System of protein-protein interaction and methods of using same

Abstract

The present invention provides a system for direct protein-protein interaction between a first cell and a second cell, including: (a) a first cell including a first polynucleotide sequence encoding a first synthetic polypeptide being a membrane anchored ligand including an extracellular portion being a ligand domain and a first membrane anchoring domain; and (b) a second cell including: (i) a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor including an extracellular portion being a binding domain having specific binding affinity to the ligand of (a), a second membrane anchoring domain, and an intracellular portion including a transcription factor; and (ii) a third polynucleotide sequence encoding a gene of interest. Further provided are methods of using the system, and a kit for preparing same.

Description

SYSTEM OF PROTEIN-PROTEIN INTERACTION AND METHODS OF USING SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 534,836, titled “SYSTEM OF PROTEIN-PROTEIN INTERACTION AND METHODS OF USING SAME”, filed 27 August 2023, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The present invention, in some embodiments, thereof, is in the field of molecular biology and genetic engineering.BACKGROUND

[0003] Currently, synthetic biology most often incorporates genetic circuits that take effect within a single cell - the cell detects changes in the internal composition and generates an output. In recent years a few genetic circuits started involving communication between cells. However, these genetic circuits either involve direct communication between two cells of the same type (e.g., mammalian cells with mammalian cells, bacteria with bacteria) or indirect communication between two different types of cells (e.g., bacteria releasing immune modulators to diffuse and be picked up by immune cells, mammalian cells releasing bacterial quorum-sensing molecules to the environment to modulate bacterial growth).

[0004] There is still a great need for systems and methods of using same allowing direct protein -protein communication, such as between bacteria and mammalian cells.SUMMARY

[0005] In the current invention, the inventors present a system allowing direct proteinprotein communication between bacteria and mammalian cells. Unlike indirect forms of communication, direct communication in the form of protein-protein interaction requires thephysical presence of both the sender and the receiver of the signal. Thus, this form of communication provides a more precise form of communication - unlike indirect communication which can result in communication with cells outside the immediate microenvironment of the cell, direct communication can only affect cells within the immediate surrounding of the cell. This flexible well-regulated platform based on efficient prokaryote-eukaryote interaction provides a new toolbox to program host cells interacting with their environments.

[0006] According to a first aspect, there is provided a system for direct protein-protein interaction between a first cell and a second cell, the system comprising: (a) a first cell comprising a first polynucleotide sequence encoding a first synthetic polypeptide being a membrane anchored ligand comprising an extracellular portion being a ligand domain and a first membrane anchoring domain, and wherein the first polynucleotide is operably linked to a first promoter sequence; and (b) a second cell comprising: i. a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to the ligand of (a), a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and ii. a third polynucleotide sequence encoding a gene of interest, wherein the third polynucleotide is operably linked to a second promoter being responsive to the transcription factor.

[0007] According to another aspect, there is provided a composition comprising the system of the invention, and an acceptable carrier.

[0008] According to another aspect, there is provided a method for expressing or repressing a gene of interest in a cell, the method comprising culturing the first cell and the second cell of the system of the invention under conditions sufficient for expression of the first synthetic polypeptide in the first cell and of the second synthetic polypeptide in the second cell, thereby, expressing or repressing the gene of interest in a cell.

[0009] According to another aspect, there is provided a method of treating a disease associated with a specific cell in a subject in need thereof, the method comprising: (a) administering to the subject a therapeutically effective amount of a first pharmaceutical composition comprising a vector having specific binding affinity to the cell, the vectorcomprising: i. a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to the ligand, a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and ii. a third polynucleotide sequence encoding a gene of interest having a therapeutic activity, wherein the third polynucleotide is operably linked to a second promoter being responsive to the transcription factor, thereby transforming the specific cell in the subject; and (b) administering to the subject a therapeutically effective amount of a second pharmaceutical composition comprising the first cell according to the system of the invention, thereby, treating the disease associated with a specific cell in the subject.

[0010] According to another aspect, there is provided a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: a first pharmaceutical composition comprising the first cell according to the system of the invention; and second pharmaceutical composition comprising the second cell according to the system of the invention, thereby, treating the disease or disorder in the subject.[Oi l] According to another aspect, there is provided a kit comprising: (a) a first polynucleotide sequence encoding a first synthetic polypeptide being a membrane anchored ligand comprising an extracellular portion being a ligand domain and a first membrane anchoring domain, and wherein the first polynucleotide is operably linked to a first promoter sequence; (b) a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to the ligand of (a), a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and (c) a third polynucleotide sequence encoding a gene of interest, wherein the third polynucleotide is operably linked to a second promoter being responsive to the transcription factor.

[0012] In some embodiments, the first promoter is a constitutive promoter or being responsive to an external signal.

[0013] In some embodiments, the first synthetic polypeptide is expressed upon activation of the first promoter.

[0014] In some embodiments, the gene of interest is expressed or repressed upon activation of the second promoter.

[0015] In some embodiments, the transcription factor is proteolytically cleaved from the second synthetic polypeptide, migrates into a nucleus of the second cell, binds DNA in the nucleus of the second cell, promotes or represses transcription in the nucleus of the second cell, or any combination thereof, upon binding of the ligand domain of the first synthetic polypeptide by the binding domain of the second synthetic polypeptide.

[0016] In some embodiments, the external signal comprises a signal generated by the second cell.

[0017] In some embodiments, the signal generated by the second cell comprises a compound expressed, secreted, or both, by the second cell.

[0018] In some embodiments, the first cell, the second cell, or both, is a transformed cell, a transgenic cell, a transduced cell, or any combination thereof.

[0019] In some embodiments, the first cell, the second cell, or both, is a prokaryote cell or a eukaryote cell.

[0020] In some embodiments, the first cell is a prokaryote cell, and the second cell is a eukaryote cell.

[0021] In some embodiments, the polynucleotide is operably linked to a third promoter being constitutively active promoter.

[0022] In some embodiments, the second cell is a transgenic cell or a transduced cell, and wherein the second polynucleotide, the third polynucleotide, or both, being introduced into the second cell by a vector.

[0023] In some embodiments, the vector is a viral vector.

[0024] In some embodiments, the vector is further encapsulated in a particle having specific binding affinity to the second cell.

[0025] In some embodiments, the second synthetic polypeptide is a synthetic Notch receptor.

[0026] In some embodiments, the ligand domain and the first membrane anchoring domain of the first synthetic polypeptide are not derived from the same gene.

[0027] In some embodiments, the binding domain having specific binding affinity to the ligand and any one of: the second membrane anchoring domain, the intracellular portion comprising the transcription factor, or both, of the second synthetic polypeptide are not derived from the same gene.

[0028] In some embodiments, the composition is a pharmaceutical composition.

[0029] In some embodiments, the gene of interest is any one of a reporter protein, and a therapeutic protein.

[0030] In some embodiments, the therapeutic protein is characterized by being capable of inducing or promoting apoptosis in a cell.

[0031] In some embodiments, the cell is a cancer cell.

[0032] In some embodiments, the specific cell is a cancer cell.

[0033] In some embodiments, therapeutic activity is anti-cancer activity.

[0034] In some embodiments, the anti-cancer activity comprises inducing or promoting cell apoptosis in a cancer cell.

[0035] In some embodiments, the gene of interest encodes a therapeutic protein.

[0036] In some embodiments, the method further comprises a step before administering of the second pharmaceutical composition, comprising transfecting, transducing, transforming, or any combination thereof, the second cell with the vector.

[0037] In some embodiments, the first cell is a prokaryote cell.

[0038] In some embodiments, the disease is a microbiome-related disease or a cell proliferation related disease.

[0039] In some embodiments, the second cell is a cell of the gastrointestinal tract of a mammalian subject.

[0040] In some embodiments, the second cell is a cell derived from the gastrointestinal tract of a mammalian subject or of a cell line of gastrointestinal origin.

[0041] In some embodiments, the cell proliferation related disease is cancer.

[0042] In some embodiments, the cell is an immune cell derived from the subject.

[0043] In some embodiments, the immune cell is a T lymphocyte.

[0044] In some embodiments, the T lymphocyte comprises a chimeric antigen receptor (CAR T).

[0045] In some embodiments, the method further comprises a step comprising producing the CAR T.

[0046] In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition to the subject.

[0047] In some embodiments, the administering of the first pharmaceutical composition and the administering of the second pharmaceutical composition are 2-7 days apart.

[0048] In some embodiments, the first polynucleotide is integrated into a first expression vector or plasmid.

[0049] In some embodiments, the second polynucleotide and the third polynucleotide are separately integrated into a second and a third expression vectors or plasmids, respectively, or are integrated into a single second expression vector.

[0050] In some embodiments, the kit further comprises at least two distinct types of cells being competent for transfection, transduction, transformation, transgenesis, or any combination thereof, with any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, and any combination thereof.

[0051] In some embodiments, the kit further comprises instruction for transfecting, transducing, transforming, or any combination thereof, at least two distinct types of cells with any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, and any combination thereof.

[0052] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplarymethods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0053] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0054] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0055] Fig. 1 includes a non-limiting scheme of a general structure of a native Notch receptor (Left) showing the cleavage sites, and the manipulations made in order to achieve ligand-dependent activation of transgenes (Right) e.g., a synthetic Notch receptor.

[0056] Figs. 2A-2B include a non-limiting scheme and a graph. (2A) Structure of the membranal mCherry in mammalian cells. (2B) An experimental result showing expression of reporter gene (tagBFP) in response to co-culture of Jurkat cells expressing an anti- mCherry synNotch receptor with NiCo21 E. coli cells with an inducer-dependent membranal mCherry. Curve 1 - without the presence of the inducer; Curve 2 - with the inducer, causing the expression of membranal mCherry and in turn a response from the engineered Jurkat cells.

[0057] Figs. 3A-3B include fluorescent micrographs showing non-induced bacteria (3A) or bacteria induced to express membranal GFP (3B). The bacteria were co-cultured with HEK293T cells in a spheroid (three-dimensional (3D)) setting. The HEK293T cells were transfected with anti-GFP synNotch receptor and expresses the reporter tagBFP in response to the activation of the synNotch receptor.DETAILED DESCRIPTIONSystem

[0058] According to the first aspect, there is provided a system for direct protein-protein interaction between a first cell and a second cell.

[0059] In some embodiments, the system comprises: (a) a first cell comprising a first polynucleotide sequence encoding a first synthetic polypeptide being a membrane anchored ligand comprising an extracellular portion being a ligand domain and a first membrane anchoring domain; and (b) a second cell comprising: (i) a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to the ligand of (a), a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and (ii) a third polynucleotide sequence encoding a gene of interest.

[0060] In some embodiments, the first polynucleotide is operably linked to a first promoter sequence.

[0061] In some embodiments, the first promoter is a constitutive promoter or being responsive to an external signal.

[0062] In some embodiments, the third polynucleotide is operably linked to a second promoter being responsive to the transcription factor.

[0063] In some embodiments, the first synthetic polypeptide is expressed upon activation of the first promoter.

[0064] In some embodiments, the gene of interest is expressed upon activation of the second promoter.

[0065] In some embodiments, the transcription factor is: proteolytically cleaved from the second synthetic polypeptide, migrates into a nucleus of the second cell, binds DNA in the nucleus of the second cell, promotes transcription in the nucleus of the second cell, or any combination thereof, upon binding of the ligand domain of the first synthetic polypeptide by the binding domain of the second synthetic polypeptide.

[0066] In some embodiments, the transcription factor promotes expression. In some embodiments, the transcription factor is repressing transcription, e.g., a repressor.

[0067] In some embodiments, an external signal comprises a signal generated by the second cell.

[0068] In some embodiments, a signal generated by the second cell comprises a compound expressed, secreted, or both, by the second cell.

[0069] In some embodiments, a first cell, a second cell, or both, comprises a transformed cell, a transgenic cell, a transduced cell, or any combination thereof.

[0070] In some embodiments, a first cell, a second cell, or both, comprises a prokaryote cell or a eukaryote cell.

[0071] In some embodiments, the first cell is a prokaryote cell, and the second cell is a eukaryote cell.

[0072] In some embodiments, the second polynucleotide is operable linked to a third promoter being constitutively active promoter.

[0073] In some embodiments, the second cell is a transgenic cell or a transduced cell, and the second polynucleotide, the third polynucleotide, or both, are introduced into the second cell by a vector or a plasmid. In one embodiment, the second polynucleotide, the third polynucleotide, or both, are incorporated into the genome of the second cell.

[0074] In some embodiments, the vector or plasmid in an expression vector.

[0075] In some embodiments, the vector is a viral vector.

[0076] In some embodiments, the vector is further encapsulated in a particle.

[0077] In some embodiments, the particle has specific binding affinity to the second cell. In some embodiments, the particle comprises an antibody or an antigen binding portion thereof, having specific binding affinity to an antigen of the second cell. In some embodiments, the antigen comprises a surface antigen of the second cell.

[0078] In some embodiments, the second synthetic polypeptide is a synthetic Notch receptor.

[0079] In some embodiments, the receptor comprises a variable fragment of an antibody or an antigen binding portion thereof.

[0080] In some embodiments, the antibody comprises a single chained antibody or a variable fragment thereof.

[0081] In some embodiments, the ligand domain and the first membrane anchoring domain of the first synthetic polypeptide are not derived from the same gene.

[0082] In some embodiments, the binding domain having specific binding affinity to the ligand and any one of: the second membrane anchoring domain, the intracellular portion comprising the transcription factor, or both, of the second synthetic polypeptide are not derived from the same gene.

[0083] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C).

[0084] The terms “nucleic acid molecule” include but not limited to singlestranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNA such as miRNA, siRNA and other short interfering nucleic acids, snoRNAs, snRNAs, tRNA, piRNA, tnRNA, small rRNA, hnRNA, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, ribozymes, viral RNA or DNA, nucleic acids of infectious origin, amplification products, modified nucleic acids, plasmidical or organellar nucleic acids and artificial nucleic acids such as oligonucleotides.

[0085] The terms "polynucleotide," "polynucleotide sequence," "nucleic acid sequence," and "nucleic acid molecule" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural, or altered nucleotide bases.

[0086] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating into cells. In one embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0087] Expressing a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector. One such example of an expression vector containing pl6-Ink4a is the mammalian expression vector pCMV pl 6 INK4A available from Addgene.

[0088] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.

[0089] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.

[0090] In some embodiments, the gene is operably linked to a promoter. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0091] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987), and / or the like.

[0092] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0093] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0094] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK- RSV and pBK-CMV which are available from Strategene, pTRES which is available from Cion tech, and their derivatives.

[0095] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0096] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression. In oneembodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0097] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Bio techniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipof ection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0098] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0099] As used herein, the term "synthetic" refers to something that is artificial or manmade. In some embodiments, a synthetic polypeptide as disclosed herein, is not present, made, or both, in nature. In some embodiments, synthetic excludes a product of nature, e.g., obtained or produced only by man.

[0100] According to another aspect, there is provided a composition comprising the system of the invention, and an acceptable carrier.

[0101] In some embodiments, the carrier is a biologically acceptable carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0102] In some embodiments, the composition is a pharmaceutical composition.

[0103] As used herein, the terms “carrier,” “excipient,” or “adjuvant” are interchangeable, and refer to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow- releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicleforming lipids which generally include neutral and negatively charged phospholipids and sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols inProtein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0104] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.Methods of use

[0105] According to another aspect, there is provided a method for expressing or repressing a gene of interest in a cell.

[0106] According to another aspect, there is provided a method of treating a disease or disorder in a subject in need thereof.

[0107] According to another aspect, there is provided a method of treating a disease associated with a specific cell in a subject in need thereof.

[0108] In some embodiments, the method comprises culturing a first cell and a second cell of the system of the invention under conditions sufficient for expression or repression of the first synthetic polypeptide in the first cell and of the second synthetic polypeptide in the second cell, thereby, expressing or repressing the gene of interest in a cell.

[0109] In some embodiments, the gene of interest is a reporter protein, or a therapeutic protein. In some embodiments, therapeutic comprises providing, inducing, enhancing, contributing, or any combination thereof, to the heath, wellbeing, or both, of a subject.

[0110] In some embodiments, the therapeutic protein is characterized by being capable of inducing or promoting apoptosis in a cell. In some embodiments, the cell is a cell of a subject. In some embodiments, the cell is a pathogenic cell of inducing or promoting a pathogenic state in the subject. In some embodiments, the cell is malicious to the subject or harming the subject.

[0111] In some embodiments, a therapeutic activity is anti-cancer activity.

[0112] In some embodiments, anti-cancer activity comprises inducing or promoting cell apoptosis in a cancer cell, cell death, cell cycle arrest, or any combination thereof.

[0113] In some embodiments, the cell is a cancer cell or a malignant cell.

[0114] In some embodiments, the method comprises: (a) administering to the subject a therapeutically effective amount of a first pharmaceutical composition comprising a vector having specific binding affinity to the cell; and (b) administering to the subject a therapeutically effective amount of a second pharmaceutical composition comprising the first cell according to the system of the invention.

[0115] In some embodiments, the method comprises providing the first pharmaceutical composition, the second pharmaceutical composition, or both.

[0116] In some embodiments, the vector comprises: (i) a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to the ligand, a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and (ii) a third polynucleotide sequence encoding a gene of interest having a therapeutic activity, wherein the third polynucleotide is operably linked to a second promoter being responsive to the transcription factor.

[0117] In some embodiments, administering the second pharmaceutical composition to the subject comprises, results, induces, or any combination thereof, transforming or transformation of a cancer in the subject.

[0118] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of: a first pharmaceutical composition comprising the first cell according to the system of the invention; and second pharmaceutical composition comprising the second cell according to the system of the invention.

[0119] In some embodiments, the second cell is a transgenic cell or a transduced cell, and the second polynucleotide, the third polynucleotide, or both, are introduced into the second cell by a vector.

[0120] In some embodiments, the method further comprises a step before administering of the second pharmaceutical composition, comprising transfecting, transducing, transforming, or any combination thereof, the second cell with the vector, thereby transforming the second cell the subject. In some embodiments, the second cell is a cancer cell of the subject. In some embodiments, the second cell is a cancer cell in the subject.

[0121] In some embodiments, the first cell is a prokaryote cell. In some embodiments, the first cell is a bacterium or a bacterial cell. In some embodiments, the first cell is a synthetic cell.

[0122] In some embodiments, the disease is a microbiome -related disease or a cell proliferation related disease.

[0123] In some embodiments, the second cell is a cell of the gastrointestinal tract of a mammalian subject.

[0124] In some embodiments, the second cell is a cell derived from the gastrointestinal tract of a mammalian subject or of a cell line of gastrointestinal origin.

[0125] In some embodiments, the cell proliferation related disease is cancer.

[0126] In some embodiments, the second cell is an immune cell derived from a subject.

[0127] In some embodiments, the immune cell is a T lymphocyte.

[0128] In some embodiments, the T lymphocyte comprises a chimeric antigen receptor (CAR T).

[0129] In some embodiments, the method further comprises a step comprising producing a CAR T.

[0130] In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition to a subject.

[0131] In some embodiments, administering of the first pharmaceutical composition and administering of the second pharmaceutical composition are 2-7 days apart.

[0132] In some embodiments, administering of the first pharmaceutical composition and administering of the second pharmaceutical composition are at least 1, 2, 3, 4, 5, 6, 7, 10, 14, or 28 days apart, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.Kit

[0133] According to another aspect, there is provided a kit comprising: (a) a first polynucleotide sequence encoding a first synthetic polypeptide being a membrane anchoredligand comprising an extracellular portion being a ligand domain and a first membrane anchoring domain; (b) a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to the ligand of (a), a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and (c) a third polynucleotide sequence encoding a gene of interest.

[0134] In some embodiments, the first polynucleotide is operably linked to a first promoter sequence being responsive to an external signal.

[0135] In some embodiments, the third polynucleotide is operably linked to a second promoter being responsive to the transcription factor.

[0136] In some embodiments, the first polynucleotide is integrated into a first vector, expression vector, or plasmid.

[0137] In some embodiments, the second polynucleotide and the third polynucleotide are separately integrated into a second and a third vectors, expression vector or plasmids, respectively, or are integrated into a single second vector, expression vector or plasmid.

[0138] In some embodiments, the kit further comprises at least two distinct types of cells being competent for transfection, transduction, transformation, transgenesis, or any combination thereof, with the first polynucleotide, the second polynucleotide, the third polynucleotide, a vector, expression vector or plasmid comprising same, or any combination thereof.

[0139] In some embodiments, the kit further comprises instruction for transfecting, transducing, transforming, or any combination thereof, at least two distinct types of cells with the first polynucleotide, the second polynucleotide, the third polynucleotide, a vector, expression vector or plasmid comprising same, or any combination thereof.

[0140] In some embodiments, the kit is for preparing the system of the invention.General

[0141] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that statedrange, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0142] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nucleobases refers to a length of 1,000 nucleobases ± 100 nucleobases.

[0143] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0144] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0145] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.

[0146] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0017] In the description and claims of the present application, each of the verbs, “comprise”, “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0018] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0019] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

[0150] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising,” indicate the inclusion of any recited integer or group of integers but not the exclusion of any other integer or group of integers.

[0151] As used herein, the term “consists essentially of’, or variations such as “consist essentially of’ or “consisting essentially of’ as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure, or composition.

[0152] As used herein, the terms "comprises", "comprising", "containing", "having" and the like can mean "includes", "including", and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. In one embodiment, the terms "comprises", "comprising", "having" are / is interchangeable with "consisting".

[0153] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.EXAMPLES

[0154] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include chemical, molecular, biochemical, computational, statistics and cell biology techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); The Organic Chemistry of Biological Pathways by John McMurry and Tadhg Begley (Roberts and Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by Richard Silverman (Academic Press, 2002); Organic Chemistry (6thEdition) by Leroy "Skip" G Wade; Organic Chemistry by T. W. Graham Solomons and, Craig Fryhle.

[0155] The current inventors approach to direct communication between bacterial and mammalian cells relies on the use of synthetic notch (synNotch) receptors. The notch receptor is a surface protein which, upon ligand binding at the extracellular domain, induces proteolytic cleavage and releases of the intracellular domain, which enters the cell nucleus to modify gene expression. By replacing the extracellular domain of a Notch- 1 receptor with an antigen-binding fragment and the intracellular domain with an orthogonal transcription factor (TF), this novel system allows the user to express premediated genes upon ligand binding (Fig. 1). The antigen-binding fragment allows recognition of a predetermined ligand, and the orthogonal TF allows the subsequent activation of a transgene which possesses fitting binding sites for the TF in its promoter region.

[0156] By designing the antigen-binding fragment to recognize a synthetic ligand expressed on the surface of bacteria, the inventors allow the mammalian cells equipped with this receptor to receive a “message” from bacteria in contact. The number of combinations of receptor and ligand are virtually endless. For example, one such system can be carried out with the expression of membrane -bound mCherrry - a fluorescent protein - in the bacteria, and the complementary recognition site on a synNotch receptor expressed in a mammalian cell.

[0157] Although mCherrry expression in bacteria does not pose a unique problem, the expression of the protein on the outer-most membrane facing the environment is a challenge. For membranal expression of mCherry in E. coli, the inventors included an E. coli signal peptide from the outer membrane protein A (OmpA), followed the mCherry amino acid sequence, and finally the transmembrane domain from two native E. coli outer membrane protein candidates - adhesin involved in diffuse adherence (AIDA-I) and pO157-encoded secreted serine protease EspP (Fig. 2A). By including these domains, the inventors ensured appropriate surface-targeting and membrane anchoring in E. coli cells.

[0158] Upon addition of a suitable inducer, expression of membranal mCherry was achieved, and in turn a response from the engineered Jurkat cells was exerted (Fig. 2B).

[0159] Similarly, the inventors showed specific recognition and subsequent activation of an anti-GFP synNotch receptor when HEK293T cells expressing the receptor were co-cultured with bacteria expressing a membrane -bound GFP (Fig. 3B).

[0160] While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

Claims

CLAIMSWhat is claimed:

1. A system for direct protein-protein interaction between a first cell and a second cell, the system comprising:(a) a first cell comprising a first polynucleotide sequence encoding a first synthetic polypeptide being a membrane anchored ligand comprising an extracellular portion being a ligand domain and a first membrane anchoring domain, and wherein said first polynucleotide is operably linked to a first promoter sequence; and(b) a second cell comprising: i. a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to said ligand of (a), a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and ii. a third polynucleotide sequence encoding a gene of interest, wherein said third polynucleotide is operably linked to a second promoter being responsive to said transcription factor.

2. The system of claim 1 , wherein said first promoter is a constitutive promoter or being responsive to an external signal.

3. The system of claim 1 or 2, wherein said first synthetic polypeptide is expressed upon activation of said first promoter.

4. The system of any one of claims 1 to 3, wherein said gene of interest is expressed or repressed upon activation of said second promoter.

5. The system of any one of claims 1 to 4, wherein said transcription factor is proteolytically cleaved from said second synthetic polypeptide, migrates into a nucleus of said second cell, binds DNA in the nucleus of said second cell, promotes or represses transcription in the nucleus of said second cell, or any combination thereof, upon binding ofsaid ligand domain of said first synthetic polypeptide by said binding domain of said second synthetic polypeptide.

6. The system of any one of claims 2 to 5, wherein said external signal comprises a signal generated by said second cell.

7. The system of any one of claims 2 to 6, wherein said signal generated by said second cell comprises a compound expressed, secreted, or both, by said second cell.

8. The system of any one of claims 1 to 7, wherein said first cell, said second cell, or both, is a transformed cell, a transgenic cell, a transduced cell, or any combination thereof.

9. The system of any one of claims 1 to 8, wherein said first cell, said second cell, or both, is a prokaryote cell or a eukaryote cell.

10. The method of any one of claims 1 to 9, wherein said first cell is a prokaryote cell, and said second cell is a eukaryote cell.

11. The system of any one of claims 1 to 10, wherein said second polynucleotide is operably linked to a third promoter being constitutively active promoter.

12. The system of any one of claims 1 to 11, wherein said second cell is a transgenic cell or a transduced cell, and wherein said second polynucleotide, said third polynucleotide, or both, being introduced into said second cell by a vector.

13. The system of claim 12, wherein said vector is a viral vector.

14. The system of claim 12 or 13, wherein said vector is further encapsulated in a particle having specific binding affinity to said second cell.

15. The system of any one of claims 1 to 14, wherein said second synthetic polypeptide is a synthetic Notch receptor.

16. The system of any one of claims 1 to 15, wherein said ligand domain and said first membrane anchoring domain of said first synthetic polypeptide are not derived from the same gene.

17. The system of any one of claims 1 to 16, wherein said binding domain having specific binding affinity to said ligand and any one of: said second membrane anchoring domain, said intracellular portion comprising said transcription factor, or both, of said second synthetic polypeptide are not derived from the same gene.

18. A composition comprising the system of any one of claims 1 to 17, and an acceptable carrier.

19. The composition of claim 18, being a pharmaceutical composition.

20. A method for expressing or repressing a gene of interest in a cell, the method comprising culturing said first cell and said second cell of the system of any one of claims 1 to 17 under conditions sufficient for expression of said first synthetic polypeptide in said first cell and of said second synthetic polypeptide in said second cell, thereby, expressing or repressing the gene of interest in a cell.

21. The method of claim 20, wherein said gene of interest is any one of a reporter protein, and a therapeutic protein.

22. The method of claim 21, wherein said therapeutic protein is characterized by being capable of inducing or promoting apoptosis in a cell.

23. The method of any one of claims 20 to 22, wherein said cell is a cancer cell.

24. A method of treating a disease associated with a specific cell in a subject in need thereof, the method comprising:(a) administering to said subject a therapeutically effective amount of a first pharmaceutical composition comprising a vector having specific binding affinity to said cell, the vector comprising: i. a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to said ligand, a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and ii. a third polynucleotide sequence encoding a gene of interest having a therapeutic activity, wherein said third polynucleotide is operably linked to a second promoter being responsive to said transcription factor, thereby transforming said specific cell in said subject; and(b) administering to said subject a therapeutically effective amount of a second pharmaceutical composition comprising said first cell according to the system of any one of claims 1 to 17,thereby, treating the disease associated with a specific cell in the subject.

25. The method of claim 24, wherein said specific cell is a cancer cell.

26. The method of claim 24 or 25, wherein said therapeutic activity is anti-cancer activity.

27. The method of claim 26, wherein said anti-cancer activity comprises inducing or promoting cell apoptosis in a cancer cell.

28. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of: a first pharmaceutical composition comprising said first cell according to the system of any one of claims 1 to 17; and second pharmaceutical composition comprising said second cell according to the system of any one of claims 1 to 17, thereby, treating the disease or disorder in the subject.

29. The method of claim 28, wherein said gene of interest encodes a therapeutic protein.

30. The method of claim 28 or 29, wherein said second cell is a transgenic cell or a transduced cell, and wherein said second polynucleotide, said third polynucleotide, or both, being introduced into said second cell by a vector.

31. The method of claim 30, further comprising a step before administering of said second pharmaceutical composition, comprising transfecting, transducing, transforming, or any combination thereof, said second cell with said vector.

32. The method of any one of claims 28 to 31 , wherein said first cell is a prokaryote cell.

33. The method of any one of claims 28 to 32, wherein said disease is a microbiome- related disease or a cell proliferation related disease.

34. The method of claim 33, wherein said second cell is a cell of the gastrointestinal tract of a mammalian subject.

35. The method of claim 33, wherein said second cell is a cell derived from the gastrointestinal tract of a mammalian subject or of a cell line of gastrointestinal origin.

36. The method of claim 33, wherein said cell proliferation related disease is cancer.

37. The method of claim 33, wherein said cell is an immune cell derived from said subject.

38. The method of claim 37, wherein said immune cell is a T lymphocyte.

39. The method of claim 38, wherein said T lymphocyte comprises a chimeric antigen receptor (CAR T).

40. The method of claim 39, further comprising a step comprising producing said CAR T.

41. The method of any one of claims 24-27, and 30-40, wherein said vector is a viral vector.

42. The method of any one of claims 29 to 40, wherein said vector is further encapsulated in a particle having specific binding affinity to said second cell.

43. The method of any one of claims 28 to 42, wherein said first pharmaceutical composition is administered before said second pharmaceutical composition to said subject.

44. The method of any one of claims 24 to 43, wherein said administering of said first pharmaceutical composition and said administering of said second pharmaceutical composition are 2-7 days apart.

45. The method of any one of claims 24 to 44, wherein said second synthetic polypeptide is a synthetic Notch receptor.

46. A kit comprising:(a) a first polynucleotide sequence encoding a first synthetic polypeptide being a membrane anchored ligand comprising an extracellular portion being a ligand domain and a first membrane anchoring domain, and wherein said first polynucleotide is operably linked to a first promoter sequence;(b) a second polynucleotide sequence encoding a second synthetic polypeptide being a receptor comprising an extracellular portion being a binding domain having specific binding affinity to said ligand of (a), a second membrane anchoring domain, and an intracellular portion comprising a transcription factor; and(c) a third polynucleotide sequence encoding a gene of interest, wherein said third polynucleotide is operably linked to a second promoter being responsive to said transcription factor.

47. The kit of claim 46, wherein said first polynucleotide is integrated into a first expression vector or plasmid.

48. The kit of claim 46 or 47, wherein said second polynucleotide and said third polynucleotide are separately integrated into a second and a third expression vectors or plasmids, respectively, or are integrated into a single second expression vector.

49. The kit of any one of claims 46 to 48, further comprising at least two distinct types of cells being competent for transfection, transduction, transformation, transgenesis, or any combination thereof, with any one of said first polynucleotide, said second polynucleotide, said third polynucleotide, and any combination thereof.

50. The kit of any one of claims 46 to 49, further comprising instruction for transfecting, transducing, transforming, or any combination thereof, at least two distinct types of cells with any one of said first polynucleotide, said second polynucleotide, said third polynucleotide, and any combination thereof.

51. The kit of any one of claims 46 to 50, wherein said first promoter is a constitutive promoter or being responsive to an external signal.

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