CCL20 promoter regulatory gene expression

Abstract

This disclosure relates to CCL20 promoters derived from regulatory regions of the CCL20 gene that stimulate gene expression in response to inflammation. These CCL20 promoters, which may be stimulated by endogenous or exogenous cytokines, can be used to control the timing of heterologous gene expression. This disclosure also relates to expression cassettes or vectors containing, for example, the CCL20 promoters of this disclosure operably ligated to a nucleic acid sequence encoding a polypeptide of interest, as well as delivery systems (e.g., viral particles, lipid vesicles, or nanoparticles), or cells containing such expression cassettes or vectors. This disclosure also relates to the use of promoters, expression cassettes, vectors, delivery systems, or cells in the treatment of inflammation-related diseases or for recombinant gene expression.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This PCT application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 491,485, filed on 21 March 2023, which is incorporated herein by reference in its entirety.

[0002] (Reference to electronically submitted sequence listings) The ST.26 sequence listing (name 5064_001PC01_SequenceListing_ST26.xml; size: 11,037 bytes, creation date: March 18, 2023), filed electronically in XML format with this application, is incorporated herein by reference in its entirety.

[0003] This disclosure provides a method relating to the expression of CCL20 promoters and CCL20 promoter-regulating genes in response to inflammation. [Background technology]

[0004] Inflammation is a broad response activated by the innate immune system upon detection of signals from damaged tissue or pathogenic microorganisms. Inflammation is generally beneficial to living organisms because it alerts the immune system to eliminate the underlying cause and restore homeostasis. Nevertheless, unregulated and excessive inflammation is harmful and can become chronic due to positive feedback mediated by inflammatory cytokines. Rapid production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and several others is characteristic of diseases with inflammatory components.

[0005] Inflammation can arise as a response to any harmful stimulus. Physical causes of inflammation include, for example, burns, frostbite, physical injury, trauma, or ionizing radiation. Biological causes of inflammation include, for example, infection by pathogens, immune responses resulting from hypersensitivity and allergies, or stress. Chemical causes of inflammation include, for example, chemical irritants or toxins. Inflammatory abnormalities underlie a wide variety of human diseases. The immune system is often involved in inflammatory disorders, as demonstrated in both allergic reactions and some myopathy, and is involved in many immune system disorders (including, for example, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis, psoriasis, or lupus erythematosus), leading to abnormal inflammation. Non-immune diseases that have a causal origin in the inflammatory process include cancer, atherosclerosis, and ischemic heart disease.

[0006] Nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are widely used and effective anti-inflammatory therapies, but they still have the potential for adverse side effects. Currently, the most advanced clinically used treatments for inflammatory diseases are TNF-α inhibitors such as adalimumab, etanercept, and infliximab, which specifically and effectively inhibit the activity of pro-inflammatory cytokines, as well as antagonists of endogenous cytokine antagonists such as anakinra (an IL-1β antagonist).

[0007] Because inflammation is an essential component of the host defense system, prolonged systemic suppression can lead to adverse side effects such as a reduced host immune response to infection and an increased risk of cancer. Anti-inflammatory therapies that can be controlled externally, while potentially consistent with the responsiveness and modulation of innate cellular responses, present both challenges and opportunities for synthetic biology. Synthetic biology allows for the construction of biological systems that are orthogonal to existing signaling components but can still read inputs from cellular processes or external signals and produce physiologically relevant outputs. Advances in this direction have already been made, such as manipulating the bacterium Lactococcus lactis to secrete IL-10, anti-TNF-α nanobodies used to treat colitis in mice in situ, or Escherichia coli that can autonomously detect intestinal inflammation through nitric oxide sensing. [Overview of the project]

[0008] This disclosure provides a polynucleotide comprising a promoter region containing a CC motif chemokine ligand 20 (CCL20) promoter operably linked to a heterogene, the CCL20 promoter inducing the expression of a heterogene (e.g., a therapeutic protein or therapeutic nucleic acid) in response to stimulation by a cytokine, such as IL-1α. In some embodiments, the CCL20 promoter comprises the CCL20 promoter sequence described in SEQ ID NO: 1. In some embodiments, the CCL20 promoter comprises the CCL20 promoter sequence described in SEQ ID NO: 2. In some embodiments, the CCL20 promoter comprises the CCL20 promoter sequence described in SEQ ID NO: 3. In some embodiments, the CCL20 promoter comprises the CCL20 promoter sequence described in SEQ ID NO: 4. In some embodiments, the CCL20 promoter comprises the CCL20 promoter sequence described in SEQ ID NO: 5.

[0009] In some embodiments, the CCL20 promoter is a functional fragment or functional variant of the CCL20 promoter sequence described in any one of SEQ ID NOs: 1-5. In some embodiments, the functional variant is a variant. In some embodiments, the functional fragment has a 5' cut, a 3' cut, or both of these with respect to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1-5. In some embodiments, the CCL20 promoter sequence has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with respect to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1-5. In some embodiments, the CCL20 promoter sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations. In some embodiments, the point mutations are conservative point mutations.

[0010] In some embodiments, heterologous genes encode proteins. In some embodiments, proteins are therapeutic proteins. In some embodiments, therapeutic proteins are chimeric antigen receptors (CARs), antibodies, tumor suppressors, apoptosis inducers, enzymes, or hormones.

[0011] The Disclosure also provides a composition comprising (a) a polynucleotide comprising a promoter region comprising one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5, operably linked to a heterogene, wherein the CCL20 promoter induces the expression of the heterogene in response to stimulation by a cytokine, e.g., IL-1α; and (b) a delivery vehicle for the delivery of the polynucleotide to a cell, e.g., a target cell, or an in vitro cell. In some embodiments, the delivery vehicle is a nucleic acid, plasmid, viral vector, prokaryotic cell, eukaryotic cell, or lipid. In some embodiments, the lipid is contained within a lipid vesicle. In some embodiments, the lipid vesicle is a micelle, liposome, lipid nanoparticle, or extracellular vesicle. In some embodiments, the viral vector is selected from the group consisting of adeno-associated viruses, adenoviruses, retroviruses, orthomyxoviruses, paramyxoviruses, papovaviruses, picornaviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, poxviruses, and alphaviruses. In some embodiments, the delivery vehicle is inserted into a cell (e.g., a target cell, a cell from another donor, or a host cell for cell culture, e.g., for the recombinant expression of a heterologous gene) together with a construct of the Disclosure, i.e., a construct containing one of SEQ ID NOs: 1-5, or a functional fragment or functional variant thereof, operably linked to a heterologous gene, the CCL20 promoter induces the expression of the heterologous gene in response to stimulation by a cytokine, e.g., IL-1α. In some embodiments, the delivery vehicle is inserted into the cell via transfection. In some embodiments, the transfection method is microinjection, lipofection, electroporation, cationic lipid transfection, microparticle transfection, magnetofection, or any preferred method known in the art. In some embodiments, the transfection can be transient. In some embodiments, the transfection can be stable.In some embodiments, a construct containing the CCL20 promoter and heterologous genes can be inserted into the cellular genome using a nuclease such as CRISPR / Cas.

[0012] The Disclosure also provides a method for producing cells expressing at least one heterogene, comprising inserting a composition (e.g., a construct or a vector containing such construct) comprising a polynucleotide including a promoter region into a cell (e.g., via transfection or microinjection), wherein the promoter region comprises a CCL20 promoter of any one of SEQ ID NOs. 1-5 or a functional fragment or functional variant thereof, operably linked to the heterogene, and the CCL20 promoter elicits the expression of the heterogene (e.g., expression of a therapeutic protein or therapeutic nucleic acid) in response to stimulation by a cytokine, e.g., IL-1α. The Disclosure also provides genetically engineered cells expressing the heterogene, the cells comprising the polynucleotides or compositions disclosed herein. Such cells can be cells administered to a target (e.g., cells in which the heterogene encodes anti-inflammatory cytokines, antibodies, CARs, etc.). The Disclosure also provides pharmaceutical compositions comprising the polynucleotides, compositions, or cells disclosed herein, and pharmaceutically acceptable carriers or excipients.

[0013] The disclosure also provides a method for inducing, regulating, or enhancing the expression of a heterogene (e.g., an anti-inflammatory cytokine, antibody, or heterogene encoding a CAR) in a subject, comprising (a) administering to a subject a polynucleotide, composition, cell, or pharmaceutical composition containing a CCL20 promoter of any one of SEQ ID NOs: 1-5 or a functional fragment or functional variant thereof operably linked to a heterogene disclosed herein, and (b) stimulating the CCL20 promoter with a cytokine, such as IL-1α. The disclosure also provides a method for inducing dose-dependent gene expression of a heterogene in a cell, comprising (a) contacting a cell with a polynucleotide, composition, cell, or pharmaceutical composition containing a CCL20 promoter of any one of SEQ ID NOs: 1-5 or a functional fragment or functional variant thereof operably linked to a heterogene disclosed herein, and (b) contacting the cell with an effective amount of a cytokine, such as IL-1α, having the ability to stimulate the CCL20 promoter, to induce the gene expression of the heterogene in a dose-dependent manner.

[0014] Furthermore, a method is provided for controlling or inducing the gene expression of a heterologous gene, the method comprising operably ligating one of the CCL20 promoters (SEQ ID NOs: 1-5) or its functional fragment or functional variant to a heterologous gene, wherein stimulation of the CCL20 promoter with a cytokine, such as IL-1α, controls or induces the expression of the heterologous gene in response to the stimulation.

[0015] This disclosure also provides a method for treating a disease or condition within a subject, or the symptoms or sequelae of such a disease or condition, comprising administering to a subject a polynucleotide, composition, cell, or pharmaceutical composition containing an effective amount of any one of SEQ ID NOs: 1-5, the CCL20 promoter, or a functional fragment or functional variant thereof, operably linked to a heterogene disclosed herein. In some embodiments, the method further includes inducing the expression of a therapeutic gene by stimulating the CCL20 promoter with a cytokine, such as IL-1α.

[0016] Also provided is a method for controlling or reducing inflammation in a subject, comprising operably ligating one of the CCL20 promoters, or a functional fragment or functional variant thereof, from sequence numbers 1 to 5, to a heterogene, wherein stimulation of the CCL20 promoter by a cytokine, such as IL-1α, induces the expression of the heterogene in response to the stimulation, and the heterogene controls or reduces inflammation by increasing or decreasing immune activation, or by increasing or decreasing the expression of another gene in the inflammatory pathway.

[0017] In some embodiments of the polynucleotides, compositions, cells, or methods disclosed herein, the cytokine stimulating the CCL20 promoter is selected from the group consisting of IL-1α, IL-1β, TNF (TNF-α or TNF-β), IL-6, sIL-6R, IL-17, CD30, or any combination thereof. In some embodiments, the cytokine stimulating the CCL20 promoter of this disclosure (i.e., one of the CCL20 promoters or its functional fragment or functional variant from SEQ ID NOs: 1-5) is an interleukin. In some embodiments, the interleukin is interleukin-1. In some embodiments, the interleukin is interleukin-1 alpha. In some embodiments, the cytokine stimulating the CCL20 promoter of this disclosure (e.g., one of the CCL20 promoters or its functional fragment or functional variant from SEQ ID NOs: 1-5) is an exogenous cytokine, i.e., it has an external source and is administered to a subject or added to a culture medium. In some embodiments, the cytokine stimulating the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is an endogenous cytokine, i.e., the cytokine is produced by the tissue of interest. In some embodiments, the endogenous cytokine stimulating the CCL20 promoter is a result of inflammation within the subject. In some embodiments, the inflammation is spontaneous. In some embodiments, the inflammation is induced. In some embodiments, for example, the cytokine stimulating the CCL20 promoter can be added to the culture medium during recombinant expression or secreted by co-cultured cells.

[0018] In some embodiments of the polynucleotides, compositions, cells, or methods disclosed herein, the use of the CCL20 promoter of this disclosure increases the expression of a heterologous gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% compared to the expression observed using a reference promoter. In some embodiments, the reference promoter is the serum amyloid A3 (SAA3) promoter.

[0019] The Disclosure also provides gene therapy vectors containing genes (e.g., CARs) that increase or decrease immune activation in a subject upon expression, wherein the genes are under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5, or a functional fragment or functional variant thereof). The Disclosure also provides cells for gene therapy containing genes (e.g., CARs) that increase or decrease immune activation in a subject upon expression, wherein the genes are under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5, or a functional fragment or functional variant thereof). In some embodiments, the cells are autologous cells, such as autologous CAR T cells. In other embodiments, the cells are allogeneic cells, such as allogeneic CAR T cells.

[0020] In some aspects, the present disclosure provides a vector comprising a CCL20 promoter comprising a sequence set forth in any one of SEQ ID NOs: 1-5, or a promoter region comprising a functional fragment or functional variant thereof. In some aspects, the vector comprises a heterologous gene operably linked to the CCL20 promoter. In some aspects, the vector does not comprise a heterologous gene operably linked to the CCL20 promoter. In some aspects, the CCL20 promoter is a functional fragment of the sequence set forth in any one of SEQ ID NOs: 1-5. In some aspects, the functional fragment has a 5' truncation, a 3' truncation, or both, relative to the CCL20 promoter sequence set forth in any one of SEQ ID NOs: 1-5. In some aspects, the CCL20 promoter is a functional variant having a sequence with at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-5. In some aspects, the sequence of the functional variant of the CCL20 promoter has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations relative to the sequence set forth in any one of SEQ ID NOs: 1-5. In some aspects, the point mutations are conservative point mutations.

Brief Description of the Drawings

[0021] [Figure 1A]Figure 1A shows the expression levels (fold increase in firefly luciferase) of the firefly luciferase reporter gene in cells transfected with the luciferase (FF-Luc) gene, (i) in the absence of a promoter, i.e., promoterless-FF-Luc (labeled "promoter control"), (ii) under the control of the CCL20 promoter, i.e., CCL20-FF-Luc (labeled "CCL20 promoter"), or (iii) under the control of the SAA3 promoter, i.e., SAA3-FF-Luc (labeled "SAA3 promoter") upon stimulation with IL-1α. The luciferase expression levels were measured at 0.5 hours, 2 hours, 4 hours, and 24 hours after IL-1α stimulation. [Figure 1B] Figure 1B shows the normalized expression levels of the firefly luciferase reporter gene in cells transfected with the luciferase (FF-Luc) gene, (i) in the absence of a promoter, i.e., promoterless-FF-Luc (labeled "promoter control"), (ii) under the control of the CCL20 promoter, i.e., CCL20-FF-Luc (labeled "CCL20 promoter"), (iii) under the control of the SAA3 promoter, i.e., SAA3-FF-Luc (labeled "SAA3 promoter"), or (iv) under the control of the SV40 promoter, i.e., SV40-FF-Luc (labeled "SV40 promoter") in the absence of stimulation or 4 hours after stimulation with IL-1α. [Figure 2] Figure 2 shows the expression levels of the anti-inflammatory cytokine interleukin receptor antagonist IL-1RA, which competes with active IL-1 and blocks the binding of both to their common activating receptor IL-1R1, secreted by cells transfected with (i) luciferase under the control of the CCL20 promoter ("CCL20-Luc"; negative control), (ii) the IL-1RA gene under the control of the CMV promoter ("CMV-IL1RA", positive control), and (iii) IL-1RA under the control of the CCL20 promoter ("CCL20-IL1RA") in response to stimulation with IL-1α. [Figure 3A]Figure 3A shows the expression levels of interleukin-8 (IL-8), a pro-angiogenic, proliferative, and inflammatory cytokine with oxidative activity, secreted by cells transfected with (i) luciferase under the control of the CCL20 promoter ("CCL20-Luc"; negative control), (ii) the IL-1RA gene under the control of the CMV promoter ("CMV-IL1RA"; positive control), and (iii) IL-1RA under the control of the CCL20 promoter ("CCL20-IL1RA"). [Figure 3B] Figure 3B shows the expression levels of interleukin-6 (IL-6), the most commonly considered pro-inflammatory cytokine involved in the acute immune response and chronic inflammation, secreted by cells transfected with (i) luciferase under the control of the CCL20 promoter ("CCL20-Luc"; negative control), (ii) the IL-1RA gene under the control of the CMV promoter ("CMV-IL1RA"; positive control), and (iii) IL-1RA under the control of the CCL20 promoter ("CCL20-IL1RA"). [Figure 3C] Figure 3C shows the expression levels of monocyte chemotactic protein-1 (MCP-1), chemokines that activate monocyte respiratory bursts and induce the expression of pro-inflammatory cytokines IL-6 and IL-1β, secreted by cells transfected with (i) luciferase under the control of the CCL20 promoter ("CCL20-Luc"; negative control), (ii) the IL-1RA gene under the control of the CMV promoter ("CMV-IL1RA"; positive control), and (iii) IL-1RA under the control of the CCL20 promoter ("CCL20-IL1RA"). [Figure 4] Figure 4 shows the IL-10 expression levels in HeLa cells transfected with the CCL20 promoter-IL10 construct after stimulation with IL-1α. [Figure 5]Figure 5 shows luciferase expression in a tunable system containing the Gal4-VP16 synthetic transcription factor under the control of the CCL20 promoter. The luciferase expression level can be controlled according to the number of upstream activating sequences (UAS) in the luciferase reporter construct. [Modes for carrying out the invention]

[0022] This disclosure provides CCL20 promoters that elicit gene expression in response to stimulation by pro-inflammatory cytokines. These inflammation-sensitive promoters can be used, for example, to control the timing of expression of heterologous genes, such as anti-inflammatory chemokines.

[0023] The CCL20 promoters disclosed herein (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or any functional fragment or variant thereof) can elicit gene expression in response to inflammation, whether spontaneous or induced, within a subject. Therefore, the CCL20 promoters of this disclosure can be used, for example, to control in a dose-dependent manner the expression levels of genes that increase or decrease immune activation, signal to another set of genes expressed in an inflammatory pathway, or alter or modify the cellular state in any way that reduces inflammation. The intensity and speed of gene expression induced by the CCL20 promoters of this disclosure create potent responses that can be manipulated for gene therapy, cell therapy, or recombinant expression.

[0024] The CCL20 promoter of this disclosure may be used, for example, in gene therapy. As a non-limiting example, an individual suffering from an inflammatory condition (e.g., cancer, neurodegenerative disease, or autoimmune disease) may receive gene therapy containing heterologous genes under the control of the CCL20 promoter of this disclosure. Endogenously released or exogenous (administered) inflammatory cytokines, such as IL-1α, stimulate the CCL20 promoter and trigger a downstream anti-inflammatory response, for example, through the expression of anti-inflammatory cytokines or anti-inflammatory molecules (e.g., antibodies) encoded by heterologous genes controlled by the CCL20 promoter. Alternatively, endogenously released or exogenous (administered) inflammatory cytokines may trigger the expression of intermediates (e.g., activators or inhibitors) encoded by heterologous genes controlled by the CCL20 promoter, which will result in an anti-inflammatory response when acting in the inflammatory pathway.

[0025] The CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or variant) can also be used to genetically engineer cells for administration to subjects suffering from an inflammatory condition (e.g., via injection or transplantation). In some embodiments, the cells are autologous cells, such as autologous CAR T cells or other autologous T cells. In other embodiments, the cells are allogeneic cells, such as allogeneic CAR T cells or other allogeneic T cells. In some embodiments, the cells can be transplanted into the subject.

[0026] Stimulation of the CCL20 promoter in administered / transplanted cells can trigger downstream anti-inflammatory responses by eliciting the expression of, for example, anti-inflammatory cytokines (e.g., IL1RA, IL-4, IL-10, IL-11, IL-13 proteins, TGF-β, leukemia inhibitors, interferon-alpha, or a combination thereof if the construct is bicistronic or polycistronic), analgesic peptides or proteins, or therapeutic proteins (e.g., antibodies or other biologics) in response to stimulation with inflammatory cytokines such as IL-1α, IL-1β, TNFα, IL-6, IL-15, IL-17, or IL-18.

[0027] In some embodiments, the CCL20 promoter of this disclosure can be indirectly activated by cytokines such as IL-6, sIL-6R, or IL-17, which leads to heterologous gene expression under the control of the CCL20 gene through increased recruitment of phosphorylated NF-κB to the CCL20 promoter.

[0028] The CCL20 promoter disclosed herein can also be used for recombinant gene expression. In some embodiments, recombinant gene expression under the control of the CCL20 promoter disclosed herein can be induced by the addition of exogenous cytokines to the cell culture medium, such as IL-1α. In other embodiments, cultured cells can be subjected to conditions that elicit cytokine production by the cultured cells or by cells co-cultured with host cells, which in turn induces recombinant gene expression under the control of the CCL20 promoter disclosed herein.

[0029] The therapeutic use of the CCL20 promoter of this disclosure is not limited to the expression of genes that trigger an anti-inflammatory response. For example, the CCL20 promoter disclosed herein can be used to trigger the expression of any protein or gene that can treat a disease or condition characterized by elevated levels of inflammatory cytokines. For example, genes encoding antimicrobial or antiviral proteins such as antibodies may be expressed under the control of the CCL20 promoter of this disclosure in subjects suffering from microbial or viral infections. Assuming dose-dependent gene expression observed upon induction of the CCL20 promoter of this disclosure, the production levels of such antimicrobial or antiviral proteins may be endogenously controlled by the severity of the inflammatory response to the disease, exogenously controlled by administering a compound (e.g., an inflammatory cytokine) that directly activates the CCL20 promoter, or indirectly controlled by administering a compound that induces the release of inflammatory cytokines, which in turn activate the CCL20 promoter.

[0030] I. Definition To facilitate understanding of this specification, certain terms are defined first. Additional definitions are provided throughout the detailed description.

[0031] It should be noted that the term “one (a)” or “one (an)” entity refers to one or more of those entities; for example, “a nucleotide sequence” is understood to represent one or more nucleotide sequences. Thus, the terms “one (a)” (or “one (an)”), “one or more,” and “at least one” can be used interchangeably herein. Furthermore, it should be noted that claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a priori basis for limitation for the use of exclusive terms such as “only,” “this only,” and similar terms in connection with the enumeration of elements of a claim or the use of negative limitations.

[0032] Furthermore, as used herein, “and / or” is taken as a specific disclosure having one or not having the other of each of two identified features or components. Thus, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0033] Whenever an aspect is described herein with the word “comprising,” it is understood that similar aspects are also provided, described in other ways in terms of “consisting of” and / or “consisting essentially of.”

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide general dictionaries for many of the terms used herein to those skilled in the art.

[0035] Units, prefixes, and symbols are shown in the Systeme International de Unites (SI) approved form. Numerical ranges include both ends of the numerical value defining the range. When ranges of values ​​are enumerated, it is understood that each intervening integer value between the upper and lower limits of the enumerated range, and each segment thereof, is also specifically disclosed along with each subrange between such values. The upper and lower limits of any range may be independently included in or excluded from a range, and each range that includes either one of the limits, neither, or both is included within this disclosure. Therefore, ranges enumerated herein are understood to be abbreviations for all values ​​within the range, including the enumerated endpoints. For example, the range 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0036] Where values ​​are explicitly enumerated, it is understood that values ​​that are substantially the same quantity or amount as the enumerated values ​​are also within the scope of this disclosure. Where combinations are disclosed, each subcombination of the elements of that combination is also specifically disclosed and is also within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of this disclosure is disclosed as having multiple substitutes, examples of this disclosure in which each substitute is excluded, either alone or in any combination with other substitutes are also disclosed herein, and two or more elements of this disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0037] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are referred herein by their commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Thus, "a" represents adenine, "c" represents cytosine, "g" represents guanine, "t" represents thymine, and "u" represents uracil.

[0038] Approximately: The term "approximately" is used herein to mean roughly, roughly, approximately, or within a certain range. When the term "approximately" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated number. Generally, the term "approximately" can be used to modify a number above and below a stated value by, for example, 10 percent, or by a difference of up or down (higher or lower).

[0039] Administration: The terms "administer", "administering", and their grammatical variants refer to introducing a composition comprising a CCL20 promoter (i.e., any one of the CCL20 promoters of SEQ ID NOs: 1-5 or a functional fragment or variant thereof), such as a polynucleotide, vector, or viral particle, into a subject via a pharmaceutically acceptable route. Introduction of the composition into the subject can be by any suitable route including oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, intratumoral, periocular, or topical. Administration includes self-administration and administration by another person. A suitable administration route enables the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein. In some embodiments, cells are administered. In some embodiments, cells can be transplanted.

[0040] Antibody: As used herein, the term "antibody" (Ab) should include, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V H and a heavy chain constant region. The heavy chain constant region comprises three constant domains, C H1 C H2 and C H3 Each light chain comprises a light chain variable region (abbreviated herein as V L and a light chain constant region. The light chain constant region comprises one constant domain, C L The V H region and the V L region can be further differentiated into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt comprises three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the traditional complement system. In some embodiments, the heterologous protein expressed via stimulation of a CCL20 promoter operably linked to a polypeptide encoding the heterologous protein contains the antibody or its antigen-binding moiety.

[0041] Antigen: The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve either antibody production or activation of certain immunologically capable cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can act as an antigen. Furthermore, antigens can originate from recombinant DNA or genomic DNA.

[0042] Antigen-binding portion: The "antigen-binding portion" of an antibody (also called the "antigen-binding fragment") refers to one or more fragments of the antibody that possess the ability to specifically bind to the antigen to which the entire antibody binds. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments included in the term "antigen-binding portion" of an antibody, for example, in the case of an anti-GD2 antibody, (i)V L , V H (ii) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of LC and CH1 domains, (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or a similar divalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region, (iii) V H Fd fragment consisting of domain and CH1 domain, (iv) single arm of antibody V L Domain and V HThe Fv fragment and (v)dAb fragment, consisting of domains (Ward et al., (1989) Nature 341:544-546), are V H (vi) an isolated complementarity-determining region (CDR) consisting of domains, and (vii) a combination of two or more isolated CDRs that can be optionally joined by a synthetic linker. Furthermore, the two domains of the Fv fragment V L and V H These are encoded by separate genes, but they can be synthesized using recombination methods. L and V H These single-chain antibodies can be linked by synthetic linkers, which allow the regions to be paired and constructed as a single protein chain forming a monovalent molecule (also known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0043] Approximately: As used herein, the term “approximately” means, when applied to one or more reference values, a value similar to the given reference value. In certain embodiments, unless otherwise stated or otherwise evident from the context, the term “approximately” means a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the given reference value (unless such numbers would exceed 100% of the possible value).

[0044] CAR: The term "chimeric antigen receptor" or, as otherwise, "CAR" refers to a pair of two polypeptides, typically in their simplest form, which, when present in an immune effector cell, provides the cell with specificity to target cells, typically cancer cells, and provides intracellular signaling. In some embodiments, a CAR includes at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to as the "intracellular signaling domain"), which include a functional signaling domain derived from stimulating and / or co-stimulating molecules as defined below. In some embodiments, a heterologous protein expressed via stimulation of a CCL20 promoter operably linked to a polypeptide encoding a heterologous protein includes a CAR.

[0045] CCL20, also known as liver activation regulatory chemokine (LARC) or macrophage inflammatory protein-3 (MIP3A), is a small cytokine belonging to the CC chemokine family. It is strongly chemotactic towards lymphocytes and weakly attracts neutrophils. CCL20 exerts its effects on its target cells by binding to and activating the chemokine receptor CCR6. CCL20 gene expression can be induced by microbial factors such as lipopolysaccharides (LPS), as well as inflammatory cytokines such as tumor necrosis factor and interferon-γ, and can be downregulated by IL-10.

[0046] CCL20 Promoter: As used herein, the terms “CCL20 promoter,” “the CCL20 promoter of this disclosure,” or “the CCL20 promoter disclosed herein” refer to nucleic acid sequences derived from the regulatory region of human CCL20 having promoter activity. The terms also encompass functional fragments and functional variants, i.e., fragments, variants, and derivatives thereof having promoter activity. In certain embodiments, the term CCL20 promoter refers to a polynucleotide comprising a nucleic acid sequence set for any one of SEQ ID NOs: 1, 2, 3, 4, or 5, or a functional variant disclosed herein.

[0047] Chemokines: The term "chemokines" refers to a family of small cytokines or signaling proteins secreted by cells that induce directional migration of other cell types, including not only leukocytes but also endothelial and epithelial cells. Chemokines are classified into four major subfamilies: CXC, CC, CX3C, and C. All of these proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, which are selectively found on the surface of their target cells.

[0048] Cytokines: The term "cytokines" refers to a broad and general category of small proteins (approximately 5–25 kDa) that are important in cellular signaling. Cytokines are peptides and cannot cross the cellular lipid bilayer to enter the cytoplasm. Cytokines have been shown to be involved in autocrine, paracrine, and endocrine signaling as immunomodulators. Examples of cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor, but generally do not include hormones or growth factors.

[0049] Complement: As used herein, the term “complement” refers to a sequence that is complementary to a reference sequence. Complementarity is a property shared between two DNA or RNA sequences, and is therefore a fundamental principle of DNA replication and transcription. It is well known that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequence become complementary, just as one would look in a mirror and see the opposite of the other. Thus, for example, the complement of the sequence 5'“ATGC”3' can be written as 3'“TACG”5' or 5'“GCAT”3'. As used herein, the terms “reverse complement,” “reverse complement,” and “reverse complementarity” are interchangeable with the terms “complement,” “complementary,” and “complementarity.” In some embodiments, the term “complementary” refers to 100% matching or complementarity (i.e., perfectly complementary) to a continuous nucleic acid sequence. In some embodiments, the term complementary refers to a match or complementarity of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% to a continuous nucleic acid sequence.

[0050] Complementary: The terms “complementary” and “complementarity” refer to two or more polynucleotides (i.e., each containing a nucleic acid base sequence) that are related to each other by the Watson-Crick base pairing rules. For example, the nucleic acid base sequence “TGA(5'→3')” is complementary to the nucleic acid base sequence “ACT(3'→5')”. Complementarity may be “partial” in that less than all of the nucleic acid bases of a given polynucleotide sequence match those of the other polynucleotide sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given polynucleotide sequence and another polynucleotide sequence may be at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. On the other hand, to continue the examples, there may be “complete” or “perfect” (100%) complementarity between a given polynucleotide sequence and another polynucleotide sequence. The degree of complementarity between polynucleotide sequences has a significant effect on the efficiency and intensity of hybridization between sequences.

[0051] Conserved: As used herein, the term “conserved” refers to a nucleotide in a polynucleotide sequence that occurs unchanged at the same position in two or more sequences being compared. Relatively conserved nucleotides are those that are conserved between related sequences more than any other nucleotides in the sequence.

[0052] Corresponding: When referring to two distinct nucleic acid sequences or nucleotide sequences, the terms “corresponding” and “corresponding” can be used to clarify regions of sequences that correspond to or are similar to each other based on homology and / or functionality, although the nucleotides of particular sequences may be numbered differently. In addition, it is recognized that different numbering systems may be employed when characterizing nucleic acids or nucleotide sequences. Furthermore, it is recognized that the nucleotide sequences of nucleic acid sequences, or different variants of nucleic acids, may be different. However, as used herein, regions of variants of nucleic acids or nucleotide sequences that share homology and / or functionality are considered to “correspond” to each other.

[0053] Culture: As used herein, the terms “culture,” “cell culture,” and “eukaryotic cell culture” refer to a population of cells, whether surface-attached or in suspension, that is maintained or grown in a culture medium (see the definition of “culture medium” below) under conditions suitable for the survival and / or proliferation of the cell population. As will be apparent to those skilled in the art, as used herein, these terms may refer to a combination including a cell population and the culture medium in which the population is suspended. As used herein, “to culture” refers to growing one or more cells in vitro under defined or controlled conditions. Examples of culture conditions that can be defined include temperature, gas mixture, time, and culture medium preparation.

[0054] Culture medium: As used herein, the terms “media,” “medium,” “cell culture medium,” “tissue culture medium,” and “growth medium” refer to solutions containing nutrients that can be used to nourish the growth of cultured host cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. Solutions may also contain components that enhance growth and / or survival beyond a minimal rate, including hormones and growth factors.

[0055] Derived: As used herein, the terms “derived” or “derivative” refer to a component isolated from a particular molecule or constructed using a particular molecule or information from a particular molecule (e.g., nucleic acid sequence). For example, a polynucleotide sequence derived from another polynucleotide sequence may contain a polynucleotide sequence identical or substantially similar to the polynucleotide sequence from which it is derived. In the case of polynucleotides, the species from which they are derived can be obtained, for example, by spontaneous mutagenesis, artificial directional mutagenesis, or artificial random mutagenesis. Mutagenesis used to induce polynucleotides can be intentionally oriented, intentionally random, or a mixture of each. Mutagenesis of a polynucleotide to produce a different polynucleotide derived from a first polynucleotide can be a random event (e.g., caused by polymerase infidelity), and the identification of the polynucleotide from which it is derived can be done by appropriate screening methods known in the art.In some embodiments, the polynucleotide sequence derived from the first polynucleotide sequence is at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, The derived polynucleotide sequence has sequence identity of at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100% identity to each of the first polynucleotide sequences, and the derived polynucleotide sequence retains the biological activity of the original polynucleotide.

[0056] Downstream / Upstream: The term "downstream" refers to a nucleotide sequence located 3' relative to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site. The term "upstream" refers to a nucleotide sequence located 5' relative to a reference nucleotide sequence.

[0057] Encoding: The term "encoding" refers to the inherent properties of a particular sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, that serve as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to encode a protein or other product of that gene or cDNA.

[0058] Unless otherwise specified, nucleotide sequences that "encode" an amino acid sequence, for example, heterologous molecules that "encode" a polynucleotide under the control of the CCL20 promoter of this disclosure, are mutually degenerate versions and include all nucleotide sequences that encode the same heterologous molecule (e.g., the same amino acid sequence).

[0059] Expression: As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide. This includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA) and the translation of mRNA into polypeptide. Expression produces a “gene product.” As used herein, a gene product can be either a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from the transcript. In some embodiments, the terms “expression” or “expressing” are used to refer to the transcription and translation that occur within a cell. The level of expression of a product gene within a host cell can be determined based on either the amount of corresponding mRNA present in the cell, or the amount of protein encoded by the product gene produced by the cell, or both.

[0060] Fragment: As used herein, the term “fragment,” for example, “fragment of the CCL20 promoter disclosed herein,” means a polynucleotide sequence of the CCL20 promoter disclosed herein that is shorter than the CCL20 promoter sequence disclosed herein (e.g., any one of the CCL20 promoters among SEQ ID NOs. 1 to 5), for example, having a deletion at the 5' end and / or 3' end, or having any portion of the polynucleotide sequence deleted compared to a naturally occurring polynucleotide.

[0061] Functional / Non-functional Fragments: As used herein, the term “functional fragment” refers to a polynucleotide fragment derived from the CCL20 promoter sequence disclosed herein that retains promoter function. As a result, in some embodiments, the functional fragments of the CCL20 promoter disclosed herein retain the ability to elicit the expression of a heterologous gene operably linked to the CCL20 promoter fragment. Conversely, a “non-functional fragment” will lack one or more of the functional properties of the parent molecule.

[0062] Whether the CCL20 promoter fragments disclosed herein are functional fragments can be evaluated by known methods of any art without requiring excessive experimentation. For example, a reporter gene (e.g., luciferase) can be operably ligated to a CCL20 promoter fragment, and the expression of luciferase in response to stimulation by IL-1α can be determined. In some embodiments, the functional fragment controls the expression of a heterologous gene (e.g., as determined by a luciferase expression assay in response to stimulation by IL-1α) by retaining, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the capacity of the CCL20 promoter disclosed herein. In some embodiments, the CCL20 promoter functional fragment consists of a sequence that is complementary to the reverse complement of any one of the CCL20 promoter sequences 1-5 and can hybridize with such reverse complement of any one of the CCL20 promoter sequences 1-5 under stringent conditions.

[0063] Functional Variants / Non-Functional Variants: As used herein, the term “functional variant” refers to a polynucleotide derived from the CCL20 promoter sequence, for example, through point mutation, insertion, deletion, etc., that retains promoter function, as disclosed herein. In some embodiments, functional variants of the CCL20 promoter disclosed herein retain the ability to elicit expression of heterologous genes operably linked to the CCL20 promoter fragment. Conversely, a “non-functional variant” lacks one or more of the functional properties of the parent molecule.

[0064] Whether a variant (e.g., a mutant) of the CCL20 promoter disclosed herein is a functional variant can be evaluated by known methods of any art without requiring excessive experimentation. For example, a reporter gene (e.g., luciferase) can be operably ligated to a CCL20 promoter variant and its luciferase expression in response to IL-1α stimulation can be determined. In some embodiments, the functional variant retains, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the capacity of the CCL20 promoter disclosed herein to control the expression of a heterologous gene (e.g., as determined by a luciferase expression assay in response to IL-1α stimulation). In some embodiments, the CCL20 promoter functional variant consists of a sequence that is complementary to the reverse complement of any one of the CCL20 promoter sequences 1-5 and can hybridize with such reverse complement sequences of the CCL20 promoter sequences 1-5 under stringent conditions.

[0065] Gene: The terms “gene,” “coding sequence,” and “encoding nucleic acid,” and their grammatical variations, are used interchangeably within this disclosure and generally refer to nucleic acids (RNA or DNA molecules) containing a nucleotide sequence that encodes a gene of interest, such as a protein, for example, a therapeutic protein such as an antibody or CAR. The coding sequence may further include start and termination signals operably linked to a promoter and a polyadenylation signal, which have the ability to direct expression within the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0066] Heterogene: As used herein, the term heterogene means a gene operably ligated to the CCL20 promoter of this disclosure, and the gene is not a naturally occurring CCL20. In some embodiments, the heterogene may be a gene encoding a therapeutic protein, such as an anti-inflammatory cytokine or antibody, or a therapeutic nucleic acid, such as an RNA interference molecule. In some embodiments, particularly for diagnostic use, the heterogene encodes a reporter molecule (e.g., luciferase).

[0067] Identical: In some embodiments, two or more sequences are said to be “completely conserved” or “identical” if they are 100% identical to each other. In some embodiments, two or more sequences are said to be “highly conserved” if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some embodiments, two or more sequences are said to be “highly conserved” if they are at least about 70% identical, at about 80% identical, at about 90% identical, at about 95% identical, at about 98% identical, or at about 99% identical to each other. In some embodiments, two or more sequences are said to be “conserved” if they are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some embodiments, two or more sequences are said to be “conserved” if they are approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to one another. Sequence conservation may apply to the entire length of a polynucleotide or polypeptide, or to a portion, region, or feature thereof.

[0068] Identity: As used herein, the term “identity” refers to the overall monomeric conservation between macromolecules, such as polypeptide molecules or polynucleotide molecules (e.g., DNA molecules and / or RNA molecules). The term “identical” without any additional modifiers, for example, “protein A is identical to protein B,” implies that the sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, “70% identical” is equivalent to describing them as having, for example, “70% sequence identity.”

[0069] The percentage of identity between two polypeptide or polynucleotide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second polypeptide or polynucleotide sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the length of the reference sequence. Then, the amino acids, or bases in the case of polynucleotides, at the corresponding amino acid positions are compared.

[0070] Molecules are identical in a given position if the position in one sequence is occupied by the same amino acid as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Sequence comparison and determination of the percentage of identity between two sequences can be achieved using mathematical algorithms.

[0071] Suitable software programs are available from various sources and can be used for both protein and nucleotide sequence alignment. One suitable program for determining sequence identity percentage is bl2seq, which is part of the BLAST suite of programs available from the BLAST website of the U.S. National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite and are available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. In one particular aspect, sequence identity corresponds to the percentage of sequence identity of global pairwise alignments determined using a program that implements the Needleman-Wunsch algorithm, for example, Needle, which is available at www.ebi.ac.uk / Tools / psa / emboss_needle / .

[0072] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, etc.

[0073] Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own sequence identity percentage. Note that the sequence identity percentage is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Also note that length values ​​are always integers.

[0074] In a particular embodiment, the percentage of identity (%ID) or the percentage of identity (%ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100 × (Y / Z), where Y is the number of amino acid residues (or nucleic acid bases) scored as a perfect match in the alignment of the first and second sequences (as aligned by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percentage of identity of the first sequence to the second sequence is higher than the percentage of identity of the second sequence to the first sequence.

[0075] Those skilled in the art will understand that the generation of sequence alignments for calculating sequence identity percentages is not limited to binary sequence-to-sequence comparisons driven exclusively by primary sequence data. It will also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structure), functional data (e.g., mutation sites), or phylogenetic data. Suitable programs for integrating heterogeneous data to generate multiple sequence alignments include T-Coffee, available at www.tcoffee.org, and alternatively, EBI, for example. It will also be understood that the final alignments used to calculate sequence identity percentages can be supervised either automatically or manually.

[0076] Inflammation: As used herein, the term “inflammation” refers to the complex biological response of vascular tissue to harmful stimuli such as pathogens, damaged cells, or irritants. Classical signs of acute inflammation are pain, heat, redness, swelling, and loss of function. Inflammation is a protective attempt by the organism to remove harmful stimuli and initiate the healing process. Inflammation is not synonymous with infection, although the two are still often correlated (the former is often a consequence of the latter). Inflammation can occur in the absence of infection, but this type of inflammation is usually maladaptive (e.g., atherosclerosis). Inflammation is a conventional response and is therefore considered a mechanism of innate immunity compared to specific adaptive immunity to each pathogen. The gradual destruction of tissue in the absence of inflammation would impair the survival of the organism. On the other hand, chronic inflammation can lead to hosts of diseases such as hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer (e.g., gallbladder cancer). For this reason, inflammation is usually strictly regulated by the body.

[0077] Inflammation can be classified as either acute or chronic. "Acute inflammation" is the body's initial response to a harmful stimulus and is achieved by an increased migration of plasma and white blood cells (especially granulocytes) from the blood to the damaged tissue. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the damaged tissue. Prolonged inflammation, also known as "chronic inflammation," leads to a gradual shift in the types of cells present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissue from the inflammatory process.

[0078] In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) can be stimulated by a pro-inflammatory cytokine released in response to inflammation, such as IL-α. In other embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) can be stimulated by inflammation or inflammatory processes, such as myocarditis, chronic myocarditis, acute myocarditis, viral myocarditis, vasculitis, pancreatitis, peritonitis, rheumatoid arthritis, inflammatory diseases of the kidney, immune-mediated kidney disease, kidney transplant rejection, immune complex-induced kidney disease, toxin-induced kidney injury, contrast-induced kidney injury, diabetic and non-diabetic nephropathy, pyelonephritis, kidney It can be used to induce or control the expression of polynucleotides encoding therapeutic agents that can treat, alleviate, or reduce the symptoms of disorders, conditions, and diseases of the cardiovascular, pulmonary, peripheral, hepatic, renal, digestive, or central nervous system, which may be associated with cysts, nephrosclerosis, hypertensive nephrosclerosis, nephrotic syndrome, chronic interstitial inflammation, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis (UC), inflammatory skin diseases, inflammatory diseases of the eye, blepharitis, dry eye syndrome, Sjögren's syndrome, or ocular fibrosis. Additional indications that can be treated using the disclosed compositions and methods are disclosed in more detail below.

[0079] As used herein, the term “spontaneous inflammation” refers to an inflammatory response that is the result of the natural course of a disease or condition, or of other causes such as exposure to an allergen or irritant. In other words, spontaneous inflammation is not the result of an external intervention to stimulate the CCL20 promoter of this disclosure, such as the injection of an exogenous interleukin. As used herein, the term “induced inflammation” refers to an external intervention to trigger an inflammatory response for the purpose of stimulating the CCL20 promoter of this disclosure, such as an increase in temperature, exposure to an antigen or allergen, or the injection of an exogenous interleukin.

[0080] Inflammatory Cytokines: The terms “inflammatory cytokines” and “pro-inflammatory cytokines” are used interchangeably and refer to types of signaling molecules (cytokines) secreted by immune cells such as helper T cells (Th) and macrophages, as well as certain other cell types that promote inflammation. These include, for example, interleukin-1 (IL-1), IL-6, IL-12, and IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and play a crucial role in mediating innate immune responses. Inflammatory cytokines are primarily produced and involved in the upregulation of inflammatory responses. Excessive chronic production of inflammatory cytokines contributes to inflammatory diseases linked to various diseases such as atherosclerosis and cancer.

[0081] As used herein, the term “exogenous cytokine” refers to a cytokine that has an external source and is administered to a subject or cell, or added to a culture medium. In some embodiments, the exogenous cytokine is an exogenous interleukin (e.g., an interleukin injected into a subject or added to a culture medium). As used herein, the term “endogenous cytokine” refers to a cytokine produced by a tissue or cell of the subject, or produced spontaneously by cells in culture. In some embodiments, the endogenous cytokine is an endogenous interleukin, e.g., an interleukin released by the tissue of the subject due to spontaneous or induced inflammation.

[0082] Immunomodulatory Factors: As used herein, the term “immunomodulatory factor” refers to drugs that modulate the immune system. Non-limiting examples of genes encoding immunomodulatory factors that can be controlled under the control of the CCL20 promoter of this disclosure include genes encoding drugs such as checkpoint inhibitor regulators, checkpoint inhibitor ligands, cytokines, their derivatives, or any combination thereof. In certain aspects of this disclosure, heterogenes under the control of the CCL20 promoter of this disclosure are immunomodulatory factors. Immunomodulatory factor genes under the control of the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters among SEQ ID NOs: 1-5 or its functional fragment or functional variant) may also include, for example, agonist proteins, antagonist proteins, antibodies, antigen-binding fragments, or polynucleotides encoding polynucleotides such as siRNA, miRNA, lncRNA, mRNA, or DNA.

[0083] Immune response: Inflammation is the immune system's response to pathogens, damaged cells, toxic compounds, or harmful stimuli such as irradiation. As used herein, “immune response” refers to the biological response within a vertebrate to an exogenous drug or abnormality, such as cancer cells, which protect the organism from these drugs and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement), which result in the selective targeting, binding to, damage to, destruction of, and / or removal from the body of the vertebrate of an invading pathogen, a pathogen-infected cell or tissue, a cancer cell or other abnormal cell, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Immune responses include, for example, activation or inhibition of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or activation or inhibition of any other cells in the immune system, such as NK cells. As a result, the immune response may include a humoral immune response (e.g., mediated by B cells), a cellular immune response (e.g., mediated by T cells), or both a humoral and cellular immune response.

[0084] In some embodiments, compositions and methods of the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or a functional fragment or functional variant thereof) can elicit an immune response that is generally an "inhibitory" immune response. An inhibitory immune response is an immune response that blocks or reduces the effect of a stimulus (e.g., an antigen). In certain embodiments, an inhibitory immune response involves the production of inhibitory antibodies against the stimulus. In some embodiments, the immune response is an "inhibitory" immune response. An inhibitory immune response is an immune response that results in the generation of effector cells (e.g., cytotoxic T lymphocytes) that can destroy and eliminate a target antigen (e.g., a tumor antigen or virus).

[0085] Interleukins: The term "interleukins," abbreviated as IL, refers to a group of cytokines (secreted proteins and signaling molecules) that are expressed and secreted not only by white blood cells (leukocytes) but also by some other somatic cells. The human genome encodes more than 50 interleukins and related proteins. As used herein, this term means, for example, interleukin-1 (interleukin-1 alpha, IL-1α, and interleukin-1 beta, IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8; CXCL8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleuk This includes interleukin 17 (IL-17), interleukin 18 (IL-18), interleukin 19 (IL-19), interleukin 20 (IL-20), interleukin 21 (IL-21), interleukin 22 (IL-22), interleukin 23 (IL-23), interleukin 24 (IL-24), interleukin 25 (IL-25), interleukin 26 (IL-26), interleukin 27 (IL-27), interleukin 28 (IL-28), interleukin 29 (IL-29), interleukin 30 (IL-30), interleukin 31 (IL-31), interleukin 32 (IL-32), interleukin 33 (IL-33), interleukin 35 (IL-35), and interleukin 36 (IL-36).

[0086] Mismatch: The term “mismatch” or “mismatch(s)” refers to one or more nucleic acid bases (whether consecutive or separated) in the oligomeric nucleic acid sequence that do not match the target premRNA according to the base pairing rules. While perfect complementarity is often desirable, some embodiments may include one or more, but preferably six, five, four, three, two, or one, mismatches with respect to the target premRNA. This includes deformations at any location within the oligomer. In certain embodiments, the antisense oligomers of this disclosure include deformations within the nucleic acid sequence near the terminals, internal deformations, and, if present, typically within about six, five, four, three, two, or one subunit at the 5' and / or 3' ends. In certain embodiments, one, two, or three nucleic acid bases may be removed and still provide on-target binding.

[0087] Nucleic acid: The terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” and “polynucleotide,” and their grammatical variations, are used interchangeably and refer to either single-stranded or double-helixed phosphate ester polymers of ribonucleosides (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecule”), or any phosphate ester analogue thereof (phosphorothioates and thioesters, etc.). Single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The terms nucleic acid molecule, and more specifically DNA molecule or RNA molecule, refer only to the primary and secondary structures of the molecule and are not limited to any specific tertiary form. Therefore, this term includes, among other things, linear or circular DNA molecules (e.g., restriction fragments), plasmids, superhelical DNA, and double-stranded DNA found within chromosomes. In considering the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the usual convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone molecular biological manipulation. Examples of DNA include, but are not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A “nucleic acid composition” of this disclosure comprises one or more nucleic acids as described herein.

[0088] Nucleic acid sequence: The terms "nucleic acid sequence" and "nucleotide sequence" are used interchangeably and refer to a continuous sequence of nucleic acids. The sequence can be either single-stranded or double-stranded DNA or RNA, for example, gRNA.

[0089] "Operatively linked": "Operatively linked" refers to a juxtaposition in which the components described in this way are in a relationship that allows them to function in the intended manner. For example, if a promoter affects its transcription or expression, the promoter is operationally linked to the coding sequence. For example, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) can be an operational linker for polynucleotides encoding, for example, anti-inflammatory proteins, antibodies, CARs, etc.

[0090] Pharmacopoeia: The terms “pharmacopoeia acceptable carrier” and “pharmacopoeia acceptable excipient,” and their grammatical variations, encompass not only any agents approved by U.S. federal regulatory authorities for use in animals, including humans, or listed in the United States Pharmacopeia, but also any carrier or diluent that does not interfere with the administration of a composition to a subject and produce undesirable physiological effects to the extent that it does not impair the biological activity and properties of the administered compound. This term includes excipients and carriers that are useful in the preparation of pharmaceutical compositions and are generally safe, non-toxic, and desirable.

[0091] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" means one or more compounds mixed with, blended with, or suspended in, one or more other chemical components, such as pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate the administration of a drug preparation to a target.

[0092] Polynucleotide: As used herein, the term “polynucleotide” refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. This term refers to the primary structure of the molecule. Therefore, this term includes not only triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid (DNA), but also triple-stranded, double-stranded, and single-stranded ribonucleic acid (RNA).

[0093] More specifically, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing a non-nucleotide (normucleotidic) backbone, such as polyamides (e.g., peptide nucleic acids “PNA”) and polymorpholinopolymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleic acid bases in a configuration that allows for base pairing and stacking, such as those found in DNA and RNA. In some aspects of this disclosure, polynucleotides may be, for example, RNA, for example, mRNA, or DNA.

[0094] Polypeptides: The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may include modified amino acids. The term also encompasses any other operations or modifications, such as naturally occurring or interventionally modified amino acid polymers, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. Polypeptides containing not only one or more analogues of amino acids (e.g., non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine) but also other modifications known in the art are also included in the definition. As used herein, the term “polypeptide” refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and analogues as described above. Polypeptides may be single polypeptides or multimolecular complexes such as dimers, trimers, or tetramers. These can also include single-chain or polychain polypeptides. Most commonly, disulfide bonds are found in polychain polypeptides. The term polypeptide can also be applied to amino acid polymers, in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some embodiments, a “peptide” can be 50 amino acid lengths or less, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid lengths.

[0095] Prevention: As used herein, the terms “prevention,” “prevent,” and their variants mean to partially or completely delay the onset of a disease, disorder, and / or condition; to partially or completely delay the onset of one or more symptoms, features, or clinical signs of a particular disease, disorder, and / or condition; to partially or completely delay the onset of one or more symptoms, features, or signs of a particular disease, disorder, and / or condition; to partially or completely delay the progression from a particular disease, disorder, and / or condition; and / or reduce the risk of progression of a pathology associated with a disease, disorder, and / or condition. In some embodiments, prevention of outcome is achieved through preventive measures. As used herein, “preventive” means a therapeutic action or course of action used to prevent the onset of a disease or condition, or to prevent or delay symptoms associated with a disease or condition. As used herein, “prevention” means measures taken to maintain health and to prevent or delay the onset of a disease or condition, or to prevent or delay symptoms associated with a disease or condition.

[0096] Recombinant: A “recombinant” polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. Polypeptides and proteins produced by recombination expressed in modified host cells are considered isolated for the purposes of this disclosure, as are natural or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any preferred technique.

[0097] Similarity: As used herein, the term “similarity” refers to the overall relationship between polymer molecules, for example, between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules), and / or between polypeptide molecules. The calculation of the percentage of similarity between polymer molecules can be carried out in the same manner as the calculation of the percentage of identity, except that the calculation of the similarity percentage takes into account conservative substitutions as understood in the art. It is understood that the percentage of similarity depends on the comparison scale used, i.e., whether amino acids are compared according to, for example, their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aroma, isoelectric point, antigenicity, or a combination thereof.

[0098] Subject: In this specification, the terms “subject,” “patient,” “individual,” and “host,” as well as their variants, are used interchangeably and refer to any mammalian subject, including but not limited to domesticated animals (e.g., dogs, cats, and similar), livestock (e.g., cattle, sheep, pigs, horses, and similar), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, and similar), to which diagnosis, treatment, or therapy is desired, and particularly to humans. The methods described herein are applicable to both human therapeutic and veterinary uses. Where used herein, the phrase “subject requiring it” includes subjects such as mammalian subjects that would benefit from the administration of a therapeutic agent, for example, a gene encoding a therapeutic protein, the gene being under the control of the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or a functional variant of SEQ ID NOs: 1-5).

[0099] Subsequence: As used herein, the term “subsequence” refers to a subset of consecutive nucleotides or amino acids within a sequence (either a physical sequence or its symbolic representation).

[0100] Therapeutic effective dose: As used herein, the term “therapeutic effective dose” means the amount of a reagent or pharmaceutical compound, e.g., a vector containing a gene encoding a therapeutic protein, the gene being under the control of the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or a functional variant of SEQ ID NOs. 1-5), which is sufficient to produce the desired therapeutic, pharmacological, and / or physiological effect in a subject requiring it. Since prevention can be considered a therapy, the therapeutic effective dose may be a “preventive effective dose.”

[0101] Treatment: As used herein, the terms “treatment,” “therapy,” or “to treat” mean, for example, reducing the severity of a disease or condition, reducing the duration of the disease course, improving or eliminating one or more symptoms associated with the disease or condition, or providing a beneficial effect to an object having the disease or condition without necessarily curing the disease or condition. The term also includes the prevention or mitigation of a disease or condition or its symptoms. In one embodiment, the terms “treatment” or “therapy” mean inducing an immune response in an object to an antigen or reducing inflammation.

[0102] Modulation: As used herein, the terms “modulation,” “modification,” and their grammatical variations generally refer to the ability to alter a particular concentration, level, expression, function, or behavior, for example, by directly or indirectly promoting / stimulating / upregulating or inhibiting / downregulating, such as in order to act as an antagonist or agonist. In some cases, a modifier may increase and / or decrease a particular concentration, level, activity, or function relative to a control, or to an average level of activity that would generally be expected, or to a control level of activity.

[0103] Vector: The terms “vector,” “expression vector,” and “plasmid,” and their grammatical variations, are used interchangeably within this disclosure and refer to an exogenous polynucleotide to the host cell genome that is inserted into a specific location within the genome of a host cell (e.g., a T cell). Generally, a plasmid comprises multiple elements, such recombination sites (e.g., homologous recombination sites and / or site-specific recombination sites), markers (e.g., detection markers and / or selection markers), one or more expression cassettes, or any combination thereof. In some embodiments, the plasmid may be a linear plasmid. In other embodiments, the plasmid may be a round plasmid, e.g., an intact round plasmid.

[0104] II. CCL20 cytokine induction promoter and its use This disclosure provides promoters derived from the regulatory region of human CC motif chemokine ligand 20 (CCL20), and their use for in-situ protein or recombinant gene expression, for example, through the regulation of CCL20 promoter activity by cytokines. The CCL20 promoters disclosed herein can be stimulated by cytokines, such as pro-inflammatory cytokines such as IL-1α, to induce the expression of heterologous genes, such as genes encoding heterologous proteins, such as anti-inflammatory cytokines, which are operably linked to the CCL20 promoter. As used herein, the terms “heterologous gene” and “heterologous protein” refer to any gene that does not encode CCL20, or a protein that is not CCL20, respectively. In some embodiments, the expression product of a heterologous gene is a therapeutic protein. In other embodiments, the expression product of a heterologous gene is a therapeutic polypeptide.

[0105] The CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) can be used in a subject to induce the expression of a heterogeneously operably linked gene, for example, to the CCL20 promoter encoding a heterogeneous protein or two or more heterogeneous proteins (e.g., controlling the expression of a gene that is part of a bicistronic or multicistronic construct) in response to stimulation of the CCL20 promoter by an inflammatory cytokine, e.g., IL-α. The inflammatory cytokine capable of inducing heterogeneous gene expression via stimulation of the CCL20 promoter of this disclosure can be endogenous (i.e., a result of spontaneous inflammation) or exogenous (i.e., a cytokine administered to a subject to elicit an inflammatory response, or a cytokine added to the culture medium in the case of recombinant expression).

[0106] In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or a functional fragment or functional variant thereof) can be used to control heterologous genes within a subject, and the process does not involve inserting the heterologous gene into the subject. For example, the CCL20 promoter of this disclosure can be inserted at a locus upstream of a heterologous gene within the genome of the subject, and the CCL20 promoter replaces a spontaneously occurring promoter, for example, using a gene editing method. In some embodiments, the substituted CCL20 promoter can be inserted at a site where a spontaneously occurring promoter is inactivated, for example, an internal site, without removing the spontaneously occurring promoter. In some embodiments, the substituted CCL20 promoter can be inserted at a site where a portion of the spontaneously occurring promoter is removed, for example, a portion of the 3' terminal region of the spontaneously occurring promoter, and where the spontaneously occurring promoter is inactivated. As a result, if, for example, the expression level of a particular gene is excessively low, or if it would be beneficial to be under external control, such as the administration of exogenous cytokines, the naturally occurring promoter may be replaced with the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant).

[0107] In some embodiments, the heterologous protein or protein expressed in response to stimulation of the CCL20 promoter by endogenous or exogenous cytokines, such as IL-α, may be an anti-inflammatory protein, such as an antibody or its antigen-binding moiety, a receptor, an antagonist, or any combination thereof. In some embodiments, the antibody or its antigen-binding moiety is an anti-TNF antibody, such as an antibody against TNF-α. In other embodiments, the antibody or its antigen-binding moiety is an anti-IL-1 antibody, such as an antibody against IL-1α. In some embodiments, the receptor is a soluble TNF receptor. In some embodiments, the receptor is a soluble IL-1 receptor. In some embodiments, the antagonist is a TNF receptor antagonist. In some embodiments, the antagonist may be an IL-1 receptor antagonist. Generally, the stimulating activity of the CCL20 promoter can be elicited by any action that increases the level of proteins that bind to the CCL20 promoter, such as NF-κB, STAT3, AP-1, AP-2, C-EBP, SP1, or ESE-1. Exemplary pathways that can result in downstream increases in levels of NF-κB, STAT3, AP-1, AP-2, C-EBP, SP1, or ESE-1 are illustrated, for example, in the signaling pathway diagrams at www.cellsignal.com / pathways / by-research / immunology-inflammation-pathways, all of which are incorporated herein in their entirety by reference.

[0108] In some embodiments, heterologous proteins or proteins expressed in response to stimulation of the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or functional fragments or functional variants of SEQ ID NOs: 1-5) by endogenous or exogenous cytokines can be protein activators or inhibitors that reduce the inflammatory response by modifying the response of the inflammatory pathway. Three common transcription factors, namely activated B cell nuclear factor-kappa light chain enhancer (NF-κB), hypoxia-induced factor-1 alpha (HIF-1α), and signaling and transcriptional activators (STATs), play major regulatory roles in the inflammatory response pathway. When inflammation becomes chronic, these factors can lead to cancer. See, for example, Bharat B. et al (2009) Clin Cancer Res 15(2): 425-430 and Chiara P. et al (2009) Immunobiology 214(2009): 761-777. Therefore, in some embodiments, the heterologous protein or protein expressed in response to stimulation of the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or its functional fragment or functional variant among SEQ ID NOs: 1-5) is an antagonist targeting NF-κB, HIF-1α, or STAT (e.g., an antibody, its antigen-binding moiety, or a molecule derived from such an antibody, e.g., CAR). In other words, stimulation of the CCL20 promoter by inflammation triggers a negative feedback mechanism that reduces inflammation.

[0109] In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) can be used to induce protein expression in vivo (e.g., after gene therapy), ex vivo, or in vitro (e.g., recombinant expression in cell culture).

[0110] In some embodiments, the Disclosure provides a polynucleotide comprising a promoter region containing the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or a functional fragment or functional variant thereof) operably ligated to a heterogene, the CCL20 promoter inducing the expression of the heterogene in response to stimulation by a cytokine, such as IL-α. In some embodiments, the cytokine used to stimulate the CCL20 promoter of the Disclosure is a pro-inflammatory cytokine.

[0111] In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by interleukin-1 (IL-1), for example, IL-1α or IL-1β. In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by interleukin-6 (IL-6). In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by interleukin-12 (IL-12). In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by interleukin-18 (IL-18). In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by tumor necrosis factor alpha (TNF-α). In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by interferon gamma (IFNγ). In some embodiments, the CCL20 promoter of this disclosure (i.e., the CCL20 promoters from SEQ ID NOs: 1-5 or their functional fragment or functional variant) is stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by soluble interleukin-6 receptor (sIL-6R). In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters among SEQ ID NOs: 1-5 or its functional fragment or functional variant) is stimulated by interleukin 17 (IL-17).In some embodiments, the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1 to 5, or a functional fragment or functional variant thereof) is stimulated by CD30.

[0112] In some embodiments, the polynucleotide comprises a CCL20 promoter comprising, consisting of, or essentially comprising, the sequence described in SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a CCL20 promoter comprising, consisting of, or essentially comprising, the sequence described in SEQ ID NO: 2. In some embodiments, the polynucleotide comprises a CCL20 promoter comprising, consisting of, or essentially comprising, the sequence described in SEQ ID NO: 3. In some embodiments, the polynucleotide comprises a CCL20 promoter comprising, consisting of, or essentially comprising, the sequence described in SEQ ID NO: 4. In some embodiments, the polynucleotide comprises a CCL20 promoter comprising, consisting of, or essentially comprising, the sequence described in SEQ ID NO: 5. [Table 1]

[0113] In some embodiments, the CCL20 promoter of this disclosure is a functional variant of the CCL20 promoter having the sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5. In some embodiments, the CCL20 promoter of this disclosure is a functional fragment of the CCL20 promoter having the sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5. As defined above, the functional fragment can be cleaved from any one of the CCL20 promoters, SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 functional fragment promoter still has the ability to induce the expression of a heterologous protein encoded by a heterologous gene operably linked to the CCL20 promoter in response to stimulation by a cytokine, such as IL-1α. Similarly, a functional variant can be a variant of any one of the CCL20 promoter sequences 1, 2, 3, 4, or 5, and the CCL20 functional variant promoter still has the ability to induce the expression of a heterologous protein encoded by a heterologous gene operably linked to the CCL20 promoter in response to stimulation by a cytokine, e.g., IL-1α. In some embodiments, the promoter activity of the functional fragment or functional variant is determined using a heterologous gene, such as a reporter gene, e.g., luciferase.

[0114] In some embodiments, the CCL20 functional fragment or functional variant promoter has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the activity of any one of the CCL20 promoters among SEQ ID NOs. 1, 2, 3, 4, or 5 under the same experimental conditions.

[0115] In some embodiments, the CCL20 functional fragment or functional variant promoter has about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the activity of any one of the CCL20 promoters among SEQ ID NOs: 1, 2, 3, 4, or 5 under the same experimental conditions.

[0116] In some embodiments, the CCL20 functional fragment or functional variant promoter has, under the same experimental conditions, about 20% to about 30%, about 25% to about 35%, about 30% to about 40%, about 35% to about 45%, about 40% to about 50%, about 45% to about 55%, about 50% to about 60%, about 55% to about 65%, about 60% to about 70%, about 65% to about 75%, about 70% to about 80%, about 75% to about 85%, about 80% to about 90%, about 85% to about 95%, and about 90% to about 100% of the activity of any one of the CCL20 promoters among SEQ ID NOs.

[0117] In some embodiments, the CCL20 functional fragment or functional variant promoter is a gain-of-function promoter, i.e., the activity of the CCL20 functional fragment or functional variant promoter is higher than the activity of any one of the CCL20 promoters from SEQ ID NOs: 1, 2, 3, 4, or 5 under the same experimental conditions.

[0118] In some embodiments, the gain-of-function CCL20 promoter has an activity that is at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% of the activity of any one of the CCL20 promoters among SEQ ID NOs. 1, 2, 3, 4, or 5 under the same experimental conditions.

[0119] In some embodiments, the gain-of-function CCL20 promoter has an activity that is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times greater than the activity of any one of the CCL20 promoters among SEQ ID NOs. 1, 2, 3, 4, or 5 under the same experimental conditions.

[0120] In some embodiments, the gain-of-function CCL20 promoter has activity that is approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than the activity of any one of the CCL20 promoters among Sequence ID No. 1, 2, 3, 4, or 5 under the same experimental conditions.

[0121] In some embodiments, the gain-of-function CCL20 promoter has an activity that is approximately 2 to 3 times, 3 to 4 times, 4 to 5 times, 5 to 6 times, 6 to 7 times, 7 to 8 times, 8 to 9 times, or 9 to 10 times greater than the activity of any one of the CCL20 promoters among SEQ ID NOs. 1, 2, 3, 4, or 5 under the same experimental conditions.

[0122] In some embodiments, the CCL20 promoter of the present disclosure has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0123] In some embodiments, the CCL20 promoter of this disclosure has approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0124] In some embodiments, the CCL20 promoter of this disclosure has approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, and approximately 95% to approximately 100% sequence identity to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 promoter has the ability to induce the expression of heterologous proteins in response to stimulation by cytokines, such as IL-1α.

[0125] In some embodiments, the CCL20 promoter is a functional fragment of the CCL20 promoter having a sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 functional fragment promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0126] In some embodiments, the CCL20 promoter is a functional fragment of the CCL20 promoter having a sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the CCL20 functional fragment promoter comprises a cleavage of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 nucleotides from 5' of the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the CCL20 functional fragment promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0127] In some embodiments, the CCL20 promoter is a functional fragment of the CCL20 promoter having a sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the CCL20 functional fragment promoter comprises a cleavage of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, or about 600 nucleotides from 5' of the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the CCL20 functional fragment promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0128] In some embodiments, the CCL20 promoter is a functional fragment of the CCL20 promoter having a sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the CCL20 functional fragment promoter includes a cleavage of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 nucleotides from 3' of the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the CCL20 functional fragment promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0129] In some embodiments, the CCL20 promoter is a functional fragment of the CCL20 promoter having a sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, wherein the CCL20 functional fragment promoter comprises a cleavage of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, or 600 nucleotides from 3' of the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 functional fragment promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0130] In some embodiments, the CCL20 promoter is a functional fragment of the CCL20 promoter having the sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 functional fragment promoter is derived from at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 of the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5. The CCL20 functional fragment promoter comprises internal cleavage of at least approximately 60, at least approximately 65, at least approximately 70, at least approximately 75, at least approximately 80, at least approximately 85, at least approximately 90, at least approximately 95, at least approximately 100, at least approximately 200, at least approximately 300, at least approximately 400, at least approximately 500, or at least approximately 600 nucleotides (i.e., cleavage that does not include the 5' or 3' ends of the sequence), and has the ability to induce the expression of heterologous proteins in response to stimulation by cytokines, such as IL-1α.

[0131] In some embodiments, the CCL20 promoter is a functional fragment of the CCL20 promoter having a sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, the CCL20 functional fragment promoter comprises an internal cleavage of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, or about 600 nucleotides from the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5 (i.e., a cleavage that does not include the 5' or 3' end of the sequence), the CCL20 functional fragment promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0132] In some embodiments, the CCL20 promoter is a functional variant of the CCL20 promoter having the sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 functional variant promoter is a variant having the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α. As used herein, the term variant means a CCL20 functional variant promoter having one or more nucleotide substitutions, insertions, or deletions to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, and the CCL20 functional variant promoter has the ability to induce the expression of a heterologous protein in response to stimulation by a cytokine, such as IL-1α.

[0133] In some embodiments, the CCL20 functional variant promoter includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 substitutions. In some embodiments, the CCL20 functional variant promoter includes at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 substitutions. In some embodiments, the CCL20 functional variant promoter includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions. In some embodiments, the CCL20 functional variant promoter includes about 20, about 25, about 30, about 35, about 40, about 45, or about 50 substitutions.

[0134] In some embodiments, the CCL20 functional variant promoter includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and at least 20 or more deletions. In some embodiments, the CCL20 functional variant promoter includes at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 deletions. In some embodiments, the CCL20 functional variant promoter includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deletions. In some embodiments, the CCL20 functional variant promoter includes approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, or approximately 50 deletions.

[0135] In some embodiments, the CCL20 functional variant promoter includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and at least 20 or more insertions. In some embodiments, the CCL20 functional variant promoter includes at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 insertions. In some embodiments, the CCL20 functional variant promoter includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 insertions. In some embodiments, the CCL20 functional variant promoter includes approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, or approximately 50 insertions.

[0136] In some embodiments, a mutation may include multiple nucleotides within the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, for example, by inserting, deleting, or substituting a subsequence containing two or more nucleotides. In some embodiments, a mutation may include a single nucleotide change within the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5, i.e., the mutation is a point mutation. In some embodiments, the CCL20 promoter sequence of this disclosure has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more point mutations with respect to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5. In some embodiments, the CCL20 promoter sequence of this disclosure has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 point mutations in the CCL20 promoter sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5. In some embodiments, the point mutations are conservative point mutations. In some embodiments, the point mutations are non-conservative point mutations. In some embodiments, the point mutations include both conservative and non-conservative point mutations.

[0137] In some embodiments, the CCL20 promoter of the present disclosure comprises at least about 50 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 350 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, or at least about 900 nucleotides from the sequence described in Sequence ID No. 1, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0138] In some embodiments, the CCL20 promoter of this disclosure comprises at least about 50 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 350 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, or at least about 900 nucleotides from the sequence described in Sequence ID No. 2, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0139] In some embodiments, the CCL20 promoter of this disclosure comprises at least about 50 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 350 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, or at least about 900 nucleotides from the sequence described in Sequence ID No. 3, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0140] In some embodiments, the CCL20 promoter of this disclosure comprises at least about 50 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 350 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, or at least about 900 nucleotides from the sequence described in Sequence ID No. 4, wherein the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0141] In some embodiments, the CCL20 promoter of this disclosure comprises at least about 50 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 350 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, or at least about 900 nucleotides from the sequence described in Sequence ID No. 5, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0142] In some embodiments, the CCL20 promoter of this disclosure comprises approximately 50 nucleotides, approximately 100 nucleotides, approximately 150 nucleotides, approximately 200 nucleotides, approximately 250 nucleotides, approximately 300 nucleotides, approximately 350 nucleotides, approximately 400 nucleotides, approximately 450 nucleotides, approximately 500 nucleotides, approximately 550 nucleotides, approximately 600 nucleotides, approximately 650 nucleotides, approximately 700 nucleotides, approximately 750 nucleotides, approximately 800 nucleotides, approximately 850 nucleotides, or approximately 900 nucleotides from the sequence described in Sequence ID No. 1, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0143] In some embodiments, the CCL20 promoter of this disclosure comprises approximately 50 nucleotides, approximately 100 nucleotides, approximately 150 nucleotides, approximately 200 nucleotides, approximately 250 nucleotides, approximately 300 nucleotides, approximately 350 nucleotides, approximately 400 nucleotides, approximately 450 nucleotides, approximately 500 nucleotides, approximately 550 nucleotides, approximately 600 nucleotides, approximately 650 nucleotides, approximately 700 nucleotides, approximately 750 nucleotides, approximately 800 nucleotides, approximately 850 nucleotides, or approximately 900 nucleotides from the sequence described in Sequence ID No. 2, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0144] In some embodiments, the CCL20 promoter of this disclosure comprises approximately 50 nucleotides, approximately 100 nucleotides, approximately 150 nucleotides, approximately 200 nucleotides, approximately 250 nucleotides, approximately 300 nucleotides, approximately 350 nucleotides, approximately 400 nucleotides, approximately 450 nucleotides, approximately 500 nucleotides, approximately 550 nucleotides, approximately 600 nucleotides, approximately 650 nucleotides, approximately 700 nucleotides, approximately 750 nucleotides, approximately 800 nucleotides, approximately 850 nucleotides, or approximately 900 nucleotides from the sequence described in Sequence ID No. 3, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0145] In some embodiments, the CCL20 promoter of this disclosure comprises approximately 50 nucleotides, approximately 100 nucleotides, approximately 150 nucleotides, approximately 200 nucleotides, approximately 250 nucleotides, approximately 300 nucleotides, approximately 350 nucleotides, approximately 400 nucleotides, approximately 450 nucleotides, approximately 500 nucleotides, approximately 550 nucleotides, approximately 600 nucleotides, approximately 650 nucleotides, approximately 700 nucleotides, approximately 750 nucleotides, approximately 800 nucleotides, approximately 850 nucleotides, or approximately 900 nucleotides from the sequence described in Sequence ID No. 4, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0146] In some embodiments, the CCL20 promoter of this disclosure comprises approximately 50 nucleotides, approximately 100 nucleotides, approximately 150 nucleotides, approximately 200 nucleotides, approximately 250 nucleotides, approximately 300 nucleotides, approximately 350 nucleotides, approximately 400 nucleotides, approximately 450 nucleotides, approximately 500 nucleotides, approximately 550 nucleotides, approximately 600 nucleotides, approximately 650 nucleotides, approximately 700 nucleotides, approximately 750 nucleotides, approximately 800 nucleotides, approximately 850 nucleotides, or approximately 900 nucleotides from the sequence described in Sequence ID No. 5, and the CCL20 promoter can induce the expression of a protein or nucleic acid encoded by a heterologous gene operably linked to the promoter in response to stimulation by a cytokine, such as IL-1α.

[0147] In some embodiments, the CCL20 promoter of this disclosure extends from the sequence described in any one of SEQ ID NOs: 1, 2, 3, 4, or 5 to approximately 50 to 100 nucleotides, approximately 100 to 150 nucleotides, approximately 150 to 200 nucleotides, approximately 200 to 250 nucleotides, approximately 250 to 300 nucleotides, approximately 300 to 350 nucleotides, approximately 350 to 400 nucleotides, and approximately 400 to 450 nucleotides. Approximately 450 nucleotides to approximately 500 nucleotides, approximately 500 nucleotides to approximately 550 nucleotides, approximately 550 nucleotides to approximately 600 nucleotides, approximately 600 nucleotides to approximately 650 nucleotides, approximately 650 nucleotides to approximately 700 nucleotides, approximately 700 nucleotides to approximately 750 nucleotides, approximately 750 nucleotides to approximately 800 nucleotides, approximately 800 nucleotides to approximately 850 nucleotides, approximately 850 nucleotides to approximately 900 nucleotides, approximately 100 nucleotides to approximately 200 nucleotides, approximately 200 nucleotides to approximately 30 0 nucleotides, approximately 300-400 nucleotides, approximately 400-500 nucleotides, approximately 500-600 nucleotides, approximately 600-700 nucleotides, approximately 700-800 nucleotides, approximately 800-900 nucleotides, approximately 100-300 nucleotides, approximately 200-400 nucleotides, approximately 300-500 nucleotides, approximately 400-600 nucleotides, approximately 500 nucleos The CCL20 promoter contains approximately 700 nucleotides, 600 to 800 nucleotides, 700 to 900 nucleotides, 100 to 400 nucleotides, 200 to 500 nucleotides, 300 to 600 nucleotides, 400 to 700 nucleotides, 500 to 800 nucleotides, or 600 to 900 nucleotides, and responds to stimulation by cytokines, such as IL-1α.It is possible to induce the expression of proteins or nucleic acids encoded by heterologous genes operablely linked to a promoter.

[0148] In some embodiments, the CCL20 promoter of this disclosure is approximately 100, approximately 125, approximately 150, approximately 175, approximately 200, approximately 225, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 425, approximately 450, approximately 475, approximately 500, approximately 525, approximately 550, approximately 575, approximately 600, approximately 625, approximately 650, approximately 675, approximately 700, approximately 725, approximately 750, approximately 775, approximately 800, approximately 825, approximately 850, approximately 875, or approximately 900 nucleotides in length. In some embodiments, the CCL20 promoter of the present disclosure has a length of at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 325, at least about 350, at least about 375, at least about 400, at least about 425, at least about 450, at least about 475, at least about 500, at least about 525, at least about 550, at least about 575, at least about 600, at least about 625, at least about 650, at least about 675, at least about 700, at least about 725, at least about 750, at least about 775, at least about 800, at least about 825, at least about 850, at least about 875, or at least about 900 nucleotides.

[0149] In some embodiments, heterogenes encode nucleic acids. In some embodiments, the nucleic acids are endogenous nucleic acids, such as miRNAs. In some embodiments, the nucleic acids are exogenous nucleic acids, such as RNA interferants, such as antisense oligonucleotides. In some embodiments, heterogenes encode proteins, such as therapeutic proteins. In some embodiments, the therapeutic proteins are, for example, chimeric antigen receptors (CARs), antibodies, tumor suppressors, apoptosis inducers, enzymes, hormones, or any combination thereof.

[0150] In some embodiments, the therapeutic protein includes an anti-inflammatory antibody or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits TNF-α. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-6. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-12. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-23. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits both IL-12 and IL-23. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-2. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits immunoglobulin E (IgE). In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits the interleukin-6 receptor (IL-6R). In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-17A. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-5. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-4RA. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-1B. In some embodiments, the anti-inflammatory antibody or its antigen-binding moiety inhibits IL-17RA.

[0151] In some embodiments, the anti-inflammatory antibody comprises infliximab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises adalimumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises ustekinumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises basiliximab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises daclizumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises omalizumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises tildrakizumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises guselkumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises sarilumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises tocilizumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises siltuximab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises secukinumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises reslizumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises mepolizumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises dupilumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises celtilizumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises canakinumab or its antigen-binding moiety. In some embodiments, the anti-inflammatory antibody comprises brodalumab or its antigen-binding moiety.

[0152] In some embodiments, the heterogene encodes a pro-inflammatory cytokine antagonist. In some embodiments, the heterogene encodes an interleukin. In some embodiments, the heterogene encodes an anti-inflammatory interleukin. In some embodiments, the heterogene encodes an inflammatory interleukin antagonist. In some embodiments, the heterogene encodes an IL-1 receptor antagonist (IL-1RA). In some embodiments, the heterogene encodes an interleukin 36 receptor antagonist (IL-36RA). In some embodiments, the heterogene encodes interleukin 37 (IL-37). In some embodiments, the heterogene encodes IL-4. In some embodiments, the heterogene encodes IL-6. In some embodiments, the heterogene encodes IL-10. In some embodiments, the heterogene encodes IL-11. In some embodiments, the heterogene encodes IL-13. In some embodiments, the heterogene encodes a specific cytokine receptor for IL-1. In some embodiments, the heterogene encodes a specific receptor for TNF-α. In some embodiments, the heterogene encodes a specific receptor for IL-18. In some embodiments, the heterogene encodes an anti-inflammatory protein.

[0153] In some embodiments, heterologous genes encode proteins or nucleic acids that can block or inhibit the expression or activity of pro-inflammatory genes (i.e., genes that promote inflammation when expressed) or their protein products, such as IL-1, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-17, IL-21, IL-22, IL-23, IL-27, IFN, CCL2, CCL3, CCL5, CCL20, CXCL5, CXCL10, CXCL12, CXCL13, or TNF-α.

[0154] In some embodiments, heterogenes encode proteins or nucleic acids that play a regulatory role in inflammatory pathways. In some embodiments, heterogenes encode regulatory proteins within inflammatory pathways. In some embodiments, heterogenes encode regulatory proteins or polypeptides that prevent the activation of pro-inflammatory pathways. In some embodiments, heterogenes encode inhibitors of inflammatory mediators. Therefore, in some embodiments, heterogenes can encode regulators or inhibitors of proteins known to be major mediators (targets) within inflammatory response pathways. Major mediator proteins that can be directly or indirectly regulated via the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or its functional variants from SEQ ID NOs: 1-5) include, for example, aggrecan, IFN-γ, LFA-1, Th1, angiopoietin-2, Mac-1, Th2, BDNF, MCP-1 / CCL2, Th9, CD23, MCP-2 / CCL8, Th17, CD30, MCP-3 / CCL7, Th22, CD40L, MIG / CXCL9, TLR 1, C-reactive protein (CRP), IKK, MIP-1α / CCL3, TLR 2, CTL, IL-1, MIP-1β / CCL4, TLR 3, E-cadherin, IL-2, MIP-2, TLR 4, EGF, IL-3, NF-κB, TLR 5, ELAM-1, IL-4, PAI-1, TLR 6, eotaxin / CCL11, IL-5, PDGF-BB, TLR 7, FGF basic, IL-6, PECAM-1g, TLR 8, G-CSF, IL-8, p-selectin, TLR 9, GDNF, IL-10, RANTES / CCL5, TLR 10, GM-CSF, IL-12, SAA, TNF-α, GRO-α, IL-13, STAT1, TNF-β, HGF, IL-15, STAT2, TGF-β1, HIF-1α, IL-16, STAT3 , TNF-RI, ICAM-1, IL-17, STAT4, TNF-RII, IFN-α, IL-18, STAT5, VCAM-1, IFN-β, IL-23, STAT6, or VEGF.

[0155] In some embodiments, the heterogene encodes a chimeric antigen receptor (CAR). In some embodiments, the heterogene encodes an enzyme, for example, an enzyme that is desirable to be expressed at levels higher than endogenous normal levels as part of enzyme replacement therapy. In some embodiments, the heterogene encodes a hormone. In some embodiments, the heterogene encodes a protein or nucleic acid that, when expressed, results in a reduction of systemic effects associated with inflammation, such as fever, leukocytosis, leukopenia, vasodilation, septic shock, swelling, pain, loss of function, redness, necrosis, or any combination thereof.

[0156] In general, to elicit the expression of any therapeutic protein or therapeutic nucleic acid within a subject, the CCL20 promoter disclosed herein (i.e., any one of the CCL20 promoters disclosed herein, or any functional fragment or functional variant of SEQ ID NOs: 1-5) can be used after administration of a vector containing the CCL20 promoter disclosed herein, which is operably ligated to a gene encoding a viral particle containing the CCL20 promoter disclosed herein, which is operably ligated to a gene encoding a viral particle containing the CCL20 promoter disclosed herein, or any one of the CCL20 promoters disclosed herein, or any functional fragment or functional variant of SEQ ID NOs: 1-5. In some embodiments, the therapeutic protein may be an antibody targeting cancer cells or a coagulation factor. In some embodiments, the heterologous gene encodes a reporter protein, such as luciferase, green fluorescent protein, red fluorescent protein, or beta-galactosidase.

[0157] In some embodiments, the disclosure provides a vector comprising a promoter region comprising a CCL20 promoter comprising the sequence described in any one of SEQ ID NOs: 1 to 5, or a functional fragment or functional variant thereof. In some embodiments, the vector comprises a heterogene operably ligated to the CCL20 promoter. In some embodiments, the vector does not comprise a heterogene operably ligated to the CCL20 promoter. In some embodiments, the CCL20 promoter is a functional fragment of the sequence described in any one of SEQ ID NOs: 1 to 5. In some embodiments, the functional fragment has a 5' cut, a 3' cut, or both with respect to the CCL20 promoter sequence described in any one of SEQ ID NOs: 1 to 5. In some embodiments, the CCL20 promoter is a functional variant having a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequence described in any one of SEQ ID NOs: 1 to 5. In some embodiments, the functional variant sequence of the CCL20 promoter has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations in the sequence described in any one of sequence numbers 1-5. In some embodiments, the point mutations are conservative point mutations.

[0158] This disclosure also: (a) A polynucleotide comprising a promoter region comprising any one of the promoters of Sequence IDs 1 to 5 or a functional fragment or functional variant thereof, operably linked to a heterogene (e.g., encoding a therapeutic protein or therapeutic nucleic acid), wherein the CCL20 promoter induces the expression of a heterogene in response to stimulation by a cytokine, e.g., IL-α, (b) The composition also includes a delivery vehicle for delivering polynucleotides to cells within a subject.

[0159] In some embodiments, the delivery vehicle comprises nucleic acids, plasmids, vectors (e.g., viral vectors), prokaryotic cells, eukaryotic cells, lipids, or any combination thereof. In some embodiments, the delivery vehicle comprises lipids in lipid vesicles (e.g., micelles), liposomes (e.g., small monolayer vesicles), lipid particles (e.g., lipid nanoparticles), or extracellular vesicles (e.g., exosomes). In some embodiments, the delivery vehicle comprises viral particles. In some embodiments, the delivery vehicle comprises a viral vector selected from the group consisting of adeno-associated viruses, adenoviruses, retroviruses, orthomyxoviruses, paramyxoviruses, papovaviruses, picornaviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, poxviruses, and alphaviruses. In some embodiments, the delivery vehicle comprises prokaryotic cells. In some embodiments, the delivery vehicle comprises eukaryotic cells.

[0160] The disclosure also provides a method for producing a cell population expressing at least one heterogene under the control of the CCL20 promoter of the disclosure (i.e., one of the CCL20 promoters

[0161] In some embodiments, the cell population is prepared for administration to a target that requires it. In other embodiments, the cell population is prepared for recombinant protein production in cell culture. In some embodiments, the cells are human cells. In some embodiments, the cells are human or non-human cell lines suitable for protein production in cell culture, e.g., CHO cells.

[0162] The Disclosure also provides cells genetically engineered to express heterologous genes, the cells comprising a polynucleotide comprising a promoter region comprising a CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5) operably linked to the heterologous gene, the CCL20 promoter inducing the expression of the heterologous gene in response to stimulation by a cytokine, such as IL-α.

[0163] In some embodiments, cells can be inserted into a device, such as an implantable device. In some embodiments, cells are microencapsulated. In some embodiments, cells are microencapsulated within alginate microcapsules. The advantage of this approach compared to conventional gene therapy is that the cells act as prosthetic devices that do not require any host genome modification.

[0164] In other embodiments, cells are implanted using alternative cell transplantation techniques, such as biocompatible hollow fibers, which may allow for easy removal of the implanted device.

[0165] This disclosure also provides pharmaceutical compositions comprising polynucleotides, compositions, or cells disclosed herein, and pharmaceutically acceptable carriers or excipients. The pharmaceutically acceptable excipients or carriers are determined not only by the particular composition being administered, but also in part by the particular method used to administer the composition.

[0166] The Disclosure also provides a method for inducing, regulating, or enhancing the expression of a heterogene within a subject, comprising (a) administering a polynucleotide, composition, cell, or pharmaceutical composition disclosed herein, containing a heterogene, to a subject under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from Sequence IDs 1 to 5 or a functional fragment or functional variant thereof), and (b) stimulating the CCL20 promoter with a cytokine, such as IL-α.

[0167] The present invention also provides a method for dose-dependent gene expression of a heterogene within a cell, comprising (a) contacting cells with a polynucleotide, composition, cell, or pharmaceutical composition disclosed herein, which includes a CCL20 promoter of the present disclosure operably linked to a heterogene (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1 to 5 or a functional fragment or functional variant thereof), and (b) contacting cells with an effective amount of cytokine, such as IL-α, which has the ability to stimulate the CCL20 promoter and induce heterogene expression in a dose-dependent manner.

[0168] The Disclosure also provides methods for controlling or inducing gene expression in a heterologous gene, including operably ligating the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or a functional fragment or functional variant thereof) to the heterologous gene, such that stimulation of the CCL20 promoter with a cytokine, e.g., IL-α, controls or induces the expression of the heterologous gene in response to the stimulation. The Disclosure also provides methods for treating diseases within a subject, including administering an effective amount of the polynucleotides, compositions, cells, or pharmaceutical compositions disclosed herein to the subject. In some embodiments, administration of a construct containing the CCL20 promoter of the Disclosure operably ligated to a heterologous gene (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or a functional fragment or functional variant thereof) includes transfecting cells from the subject or from a donor with the construct. Transfection of cells with the construct can be carried out using any molecular biology method known in the Art. In some embodiments, the construct is inserted into the cell's genome using a nuclease system, such as CRISPR / Cas9.

[0169] The Disclosure also provides a method for controlling inflammation in a subject requiring it, comprising operably linking the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5) to a heterogene, wherein stimulation of the CCL20 promoter with cytokines, e.g., IL-1α, IL-1β, TNF (TNF-α or TNF-β), IL-6, sIL-6R, IL-17, or CD30, induces the expression of the heterogene in response to the stimulation, and the heterogene reduces immune activation directly or indirectly through increased or decreased expression of another gene (e.g., a regulatory gene in the inflammatory pathway). In some embodiments, the expression of the heterogene induced by the CCL20 promoter functionally results in a reduction of systemic effects associated with inflammation. For example, fever, leukocytosis, leukopenia, vasodilation, septic shock, swelling, pain, loss of function, redness, necrosis, or any combination thereof can be reduced.

[0170] As discussed above, the cytokines used to stimulate the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or its functional fragment or functional variant among SEQ ID NOs: 1-5) can be exogenous (i.e., the source of the cytokine is external) or endogenous (i.e., the cytokine has an internal source). In some embodiments, the endogenous source of the cytokine is an ongoing inflammatory state within the subject, e.g., a chronic state. In some embodiments, the endogenous source of the cytokine is a change in the inflammatory state, e.g., a recurrence or exacerbation of a particular inflammatory state or infection. In these two cases, the inflammation would be spontaneous, i.e., not induced to trigger an increase in endogenous cytokines. In other embodiments, the inflammation is induced, i.e., there is an external cause that intentionally causes inflammation, which in turn triggers the endogenous release of inflammatory cytokines.

[0171] In some embodiments, the cytokines used to stimulate the CCL20 promoter are exogenous, for example, administered to a subject to stimulate the promoter. When the CCL20 promoter of this disclosure is used for recombinant gene expression, the cytokines used to stimulate the CCL20 promoter can be added to the culture medium.

[0172] In some embodiments, polynucleotides, compositions, cells, or methods comprising the use of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or a functional fragment or functional variant thereof) can result in improved heterogeneous gene expression compared to the expression of the same heterogene under the control of a reference promoter, such as the serum amylose 3 (SAA3) promoter, which is a promoter that can be activated by a reference promoter, e.g., IL-1 and IL-6. As a result, in some embodiments, the Disclosure provides a method for improving heterogeneous gene expression, comprising expressing such heterogenes under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or a functional fragment or functional variant thereof), wherein the expression under the control of the CCL20 promoter reduces the heterogeneous gene expression by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 60% compared to the expression observed using the reference promoter. The improvement is 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 325%, at least about 350%, at least about 375%, at least about 400%, at least about 425%, at least about 450%, at least about 475%, or at least about 500%, and the reference promoter is the SAA3 promoter.

[0173] In certain embodiments, the Disclosure provides a method for improving the expression of heterogeneous genes, which includes expressing such heterogeneous genes under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5), wherein the expression under the control of the CCL20 promoter reduces the expression of the heterogeneous gene to approximately 20%, approximately 30%, and approximately the expression observed using the reference promoter. The improvement is 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 110%, approximately 120%, approximately 130%, approximately 140%, approximately 150%, approximately 160%, approximately 170%, approximately 180%, approximately 190%, approximately 200%, approximately 225%, approximately 250%, approximately 275%, approximately 300%, approximately 325%, approximately 350%, approximately 375%, approximately 400%, approximately 425%, approximately 450%, approximately 475%, or approximately 500%, and the reference promoter is the SAA3 promoter.

[0174] In certain embodiments, the Disclosure provides a method for improving the expression of heterogeneous genes, comprising expressing such heterogeneous genes under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5), wherein expression under the control of the CCL20 promoter improves the expression of the heterogeneous gene by about 20% to about 30% compared to the expression observed using a reference promoter. Approximately 30% to 40%, approximately 40% to 50%, approximately 50% to 60%, approximately 60% to 70%, approximately 70% to 80%, approximately 80% to 90%, approximately 90% to 100%, approximately 100% to 110%, approximately 110% to 120%, approximately 120% to 130%, approximately 130% to 140%, approximately 140% to 150%, approximately 150% to 160%, approximately 160% to 170%, approximately 170% to 180%, approximately 180% to 190%, approximately 1 Improvements were observed from 90% to approximately 200%, from approximately 200% to approximately 225%, from approximately 225% to approximately 250%, from approximately 250% to approximately 275%, from approximately 275% to approximately 300%, from approximately 300% to approximately 325%, from approximately 325% to approximately 350%, from approximately 350% to approximately 375%, from approximately 375% to approximately 400%, from approximately 400% to approximately 425%, from approximately 425% to approximately 450%, from approximately 450% to approximately 475%, and from approximately 475% to approximately 500%, with the reference promoter being the SAA3 promoter.

[0175] The Disclosure also provides gene therapy vectors containing heterologous genes that increase or decrease immune activation in a subject upon expression, wherein the genes are under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5, or a functional fragment or functional variant thereof). The Disclosure also provides gene therapy cells containing heterologous genes that increase or decrease immune activation in a subject upon expression, wherein the genes are under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5, or a functional fragment or functional variant thereof). The Disclosure also provides personalized medicine methods comprising administering gene therapy vectors or cells containing heterologous genes that increase or decrease immune activation in a subject upon expression, wherein the genes are under the control of the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5, or a functional fragment or functional variant thereof).

[0176] The Disclosure also provides diagnostic sensors comprising the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1-5 or a functional fragment or functional variant thereof). As used herein, the term “diagnostic sensor” refers to any compound, material, or device that can be used to indicate the level of inflammation when exposed to an animal body sample containing inflammatory cytokines capable of stimulating the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1-5 or a functional fragment or functional variant thereof). In some embodiments, the diagnostic sensor comprises the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1-5 or a functional fragment or functional variant thereof), a reporter gene, such as a responsive linker to luciferase. In some embodiments, the presence of inflammatory cytokines such as IL-1α, IL-1β, TNF-α, TNF-β, IL-6, sIL-6R, IL-17, or CD30 in a sample triggers the expression of a reporter gene, such as luciferase, when the diagnostic sensor comes into contact with the sampler. Consequently, the disclosure also provides a method for determining the level of inflammatory cytokines or inflammation, which involves contacting a sample from a subject with the diagnostic sensor disclosed herein.

[0177] The disclosure also provides a tunable regulatory system comprising a construct containing a GAL4-VP16 synthetic transcription factor under the control of the CCL20 promoter of the disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1-5 or a functional fragment or functional variant thereof), GAL4-VP16 promotes the transcription of downstream heterologous genes when one or more UAS (upstream activation sequences), which are among its homogeneous DNA binding sites, are present. The number of UAS elements can control the amount of transcription of downstream genes. Therefore, the number of UAS elements can be varied to control the level of expression of heterologous genes located downstream of the UAS elements. In some embodiments, the tunable regulatory system of the disclosure comprises a single polynucleotide containing the CCL20 promoter of the disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1-5 or a functional fragment or functional variant thereof), operably ligated to the GAL4-VP16 synthetic transcription factor, and further downstream in the construct, one or more UAS elements operably ligated to nucleic acids encoding heterologous proteins. In some embodiments, the adjustable regulatory system of the present disclosure comprises two polynucleotides: (1) a first polynucleotide comprising the CCL20 promoter of the present disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5) operably linked to a GAL4-VP16 synthetic transcription factor; and (2) a second polynucleotide comprising one or more UAS sites operably linked to a nucleic acid encoding a heterologous protein.

[0178] In some embodiments, the disclosure provides a method for controlling the expression of a heterologous protein by using a tunable regulatory system disclosed herein, wherein the expression level of the heterologous protein correlates with the number of UAS elements in the construct. In some embodiments, the tunable regulatory system may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten UAS elements. In some embodiments, the tunable regulatory system may include one, two, three, four, five, six, seven, eight, nine, or ten UAS elements.

[0179] III. Adaptation This disclosure provides a method for treating an inflammatory disease or condition requiring such treatment, comprising administering to a subject a construct comprising the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5) operably linked to a heterogene, wherein stimulation of the CCL20 promoter with cytokines, e.g., IL-1α, IL-1β, TNF (TNF-α or TNF-β), IL-6, sIL-6R, IL-17, or CD30, induces heterogeneous gene expression in response to the stimulation. The Disclosure also provides a method for preventing or improving symptoms of a disease or condition in a subject requiring it, comprising administering to a subject a construct comprising the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters or a functional variant of SEQ ID NOs: 1-5) operably linked to a heterogene, wherein stimulation of the CCL20 promoter with cytokines, e.g., IL-1α, IL-1β, TNF (TNF-α or TNF-β), IL-6, sIL-6R, IL-17, or CD30, induces the expression of a heterogene in response to the stimulation.

[0180] The Disclosure also provides a method for preventing and / or treating a disease or disorder in a subject requiring it, comprising administering to the subject a construct comprising the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5) operably ligated to a heterologous gene, wherein stimulation of the CCL20 promoter with cytokines, e.g., IL-1α, IL-1β, TNF (TNF-α or TNF-β), IL-6, sIL-6R, IL-17, or CD30, induces the expression of a heterologous gene in response to the stimulation. In some embodiments, the treatment is preventive.

[0181] In other embodiments, a construct comprising the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or a functional fragment or functional variant thereof) operably linked to a heterogene can be administered to a subject, and the stimulation of the CCL20 promoter by cytokines, such as IL-1α, IL-1β, TNF (TNF-α or TNF-β), IL-6, sIL-6R, IL-17, or CD30, which induces the expression of a heterogene in response to the stimulation, can be used to induce an immune response. Therefore, in some embodiments, a subject can be vaccinated using a construct comprising the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs: 1-5 or a functional fragment or functional variant thereof) operably linked to a heterogene.

[0182] In some embodiments, the compositions and methods disclosed herein are for the treatment of inflammatory disorders of the central nervous system (CNS), such as encephalitis, myelitis, meningitis, or arachnoiditis; inflammatory disorders of the peripheral nervous system (PNS), such as neuritis, ocular inflammatory disorders, dacryodenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, or uveitis; ocular inflammatory disorders, such as otitis externa, otitis media, otitis interna, mastoiditis, carditis, endocarditis, myocarditis, pericarditis, vasculitis, and arteritis. Inflammatory disorders of the cardiovascular system such as phlebitis or capillary vasculitis; inflammatory disorders of the respiratory system such as sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleurisy, or mediastinitis; stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis / parotitis, cheilitis, pulpitis, mandibular inflammation, esophagitis, gastritis, gastroenteritis, enteritis, colitis, small intestine-colitis, duodenitis, ileitis, cecumitis, appendicitis, diabetes, proctitis, hepatitis, ascending cholangitis, gallbladder Inflammatory disorders of the digestive system and auxiliary digestive organs, such as inflammation, pancreatitis, or peritonitis; inflammatory disorders of the skin, such as dermatitis, folliculitis, cellulitis, or hidradenitis; inflammatory disorders of the musculoskeletal system, such as arthritis, dermatomyositis, myositis, synovitis, tenosynovitis, bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, tendinitis, panniculitis, osteochondritis, osteitis, osteomyelitis, spondylitis, periostitis, or chondritis; nephritis, glomerulonephritis, pyelonephritis, ureteritis, cystitis, or urethritis, etc. It can be used to treat inflammatory disorders of the urinary tract; inflammatory disorders of the reproductive system such as oophoritis, salpingitis, endometritis, paraconnective tissue inflammation, cervicitis, vaginitis, vulvitis, mastitis, orchitis, epididymitis, prostatitis, seminal vesicle inflammation, balanitis, balanitis, or balanoposthitis; inflammatory disorders of the endocrine system such as isletitis, hypophysitis, thyroiditis, parathyroiditis, or adrenalitis; or inflammatory disorders of the lymphatic system such as lymphangitis or lymphadenitis.

[0183] Inflammatory abnormalities are a major group of disorders underlying a wide variety of human diseases. The immune system is often involved in inflammatory disorders, as demonstrated in both allergic reactions and some myopathy, and numerous immune system disorders result in abnormal inflammation. In some embodiments, the compositions and methods disclosed herein can be used to treat non-immune diseases that have a causal origin within the inflammatory process, including cancer, atherosclerosis, and ischemic heart disease.Inflammatory disorders include acne vulgaris, rosacea, asthma, atopic eczema, allergic rhinitis, autoimmune diseases (e.g., celiac disease, irritable bowel disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, Graves' disease, psoriasis, or aplastic anemia), autoinflammatory diseases (e.g., adult-onset Still's disease, systemic juvenile idiopathic arthritis, Schnitzler syndrome, chronic relapsing multifocal osteomyelitis, familial Mediterranean fever (FMF), hyperimmune globulin D emia with relapsing fever (HIDS), TNF receptor-associated periodic syndromes (TRAPS), Mackle-Wells syndrome (urticaria-deafness amyloidosis), familial cold urticaria, neonatal onset multiorgan inflammatory disease (NOMID), periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA syndrome), Blau syndrome, suppurative aseptic arthritis, pyoderma gangrenosum, acne (PAPA), interleukin-1 receptor antagonist deficiency (DIRA), or Yao syndrome), chronic prostatitis, colitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, Behçet's disease, microscopic colitis, fecal diarrhea) (Changing colitis, or unclassifiable colitis), interstitial cystitis, lichen planus, mast cell activation syndrome, mastocytosis, otitis, pelvic inflammatory disease, marginal ulcerative keratitis, pneumonia, reperfusion injury, rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, transplant rejection, vasculitis, atherosclerosis, allergies, anaphylaxis, myopathy (e.g., systemic sclerosis, dermatomyositis, polymyositis, or inclusion body myositis), leukocyte deficiency (e.g., Chediak-Higashi syndrome or chronic granulomatosis), pharmacologically induced inflammation, vitamin A deficiency, Other conditions include HIV and AIDS, or cancer (e.g., cancers associated with chronic inflammation such as mesothelioma, lung cancer, bladder cancer, oral squamous cell carcinoma, colorectal cancer, vulvar squamous cell carcinoma, pancreatic cancer, esophageal cancer, salivary gland cancer, MALT lymphoma, or melanoma; and cancers associated with infectious agents such as cholangiosarcoma, colon cancer, gallbladder cancer, gastric adenocarcinoma, hepatocellular carcinoma, B-cell non-Hodgkin lymphoma, Burkitt lymphoma, non-Hodgkin lymphoma, squamous cell carcinoma, Kaposi's sarcoma, skin cancer, ovarian cancer, cervical cancer, anal cancer, bladder cancer, liver cancer, rectal cancer, or follicular lymphoma of the spleen).

[0184] In some embodiments, inflammation is caused by pathogens, i.e., pathogenic fungi or microorganisms such as viruses, bacteria, plia, fungi, or parasites that cause disease in humans. In some embodiments, inflammation is caused by viral diseases, such as the pathogenicity of adenoviridae, picornaviridae, herpesviridae, hepadnaviridae, coronavirusidae, flaviviridae, retroviridae, orthomyxoviridae, paramyxoviridae, papovaviridae, polyomaviruses, poxviridae, rhabdoviridae, and togaviridae. Some notable viral diseases include smallpox, influenza, mumps, measles, chickenpox, Ebola, rubella, Zika fever, yellow fever, West Nile fever, viral pneumonia, shingles, COVID-19, SARS, MERS, rotavirus infection, Rift Valley fever, respiratory syncytial virus infection, rabies, poliomyelitis, norovirus infection, Epstein-Barr virus mononucleosis (mono), herpes, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hantavirus pulmonary syndrome, Ebola, dengue fever, the common cold, AIDS, or adenovirus infection.In some cases, inflammation is caused by bacterial diseases, such as anthrax (infection with Bacillus anthracis), bacterial meningitis, bacterial pneumonia, bubonic plague (infection with Yersinia pestis), botulism (infection with Clostridium botulinum), cholera (infection with Vibrio cholerae), diphtheria (infection with Corynebacterium diphtheriae), typhus (infection with Rickettsia prowazekii), glanders (infection with Burkholderia mallei), meningitis (infection with Neisseria meningitidis), and pertussis (wheezing cough) (with Bordetella pertussis). It can be caused by pertussis (infection), salmonellosis, scarlet fever (Group A Streptococcus infection), sepsis, dysentery (bacterial dysentery) (Shigella infection), toxic shock syndrome (TSS) (Staphylococcus aureus or Streptococcus pyogenes infection), tuberculosis (Mycobacterium tuberculosis infection), or typhoid fever (infection by Salmonella enterica subsp. enterica or Salmonella serovar typhi).

[0185] In some embodiments, the disease or disorder that can be treated using the compositions or methods disclosed herein is cancer. The process involves administering a construct to a subject comprising the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters or a functional fragment or functional variant of SEQ ID NOs: 1-5) operably linked to a heterogene, wherein stimulation of the CCL20 promoter by cytokines, e.g., IL-1α, IL-1β, TNF (TNF-α or TNF-β), IL-6, sIL-6R, IL-17, or CD30, induces the expression of the heterogene in response to the stimulation. When administered to a subject with cancer, in certain embodiments, the expression of the heterogene product can upregulate the immune response and enhance the tumor targeting of the subject's immune system. In other words, in some cases, the constructs disclosed herein may be used to reduce inflammation associated with cancer, while in other cases, the constructs disclosed herein may be used to increase inflammation to improve tumor targeting. In the first scenario, the presence of endogenous inflammatory cytokines would, for example, induce the expression of heterologous genes encoding anti-inflammatory cytokines. In the second scenario, the presence of low levels of endogenous inflammatory cytokines may be amplified by many of the constructs of this disclosure, and stimulation of the CCL20 promoter by endogenous inflammatory cytokines would induce the expression of pro-inflammatory cytokines.

[0186] IV. Kits and manufactured products The Disclosure also provides kits and products comprising polynucleotides, for example, vectors, that encode the polynucleotides disclosed herein, such as the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or its functional fragment or functional variant).

[0187] In some embodiments, the Disclosure provides a kit or product comprising (i) a CCL20 promoter comprising the sequence or a functional fragment or functional variant described in any one of SEQ ID NOs: 1 to 5, (ii) a vector comprising a CCL20 promoter comprising the sequence or a functional fragment or functional variant described in any one of SEQ ID NOs: 1 to 5, or (iii) a diagnostic construct or sensor comprising a CCL20 promoter comprising the sequence or a functional fragment or functional variant described in any one of SEQ ID NOs: 1 to 5, operably ligated to a reporter gene, and instructions for use.

[0188] In some embodiments, a CCL20 promoter containing the sequence described in any one of SEQ ID NOs: 1-5, or a functional fragment or functional variant thereof, is operably ligated to a heterogene. In some embodiments, a vector containing a CCL20 promoter containing the sequence described in any one of SEQ ID NOs: 1-5, or a functional fragment or functional variant thereof, contains a heterogene operably ligated to the CCL20 promoter. In some embodiments, a diagnostic construct or sensor comprising a CCL20 promoter contains the sequence described in any one of SEQ ID NOs: 1-5, or a functional fragment or functional variant thereof, operably ligated to a reporter gene, and also contains a further heterogene (e.g., encoding a therapeutic protein or therapeutic nucleotide) operably ligated to CCL20 (e.g., in a bicistronic construct).

[0189] In some embodiments, the CCL20 promoter, or a functional fragment or functional variant thereof, containing the sequence described in any one of SEQ ID NOs: 1-5, is not operably ligated to a heterogene. In some embodiments, a vector containing the CCL20 promoter, containing the sequence described in any one of SEQ ID NOs: 1-5, or a functional fragment or functional variant thereof, does not contain a heterogene operably ligated to the CCL20 promoter. In some embodiments, a diagnostic construct or sensor comprising the CCL20 promoter contains the sequence described in any one of SEQ ID NOs: 1-5, or a functional fragment or functional variant thereof, operably ligated to a reporter gene, but this also does not contain any further heterogene (e.g., encoding a therapeutic protein or therapeutic nucleotide) operably ligated to the CCL20 promoter.

[0190] In certain embodiments, the Disclosure provides a kit or article preparation comprising (i) a polynucleotide disclosed herein, for example, a polynucleotide encoding the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or a functional fragment or functional variant thereof), for example, a vector containing such promoter; (ii) optionally, a solvent and / or other reagents; and (iii) instructions describing a method for generating a construct containing a heterogene under the control of the CCL20 promoter of the Disclosure.

[0191] In some embodiments, the Disclosure provides a diagnostic sensor of the Disclosure and, optionally, a kit or article comprising instructions for use. In some embodiments, the diagnostic sensor comprises the CCL20 promoter of the Disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or a functional fragment or functional variant thereof), a reporter gene, for example, a responsive linker to luciferase.

[0192] In some embodiments, the Disclosure provides the production of a tunable expression system of the Disclosure and, optionally, a kit or article including instructions for use. In some embodiments, the tunable regulatory system comprises a single polynucleotide comprising the CCL20 promoter of the Disclosure (i.e., one of the CCL20 promoters or a functional fragment or functional variant of any of SEQ ID NOs: 1-5) operably linked to a GAL4-VP16 synthetic transcription factor, and one or more UAS elements further downstream in the construct operably linked to a nucleic acid encoding a heterologous protein. In some embodiments, the tunable regulatory system comprises two polynucleotides: (1) a first polynucleotide comprising the CCL20 promoter of the Disclosure (i.e., one of the CCL20 promoters or a functional fragment or functional variant of any of SEQ ID NOs: 1-5) operably linked to a GAL4-VP16 synthetic transcription factor, and (2) a second polynucleotide comprising one or more UAS sites operably linked to a nucleic acid encoding a heterologous protein.

[0193] Such kits and products may include containers each having one or more of the various components used in the manner disclosed herein, such as polynucleotides disclosed herein, for example, polynucleotides encoding the CCL20 promoter of this disclosure (i.e., any one of the CCL20 promoters from SEQ ID NOs. 1 to 5 or a functional fragment or functional variant thereof), for example, vectors containing such promoters. In some embodiments, the kit includes printed instructions. Therefore, kits provided in accordance with this disclosure may also include a brochure or instructions. Instructions included in the kit may be affixed to the packaging material or included as accompanying documentation. Instructions are typically written or printed materials, but are not limited thereto. Any medium capable of storing such instructions and distributing them to end users is intended. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), and similar media. As used herein, the term “instructions” may include the address of an internet site providing the instructions.

[0194] The following examples are provided for illustrative purposes only and not for limiting purposes. [Examples]

[0195] Example 1 Increased expression of genes (operably ligated to the CCL20 promoter) under the control of the CCL20 promoter in response to inflammation.

[0196] Synthetic constructs encoding the firefly luciferase gene without any promoter sequence (designated as promoterless FF-Luc or promoterless control) were also obtained (Promega pGL4.10[luc2]). Synthetic constructs encoding the firefly luciferase gene (operably ligated) under the control of the CCL20 promoter of Sequence ID No. 1 (designated as CCL20-FF-Luc or CCL20-Luc) were generated from pGL4.10[luc2]. A third synthetic construct encoding firefly luciferase (operably ligated) under the control of the SAA3 promoter (designated as SAA3-FF-Luc) was also generated from pGL4.10[luc2]. In addition, a synthetic control construct encoding firefly luciferase (operably ligated) under the control of the SV40 constitutive activity promoter (designated as SV40-FF-Luc) was also obtained (Promega pGL4.35[luc2 / SV40]).

[0197] Four groups of HeLa cells were transfected with Lipofectamine 3000 (Invitrogen) using the CCL20-FF-Luc construct, promoterless-FF-Luc construct, SAA3-FF-Luc construct, or SV40-FF-Luc construct, and the construct pGL4.75[hRluc / CMV] to normalize transfection dispersion. To mimic an inflammatory state, the inflammatory cytokine interleukin-1-alpha (IL-1α) was added to each cell culture at a concentration of 5 ng / ml, and luciferase expression (i.e., luciferase reporter assay) was measured using a luciferase reporter assay at 0.5, 2, 4, and 24 hours after IL-1α stimulation. The expression of firefly luciferase and sea urchin luciferase was determined using the Promega Dual-Glo luciferase assay system and read on a plate reader capable of measuring luminescence.

[0198] Figures 1A and 1B show diagrams describing the polyinduction of gene expression induced by each promoter (CCL20, SAA3, or SV40), or promoter-less expression, as measured by luciferase expression. Luciferase expression levels increased 2 and 4 hours after IL-1α stimulation in cells transfected with the CCL20-FF-Luc and SAA3-FF-Luc constructs, whereas luciferase levels remained unchanged at all time points in cells transfected with the promoter-less FF-Luc construct (Figure 1A).

[0199] At 4 hours after IL-1α stimulation, the CCL20 promoter produced 117-fold higher levels of luciferase than in the absence of IL-1α, while the SAA3 promoter produced 27-fold higher levels of luciferase than in the absence of IL-1α. Promoter-less constructs did not produce any difference between the presence or absence of IL-1α. The SV40 promoter produced high levels of luciferase expression in both the presence and absence of IL-1α (Figure 1B). The results of the above luciferase reporter assay indicate that the CCL20 promoter is more efficient than the SAA3 promoter in inducing downstream gene expression in the presence of IL-1α (a drug that mimics an inflammatory state). The results also show that the CCL20 promoter can effectively induce gene expression in response to stimulation by inflammatory cytokines.

[0200] To further investigate the CCL20 promoter's ability to regulate specific gene expression levels in response to inflammatory signals (in this case, IL-1α stimulation), we generated a synthetic fusion construct (CCL20-1RA) encoding the interleukin-1 receptor antagonist (IL-1RA) under the control (operably linked) of the CCL20 promoter. We also generated a second synthetic construct (CMV-IL-1RA) encoding IL-1RA under the control of the constitutively active CMV promoter.

[0201] HeLa cells were transfected with Lipofectamine 3000 (Invitrogen) using the CCL20-IL-1RA, CMV-IL-1RA, or CCL20-FF-Luc construct, and then stimulated with 5 ng / ml IL-1α. IL-1RA expression levels were measured from 0 to 25 hours after IL-1α stimulation. Cells transfected with the CCL20-FF-Luc construct, a control construct that does not encode IL-1RA, showed no IL-1RA expression. Cells transfected with CMV-IL-1RA (IL-1RA under the control of the constitutively active CMV promoter) showed a constant high level of IL-1RA expression and did not show any change in IL-1RA expression with IL-1α stimulation. Cells transfected with the CCL20-IL-1RA construct showed an increase in IL-1RA expression levels with IL-1α stimulation (Figure 2). These results demonstrate that the CCL20 promoter has the ability to modulate IL-1RA expression in response to inflammatory signals (in this case, IL-1α).

[0202] Example 2 Decreased levels of pro-inflammatory cytokine expression under the control of the CCL20 promoter and in the presence of pro-inflammatory cytokines under exposure to inflammatory signals.

[0203] To address whether IL-1RA expression under the control of the CCL20 promoter has the ability to reduce the expression levels of pro-inflammatory cytokines, and whether this reduction exhibits any dose-dependent relationship with respect to the amount of induced cytokine used, HeLa cells were transfected with Lipofectamine 3000 (Invitrogen) with CCL20-IL-1RA, CMV-IL-1RA, or CCL20-FF-Luc constructs, stimulated with 5 ng / ml IL-1, and the levels of IL-8, IL-6, and MCP-1 were measured in the extracellular environment using the Biolegend Legendplex Human Inflammation Panel 1 for 0–25 hours of IL-1α stimulation.

[0204] IL-8, IL-6, and MCP-1 expression levels remained high upon IL-1α stimulation in cells transfected with CCL20-FF-Luc, a construct that does not encode IL-1RA (Figures 3A-3C). IL-8, IL-6, and MCP-1 expression levels remained low (did not increase) in response to IL-1α stimulation in cells transfected with the CMV-IL-1RA construct, i.e., cells constitutively expressing high levels of IL-1RA under the control of a constitutively active CMV promoter (Figures 3A-3C). IL-8, IL-6, and MCP-1 expression levels also remained low (did not increase) in response to IL-1α stimulation in cells transfected with the CCL20-IL-1RA construct, demonstrating the ability of the CCL20 promoter to produce high gene expression levels of IL-1RA upon IL-1α stimulation (Figures 3A-3C), which can then inhibit downstream inflammatory signaling.

[0205] These results demonstrated that immune activation can be reduced by expressing pro-inflammatory cytokine antagonists under the control of the CCL20 promoter. Furthermore, this regulation was equivalent to producing high levels of pro-inflammatory cytokine antagonists present at least at the initiation of pro-inflammatory signaling (IL-1α stimulation).

[0206] Example 3 IL-10 expression in response to IL-1α stimulation via the CCL20 promoter

[0207] HeLa cells were transfected with the CCL20-IL10 construct using Lipofectamine 3000 (Invitrogen) and then stimulated with 5 ng / ml IL-1α. IL-10 expression levels were measured 24 hours after IL-1α stimulation. Unstimulated cells produced only low levels of IL-10 immunomodulation, while stimulation led to high levels of IL-10 stimulation as measured by IL-10 ELISA. See Figure 4.

[0208] Example 4 Adjustable second level control using the CCL20 promoter

[0209] Under the control of the CCL20 promoter, constructs were created using the synthetic transcription factor GAL4-VP16. GAL4-VP16 promotes downstream gene transcription when its allogeneic DNA binding sites (UASs) are present. The number of UAS elements can control the amount of downstream gene transcription. A construct containing nine UAS sites (Promega pGL4.35[luc2P / 9XGAL4UAS / Hygro]) was obtained and used to create constructs with four UAS sites and constructs with two UAS sites. Each UAS construct was transfected into HeLa cells in cooperation with the CCL20-GAL4-VP16 construct and the CMV-Cyclops luciferase construct. Next, cells were stimulated for 4 hours with either 0, 0.05, 0.1, 0.5, 1, 5, or 10 ng / ml of IL-1α, and luciferase levels were determined by the Dual Glo luciferase assay system, normalized to sea urchin luciferase to account for any transfection variability, and then the multiplicity in luciferase expression was determined compared to the 0 ng / ml stimulation level. The constructs exhibited a dose-dependent response compared to 1) the concentration of IL-1α stimulation and 2) the number of UAS elements present in the reporter construct. See Figure 5.

[0210] It should be understood that the section on embodiments for carrying out the invention is intended to be used for interpreting the claims, rather than the section on the summary and abstract of the invention. The section on the summary and abstract of the invention may describe one or more exemplary embodiments of the invention as contemplated by the inventor, but not all, and is therefore not intended to limit the invention and the appended claims in any way.

[0211] The present invention has been described above with the help of functional configuration blocks illustrating the implementation of the specified functions and their relationships. The boundaries of these functional configuration blocks are arbitrarily defined herein for the sake of convenience of description. Alternative boundaries can be defined, provided that the specified functions and their relationships are adequately implemented.

[0212] The foregoing descriptions of specific embodiments so fully reveal the general nature of the invention that others can readily modify and / or adapt such specific embodiments for various uses without departing from the general concept of the invention, without requiring any experimentation beyond what is necessary, by applying knowledge within the scope of the art of the art. Therefore, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. It is understood that the language or terminology used herein is for descriptive purposes and not for restrictive purposes, and thereby the language or terminology used herein should be interpreted by those skilled in the art in light of the teachings and guidance.

[0213] The breadth and scope of the present invention should not be limited by any of the exemplary embodiments described above, but rather should be defined solely by the following claims and their equivalents.

[0214] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Similar or equivalent methods and materials may be used in the practice or testing of the present invention, but preferred methods and materials are described herein.

[0215] All publications, patent applications, patents, and other reference materials described herein are incorporated in their entirety by reference. Database entries and electronic publications disclosed in this disclosure are incorporated in their entirety by reference. The versions of database entries or electronic publications incorporated by reference in this application are the most recent versions of the database entries or electronic publications that were publicly available at the time this application was filed. Database entries corresponding to gene or protein identifiers disclosed in this application (e.g., genes or proteins identified by accession numbers or database identifiers in public databases such as Genbank, Refseq, or Uniprot) are incorporated in their entirety by reference. The incorporated gene or protein-related information is not limited to the sequence data contained within the database entry. The incorporated information includes the entire contents of the database entry in the most recent version of the database that was publicly available at the time this application was filed. In case of any inconsistency, this specification, including definitions, shall prevail. In addition, materials, methods, and examples are illustrative and not intended to limit the scope of this application.

Claims

1. A polynucleotide comprising a promoter region containing a C-C motif chemokine ligand 20 (CCL20) promoter operably linked to a heterologous gene, wherein the CCL20 promoter induces the expression of the heterologous gene in response to cytokine stimulation.

2. The polynucleotide according to claim 1, wherein the CCL20 promoter includes a sequence described in any one of sequence numbers 1 to 5.

3. The polynucleotide according to claim 1, wherein the CCL20 promoter is a functional fragment or functional variant of the sequence described in any one of SEQ ID NOs: 1 to 5.

4. The polynucleotide according to claim 3, wherein the CCL20 promoter is a functional fragment.

5. The polynucleotide according to claim 4, wherein the functional fragment has a 5' cleavage, a 3' cleavage, or both of the CCL20 promoter sequence described in any one of SEQ ID NOs: 1 to 5.

6. The polynucleotide according to claim 3, wherein the CCL20 promoter is a functional variant having a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequence described in any one of sequence numbers 1 to 5.

7. The polynucleotide according to claim 3, wherein the sequence of the functional variant of the CCL20 promoter has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations.

8. The polynucleotide according to claim 7, wherein the point mutation is a conservative point mutation.

9. The polynucleotide according to claim 1, wherein the heterogene encodes a protein.

10. The polynucleotide according to claim 9, wherein the protein is a therapeutic protein.

11. The polynucleotide according to claim 10, wherein the therapeutic protein is a chimeric antigen receptor (CAR), an antibody, a tumor suppressor, an apoptosis inducer, an enzyme, or a hormone.

12. A composition comprising: (a) a polynucleotide comprising a promoter region comprising a CCL20 promoter operably linked to a heterogene, wherein the CCL20 promoter induces the expression of the heterogene in response to cytokine stimulation; and (b) a delivery vehicle for delivering the polynucleotide to a target cell.

13. The composition according to claim 12, wherein the delivery vehicle is a nucleic acid, plasmid, vector, prokaryotic cell, eukaryotic cell, or lipid.

14. The composition according to claim 13, wherein the lipid is contained in the lipid vesicle.

15. The composition according to claim 13, wherein the lipid vesicle is a micelle, liposome, lipid nanoparticle, or extracellular vesicle.

16. The composition according to claim 13, wherein the vector is a viral vector selected from the group consisting of adeno-associated virus, adenovirus, retrovirus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, and alphavirus.

17. A vector comprising a promoter region containing the CCL20 promoter, wherein the vector comprises the sequence described in any one of sequence numbers 1 to 5, or a functional fragment or functional variant thereof.

18. The vector according to claim 17, wherein the CCL20 promoter is a functional fragment.

19. The vector according to claim 18, wherein the functional fragment has a 5' cut, a 3' cut, or both of the CCL20 promoter sequence described in any one of sequence numbers 1 to 5.

20. The vector according to claim 17, wherein the CCL20 promoter is a functional variant having a sequence that has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequence described in any one of sequence numbers 1 to 5.

21. The vector according to claim 17, wherein the sequence of the functional variant of the CCL20 promoter has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations in the sequence described in any one of sequence numbers 1 to 5.

22. The vector according to claim 21, wherein the point mutation is a conservative point mutation.

23. A method for producing cells expressing at least one heterologous gene, comprising introducing into the cells a composition comprising a polynucleotide comprising a promoter region including a CCL20 promoter operably linked to the heterologous gene, wherein the CCL20 promoter induces the expression of the heterologous gene in response to cytokine stimulation.

24. Cells genetically engineered to express heterologous genes, comprising a polynucleotide according to any one of claims 1 to 11, a composition according to any one of claims 12 to 16, or a vector according to any one of claims 17 to 22.

25. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 11, a composition according to any one of claims 12 to 16, a vector according to any one of claims 17 to 22, or a cell according to claim 24, and a pharmaceutically acceptable carrier or excipient.

26. A method for inducing, regulating, or enhancing the expression of a heterologous gene within a subject, comprising: (a) administering to the subject a polynucleotide according to any one of claims 1 to 11, a composition according to any one of claims 12 to 16, a vector according to any one of claims 17 to 22, a cell according to claim 24, or a pharmaceutical composition according to claim 25; and (b) stimulating the CCL20 promoter with a cytokine.

27. A method for dose-dependently inducing the gene expression of a heterogene within a cell, comprising: (a) contacting the cell with a polynucleotide according to any one of claims 1 to 11, a composition according to any one of claims 12 to 16, a vector according to any one of claims 17 to 22, a cell according to claim 24, or a pharmaceutical composition according to claim 25; and (b) contacting the cell with an effective amount of cytokine having the ability to stimulate the CCL20 promoter to induce the expression of the heterogene in a dose-dependent manner.

28. A method for controlling or inducing the gene expression of a heterogene, comprising stimulating a CCL20 promoter operably linked to the heterogene, wherein the stimulation of the CCL20 promoter by a cytokine controls or induces the expression of the heterogene in response to the stimulation.

29. A method for treating a disease or condition in a subject, comprising administering to the subject an effective amount of a polynucleotide according to any one of claims 1 to 11, a composition according to any one of claims 12 to 16, a vector according to any one of claims 17 to 22, a cell according to claim 24, or a pharmaceutical composition according to claim 25.

30. The method according to claim 29, further comprising stimulating the CCL20 promoter with an exogenous cytokine.

31. A method for controlling inflammation within a subject, comprising operably linking the CCL20 promoter to a heterogene, wherein cytokine stimulation of the CCL20 promoter induces the expression of the heterogene in response to the stimulation, and the heterogene controls inflammation by increasing or decreasing immune activation or by increasing or decreasing the expression of a gene that modulates an inflammatory pathway.

32. The polynucleotide, composition, vector, cell, or method according to any one of claims 1 to 31, wherein the cytokine stimulating the CCL20 promoter is selected from the group consisting of IL-1α, IL-1β, TNF-α, IL-6, sIL-6R, IL-17, CD30, or any combination thereof.

33. The polynucleotide, composition, vector, cell, or method according to any one of claims 1 to 31, wherein the cytokine stimulating the CCL20 promoter is an interleukin.

34. The polynucleotide, composition, vector, cell, or method according to claim 33, wherein the interleukin is interleukin-1.

35. The polynucleotide, composition, vector, cell, or method according to claim 34, wherein the interleukin-1 is IL-1α.

36. The polynucleotide, composition, vector, cell, or method according to any one of claims 1 to 35, wherein the cytokine stimulating the CCL20 promoter is an exogenous cytokine.

37. The polynucleotide, composition, vector, cell, or method according to any one of claims 1 to 35, wherein the cytokine stimulating the CCL20 promoter is an endogenous cytokine.

38. The polynucleotide, composition, vector, cell, or method according to claim 37, wherein the endogenous cytokine stimulating the CCL20 promoter is a result of the inflammation.

39. The polynucleotide, composition, vector, cell, or method according to claim 38, wherein the inflammation is spontaneous.

40. The polynucleotide, composition, vector, cell, or method according to claim 38, wherein the aforementioned inflammation is induced.

41. The polynucleotide, composition, vector, cell, or method according to any one of claims 1 to 40, wherein the use of the CCL20 promoter improves the expression of the heterologous gene by at least about 20%, at least about 40%, at least about 60%, at least about 80%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% compared to the expression observed using a reference promoter.

42. The polynucleotide, composition, vector, cell, or method according to claim 41, wherein the reference promoter is serum amyloid A3 (SAA3 promoter).

43. A gene therapy vector or cell comprising a gene that increases or decreases immune activation within a target upon expression, wherein the gene is under the control of the CCL20 promoter.

44. A gene therapy vector or cell comprising a gene that increases or decreases an inflammatory response within a target when expressed, wherein the gene is under the control of the CCL20 promoter.

45. A method for producing a recombinant protein, comprising culturing cells containing a polynucleotide including a CCL20 promoter operably linked to a heterologous gene encoding the recombinant protein, wherein the CCL20 promoter induces the expression of the heterologous gene in response to cytokine stimulation.

46. A method for determining the presence of one or more inflammatory cytokines in a sample from a subject, comprising: (i) contacting the sample with a polynucleotide or cell containing a CCL20 promoter operably linked to a heterologous gene encoding a reporter protein; and (ii) determining the expression of the reporter protein in response to stimulation of the CCL20 promoter by an inflammatory cytokine in the sample.

47. A kit or manufactured product, (i) A CCL20 promoter comprising the sequence described in any one of Sequence IDs 1 to 5, or a functional fragment or functional variant thereof, (ii) A vector comprising a CCL20 promoter containing the sequence described in any one of sequence numbers 1 to 5, or a functional fragment or functional variant thereof, or (iii) A diagnostic construct or sensor comprising a CCL20 promoter, comprising a sequence described in any one of sequence numbers 1 to 5, or a functional fragment or functional variant thereof, operably linked to a reporter gene, A kit or manufactured product that includes an instruction manual.

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