed can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

[0178] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0179] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0180] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0181] Sequences

[0182] SEQ ID NO: 1 Wild-type (wt) Tetracycline Repressor (TetR) – locations for mutation of amino acids are bolded and underlined: V36, E37 and P39 are present in the -39- 4871-7792-0235.1Atty Dkt.: 114198-3110 wild-type repressor. Amino acids 1-49 represent the DBD domain, and amino acids 50-207 represent the LBD domain.

[0183] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALL DALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEK QYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDS MPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSGSREFRSY

[0184] SEQ ID NO 2: TetR-V36AE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the LBD domain. Locations for mutated amino acids are bolded and underlined.

[0185] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGAAQKTLYWHVKNKRAL LDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTE KQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSGSREFRSY

[0186] SEQ ID NO 3: L15-20. Amino acids 1-49 represent the wild-type DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0187] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALL DAMAIEMLDPHKIHYLPLEGESWQDFLRNRAKSMRNALLSHRDGAKVCLGTGFTE RQYETAENTLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETPTT DSMPPLVRQAYELADHQGAEPAFLFGLELIISGLEKQLKAESGSAYSGSREFRSY

[0188] SEQ ID NO 4: L13-1-9. Amino acids 1-49 represent the wild-type DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0189] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALL DAMAIEMLDRHATHYCPLEGESWQDFLRNKAKSMRNALLSHRDGAKVALGTGFT EQQYETMENSLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETPT TDSMPPLVRQAYELKDHQGAEPAFLFGLELIICGLEKQLKAESGSAYSGSREFRSY

[0190] SEQ ID NO 5: CsL4.2-20. Amino acids 1-49 represent the wild-type DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0191] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALL DALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRLALLSHRDGAKVSLGTRWTE -40- 4871-7792-0235.1Atty Dkt.: 114198-3110 QQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETPT PDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0192] SEQ ID NO 6: CsL4.2-15. Amino acids 1-49 represent the wild-type DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0193] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALL DALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRRALLSHRDGAKVSLGTRWTE QQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETPT TDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0194] SEQ ID NO: 7: (TetO sequence) TCC CTA TCA GTG ATA GAG A.

[0195] SEQ ID NO: 8: (TetO-4C5G sequence) TCC CCG TCA GTG ACG GAG A.

[0196] SEQ ID NO 9: TetR-E37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the LBD domain.

[0197] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVAQKTLYWHVKNKRAL LDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTE KQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSGSREFRSY

[0198] SEQ ID NO 10: TetR-V36FE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the LBD domain.

[0199] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGFAQKTLYWHVKNKRAL LDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTE KQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSGSREFRSY

[0200] SEQ ID NO 11: L15-20-E37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0201] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVAQKTLYWHVKNKRAL LDAMAIEMLDPHKIHYLPLEGESWQDFLRNRAKSMRNALLSHRDGAKVCLGTGFT ERQYETAENTLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETPT TDSMPPLVRQAYELADHQGAEPAFLFGLELIISGLEKQLKAESGSAYSGSREFRSY -41- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0202] SEQ ID NO 12: L15-20-V36AE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0203] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGAAQKTLYWHVKNKRAL LDAMAIEMLDPHKIHYLPLEGESWQDFLRNRAKSMRNALLSHRDGAKVCLGTGFT ERQYETAENTLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETPT TDSMPPLVRQAYELADHQGAEPAFLFGLELIISGLEKQLKAESGSAYSGSREFRSY

[0204] SEQ ID NO 13: L15-20-V36FE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0205] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGFAQKTLYWHVKNKRAL LDAMAIEMLDPHKIHYLPLEGESWQDFLRNRAKSMRNALLSHRDGAKVCLGTGFT ERQYETAENTLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETPT TDSMPPLVRQAYELADHQGAEPAFLFGLELIISGLEKQLKAESGSAYSGSREFRSY

[0206] SEQ ID NO 14: L13-1-9-E37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0207] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVAQKTLYWHVKNKRAL LDAMAIEMLDRHATHYCPLEGESWQDFLRNKAKSMRNALLSHRDGAKVALGTGF TEQQYETMENSLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETP TTDSMPPLVRQAYELKDHQGAEPAFLFGLELIICGLEKQLKAESGSAYSGSREFRSY

[0208] SEQ ID NO 15: L13-1-9-V36AE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0209] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGAAQKTLYWHVKNKRAL LDAMAIEMLDRHATHYCPLEGESWQDFLRNKAKSMRNALLSHRDGAKVALGTGF TEQQYETMENSLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETP TTDSMPPLVRQAYELKDHQGAEPAFLFGLELIICGLEKQLKAESGSAYSGSREFRSY

[0210] SEQ ID NO 16: L13-1-9-V36FE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0211] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGFAQKTLYWHVKNKRAL LDAMAIEMLDRHATHYCPLEGESWQDFLRNKAKSMRNALLSHRDGAKVALGTGF -42- 4871-7792-0235.1Atty Dkt.: 114198-3110 TEQQYETMENSLAFLTQQGFSLENALYAFQAVGIYTLGCVLLDQELQVAKEERETP TTDSMPPLVRQAYELKDHQGAEPAFLFGLELIICGLEKQLKAESGSAYSGSREFRSY

[0212] SEQ ID NO 17: CsL4.2-20-E37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0213] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVAQKTLYWHVKNKRAL LDALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRLALLSHRDGAKVSLGTRWT EQQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETP TPDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0214] SEQ ID NO 18: CsL4.2-20-V36AE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0215] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGAAQKTLYWHVKNKRAL LDALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRLALLSHRDGAKVSLGTRWT EQQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETP TPDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0216] SEQ ID NO 19: CsL4.2-20-V36FE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0217] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGFAQKTLYWHVKNKRAL LDALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRLALLSHRDGAKVSLGTRWT EQQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETP TPDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0218] SEQ ID NO 20: CsL4.2-15-E37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0219] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVAQKTLYWHVKNKRAL LDALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRRALLSHRDGAKVSLGTRWT EQQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETP TTDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0220] SEQ ID NO 21: CsL4.2-15-V36AE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain. -43- 4871-7792-0235.1Atty Dkt.: 114198-3110

[0221] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGAAQKTLYWHVKNKRAL LDALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRRALLSHRDGAKVSLGTRWT EQQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETP TTDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0222] SEQ ID NO 22: CsL4.2-15-V36FE37AP39K. Amino acids 1-49 represent the mutated DBD domain, and amino acids 50-207 represent the mutated LBD domain.

[0223] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGFAQKTLYWHVKNKRAL LDALAIEMHDRHQTHYLPLEGESWQDFLRNFAKSMRRALLSHRDGAKVSLGTRWT EQQYETAENMLAFLTQQGFSLENALYATDAVRVFTLGAVLLDQEQQVAKEERETP TTDSMPPLLRQAWELKVHQGAEPAFLFGLELIIAGLEKQLKRESGSAYSGSREFRSY

[0224] Other aspects are set forth within the following claims. -44- 4871-7792-0235.1Atty Dkt.: 114198-3110 REFERENCES 1 Doshi, A., Sadeghi, F., Varadarajan, N. & Cirino, P. C. Small-molecule inducible transcriptional control in mammalian cells. Critical reviews in biotechnology 40, 1131-1150 (2020). 2 Stebbins, M. J. et al. Tetracycline-inducible systems for Drosophila. Proceedings of the National Academy of Sciences 98, 10775-10780 (2001). 3 Mao, S. et al. A Tet / Q hybrid system for robust and versatile control of transgene expression in C. elegans. IScience 11, 224-237 (2019). 4 Esengil, H. & Chen, J. K. Gene regulation technologies in zebrafish. Molecular BioSystems 4, 300-308 (2008). 5 Le Guiner, C. et al. Transgene regulation using the tetracycline-inducible TetR- KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates. PloS one 9, e102538 (2014). 6 Kallunki, T., Barisic, M., Jäättelä, M. & Liu, B. How to choose the right inducible gene expression system for mammalian studies? Cells 8, 796 (2019). 7 Mullick, A. et al. The cumate gene-switch: a system for regulated expression in mammalian cells. BMC biotechnology 6, 1-18 (2006). 8 Rivera, V. M. et al. A humanized system for pharmacologic control of gene expression. Nature medicine 2, 1028-1032 (1996). 9 Gosti, F. et al. ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling. The Plant Cell 11, 1897-1909 (1999). 10 Del Prato, S. & Pulizzi, N. The place of sulfonylureas in the therapy for type 2 diabetes mellitus. Metabolism 55, S20-S27 (2006). 11 Krueger, M., Scholz, O., Wisshak, S. & Hillen, W. Engineered Tet repressors with recognition specificity for the tetO-4C5G operator variant. Gene 404, 93-100 (2007). 12 Wang, X. et al. Glimepiride and glibenclamide have comparable efficacy in treating acute ischemic stroke in mice. Neuropharmacology 162, 107845 (2020). 13 Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences 89, 5547-5551 (1992). -45- 4871-7792-0235.1

Claims

Atty Dkt.: 114198-3110 WHAT IS CLAIMED IS:

1. A multiple gene repressor system comprising two or more gene repressor systems, wherein each of the gene repressor systems comprises: a ligand-binding domain (LBD); b. a DNA-binding domain (DBD); and c. an operator nucleotide that controls expression of a target polynucleotide, and optionally wherein LBD, DBD, or the operator nucleotide comprise one or more mutations that change the DNA-binding specificity of the gene repressor system, and further optionally wherein each of the two or more gene repressor systems work concurrently to modulate distinct target gene sequences controlled by a wild-type operator nucleotide or a mutated operator nucleotide.

2. The multiple gene expression system of claim 1, wherein at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) and the ligand-binding domain binds to tetracycline (Tet).

3. The multiple gene expression system of claim 1 or 2, wherein each of the two or more gene expression systems are on a single contiguous polynucleotide.

4. The multiple gene expression system of any one of claims 1-3, further comprising a polynucleotide comprising a target gene sequence operably linked to a promoter comprising at least one operator sequence.

5. The multiple gene expression system of any one of claims 1-4, wherein the operator nucleotide comprises wild-type TetO (SEQ ID NO: 7) or an operator variant, optionally the TetO-4C5G (SEQ ID NO: 8) variant.

6. The multiple gene expression system of any one of claims 1-5, wherein the LBD comprises one or more mutations that alter the ligand binding activity of the LBD to bind to other molecules other than the LBD wild-type binding molecule.

7. The multiple gene expression system of claim 6, wherein the LBD binds to a repressor agent selected from tetracycline (Tet), a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs). -46- 4871-7792-0235.1Atty Dkt.: 114198-3110 8. The multiple gene expression system of any one of claims 1-7, wherein the mutated DBD comprises a mutated polypeptide as compared to amino acids 1-49 of SEQ ID NO: 1 and / or the mutated LBD polypeptide comprises a mutated polypeptide as compared to amino acids 50-207 of SEQ ID NO:

1.

9. The multiple gene expression system of any one of claims 1-8, wherein the mutated DNA-binding domain (DBD) and / or the mutated LBD comprise a fusion protein with the individual LBD and DBD selected from those DBD and / or LBD domains identified in any polypeptides of SEQ ID NOs: 2-6 or 9-22, and equivalents of each thereof.

10. The multiple gene expression system of any one of claims 1-9, comprising TetO- 4C5G operator and TetR-V36FE37AP39K repressor; or TetO operator and L13-1-9 repressor.

11. The multiple gene expression system of any one of claims 1-9, comprising TetO operator and wtTetR repressor; or TetO-4C5G operator and L15-20-V36AE37AP39K repressor.

12. The multiple gene expression system of any one of claims 1-11, wherein the DBD and LBDs are selected from any one of SEQ ID NOs: 2-6 or 9-22, and equivalents of each thereof.

13. The multiple gene expression system of any one of claims 1-11, wherein one or more, two or more or all three of the ligand-binding domain (LBD); the DNA-binding domain (DBD); or the operator nucleotide is detectably labeled.

14. A gene repressor system comprising: a. a ligand-binding domain (LBD); b. a mutated DNA-binding domain (DBD); and c. an operator nucleotide that controls expression of a target polynucleotide, and optionally wherein LBD or the operator nucleotide comprise one or more mutations that change the DNA-binding specificity of the gene repressor system.

15. The system of claim 14, further comprising a polynucleotide comprising a target gene sequence operably linked to a promoter comprising at least one operator sequence. -47- 4871-7792-0235.1Atty Dkt.: 114198-3110 16. The system of claim 14 or 15, wherein the LBD comprises one or more mutation that changes the DNA-binding specificity of the gene repressor system.

17. The gene expression system of any one of claims 14-16, wherein the gene repressor system is a tetracycline repressor system (TetR) and the ligand-binding domain binds to tetracycline (Tet) repressor agent.

18. The gene expression system of any one of claims 14-17, wherein the operator nucleotide comprises wild-type TetO (SEQ ID NO: 7) or an operator variant, optionally the TetO-4C5G (SEQ ID NO: 8) variant.

19. The gene expression system of any one of claims 14-18, wherein the LBD comprises one or more mutations that alter the ligand binding activity of the LBD to bind to other molecules other than the LBD wild-type binding molecule.

20. The gene expression system of claim 19, wherein the LBD binds to a repressor agent selected from tetracycline (Tet), a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

21. The gene expression system of any one of claims 14-20, wherein the mutated DBD comprises a mutated polypeptide as compared to amino acids 1-49 of SEQ ID NO: 1 and / or the mutated LBD polypeptide comprises a mutated polypeptide as compared to amino acids 50-207 of SEQ ID NO:

1.

22. The gene expression system of any one of claims 14-21, wherein the mutated DNA- binding domain (DBD) and / or the mutated LBD comprise a fusion protein with the individual LBD and DBD domains identified in any one of the polypeptides of SEQ ID NOs: 2-6 or 9- 22, and equivalents of each thereof.

23. The gene expression system of any one of claims 14-22, wherein the LBD and the DBD are fusion polypeptides selected from any one of SEQ ID NOs: 2-6 or 9-22.

24. The gene expression system of any one of claims 14-23, comprising TetO-4C5G operator and TetR-V36FE37AP39K repressor; or TetO operator and L13-1-9 repressor.

25. The gene expression system of any one of claims 14-24, comprising TetO operator and wtTetR repressor; or TetO-4C5G operator and L15-20-V36AE37AP39K repressor. -48- 4871-7792-0235.1Atty Dkt.: 114198-3110 26. The gene expression system of any one of claims 14-25, wherein one or more, two or more or all three of the ligand-binding domain (LBD); the DNA-binding domain (DBD); or the operator nucleotide is detectably labeled.

27. A multiple gene repressor system comprising two or more gene repressor systems, wherein each of the gene repressor systems comprises: a ligand-binding domain (LBD); b. a DNA-binding domain (DBD); and c. an operator nucleotide that controls expression of a target polynucleotide, and optionally wherein LBD, DBD, or the operator nucleotide comprise one or more mutations that change the DNA-binding specificity of the gene repressor system, or alternatively, wherein the two or more gene repressor systems are defined by any one of claims 14-26, and further optionally wherein each of the two or more gene repressor systems work concurrently to modulate distinct target gene sequences controlled by a wild-type operator nucleotide or a mutated operator nucleotide.

28. The multiple gene expression system of claim 27, wherein at least one of the two or more gene repressor systems is a tetracycline repressor system (TetR) and the ligand-binding domain binds to tetracycline (Tet).

29. The multiple gene expression system of claim 27 or 28, wherein each of the two or more gene expression systems are on a single contiguous polynucleotide.

30. An isolated fusion polypeptide selected from any one of SEQ ID NOs: 2-6 or 9-22, and equivalents of each thereof.

31. An isolated polynucleotide encoding the isolated fusion polypeptide of claim 30, or an equivalent polynucleotide.

32. An isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2 to 6 or 9-22, and polynucleotides encoding equivalents having at least 90% sequence identity to any one of SEQ ID NO: 2 to 6 or 9 to 22 when evaluated under BLAST, or having at least 90% sequence identity to a nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2 to 6 or 9 to 22, with the proviso that the equivalent excludes the wild-type nucleic acid sequence individually for the -49- 4871-7792-0235.1Atty Dkt.: 114198-3110 respective nucleic acid sequence encoding a polypeptide selected from SEQ ID NO: 2, 3-6, or 9-22.

33. An isolated polynucleotide encoding one or more of elements a through c of the gene expression system of any one of claims 1-29.

34. A vector comprising an isolated polynucleotide of any one of claims 31-33.

35. The vector of claim 34, wherein the vector is a viral vector or a plasmid vector.

36. An isolated host cell comprising one or more of the gene expression system of any of claims 1-29, the fusion polypeptide of claim 30, the isolated polynucleotide of any one of claims 31-33, or the vector of claim 34 or 35.

37. The isolated host cell of claim 36, wherein the cell is a procaryotic or a eukaryotic cell.

38. A method for controlling expression of a target gene linked to an operator nucleotide that controls expression of a target polynucleotide, comprising expressing a polynucleotide encoding the gene expression system of any of claims 1-29 in a host cell, and optionally contacting the cell with a repressor agent.

39. The method of claim 38, further comprising a target polynucleotide.

40. The method of claim 38 or 39, wherein the host cell is an animal cell, optionally a mammalian cell.

41. The method of claim any one of claims 38-40, wherein the repressor agent is selected from tetracycline (Tet), a sulfonylurea (SU) herbicide, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs).

42. A method of regulating transcription of a polynucleotide of interest in an animal host cell comprising: a) providing a repressor agent selected from a sulfonylurea herbicide compound, ethametsulfuron-methyl (Es), or chlorsulfuron (Cs) to a host cell of any one of claim 36 or 37such that the repressor agent binds to form a complex that modifies the binding properties to the operator. -50- 4871-7792-0235.1Atty Dkt.: 114198-3110 43. The method of claim 42, wherein the host cell is an animal cell, optionally a mammalian cell. -51- 4871-7792-0235.1

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