EP4694906A1 - Inflammation-inducible promoters - Google Patents

Inflammation-inducible promoters

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Publication number
EP4694906A1
EP4694906A1 EP24718226.4A EP24718226A EP4694906A1 EP 4694906 A1 EP4694906 A1 EP 4694906A1 EP 24718226 A EP24718226 A EP 24718226A EP 4694906 A1 EP4694906 A1 EP 4694906A1
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European Patent Office
Prior art keywords
seq
sequence
nos
vector
inflammation
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EP24718226.4A
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German (de)
French (fr)
Inventor
Janneke Meulenberg
Sabine VAN DER SANDEN
Kassiani KYTIDOU
Marleen Van Loenen
Joris VAN ARENSBERGEN
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Meiragtx Gene Regulation Ltd
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Meiragtx Gene Regulation Ltd
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Publication of EP4694906A1 publication Critical patent/EP4694906A1/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • the application relates to promoters that are able to enhance expression of genes in response to inflammatory stimuli.
  • the application further relates to methods employing these promoters and uses of these promoters.
  • Expression cassettes and vectors containing these promoters are also disclosed. These are particularly useful for applications using gene therapy.
  • Gene therapy vectors including adeno-associated virus (AAV)-based gene therapy vectors, are promising candidates for the treatment of inflammatory diseases that result from a biological response of the immune system triggered by a variety of different factors.
  • Regulatory elements including promoters and enhancers, can be engineered for use in AAV vectors to optimize the strength, kinetics, and specificity of transgene expression.
  • the incorporation of promoters inducible by inflammation can help to reduce the risk for side effects due to overexpression of and/or continuous exposure to the anti-inflammatory therapeutic protein by such AAV vectors.
  • inflammation-inducible hybrid promoters constructs comprising such promoters, and methods of using thereof, including, but not limited to, the regulation of gene expression in response to inflammatory stimulation.
  • an inflammation-inducible promoter comprising: (a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
  • the inflammation-inducible promoter comprises from 5’ to 3’ :
  • the inflammation-inducible promoter comprises:
  • the inflammation-inducible promoter comprises from 5’ to 3’ :
  • the inflammation-inducible promoter comprises:
  • the inflammation-inducible promoter comprises from 5’ to 3’ :
  • the inflammation-inducible promoter comprises:
  • the inflammation-inducible promoter comprises:
  • the inflammation-inducible comprises any one of SEQ ID NOs:l, 7, 8, 14, 17, 18, 20, 22, 24, 27, 29, 31, 33, 35, or 36.
  • an expression construct comprising (1) an inflammation-inducible promoter disclosed herein; and (2) an operatively linked transgene.
  • the transgene encodes a therapeutic protein.
  • the transgene encodes a therapeutic nucleic acid.
  • a recombinant nucleic acid comprising an inflammation-inducible promoter disclosed herein or an expression construct disclosed herein.
  • the vector comprising an inflammation-inducible promoter disclosed herein or an expression construct disclosed herein.
  • the vector is a viral vector.
  • the viral vector is selected from the group consisting of adenoviral vector, adeno-associated virus (AAV) vector, and lentiviral vector.
  • the vector comprises an AAV2 genome.
  • the vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV.rh8, AAV.rhlO, AAV.rh39, AAV.43, AAV2/2-66, AAV2/2-84, or AAV2/2-125 capsid.
  • the vector comprises a capsid protein comprising any one of SEQ ID NOs:40-47.
  • the vector is a non-viral vector.
  • the vector is a plasmid, bacmid, cosmid, or, closed-ended linear DNA (ceDNA).
  • composition comprising a vector disclosed herein and a pharmaceutically acceptable excipient.
  • a cell comprising an inflammation-inducible promoter disclosed herein, an expression construct disclosed herein, or a vector disclosed herein.
  • the cell is an isolated cell.
  • the cell is a fibroblast-like synoviocyte, cartilage cell, chondrocyte, or chondroblast.
  • a method of inducing the expression of a transgene comprising: (1) providing a cell comprising an inflammation-inducible promoter disclosed herein, an expression construct disclosed herein, or a vector disclosed herein; and (2) contacting the promoter with one or more inflammatory stimuli.
  • the one or more inflammatory stimuli are selected from the group consisting of inflammatory cytokines, inflammatory chemokines, growth factors, pathogen associated molecular patterns (PAMP), or damage associated molecular patterns (DAMP).
  • the one or more inflammatory stimuli are one or more inflammatory cytokines or chemokines.
  • the one or more inflammatory stimuli are lipopolysaccharides.
  • one or more inflammatory stimuli are IL-1, IL-6, IL-8, IL- 17, IL-23, TNF-a, (IP- 10), or combinations thereof.
  • Fig. 1 illustrates the genetic components of the AAV vector incorporated in the plasmids used for transfection.
  • ITR Inverted Terminal Repeat of AAV2;
  • LUC2P firefly luciferase reporter gene;
  • hGHpA human growth hormone polyadenylation signal.
  • Figs. 2A, 2B, 2C, 2D, and 2E illustrate inflammation-inducible expression of hybrid promoter gl90.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • Fig. 2A HT1080 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters (left panel; transfection).
  • HEK293T/17 cells were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters (right panel; transduction).
  • Fig. 2B illustrate inflammation-inducible expression of hybrid promoter gl90.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL
  • C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 2C Murine fibroblast-like synoviocytes (mFLS) were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 2D Human rheumatoid arthritis (RA)-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 2E Human rheumatoid arthritis
  • C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Stimulation 1 IL1-P: 10 pg/mL; TNFa: 5 pg/mL, IL-6: 50 pg/mL.
  • Stimulation 2 IL1-P: 50 pg/mL; TNFa: 100 pg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
  • Figs. 3A, 3B, 3C, and 3D illustrate inflammation-inducible expression of hybrid promoter g202.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • Fig. 3A HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 3B illustrate inflammation-inducible expression of hybrid promoter g202.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • Fig. 3A HEK293T/17 cells were lipof
  • mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 3C Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 3D C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Stimulation 1 IL1-P: 10 pg/mL; TNFa: 5 pg/mL; IL-6: 50 pg/mL.
  • Stimulation 2 IL1-P: 50 pg/mL; TNFa; lOOpg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
  • Figs. 4A, 4B, 4C, 4D, and 4E illustrate inflammation-inducible expression of hybrid promoter g210.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • Fig. 4A HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 4B illustrate inflammation-inducible expression of hybrid promoter g210.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • Fig. 4A HEK293T/17 cells
  • C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 4C mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 4D Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 4E C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Stimulation 1 IL1-P: 10 pg/mL; TNFa: 5 pg/mL; IL-6: 50 pg/mL.
  • Stimulation 2 IL1-P: 50 pg/mL; TNFa: 100 pg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
  • Figs. 5A, 5B, 5C, and 5D illustrate inflammation-inducible expression of hybrid promoter g211.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • Fig. 5A HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 5B illustrate inflammation-inducible expression of hybrid promoter g211.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • Fig. 5A HEK293T/17 cells were lipof
  • mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 5C Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 5D C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Stimulation 1 IL1-P: 10 pg/mL; TNFa: 5pg/mL; IL-6: 50 pg/mL.
  • Figs. 6A, 6B, 6C, and 6D illustrate inflammation-inducible expression of hybrid promoter g221.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise).
  • HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 6B mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 6C Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 6D Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Stimulation 1 IL1-P: 10 pg/mL; TNFa: 5pg/mL; IL-6: 50 pg/mL.
  • Stimulation 2 IL1-P: 50 pg/mL; TNFa: 100 pg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
  • Figs. 7A, 7B, and 7C illustrate inflammation-inducible expression of hybrid promoter g231.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • Fig. 7A HEK293T/17 cells were lipofected with plasmids (left panel; nucleofection/transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 7B illustrate inflammation-inducible expression of hybrid promoter g231.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • Fig. 7A HEK293T/17 cells were lipofected with plasmid
  • mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters.
  • FIG. 7C Human RA-FLS were nucleofected with plasmids (left panel; nucleofection/transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
  • Figs. 8A, 8B, and 8C illustrate inflammation-inducible expression of hybrid promoter gl87.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • HT1080 cells Fig. 8A
  • C2C12 Fig. 8B
  • human RA-FLS Fig. 8C
  • Fig. 8C were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Figs. 9A and 9B illustrate inflammation-inducible expression of hybrid promoter g201.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • Fig. 9A HEK293T/17 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 9B Human RA-FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Figs. 10A, 10B, and 10C illustrate inflammation-inducible expression of hybrid promoter ng007.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • HT1080 cells Fig. 10A
  • C2C12 Fig. 10B
  • Human RA- FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Figs. HA, 11B, and 11C illustrate inflammation-inducible expression of hybrid promoter ng008.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • HT1080 cells Fig. HA
  • C2C12 Fig. 11B
  • Human RA-FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Figs. 12A, 12B, and 12C illustrate inflammation-inducible expression of hybrid promoter ng012.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • HT1080 cells Fig. 12A
  • C2C12 Fig. 12B
  • Human RA- FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
  • Figs. 13A, and 13B illustrate inflammation-inducible expression of hybrid promoter g229.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • Fig. 13A HT1080 cells lipofected with plasmids (left panel; transfection) comprising the reporter gene luciferase under the control of the indicated promoters.
  • HEK293T/17 cells were transduced with an AAV vector (right panel; transduction) comprising the reporter gene luciferase under the control of the indicated promoters.
  • Fig. 13B illustrate inflammation-inducible expression of hybrid promoter g229.
  • the NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip.
  • Fig. 13A
  • Fig. 14 illustrates NFKB activation perpetuates chronic inflammation by targeting genes involved in inflammation during RA development. Adopted from Nejatbakhsh Samimi et al., NF-KB signaling in rheumatoid arthritis with focus on fibroblast-like synoviocytes. Auto Immun Highlights. 2020 Aug 8; 11(1): 11, incorporated herein in its entirety.
  • inflammation-inducible hybrid promoters constructs comprising such promoters, and methods of using thereof, including, but not limited to, the regulation of gene expression in response to inflammatory stimulation.
  • promoters are defined as DNA regions where transcription is initiated.
  • the promoter is typically located at the 5' end of the gene (or transcription unit) that the promoter regulates. Promoters contain DNA sequences and response elements that provide an initial binding site for the RNA transcriptional machinery (including RNA polymerase and transcription factors that recruit the RNA polymerase). Transcription factors bind specific activator and repressor sequences that attach to certain promoters and regulate gene expression. Since the promoter region controls when and where a gene of interest is expressed in an organism, promoters are crucial elements for regulating the level and specificity of transgene expression, particularly in the context of gene therapy.
  • hybrid promoter comprises multiple elements, including, but not limited to, multiple synthetic elements, or elements from one or more different promoter regions.
  • a hybrid promoter can contain one or more enhancer elements and one or more activation sequences, and/or elements that are differentially regulated.
  • an “inflammation-inducible promoter” is a promoter that increases the expression of a gene operably linked to the promoter in response to inflammatory stimuli.
  • the word “inflammation” or “inflammatory” can refer to a local increase in lymphocytes such as monocytes, T-cells, B-cells, leukocytes, natural-killer (NK) cells, macrophages or the like.
  • the word “inflammation” or “inflammatory” can refer to a local increase of the concentration of the interleukins, interferons, tumor necrosis factor or prostaglandins, leukotrienes or other small signaling molecules. Inflammation can also refer to a systemic response.
  • a systemic inflammatory reaction may arise as a response to a range of insults to the host.
  • Inflammatory agents include toxins or pathogens such as virus or bacteria. Inflammation may also arise from an autoimmune response to the host’s own proteins; in reaction to injury; or as an immune response to allergens. Acute inflammation may be serious and result in pathologic consequences.
  • Some of the specific cytokines known to be involved in an acute inflammatory response include IL-1, TNF-a and IL-6. Ways of testing whether a promoter is inducible by inflammation are known in the art.
  • the person skilled in the art may compare expression of a transgene operatively linked to the inflammation-inducible promoter in the presence or absence of one or more inflammatory stimuli, including, but not limited to, inflammatory cytokines, inflammatory chemokines, or LPS.
  • inflammatory stimuli including, but not limited to, inflammatory cytokines, inflammatory chemokines, or LPS.
  • Non-limiting examples of measuring whether a promoter is inducible by inflammation are provided in the Examples.
  • operatively linked refers to a first molecule joined to a second molecule, wherein the two molecules are so arranged that the first molecule affects the function of the second molecule.
  • the two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent.
  • a promoter is operatively linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell.
  • two portions of a transcription regulatory element are operatively linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion.
  • Two transcription regulatory elements may be operatively linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operatively linked to one another with no intervening nucleotides present.
  • the inflammation-inducible hybrid promoters disclosed herein can be used for the expression of transgenes in particular tissues in different pathological situations in which an inflammatory component is present.
  • the inflammation-inducible hybrid promoters disclosed herein are used for expression of transgenes in fibroblast-like synoviocytes (FLS), cartilage cells, chondrocytes, or chondroblasts.
  • FLS fibroblast-like synoviocytes
  • the inflammation-inducible hybrid promoters disclosed herein are useful for the expression of transgenes for the treatment of arthritis, including, but not limited to, rheumatoid arthritis (RA) and osteoarthritis (OA).
  • RA rheumatoid arthritis
  • OA osteoarthritis
  • the pathology of arthritis involves joint swelling and synovial inflammation (Fig. 14).
  • the inflammation-inducible hybrid promoters disclosed herein can also be used to control the rate and extent of expression of a gene encoding an antiinflammatory protein in a negative feedback manner.
  • an inflammation-inducible hybrid promoters disclosed herein is operatively linked to a gene encoding an antiinflammatory protein. In the absence of inflammation, little or no anti-inflammatory protein will be produced. When inflammation occurs, the anti-inflammatory protein will be produced according to the intensity of the inflammatory reaction (c.g, in response to circulating inflammatory cytokines or other mediators). Anti-inflammatory protein production will limit the extent and duration of the inflammatory reaction, avoiding dangerous (toxic) excess. As the inflammatory reaction wanes, production of the anti-inflammatory protein will decrease, thereby avoiding prolonged immunosuppression.
  • an inflammation-inducible hybrid promoter comprising one or more elements disclosed in Tables 2 or 3.
  • a person skilled in the art will appreciate that certain modifications can be made to the inflammation-inducible hybrid promoters disclosed herein without eliminating the inflammation-inducible property of the hybrid promoter.
  • provided are inflammation-inducible hybrid promoters that comprise the elements of another inflammation-inducible hybrid promoter disclosed herein, wherein the 5’ to 3’ order of the elements has been changed.
  • the orientation of one or more of the elements of a hybrid promoter disclosed herein can be changed such that the reverse version or the reverse compliment version of a given element is used in a hybrid promoter.
  • inflammation-inducible hybrid promoters that include sequence in addition (e.g., added at either end or inserted within the hybrid promoter sequence) to the elements of a hybrid promoter disclosed herein.
  • inflammation-inducible hybrid promoters in which certain sequence has been removed from the hybrid promoters disclosed herein (e.g., as a terminal or internal deletion).
  • the inflammationinducible hybrid promoter may comprise any of the elements disclosed in Tables 2 or 3 in the 5’-> 3’ orientation indicated in Tables 2 or 3 or a reverse or a reverse compliment form.
  • inflammation-inducible hybrid promoters that share certain percent sequence identity with an inflammation-inducible hybrid promoter disclosed herein.
  • identity refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position. A degree identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions.
  • an inflammation-inducible hybrid promoter comprising:
  • an inflammation-inducible hybrid promoter comprising:
  • an inflammation-inducible hybrid promoter comprising:
  • an inflammation-inducible hybrid promoter comprising:
  • an inflammation-inducible hybrid promoter comprising:
  • an inflammation-inducible hybrid promoter comprising: (a) SEQ ID N0:2, SEQ ID N0:3, and any one of SEQ ID NOs:4-6, 16, or 28;
  • an inflammation-inducible hybrid promoter comprising:
  • an inflammation-inducible hybrid promoter comprising from 5’ to 3’ :
  • an inflammation-inducible hybrid promoter comprising from 5’ to 3’ :
  • an inflammation-inducible hybrid promoter comprising from 5’ to 3’ :
  • an inflammation-inducible hybrid promoter comprising from 5’ to 3’ :
  • an inflammation-inducible hybrid promoter comprising from 5’ to 3’ :
  • an inflammation-inducible hybrid promoter comprising from 5’ to 3’ :
  • an inflammation-inducible hybrid promoter comprising from 5’ to 3’ :
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.
  • the hybrid promoter comprises SEQ ID NO: 1.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3, and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7 or SEQ ID NO: 8.
  • the hybrid promoter comprises SEQ ID NO: 7 or SEQ ID NO: 8.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:9- 12 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical SEQ ID NO: 13.
  • the hybrid promoter comprises (a) any one of SEQ ID NOs:9-12 and (b) SEQ ID NO: 13.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.
  • the hybrid promoter comprises SEQ ID NO: 14.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28.
  • the hybrid promoter comprises (a) SEQ ID NO: 15 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17 or SEQ ID NO: 18.
  • the hybrid promoter comprises SEQ ID NO: 17 or SEQ ID NO: 18.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 19, 39, or 48 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises (a) any one of SEQ ID NOs: 19, 39, or 48 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20.
  • the hybrid promoter comprises SEQ ID NO:20.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28.
  • the hybrid promoter comprises (a) SEQ ID NO:21 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22.
  • the hybrid promoter comprises SEQ ID NO:22.
  • the hybrid promoter comprises (a) comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and (b) comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises (a) SEQ ID NO:23 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24.
  • the hybrid promoter comprises SEQ ID NO:24.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25, (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:26, and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises (a) a
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:27.
  • the hybrid promoter comprises SEQ ID NO:27.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13.
  • the hybrid promoter comprises (a) any one of SEQ ID NOs:4- 6, 16, or 28 and (b) SEQ ID NO: 13.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:29.
  • the hybrid promoter comprises SEQ ID NO:29.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28.
  • the hybrid promoter comprises (a) SEQ ID NO:30 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31.
  • the hybrid promoter comprises SEQ ID NO:31.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:32 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28.
  • the hybrid promoter comprises (a) SEQ ID NO:32 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33.
  • the hybrid promoter comprises SEQ ID NO:33.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:34 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28.
  • the hybrid promoter comprises (a) SEQ ID NO:34 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:35 or SEQ ID NO:36.
  • the hybrid promoter comprises SEQ ID NO:35 or SEQ ID NO:36.
  • the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:37, 38, or 49 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises (a) any one of SEQ ID NOs:37, 38, or 49 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
  • the hybrid promoter comprises one or more sequences sequence that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences in Tables 2 or 3.
  • the hybrid promoter comprises one or more sequences disclosed in Tables 2 or 3.
  • nucleic acid constructs and their use for the expression of a transgene in a cell are also provided.
  • recombinant vectors and their use for the introduction of a transgene or an expression construct into a cell are also provided.
  • Nucleic acid constructs include expression constructs including plasmids.
  • expression construct refers to a recombinant polynucleotide construct that includes a nucleic acid coding for an RNA capable of being transcribed in a cell. Methods for constructing expression constructs and plasmids through standard recombinant techniques are known in the art.
  • the nucleic acid constructs or vectors disclosed herein comprise recombinant nucleic acid constructs that include additional DNA elements, including DNA segments that provide for the replication of the DNA in a host cell and expression of the target gene in target cells at appropriate levels.
  • Vector means a vehicle that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.
  • Nonlimiting examples of vectors include a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini-circle, or a virus (including virus derived sequences).
  • a vector may also refer to a virion comprising a nucleic acid to be delivered into a host cell, either in vitro or in vivo.
  • a vector refers to a virion comprising a recombinant viral genome, wherein the viral genome comprises one or more ITRs and a transgene.
  • the recombinant vector is a viral vector or a combination of multiple viral vectors.
  • a vector comprising any of the expression constructs disclosed herein.
  • nucleic acid constructs and vectors comprising an inflammationinducible hybrid promoter disclosed herein operatively linked to a transgene.
  • the nucleic acid constructs or vectors disclosed herein comprise additional regulatory elements, including, but not limited to, promoters, enhancers, translation initiation signals, introns, and/ or splicing enhancers.
  • the nucleic acid constructs or vectors disclosed herein comprise a polyadenylation sequence, which may include splice donor and acceptor sites.
  • nucleic acid constructs or vectors disclosed herein comprise an internal ribosome entry site (IRES).
  • An IRES sequence may be used to produce more than one polypeptide from a single gene transcript.
  • An IRES (or other suitable sequence) is used to produce a protein that contains more than one polypeptide chain or to express two different proteins from or within the same cell.
  • An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in photoreceptors, RPE and ganglion cells. In one embodiment, the IRES is located 3' of the transgene.
  • transgene is used to refer to a polynucleotide that can be introduced into a cell or organism to express a gene product of interest.
  • the transgene can encode a “therapeutic polypeptide” or “therapeutic protein,” which are to be understood herein as a polypeptide or protein that can have a beneficial effect on a subject.
  • the individual may be a human.
  • the subject suffers from a disease.
  • Such therapeutic polypeptide may be selected from, but is not limited to, the group consisting of an enzyme, a co-factor, a cytokine, an antibody, a growth factor, a hormone and an anti-inflammatory protein.
  • the gene product of interest treats, prevents or suppresses symptoms associated with an arthritic disease. In embodiments, the gene product of interest treats, prevents or suppresses symptoms associated with an arthritic disease, wherein a subject experiences these symptoms in the joints. It is understood herein that the gene product of interest may include biosimilars of said gene product of interest.
  • the gene product of interest is a nucleic acid. In some embodiments, the transgene encodes an antisense oligonucleotide, an siRNA, or an RNAi.
  • the gene product of interest is an immunosuppressant.
  • the gene product of interest is selected from the group consisting of ANP32A, Nrf2, IL-6 inhibitor, soluble IL-6 receptor, IL-6 receptor antagonist, humanized anti-IL-6 monoclonal antibody, chimeric anti-IL-6 monoclonal antibody, humanized rabbit anti-IL-6 monoclonal antibody, interleukin 1 (IL-1) inhibitor, tumor necrosis factor alpha (TNFa) inhibitor, IL-1 receptor antagonist, soluble IL-1 receptor, IL- 17 inhibitor, IL-12/IL-23 inhibitor, T-cell costimulation inhibitor, B cell depleting and inhibiting agents, IL-15 inhibitor, IL-22 inhibitor, inhibitor of GM-CSF, insulin-like growth factor (IGF-1), fibroblast-growth factor (FGF), receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor, complement 5a inhibitor, bone morphogenetic protein family member (BMPs), transforming growth
  • IL-1 tumor necrosis factor
  • the gene product of interest is selected from the group consisting of tocilizumab, sarilumab, olokizumab, sirukumab, Siltucimab, Clazakizumab, anakinra, canakinumab, rilonacept, gevokizumab and lutikizumab, etanercept, infliximab, adalimumab, certilizumab pegol, golimumab, secukinumab, brodalumab, ixekizumab, ustekinumab, risankizumab, guselkumab, tildrakizumab, abatacept, rituximab, belimumab, ianalumab, tabalumab, AMG-714, Fezakunimab, lenzilumab, namilumab, rhFGF
  • the gene product of interest is an interleukin 6 (IL-6) inhibitor.
  • the gene product of interest is a soluble IL-6 receptor.
  • the gene product of interest is an IL-6 receptor antagonist.
  • the IL-6 receptor antagonist is at least one of tocilizumab and sarilumab.
  • the gene product of interest is a humanized or a chimeric anti-IL-6 monoclonal antibody.
  • the anti-IL-6 antibody is olokizumab or sirukumab.
  • the chimeric anti-IL-6 monoclonal antibody is siltucimab.
  • the humanized rabbit anti-IL-6 monoclonal antibody is clazakizumab.
  • the gene product of interest is an interleukin 1 (IL-1) inhibitor.
  • the IL-1 inhibitor is selected from the group consisting of anakinra, canakinumab, rilonacept, gevokizumab and lutikizumab.
  • the IL-1 inhibitor is selected from the group consisting of (i) an IL-1 receptor antagonist (ii) an anti-IL-1 monoclonal antibody, (iii) a human dimeric anti-IL-1 fusion protein, and (iv) a human dual variable domain anti-IL-1 immunoglobulin.
  • the IL-1 receptor antagonist is a human IL-1 receptor antagonist. In one embodiment, the human IL-1 receptor antagonist anakinra.
  • the anti-IL-1 monoclonal antibody is a human anti-IL-1 monoclonal antibody.
  • the human IL-1 monoclonal antibody canakinumab or gevokizumab.
  • the human dimeric anti-IL-1 fusion protein is the human dimeric anti-IL-1 fusion protein rilonacept.
  • the human dual variable domain anti-IL-1 immunoglobulin is the human dual variable domain anti-IL-1 immunoglobulin lutikizumab.
  • the gene product of interest is a tumor necrosis factor alpha (TNFa) inhibitor.
  • TNFa tumor necrosis factor alpha
  • the TNFa inhibitor is selected from the group consisting of etanercept, infliximab, adalimumab, certilizumab pegol and golimumab.
  • the TNFa inhibitor is etanercept.
  • the gene product of interest is an IL-1 receptor antagonist. In one embodiment, the gene product of interest is a soluble IL-1 receptor.
  • the gene product of interest is an IL- 17 inhibitor.
  • the IL- 17 inhibitor is selected from the group consisting of secukinumab, brodalumab and ixekizumab.
  • the gene product of interest is an IL-12/IL-23 inhibitor.
  • the IL-12/IL-23 inhibitor is selected from the group consisting of ustekinumab, risankizumab, guselkumab and tildrakizumab.
  • the gene product of interest is a T-cell costimulation inhibitor. In one embodiment, the T-cell costimulation inhibitor is abatacept. [0093] In one embodiment, the gene product of interest is a B cell depleting and inhibiting agent. In one embodiment, the B cell depleting and inhibiting agent is selected from the group consisting of rituximab, belimumab, ianalumab and tabalumab.
  • the gene product of interest is an IL- 15 inhibitor.
  • the IL-15 inhibitor is AMG-714.
  • the gene product of interest is an IL-22 inhibitor.
  • the IL-22 inhibitor is fezakunimab.
  • the gene product of interest is an inhibitor of GM-CSF.
  • the inhibitor of GM-CSF is lenzilumab or namilumab.
  • the gene product of interest is an insulin-like growth factor (IGF-1).
  • IGF-1 insulin-like growth factor
  • the gene product of interest is a fibroblast-growth factor (FGF).
  • FGF fibroblast-growth factor
  • the fibroblast-growth factor (FGF) is rhFGF-18/sprifermin.
  • the gene product of interest is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor.
  • the RANKL inhibitor is denosumab.
  • the gene product of interest is a complement 5a inhibitor.
  • the complement 5a inhibitor is C5aR-151.
  • the gene product of interest is a bone morphogenetic protein family member (BMPs).
  • BMPs bone morphogenetic protein family member
  • the gene product of interest is a transforming growth factor-beta (TGF-p).
  • TGF-p transforming growth factor-beta
  • the gene product of interest is a growth differentiation factor family member (GDFs).
  • GDFs growth differentiation factor family member
  • the gene product of interest is an interleukin- 18 inhibitor.
  • the interleukin- 18 inhibitor is Tadekinig alfa/ IL-18 binding protein.
  • the gene product of interest is an IL-2 inhibitor.
  • the IL-2 inhibitor is basiliximab or daclizumab.
  • the gene product of interest is a soluble TNFa (sTNFa) receptor p55.
  • sTNFa soluble TNFa
  • the gene product of interest is a sTNFa receptor p75. In one embodiment, the gene product of interest is a sTNFa receptor fused with an IgG. In one embodiment, the gene product of interest is an inhibitor of TNFa receptor p55. In one embodiment, the gene product of interest is an inhibitor of sTNFa receptor p75. [0108] In one embodiment, the gene product of interest is a dominant negative IxE-kinase (dn-IKK-p).
  • the gene product of interest is an interleukin-4 (IL-4).
  • IL-4 interleukin-4
  • the gene product of interest is an interleukin- 10 (IL- 10).
  • IL- 10 is F8IL10/Dekavil.
  • the gene product of interest is an interleukin- 13 (IL-13).
  • the gene product of interest is an IL-33 inhibitor.
  • the gene product of interest is a CCL17-inhibitor
  • the gene product of interest is an interferon beta (IFN-P).
  • IFN-P interferon beta
  • the gene product of interest is a tissue inhibitor of MMP family member (TIMPs),
  • the gene product of interest is a plasminogen-activator inhibitor (PAIs).
  • PAIs plasminogen-activator inhibitor
  • the gene product of interest is a serine protease inhibitor (serpins).
  • the gene product of interest is a signaling molecule/transcription factor.
  • the signaling molecule/transcription factor is selected from the group consisting of SMAD, Sox9 and IkB.
  • the gene product of interest is an extracellular matrix component.
  • the extracellular matrix component is selected from the group consisting of collagen, cartilage oligomeric matrix protein (COMP), proteoglycans and elastin.
  • the gene product of interest is a vasoactive intestinal peptide (VIP).
  • the gene product of interest is a Cluster of Differentiation 39 (CD39).
  • the gene product of interest is a Cluster of Differentiation 73 (CD73).
  • the gene product of interest is a superoxide dismutase (SOD).
  • SOD superoxide dismutase
  • the gene product of interest is a sequence-specific nuclease
  • Functional genome editing systems for use in all embodiments of the invention are known to the person skilled in the art and include: Transcription Activator-Like Effector Nucleases (TALENs, see, e.g., Gaj et al., ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013 Jul;31(7):397-405), zinc-finger nucleases (ZFNs, see., e.g., Gaj et al.
  • TALENs Transcription Activator-Like Effector Nucleases
  • ZFNs zinc-finger nucleases
  • meganucleases such as I-Scel (Arnould et al., Engineered I-Crel derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol. 2007 Aug 3 ;371(l):49-65; Takeuchi et al., Tapping natural reservoirs of homing endonucleases for targeted gene modification. Proc Natl Acad Sci U S A. 2011 Aug 9;108(32): 13077-82.), RNA-guided endonuclease systems like CRISPR/Cas (see, e.g., Mali et al., Cas9 as a versatile tool for engineering biology.
  • a preferred gene editing system may be CRISPR (comprising CRISPR/Cpfl and CRISPR-Cas), because it is quicker and cheaper than other methods.
  • CRISPR can be easily repurposed to target different DNA sequences using the CRISPR single guide RNAs.
  • a nucleic acid construct that comprises at least one of (i) a polynucleotide comprising a sequence encoding at least one guide RNA (gRNA); wherein the guide RNA is substantially complementary - in some embodiments complementary - to a target polynucleotide sequence(s) in a genome; and (ii) a polynucleotide comprising a sequence encoding a nuclease; wherein the nuclease forms a ribonuclease complex with the guide RNA, and wherein the ribonuclease complex makes site-specific double-stranded DNA breaks in the genome.
  • gRNA guide RNA
  • the gene product of interest is an antibody, antigen-binding fragment thereof, or a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • Viral vectors for the expression of a target gene in a target cell, tissue, or organism include, for example, an AAV vector, adenovirus vector, lentivirus vector, retrovirus vector, poxvirus vector, baculovirus vector, herpes simplex virus vector, vaccinia virus vector, or a synthetic virus vector (e.g., a chimeric virus, mosaic virus, or pseudotyped virus, and/or a virus that contains a foreign protein, synthetic polymer, nanoparticle, or small molecule).
  • AAV vector e.g., adenovirus vector, lentivirus vector, retrovirus vector, poxvirus vector, baculovirus vector, herpes simplex virus vector, vaccinia virus vector, or a synthetic virus vector (e.g., a chimeric virus, mosaic virus, or pseudotyped virus, and/or a virus that contains a foreign protein, synthetic polymer, nanoparticle, or small molecule).
  • AAV are small, single-stranded DNA viruses which require helper virus to facilitate efficient replication.
  • the 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITR) and two open reading frames which encode the Rep proteins and Cap proteins, respectively.
  • the Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD, and 40 kD. These proteins function mainly in regulating AAV replication and rescue and integration of the AAV into a host cell's chromosomes.
  • the Cap reading frame encodes three structural proteins of molecular weight 85 kD (VP 1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid.
  • More than 80% of total proteins in AAV virion comprise VP3. Flanking the rep and cap open reading frames at the 5' and 3' ends are about 145 bp long inverted terminal repeats (ITRs). The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene.
  • ITRs inverted terminal repeats
  • Recombinant adeno-associated virus “rAAV” vectors include any vector derived from any AAV serotype. rAAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, in some embodiments, the Rep and/or Cap genes, but retain functional flanking ITR sequences.
  • the viral vector is an rAAV virion, which comprises an rAAV genome and one or more capsid proteins.
  • the rAAV genome comprises an expression construct disclosed herein.
  • the viral vector disclosed herein comprises a nucleic acid comprising an AAV 5' ITR and 3' ITR located 5' and 3' of the transgene, respectively.
  • the nucleic acid may be desirable for the nucleic acid to contain the 5 ' ITR and 3' ITR sequences arranged in tandem, e.g., 5' to 3' or a head-to-tail, or in another alternative configuration.
  • the ITRs sequences may be located immediately upstream and/or downstream of the heterologous molecule, or there may be intervening sequences.
  • the ITRs need not be the wild-type nucleotide sequences, and may be altered (e.g., by the insertion, deletion, or substitution of nucleotides) so long as the sequences provide for functional rescue, replication, and packaging.
  • the ITRs may be selected from AAV2, or from among the other AAV serotypes, as described herein.
  • a vector comprising a nucleic acid sequence comprising (i) an expression construct disclosed herein and (ii) one or more inverted terminal repeats (ITR).
  • the nucleic acid sequence comprises a 5’ ITR and a 3’ ITR.
  • the 5’ ITR and a 3’ ITR are derived from AAV serotype AAV2.
  • the 5’ ITR and a 3’ ITR are derived from AAV serotype AAV5.
  • the viral vector is an AAV vector, such as an AAV1 (i.e., an AAV containing AAV1 ITRs and AAV1 capsid proteins), AAV2 (z.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (z.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV1 (i.e., an
  • the viral vector is a pseudotyped AAV vector, containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype.
  • the pseudotyped AAV is AAV2/9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins).
  • the pseudotyped AAV is AAV2/10 (i.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins).
  • the pseudotyped AAV is AAV2/7m8 (i.e., an AAV containing AAV2 ITRs and AAV7m8 capsid proteins).
  • the AAV vector contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more of capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10.
  • the capsid protein is a variant AAV capsid protein.
  • the capsid protein is AAV2 variant rAAV2-retro (SEQ ID NO:44 from WO 2017/218842, incorporated herein by reference in its entirety).
  • the capsid protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence disclosed in WO 2017/218842.
  • an AAV with tropism for joint tissues is used, including, but not limited to an AAV comprising a viral capsid protein disclosed in International Patent Publication No. WO2019/141765, entitled “A modified raav capsid protein for gene therapy” or International Patent Publication No.
  • the capsid protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence disclosed in International Patent Publication Nos. WO2019/141765 or WO202 1/009684.
  • the AAV particle comprises a capsid protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence in Table 1.
  • the AAV vector contains two or more capsid proteins selected from different serotypes.
  • the AAV vector contains a capsid from a first serotype and a capsid protein from a second serotype.
  • the AAV vector contains capsid proteins from a first and a second AAV serotype, respectively, in aratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.
  • the AAV vector contains capsid proteins from a first and a second AAV serotype, respectively, in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.
  • a mixture of (1) AAV vectors comprising capsid proteins from a first serotype and (2) AAV vectors comprising capsid proteins from a second serotype is used.
  • a ratio of the (1) AAV vectors comprising capsid proteins from a first serotype and (2) AAV vectors comprising capsid proteins from a second serotype, respectively, of 1 : 1, 1 :2, 1:3, 1 :4, 1 :5, 1 :6, 1 :7, 1:8, 1 :9, 1 : 10, 1 : 15, 1 :20, 1:25, 1 :30, 1 :35, 1 :40, 1 :45, or 1 :50 is used.
  • a ratio of the (1) AAV vectors comprising capsid proteins from a first serotype and (2) AAV vectors comprising capsid proteins from a second serotype, respectively, of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1:6, 1:7, 1 :8, 1 :9, 1 : 10, 1 : 15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, or 1 :50 is used.
  • viral vectors include adenoviral (AV) vectors, for example, those based on human adenovirus type 2 and human adenovirus type 5 that have been made replication defective through deletions in the El and E3 regions.
  • the transcriptional cassette can be inserted into the El region, yielding a recombinant E1ZE3 -deleted AV vector.
  • Adenoviral vectors also include helper-dependent high-capacity adenoviral vectors (also known as high- capacity, “gutless” or “gutted” vectors), which do not contain viral coding sequences.
  • helper-dependent adenoviral vectors also known as high- capacity, “gutless” or “gutted” vectors
  • These vectors contain the cis-acting elements needed for viral DNA replication and packaging, mainly the inverted terminal repeat sequences (ITR) and the packaging signal (CY).
  • ITR inverted terminal repeat sequences
  • CY packaging signal
  • Lentiviral-based systems can transduce nondividing as well as dividing cells making them useful for applications targeting, for examples, the nondividing cells of the CNS.
  • Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome providing the potential for very long-term gene expression.
  • Polynucleotides including plasmids, YACs, minichromosomes and minicircles, carrying the target gene containing the expression cassette can also be introduced into a cell or organism by nonviral vector systems using, for example, cationic lipids, polymers, or both as carriers.
  • Conjugated poly-L-lysine (PLL) polymer and polyethylenimine (PEI) polymer systems can also be used to deliver the vector to cells.
  • Other methods for delivering the vector to cells includes hydrodynamic injection and electroporation and use of ultrasound, both for cell culture and for organisms.
  • the rAAV virions disclosed herein may be constructed and produced using the materials and methods described herein, as well as those known to those of skill in the art.
  • Such engineering methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). Further, methods suitable for producing a rAAV cassette in an adenoviral capsid have been described in U.S. Pat. Nos. 5,856,152 and 5,871,982.
  • a host cell that contains sequences necessary to express AAV rep and AAV cap or functional fragments thereof as well as helper genes essential for AAV production.
  • the AAV rep and cap sequences are obtained from an AAV source as identified herein.
  • the AAV rep and cap sequences may be introduced into the host cell in any manner known to one in the art, including, without limitation, transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion.
  • the rep and cap sequences may be transfected into the host cell by one or more nucleic acid molecules and exist stably in the cell as an episome.
  • the rep and cap sequences are stably integrated into the genome of the cell.
  • Another embodiment has the rep and cap sequences transiently expressed in the host cell.
  • a useful nucleic acid molecule for such transfection comprises, from 5' to 3', a promoter, an optional spacer interposed between the promoter and the start site of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.
  • the rep and cap sequences may be supplied on a single vector, or each sequence may be supplied on its own vector.
  • the rep and cap sequences are supplied on the same vector.
  • the rep and cap sequences may be supplied on a vector that contains other DNA sequences that are to be introduced into the host cells.
  • the promoter used in this construct may be any suitable constitutive, inducible or native promoters known to one of skill in the art.
  • the molecule providing the rep and cap proteins may be in any form which transfers these components to the host cell. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as those for marker genes.
  • This molecule does not contain the AAV ITRs and generally does not contain the AAV packaging sequences. To avoid the occurrence of homologous recombination, other virus sequences, particularly those of adenovirus, are avoided in this plasmid.
  • This plasmid is desirably constructed so that it may be stably transfected into a cell.
  • the molecule providing rep and cap may be transiently transfected into the host cell, it may be preferred that the host cell be stably transformed with sequences necessary to express functional rep/cap proteins in the host cell, e.g., as an episome or by integration into the chromosome of the host cell. Depending upon the promoter controlling expression of such stably transfected host cell, the rep/cap proteins may be transiently expressed (e.g., through use of an inducible promoter).
  • the methods employed for constructing embodiments of this disclosure are conventional genetic engineering or recombinant engineering techniques such as those described in the references above.
  • the rAAV may be produced utilizing a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif), according to manufacturer’s instructions. See also Herzog et al., Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat Med.
  • plasmid with the transgene, a helper plasmid containing AAV rep and cap, and a plasmid supplying adenovirus helper functions of E2A, E40rf6 and VA.
  • a two- plasmid system is used, e.g., such as the system described in Grimm et al., Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6.
  • Molecular therapy the journal of the American Society of Gene Therapy. 2003;7(6):839-50.
  • the rAAV virions are then produced by culturing a host cell containing a rAAV virus as described herein which contains a rAAV genome to be packaged into a rAAV virion, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof.
  • Suitable viral helper genes e.g., adenovirus E2A, E40rf6 and VA, among other possible helper genes, may be provided to the culture in a variety of ways known to the art, including on a separate plasmid.
  • the recombinant AAV virion which directs expression of the transgene is isolated from the cell or cell culture in the absence of contaminating helper virus or wildtype AAV.
  • RNA expression may be monitored in ways known in the art.
  • a target cell may be infected in vitro, and the number of copies of the transgene in the cell monitored by Southern blotting or quantitative polymerase chain reaction (PCR).
  • the level of RNA expression may be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and the level of protein expression may be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or by the specific methods detailed below in the Examples.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • the nucleic acid constructs and vectors disclosed herein are used to deliver an inflammation-inducible promoter operatively linked to a transgene to a cell.
  • cells comprising a promoter, a nucleic acid construct, or a vector disclosed herein.
  • the cell is a fibroblast-like synoviocyte (FLS), cartilage cell, chondrocyte, chondroblast, adipocyte, osteoclast, T-lymphocyte, macrophage, monocyte, neutrophil, osteoblast, dendritic cell, plasma cell, mast cell, (skeletal) muscle cell, cardiomyocyte, or B lymphocyte.
  • the cell is a synovial cell.
  • the “synovium” or “synovial tissue” or “synovial cells” as used herein refers to the cellular lining covering the non-cartilaginous surfaces of the synovial joints, as further described in Tak (2000, Examination of the synovium and synovial fluid. In: Firestein GS, Panyani GS, Wollheim FA editors. Rheumatoid Arthritis. New York: Oxford Univ. Press, Inc. 55-68) and incorporated herein by reference.
  • the synovium consists of the intimal lining layer (or synovial lining layer) and the synovial sublining (subsynovium), which merges with the joint capsule.
  • the intimal lining layer comprises intimal macrophages (or macrophage-like synoviocytes or type A synoviocytes) and FLS (or type B synoviocytes).
  • “Synovium” may therefore be replaced by, or is synonymous with, “synovial tissue”.
  • a synovial cell can include any cell present in the synovium including FLS and macrophage-like synoviocytes.
  • a synoviocyte cell may also be a neutrophil, T, B cells and/or connective tissue cells, which may all be present in the synovium.
  • Fibroblast-like synoviocytes FLS are cells of mesenchymal origin that display many characteristics that are in common with fibroblasts, such as expression of specific proteins, such as for example several types of collagens.
  • FLS also secrete proteins that are normally absent in other fibroblast lineages, such as for example lubricin.
  • FLS express molecules that are important for the mediation of cell adhesion, such as cadherin- 11, VCAM-1, several integrins and their receptors. Specific for FLS is the expression of CD55 and this protein is therefore typically used to identify FLS in the synovium by immunohistochemistry.
  • FLS represent a specialized cell type located inside joints in the synovium, whose cells play a crucial role in the pathogenesis of chronic inflammatory diseases, such as rheumatoid arthritis (RA).
  • RA chronic inflammatory diseases
  • rheumatoid synovium or “rheumatoid synovial cells” or “rheumatoid synovial tissue” refers to the inflamed synovium of the joints of an individual suffering from RA.
  • the rheumatoid synovium is characterized by intimal lining hyperplasia and by accumulation of FLS, T-cells, plasma cells, macrophages, B-cells, natural killer cells and dendritic cells in the synovial sublining. These accumulated cells are comprised in the definition of rheumatoid synovial cells.
  • the synovial tissue becomes a place where constant inflammatory processes take place, which can eventually lead to cartilage damage and joint destruction and deformation.
  • FLS that are present in the synovium during RA have been reported to display an altered phenotype compared to the FLS present in normal tissues.
  • the FLS in rheumatoid synovium lose “contact inhibition”, z.e., they lose the property to arrest their growth when more cells come into contact with each other. In addition, they lose the dependency to grow on adhesive surfaces. As a result, the number of FLS in the diseased synovium increases.
  • the inflammation is further enhanced by the production of several pro-inflammatory signaling molecules, particularly interleukins IL-6 and IL-8, prostanoids and matrix metalloproteinases (MMPs).
  • MMPs matrix metalloproteinases
  • compositions comprising a nucleic acid construct or vector disclosed herein and a pharmaceutically acceptable excipient.
  • nucleic acid construct or vector disclosed herein may be assessed for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for storage and/or administration to a patient.
  • Formulations of the nucleic acid constructs or vectors disclosed herein disclosed herein involve the use of a pharmaceutically and/or physiologically acceptable vehicle or carrier, such as buffered saline or other buffers, e.g., PBS or HEPES, to maintain pH at appropriate physiological levels.
  • a pharmaceutically and/or physiologically acceptable vehicle or carrier such as buffered saline or other buffers, e.g., PBS or HEPES
  • the nucleic acid constructs or vectors disclosed herein can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the vector, a pharmaceutically and/or physiologically acceptable excipient, carrier, buffer, stabilizer, antioxidants, preservative, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may be determined by the skilled person according to the route of administration.
  • the pharmaceutical composition is typically in liquid form.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Additional carriers are provided in International Patent Publication No. W02000/015822, entitled “Methods for treatment of degenerative retinal diseases,” incorporated herein by reference.
  • Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • a surfactant such as pluronic acid (PF68) 0.001% may be used.
  • Ringer's Injection, Lactated Ringer's Injection, or Hartmann's solution is used.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
  • compositions comprising a nucleic acid construct or vector disclosed herein may formulated with one or more pharmaceutically-acceptable excipients, which can be a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and/or a preservative.
  • a pharmaceutically-acceptable excipients such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and/or a preservative.
  • materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato star
  • a bulking agent is a compound which adds mass to a pharmaceutical formulation and contributes to the physical structure of the formulation in lyophilized form.
  • Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol and sorbitol.
  • a surfactant can reduce aggregation of the reconstituted protein and/or reduce the formation of particulates in the reconstituted formulation.
  • the amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution.
  • Suitable surfactants according to the present invention include polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g.
  • poloxamer 188 Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl- sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.
  • lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol e.g. Pluronics, PF68, etc.).
  • Preservatives may be used in formulations of invention. Suitable preservatives for use in the formulation of the invention include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 -pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, e.g., in "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995).
  • nucleic acid construct or vector disclosed herein may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.
  • a vector If a vector is to be stored long-term, it may be frozen in the presence of glycerol.
  • a method of inducing the expression of transgene comprising providing a cell comprising (i) a nucleic acid construct comprising an inflammation-inducible hybrid promoter disclosed herein operatively linked to a transgene or (ii) a vector comprising an inflammation-inducible hybrid promoter disclosed herein operatively linked to a transgene, and contacting the promoter with one or more inflammatory stimuli.
  • gene expression is induced by delivering an inflammationinducible hybrid promoter disclosed herein operatively linked to a transgene to a cell in a subject, wherein the subject produces inflammatory stimuli (including, but not limited to, cytokines or chemokines) that induce the expression of the transgene.
  • gene expression is induced by delivering an inflammation-inducible hybrid promoter disclosed herein operatively linked to a transgene to a cell in a subject, wherein the cell in the subject is exposed to exogenous inflammatory stimuli (including, but not limited to, LPS) that induce the expression of the transgene.
  • the inflammatory stimulus is a cytokine, a chemokine, a growth factor, a pathogen associated molecular pattern (PAMP), or a damage associated molecular pattern (DAMP).
  • the inflammation-inducible hybrid promoter responds to one or more of IL-1, IL-alpha, IL-1 beta, IL-2, IL-5, IL-4, IL-6, IL-7, IL-8, IL- 10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-25, tumor necrosis factor alpha (TNF-a), interferon-alpha (IFN-a), interferon-P (IFN-beta), interferon gamma (IFNy), granulate colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nuclear factor kappa-light-chain
  • TNF-a tumor necrosis
  • the inflammation-inducible hybrid promoter responds to lipopolysaccharides (LPS).
  • LPS lipopolysaccharides
  • the inflammation-inducible hybrid promoter responds to PAMPs (including, but not limited to, dsRNA, ssRNA, CpG DNA, peptidoglycan, flagellin, lipoteichoic acid (LT A), and diacyl lipopeptide).
  • the inflammation-inducible hybrid promoter responds to DAMPs (including, but not limited to, DNA, RNA, ATP, ECM components (glycoproteins, proteoglycans, glycosaminoglycans), defensins, heat shock proteins (HSP), HMGB-1, S-100 proteins, uric acid and uric acid crystals, mtDNA, and fatty acids).
  • DAMPs including, but not limited to, DNA, RNA, ATP, ECM components (glycoproteins, proteoglycans, glycosaminoglycans), defensins, heat shock proteins (HSP), HMGB-1, S-100 proteins, uric acid and uric acid crystals, mtDNA, and fatty acids).
  • Example 1 Identification of Inflammation-Inducible Promoters
  • a barcoded library containing around 300 million human genome DNA elements with an average insert size of about 300 base pairs was screened in HT1080, a human fibrosarcoma cell line. Elements that showed increased expression in the presence of TNF alpha (TNFa) and IL-1 beta (IL-1 (3) were subsequently used to create hybrid combinations, either by rationale design and fusing them to the NFkB-responsive CMV promoter by cloning, or randomly by creating a new library of hybrids of 96 elements and the NFkB-responsive CMV promoter, containing over 40,000 hybrid combinations on total which were then again screening using SuRE.
  • TNF alpha TNF alpha
  • IL-1 beta IL-1 (3)
  • NFkB-CMV promoter The NFkB-responsive CMV promoter, herein referred to as NFkB-CMV promoter (Khoury et al., Inflammation-inducible anti-TNF gene expression mediated by intra-articular injection of serotype 5 adeno-associated virus reduces arthritis. J Gene Med. 2007 Jul;9(7)), was used as a control. The most promising hybrid promoter candidates were subjected to further analysis (Table 2).
  • SEQ ID NO: 1 includes part of bacterial plasmid backbone.
  • SEQ ID NO: 10 is missing a 3’ “G” as compared to SEQ ID NO:9.
  • SEQ ID NO: 16 is missing 5’ “CTAGCA” as compared to SEQ ID NO:4.
  • SEQ ID NO:24 includes part of bacterial plasmid backbone.
  • SEQ ID NO:28 is missing 5’ “GT” as compared to SEQ ID NO:4.
  • SEQ ID NO:38 comprises a point mutation as compared to SEQ ID NO:37.
  • SEQ ID NO:39 comprises three point mutations as compared to SEQ ID NO: 19. Constructs marked with * were used in the Examples.
  • SEQ ID NOs:4-6, 16, and 28 may be replaced a different sequence selected from a group comprising SEQ ID NOs:4-6, 16, and 28.
  • SEQ ID NOs:9-12 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:9-12.
  • SEQ ID NOs: 19, 39, and 48 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:19, 39, and 48.
  • SEQ ID NOs:37, 38, and 49 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:37, 38, and 49.
  • SIN SEQ ID NO. [0174]
  • Example 2 Induction of Selected Promoters by Inflammatory Cytokines (Plasmid Transfections)
  • hybrid promoters were cloned into plasmids containing a single stranded AAV2 genome with luciferase as a reporter gene. Subsequently, the plasmids were transfected into HEK293T/17 cells (human embryonic kidney cells stably expressing the adenoviral El A and ElB-55k proteins and the SV40 large T antigen), HT1080 cells (human fibrosarcoma cell line), C2C12 cells (mouse myoblast cell line) cells, and primary fibroblast-like synoviocytes from rheumatoid arthritis patients (RA-FLS cells), respectively. The cells were stimulated with cytokines and luciferase expression was determined.
  • hybrid promoters g201, g202, g210, g211, g221, and g229 were PCR amplified using primers flanking Mlul and Hindlll restriction sites. After PCR amplification, the DNA was loaded and run on a 0.6% agarose gel to isolate fragments with the right size, and after gel extraction and DNA isolation the DNA was digested with Mlul and Hindlll restriction enzymes. Digested elements were then put again on DNA gel electrophoresis, 0.6% agarose, and bands were successfully isolated (-500 bp).
  • the hybrids were ligated into cut pART548 backbone.
  • the ligation mixture was transformed to E. coli XL 10 Gold cells and the constructs were checked by restriction digestions and sequencing.
  • the ITRs and expression cassette were sequenced confirming the presence of the ITRs and to confirm the sequence of the hybrid promotes.
  • the final recombinant AAV plasmids contained AAV2 ITRs, a hybrid promoter of interest, a LUC2P reporter gene and the human growth hormone polyadenylation signal (hGHpA) (Fig. 1).
  • Hybrid promoters gl87, gl90, g231, ng007, ng008 and ng012 were cloned in the EcoRV site of plasmid pUC57. Subsequently, these plasmids were digested with Mlul and Hindlll restriction enzymes and ligated in pART548 digested with the same enzymes, as described above. The ITRs and expression cassette were sequenced confirming the presence of the ITRs and the sequence of the hybrid promoters.
  • HEK293T/17 and HT1080 were seeded at a density of 50,000 cells per well in 200 pl total volume in 96 well plates, whereas C2C12 cells were seeded at 10,000 cells per well in 300 pl total volume in 48 well plates. The cells were incubated at 37 °C, 5% CO2. For all cell lines, DMEM + GlutaMAX-I (Gibco) supplemented 10% (v/v) Fetal Bovine Serum (FBS), and 1% (v/v) Gibco® Antibiotic- Antimycotic was used.
  • HEK293T/17 and HT1080 cells were transfected using the Invitrogen LipofectamineTM 2000 Transfection Reagent, and C2C12 cells using the Invitrogen LipofectamineTM 3000 Transfection Reagent by adding the Lipofectamine/plasmid DNA/Opti-MEM® I medium (Gibco) mixture, according to the manufacturer’s instructions. After 48 h of incubation with the Lipofectamine/plasmid DNA complexes at 37 °C, 5% CO2, cells were stimulated with cytokines (10 ng/mL TNFa and 1 ng/mL IL-ip).
  • the medium of the cells containing the plasmid and lipofection reagent was exchanged for fresh DMEM + GlutaMAX-I medium supplemented 10% (v/v) Fetal Bovine Serum (FBS), and 1% (v/v) Gibco® Antibiotic- Antimycotic with or without the cytokines.
  • the medium composition for cytokine stimulation was DMEM + GlutaMAX-I (Gibco) supplemented 1% (v/v) Fetal Bovine Serum (FBS), and 0.5% (v/v) Gibco® Antibiotic- Antimycotic, with or without the cytokines.
  • Cells were incubated for another 24 h and were either directly harvested and lysed to measure luciferase activity, or first assessed for cell death and toxicity using the Cell Counting kit-8 (CCK8) from Dojindo.
  • CCK8 reagent 30 pL was added to each well, the plates were incubated for 2 h at 37 °C, and absorbance of the plate measured at 450 nm. After measurement of cell toxicity/cell viability, the media of the cells was removed, and the cells were all washed with PBS buffer, except HEK293T/17 cells since they are prone to detachment. After washing, cells were subsequently harvested and lysed using passive lysis buffer from Promega. Finally, luciferase activity was measured in the lysates using the One gio substrate from Promega. The luciferase signal was measured with the Omega fluostar machine.
  • a separate transfection experiment was performed with C2C12 cells and plasmids containing the gl90, g202, g210, g211 and g221 hybrid promoters. To mimic cytokine levels in different inflammatory murine models, the amount and type of cytokines was varied. In addition, Lipopolysaccharide (LPS) was used as an inflammatory component. C2C12 cells were seeded at a density of 5,000 cells per well in 200 pl total volume in 96 well plates, 24 h prior to transfection. Transfection was performed using Invitrogen LipofectamineTM 3000 Transfection Reagent according to manufacturer’s instructions.
  • LPS Lipopolysaccharide
  • cells were stimulated with cytokines, using three different stimulation conditions, namely 10 pg/mL IL-ip, 5 pg/mL TNFa, and 50 pg/mL IL-6; or 50 pg/mL IL-ip, 100 pg/mL TNFa, and 50,000 pg/mL IL-6; or 5 pg/mL LPS.
  • FBS Fetal Bovine Serum
  • Gibco® Antibiotic-Antimycotic 1% (v/v) Gibco® Antibiotic-Antimycotic, with or without the stimulatory agents.
  • cells were harvested and lysed using passive lysis buffer from Promega.
  • luciferase activity was measured in the lysates using the One gioTM substrate from Promega, similar to prior experiments. The Luciferase signal was measured with the Omega fluostar machine.
  • RA-FLS cells were cultured in T175 flasks in DMEM+GlutaMAX-I (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) IM Hepes (Gibco) and 1% (v/v) Gibco® Antibiotic- Antimycotic at 37 °C, 5% CO2 prior to nucleofection experiments in order to reach proper cell densities for nucleofections. Once the cells had reached proper cell densities for nucleofection experiments, they were trypsinized and counted. Subsequently, the cells were centrifuged, the supernatant was removed, and the cells were resuspended.
  • cells were plated in 24 well plates at densities of 30,000, 60,000 or 40,000 cells per well in the in DMEM+ GlutaMAX-I (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) IM Hepes (Gibco) and 1% (v/v) Gibco® Antibiotic- Antimycotic and incubated at 37 °C, 5% CO2.
  • the next day the medium of the cells was refreshed. 48 h after transfection, cells were stimulated with cytokines. To this end, the medium of the cells was refreshed with new medium with or without the cytokines (10 ng/mL TNFa and 1 ng/mL IL-ip).
  • cells were plated in 48 well plates, at 20,000 cells per well in DMEM+GlutaMAX-I (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) IM Hepes (Gibco) and 1% (v/v) Gibco® Antibiotic-Antimycotic and incubated at 37 °C, 5% CO2. 48 h after transfection, cells were stimulated with cytokines.
  • the medium of the cells was refreshed with new DMEM+GlutaMAX-I (Gibco) medium supplemented 1% (v/v) Fetal Bovine Serum (FBS), and 0.5% (v/v) Gibco® Antibiotic- Antimycotic with or without the cytokines (10 ng/mL TNFa and 1 ng/mL IL-ip).
  • hybrid promoters For hybrid promoters, gl90, g201, g202, g210, g211, g221, g229, and g231, cells were incubated for another 48 hand subsequently they were lysed using passive lysis buffer from Promega. Finally, luciferase activity was measured in the lysates using the One gioTM substrate from Promega. The Luciferase signal was measured with the Omega fluostar machine. For hybrid promoters, gl87, ng007, ng008 and ng012, cells were incubated for 48 h, following a cell death and toxicity assessment, using the Cell Counting kit-8 (CCK8) from Dojindo.
  • CCK8 Cell Counting kit-8
  • CCK8 reagent 30 pL was added to each well, the plates were incubated for 2 h at 37 °C, and absorbance of the plate measured at 450 nm. After measurement of cell toxicity/cell viability, the media of the cells was removed, and the cells were all washed with PBS buffer. After washing, cells were lysed and Luciferase signal was measured similar to previous group of hybrid promoters.
  • Inflammation-inducible promoters show increased reporter gene expression in the presence of inflammatory stimulants in a variety of cell types.
  • Results for HEK293T/17 cells are shown in Figs. 3A, 4A, 5A, 6A, 7A, and 9A.
  • Results for HT1080 cells are shown in Figs. 2A, 8A, 10A, 11A, 12A, and 13A.
  • Results for C2C12 cells are shown in Figs. 2B, 2E, 3D, 4B, 4E, 5D, 6D, 8B, 10B, 11B, and 12B.
  • Results for primary RA-FLS are shown in Figs. 2D, 3C, 4D, 5C, 6C, 7C, 8C, 9B, 10C, 11C, 12C, and 13B.
  • Inflammation-inducible promoters show increased reporter gene expression in the presence of different inflammatory stimulants (see, e.g., Figs. 2E, 3D, 4E, 5D, and 6D).
  • Example 3 Induction of Selected Promoters by Inflammatory Cytokines (AAV Vector Transduction)
  • the AAV2 genomes containing a s luciferase reporter gene under the control of selected hybrid promoters were packed into AAV5 capsids.
  • the AAV2/5 viral particles were used to transduce HEK293T/17, mFLS AXR7, or RA-FLS, respectively.
  • Viruses were formulated in PBS formulation buffer pH 7.3 with 0.001% Pluronic®F- 68. Titers (Vector Genomes/mL) was determined by qPCR.
  • AAV transduction experiments were done in a 48 well plate format. The following cell lines with their specific seeding cell densities were used: HEK293T/17 cells were seeded at 20,000 cells per well, in 300 pL total volume; mouse FLS AXR7 and RA-FLS were seeded at 8,000 cells per well in 300 pL total volume. After 24 hours, cells were transduced with the rAAV vectors (diluted in DMEM + GlutaMAX-I (Gibco), without FBS and without Gibco® Antibiotic- Antimycotic ) at a multiplicity of infection in terms of VG/cell (MOI) of 200,000.
  • DMEM + GlutaMAX-I Gibco
  • MOI Gibco® Antibiotic- Antimycotic
  • the cytokines were either human or mouse IL-ip and TNFa, depending on the cell type (1 ng/mL IL-ip and 10 ng/mL TNFa in DMEM + GlutaMAX-I (Gibco) supplemented with 1% (v/v) Fetal Bovine Serum, and 0.5% (v/v) Gibco® Antibiotic- Antimycotic). If applicable, cells were incubated with or without these cytokines for 72 h and cell toxicity was assessed using the Cell Counting kit-8 (CCK8) from Dojindo.
  • CCK8 Cell Counting kit-8
  • CCK8 reagent 30 pL was added to each well, the plates were incubated for 2 h at 37 °C, and absorbance of the plate measured at 450 nm. After measurement of cell toxicity/cell viability, the media of the cells was removed, and the cells were all washed with PBS buffer except HEK293T/17 cells since they are prone to detachment. After washing, cells were lysed using 60 pL passive lysis buffer from Promega, at each well. Then samples were used for One gioTM luciferase measurement, using One gioTM substrate from Promega. The Luciferase signal was measured with the Omega fluostar machine. The measurement was done at different machine gain values due to the differences of the signal found in different cell lines and donors.
  • Inflammation-inducible promoters show increased reporter gene expression in the presence of inflammatory stimulants in a variety of cell types.
  • Results for HEK293T/17 cells are shown in Figs. 2A, 3A, 4A, 5A, 6A, 7A, and 13A.
  • Results for murine FLS are shown in Figs. 2C, 3B, 4C, 5B, 6B, and 7B.
  • Results for primary RA-FLS are shown in Figs. 2D, 3C, 4D, 5C, 6C, 7C, and 13B
  • SIN SEQ ID NO. Underlined (i.e., (1) simply underlined or (2) bold and underlined) sequence may be replaced fully or partially by an alternative sequence or may be omitted.
  • SEQ ID NOs:4-6, 16, and 28 may be replaced a different sequence selected from a group comprising SEQ ID NOs:4-6, 16, and 28.
  • SEQ ID NOs:9-12 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:9-12.
  • SEQ ID NOs:19, 39, and 48 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs: 19, 39, and 48.
  • SEQ ID NOs:37, 38, and 49 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:37, 38, and 49,

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Abstract

Provided herein are inflammation-inducible hybrid promoters. Further provided are constructs as well as vectors comprising such promoters. Also provided herein are methods of using the inflammation-inducible hybrid promoters, constructs, and vectors disclosed herein, including, but not limited to, methods for the regulation of gene expression in response to inflammatory stimulation.

Description

INFLAMMATION-INDUCIBLE PROMOTERS
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0001] This application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on April 4, 2024, is named SeqList -162027-51676. xml and is 62,557 bytes in size.
FIELD OF THE INVENTION
[0002] The application relates to promoters that are able to enhance expression of genes in response to inflammatory stimuli. The application further relates to methods employing these promoters and uses of these promoters. Expression cassettes and vectors containing these promoters are also disclosed. These are particularly useful for applications using gene therapy.
BACKGROUND
[0003] Gene therapy vectors, including adeno-associated virus (AAV)-based gene therapy vectors, are promising candidates for the treatment of inflammatory diseases that result from a biological response of the immune system triggered by a variety of different factors. Regulatory elements, including promoters and enhancers, can be engineered for use in AAV vectors to optimize the strength, kinetics, and specificity of transgene expression. Specifically, the incorporation of promoters inducible by inflammation can help to reduce the risk for side effects due to overexpression of and/or continuous exposure to the anti-inflammatory therapeutic protein by such AAV vectors.
[0004] Accordingly, inflammation-inducible promoters or c/.s-regulatory elements that show improved inducibility in response to inflammatory stimuli are urgently needed.
SUMMARY OF THE INVENTION
[0005] Provided herein are inflammation-inducible hybrid promoters, constructs comprising such promoters, and methods of using thereof, including, but not limited to, the regulation of gene expression in response to inflammatory stimulation.
[0006] Provided herein is an inflammation-inducible promoter, the promoter comprising: (a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to any one of SEQ ID NOs:19, 39, or 48, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13;
(i) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 80% identical to any one of SEQ ID NOs:37, 38, or 49, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0007] In embodiments, the inflammation-inducible promoter comprises from 5’ to 3’ :
(a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; (d) a sequence that is at least 80% identical to any one of SEQ ID NOs:19, 39, or 48, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13;
(i) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 80% identical to any one of SEQ ID NOs:37, 38, or 49, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0008] In embodiments, the inflammation-inducible promoter comprises:
(a) a sequence that is at least 90% identical to SEQ ID NO:2, a sequence that is at least 90% identical to SEQ ID NO:3, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 90% identical SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO: 15 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to any one of SEQ ID NOs:19, 39, or 48, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 90% identical to SEQ ID NO:21 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 90% identical to SEQ ID NO:23 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; (g) a sequence that is at least 90% identical to SEQ ID NO:25, a sequence that is at least 90% identical to SEQ ID NO:26, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 90% identical to SEQ ID NO: 13;
(i) a sequence that is at least 90% identical to SEQ ID NO:30 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 90% identical to SEQ ID NO:32 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 90% identical to SEQ ID NO:34 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 90% identical to any one of SEQ ID NOs:37, 38, or 49, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0009] In embodiments, the inflammation-inducible promoter comprises from 5’ to 3’ :
(a) a sequence that is at least 90% identical to SEQ ID NO:2, a sequence that is at least 90% identical to SEQ ID NO:3, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 90% identical SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO: 15 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to any one of SEQ ID NOs:19, 39, or 48, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 90% identical to SEQ ID NO:21 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 90% identical to SEQ ID NO:23 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 90% identical to SEQ ID NO:25, a sequence that is at least 90% identical to SEQ ID NO:26, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 90% identical to SEQ ID NO: 13;
(i) a sequence that is at least 90% identical to SEQ ID NO:30 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; (j) a sequence that is at least 90% identical to SEQ ID NO:32 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 90% identical to SEQ ID NO:34 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 90% identical to any one of SEQ ID NOs:37, 38, or 49, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0010] In embodiments, the inflammation-inducible promoter comprises:
(a) SEQ ID NO:2, SEQ ID NO:3, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs:9-12 and SEQ ID NO: 13;
(c) SEQ ID NO: 15 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) any one of SEQ ID NOs: 19, 39, or 48, and any one of SEQ ID NOs:4-6, 16, or 28;
(e) SEQ ID NO:21 and any one of SEQ ID NOs:4-6, 16, or 28;
(f) SEQ ID NO:23 and any one of SEQ ID NOs:4-6, 16, or 28;
(g) SEQ ID NO:25, SEQ ID NO:26, and any one of SEQ ID NOs:4-6, 16, or 28;
(h) any one of SEQ ID NOs:4-6, 16, or 28, and SEQ ID NO: 13;
(i) SEQ ID NO:30 and any one of SEQ ID NOs:4-6, 16, or 28;
(j) SEQ ID NO:32 and any one of SEQ ID NOs:4-6, 16, or 28;
(k) SEQ ID NO:34 and any one of SEQ ID NOs:4-6, 16, or 28; or
(l) any one of SEQ ID NOs:37, 38, or 49, and any one of SEQ ID NOs:4-6, 16, or 28.
[0011] In embodiments, the inflammation-inducible promoter comprises from 5’ to 3’ :
(a) SEQ ID NO:2, SEQ ID NO:3, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs: 9- 12 and SEQ ID NO: 13;
(c) SEQ ID NO: 15 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) any one of SEQ ID NOs: 19, 39, or 48, and any one of SEQ ID NOs:4-6, 16, or 28;
(e) SEQ ID NO:21 and any one of SEQ ID NOs:4-6, 16, or 28;
(f) SEQ ID NO:23 and any one of SEQ ID NOs:4-6, 16, or 28;
(g) SEQ ID NO:25, SEQ ID NO:26, and any one of SEQ ID NOs:4-6, 16, or 28;
(h) any one of SEQ ID NOs:4-6, 16, or 28, and SEQ ID NO: 13;
(i) SEQ ID NO:30 and any one of SEQ ID NOs:4-6, 16, or 28;
(j) SEQ ID NO:32 and any one of SEQ ID NOs:4-6, 16, or 28;
(k) SEQ ID NO:34 and any one of SEQ ID NOs:4-6, 16, or 28; or
(l) any one of SEQ ID NOs:37, 38, or 49, and any one of SEQ ID NOs:4-6, 16, or 28.
[0012] In embodiments, the inflammation-inducible promoter comprises:
(a) a sequence that is at least 80% identical to SEQ ID NO: 1; (b) a sequence that is at least 80% identical to SEQ ID NO:7 or SEQ ID NO:8;
(c) a sequence that is at least 80% identical to SEQ ID NO: 14;
(d) a sequence that is at least 80% identical to SEQ ID NO: 17 or SEQ ID NO: 18;
(e) a sequence that is at least 80% identical to SEQ ID NO:20;
(f) a sequence that is at least 80% identical to SEQ ID NO:22;
(g) a sequence that is at least 80% identical to SEQ ID NO:24;
(h) a sequence that is at least 80% identical to SEQ ID NO:27;
(i) a sequence that is at least 80% identical to SEQ ID NO:29;
(j) a sequence that is at least 80% identical to SEQ ID NO:31;
(k) a sequence that is at least 80% identical to SEQ ID NO:33; or
(l) a sequence that is at least 80% identical to SEQ ID NO :35 or SEQ ID NO:36.
[0013] In embodiments, the inflammation-inducible promoter comprises:
(a) a sequence that is at least 90% identical to SEQ ID NO: 1;
(b) a sequence that is at least 90% identical to SEQ ID NO:7 or SEQ ID NO:8;
(c) a sequence that is at least 90% identical to SEQ ID NO: 14;
(d) a sequence that is at least 90% identical to SEQ ID NO: 17 or SEQ ID NO: 18;
(e) a sequence that is at least 90% identical to SEQ ID NO:20;
(f) a sequence that is at least 90% identical to SEQ ID NO:22;
(g) a sequence that is at least 90% identical to SEQ ID NO:24;
(h) a sequence that is at least 90% identical to SEQ ID NO:27;
(i) a sequence that is at least 90% identical to SEQ ID NO:29;
(j) a sequence that is at least 90% identical to SEQ ID NO:31;
(k) a sequence that is at least 90% identical to SEQ ID NO:33; or
(l) a sequence that is at least 90% identical to SEQ ID NO:35 or SEQ ID NO:36.
[0014] In embodiments, the inflammation-inducible comprises any one of SEQ ID NOs:l, 7, 8, 14, 17, 18, 20, 22, 24, 27, 29, 31, 33, 35, or 36.
[0015] Provided herein is an expression construct comprising (1) an inflammation-inducible promoter disclosed herein; and (2) an operatively linked transgene. In one embodiment, the transgene encodes a therapeutic protein. In one embodiment, the transgene encodes a therapeutic nucleic acid.
[0016] Provided herein is a recombinant nucleic acid comprising an inflammation-inducible promoter disclosed herein or an expression construct disclosed herein.
[0017] Provided herein is a vector comprising an inflammation-inducible promoter disclosed herein or an expression construct disclosed herein. In one embodiment, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of adenoviral vector, adeno-associated virus (AAV) vector, and lentiviral vector. In one embodiment, the vector comprises an AAV2 genome. In some embodiments, the vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV.rh8, AAV.rhlO, AAV.rh39, AAV.43, AAV2/2-66, AAV2/2-84, or AAV2/2-125 capsid. In some embodiments, the vector comprises a capsid protein comprising any one of SEQ ID NOs:40-47. In one embodiment, the vector is a non-viral vector. In some embodiments, the vector is a plasmid, bacmid, cosmid, or, closed-ended linear DNA (ceDNA).
[0018] Provided herein is a composition comprising a vector disclosed herein and a pharmaceutically acceptable excipient.
[0019] Provided herein is a cell comprising an inflammation-inducible promoter disclosed herein, an expression construct disclosed herein, or a vector disclosed herein. In one embodiment, the cell is an isolated cell. In one embodiment, the cell is a fibroblast-like synoviocyte, cartilage cell, chondrocyte, or chondroblast.
[0020] Provided herein is a method of inducing the expression of a transgene, the method comprising: (1) providing a cell comprising an inflammation-inducible promoter disclosed herein, an expression construct disclosed herein, or a vector disclosed herein; and (2) contacting the promoter with one or more inflammatory stimuli. In some embodiments, the one or more inflammatory stimuli are selected from the group consisting of inflammatory cytokines, inflammatory chemokines, growth factors, pathogen associated molecular patterns (PAMP), or damage associated molecular patterns (DAMP). In some embodiments, the one or more inflammatory stimuli are one or more inflammatory cytokines or chemokines. In embodiments, the one or more inflammatory cytokines or chemokines are selected from the group consisting of IL-1, IL-2, IL-5, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL- 22, IL-23, IL-25, tumor necrosis factor alpha (TNF-a), interferon-alpha (IFN-a), interferon-P (IFN-beta), interferon gamma (IFNy), granulate colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nuclear factor kappa-light- chain-enhancer of activated B cells (NF-KB), macrophage inflammatory protein- la (MIP-la = C-X-C motif chemokine 2 (CCL2)), CCL3, CCL4, CCL5, RANTES, stromal cell-derived factor 1 (SDF-1 = C-X-C motif chemokine 12 (CXCL12)), Neutrophil-activating protein 3 (NAP-3 (CXCL1)), monocyte chemoattractant protein (MCP-1), CXCL8, IFN-y-induced protein (IP- 10 (CXCL-10)), lymphotactin, fractalkine, and combinations thereof. In some embodiments, the one or more inflammatory stimuli are lipopolysaccharides. [0021] In some embodiments, one or more inflammatory stimuli are IL-1, IL-6, IL-8, IL- 17, IL-23, TNF-a, (IP- 10), or combinations thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0022] Fig. 1 illustrates the genetic components of the AAV vector incorporated in the plasmids used for transfection. ITR= Inverted Terminal Repeat of AAV2; LUC2P = firefly luciferase reporter gene; hGHpA = human growth hormone polyadenylation signal.
[0023] Figs. 2A, 2B, 2C, 2D, and 2E illustrate inflammation-inducible expression of hybrid promoter gl90. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise). Fig. 2A. HT1080 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters (left panel; transfection). HEK293T/17 cells were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters (right panel; transduction). Fig. 2B. C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 2C. Murine fibroblast-like synoviocytes (mFLS) were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 2D. Human rheumatoid arthritis (RA)-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 2E. C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Stimulation 1 : IL1-P: 10 pg/mL; TNFa: 5 pg/mL, IL-6: 50 pg/mL. Stimulation 2: IL1-P: 50 pg/mL; TNFa: 100 pg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
[0024] Figs. 3A, 3B, 3C, and 3D illustrate inflammation-inducible expression of hybrid promoter g202. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise). Fig. 3A. HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 3B. mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 3C. Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 3D. C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Stimulation 1 : IL1-P: 10 pg/mL; TNFa: 5 pg/mL; IL-6: 50 pg/mL. Stimulation 2: IL1-P: 50 pg/mL; TNFa; lOOpg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
[0025] Figs. 4A, 4B, 4C, 4D, and 4E illustrate inflammation-inducible expression of hybrid promoter g210. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise). Fig. 4A. HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 4B. C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 4C. mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 4D. Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 4E. C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Stimulation 1 : IL1-P: 10 pg/mL; TNFa: 5 pg/mL; IL-6: 50 pg/mL. Stimulation 2: IL1-P: 50 pg/mL; TNFa: 100 pg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
[0026] Figs. 5A, 5B, 5C, and 5D illustrate inflammation-inducible expression of hybrid promoter g211. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise). Fig. 5A. HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 5B. mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 5C. Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 5D. C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Stimulation 1 : IL1-P: 10 pg/mL; TNFa: 5pg/mL; IL-6: 50 pg/mL. Stimulation 2: IL1-P: 50 pg/mL; TNFa: lOOpg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL. [0027] Figs. 6A, 6B, 6C, and 6D illustrate inflammation-inducible expression of hybrid promoter g221. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip (unless indicated otherwise). Fig. 6A. HEK293T/17 cells were lipofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 6B. mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 6C. Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 6D. C2C12 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Stimulation 1 : IL1-P: 10 pg/mL; TNFa: 5pg/mL; IL-6: 50 pg/mL. Stimulation 2: IL1-P: 50 pg/mL; TNFa: 100 pg/mL; IL-6: 50000 pg/mL; LPS: 5 pg/mL.
[0028] Figs. 7A, 7B, and 7C illustrate inflammation-inducible expression of hybrid promoter g231. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip. Fig. 7A. HEK293T/17 cells were lipofected with plasmids (left panel; nucleofection/transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 7B. mFLS were transduced with an AAV vector comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 7C. Human RA-FLS were nucleofected with plasmids (left panel; nucleofection/transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters.
[0029] Figs. 8A, 8B, and 8C illustrate inflammation-inducible expression of hybrid promoter gl87. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip. HT1080 cells (Fig. 8A) or C2C12 (Fig. 8B), respectively, were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters, human RA-FLS (Fig. 8C) were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
[0030] Figs. 9A and 9B illustrate inflammation-inducible expression of hybrid promoter g201. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip. Fig. 9A. HEK293T/17 cells were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 9B. Human RA-FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
[0031] Figs. 10A, 10B, and 10C illustrate inflammation-inducible expression of hybrid promoter ng007. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip. HT1080 cells (Fig. 10A) or C2C12 (Fig. 10B), respectively, were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 10C. Human RA- FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
Figs. HA, 11B, and 11C illustrate inflammation-inducible expression of hybrid promoter ng008. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip. HT1080 cells (Fig. HA) or C2C12 (Fig. 11B), respectively, were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 11C. Human RA-FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
[0032] Figs. 12A, 12B, and 12C illustrate inflammation-inducible expression of hybrid promoter ng012. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip. HT1080 cells (Fig. 12A) or C2C12 (Fig. 12B), respectively, were lipofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 12C. Human RA- FLS were nucleofected with plasmids comprising the reporter gene luciferase under the control of the indicated promoters.
[0033] Figs. 13A, and 13B illustrate inflammation-inducible expression of hybrid promoter g229. The NFkB-CMV promoter served as a control. Reporter gene expression is shown with and without stimulation of 10 ng/mL TNFa and 1 ng/mL IL-ip. Fig. 13A. HT1080 cells lipofected with plasmids (left panel; transfection) comprising the reporter gene luciferase under the control of the indicated promoters. HEK293T/17 cells were transduced with an AAV vector (right panel; transduction) comprising the reporter gene luciferase under the control of the indicated promoters. Fig. 13B. Human RA-FLS were nucleofected with plasmids (left panel; transfection) or transduced with an AAV vector (right panel; transduction), respectively, comprising the reporter gene luciferase under the control of the indicated promoters. [0034] Fig. 14 illustrates NFKB activation perpetuates chronic inflammation by targeting genes involved in inflammation during RA development. Adopted from Nejatbakhsh Samimi et al., NF-KB signaling in rheumatoid arthritis with focus on fibroblast-like synoviocytes. Auto Immun Highlights. 2020 Aug 8; 11(1): 11, incorporated herein in its entirety.
DETAILED DESCRIPTION
[0035] Provided herein are inflammation-inducible hybrid promoters, constructs comprising such promoters, and methods of using thereof, including, but not limited to, the regulation of gene expression in response to inflammatory stimulation.
[0036] Promoters
[0037] Traditionally, promoters are defined as DNA regions where transcription is initiated. The promoter is typically located at the 5' end of the gene (or transcription unit) that the promoter regulates. Promoters contain DNA sequences and response elements that provide an initial binding site for the RNA transcriptional machinery (including RNA polymerase and transcription factors that recruit the RNA polymerase). Transcription factors bind specific activator and repressor sequences that attach to certain promoters and regulate gene expression. Since the promoter region controls when and where a gene of interest is expressed in an organism, promoters are crucial elements for regulating the level and specificity of transgene expression, particularly in the context of gene therapy.
[0038] Incorporation of stronger promoters into viral gene expression constructs can increase potency and efficacy at lower viral vector doses, thereby potentially decreasing safety risks, immune responses, and reducing cost of vector production. Likewise, the use of promoters that are activated by signaling cues from the microenvironment (e.g., the presence of inflammatory cytokines) allows for expression of therapeutic transgenes when and where they are needed, thereby minimizing the side effects caused by constitutive expression.
[0039] Provided herein are inflammation-inducible hybrid promoters. As used herein, a “hybrid promoter” comprises multiple elements, including, but not limited to, multiple synthetic elements, or elements from one or more different promoter regions. A hybrid promoter can contain one or more enhancer elements and one or more activation sequences, and/or elements that are differentially regulated.
[0040] As used herein an “inflammation-inducible promoter” is a promoter that increases the expression of a gene operably linked to the promoter in response to inflammatory stimuli. The word “inflammation” or “inflammatory” can refer to a local increase in lymphocytes such as monocytes, T-cells, B-cells, leukocytes, natural-killer (NK) cells, macrophages or the like. The word “inflammation” or “inflammatory” can refer to a local increase of the concentration of the interleukins, interferons, tumor necrosis factor or prostaglandins, leukotrienes or other small signaling molecules. Inflammation can also refer to a systemic response. For example, a systemic inflammatory reaction may arise as a response to a range of insults to the host. Inflammatory agents include toxins or pathogens such as virus or bacteria. Inflammation may also arise from an autoimmune response to the host’s own proteins; in reaction to injury; or as an immune response to allergens. Acute inflammation may be serious and result in pathologic consequences. Some of the specific cytokines known to be involved in an acute inflammatory response include IL-1, TNF-a and IL-6. Ways of testing whether a promoter is inducible by inflammation are known in the art. For example, the person skilled in the art may compare expression of a transgene operatively linked to the inflammation-inducible promoter in the presence or absence of one or more inflammatory stimuli, including, but not limited to, inflammatory cytokines, inflammatory chemokines, or LPS. Non-limiting examples of measuring whether a promoter is inducible by inflammation are provided in the Examples.
[0041] As used herein, “operatively linked” refers to a first molecule joined to a second molecule, wherein the two molecules are so arranged that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operatively linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell. Additionally, two portions of a transcription regulatory element (such as a promoter) are operatively linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two transcription regulatory elements may be operatively linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operatively linked to one another with no intervening nucleotides present.
[0042] In embodiments, the inflammation-inducible hybrid promoters disclosed herein can be used for the expression of transgenes in particular tissues in different pathological situations in which an inflammatory component is present. In embodiments, the inflammation-inducible hybrid promoters disclosed herein are used for expression of transgenes in fibroblast-like synoviocytes (FLS), cartilage cells, chondrocytes, or chondroblasts. In embodiments, the inflammation-inducible hybrid promoters disclosed herein are useful for the expression of transgenes for the treatment of arthritis, including, but not limited to, rheumatoid arthritis (RA) and osteoarthritis (OA). The pathology of arthritis involves joint swelling and synovial inflammation (Fig. 14).
[0043] In other embodiments, the inflammation-inducible hybrid promoters disclosed herein can also be used to control the rate and extent of expression of a gene encoding an antiinflammatory protein in a negative feedback manner. For this, an inflammation-inducible hybrid promoters disclosed herein is operatively linked to a gene encoding an antiinflammatory protein. In the absence of inflammation, little or no anti-inflammatory protein will be produced. When inflammation occurs, the anti-inflammatory protein will be produced according to the intensity of the inflammatory reaction (c.g, in response to circulating inflammatory cytokines or other mediators). Anti-inflammatory protein production will limit the extent and duration of the inflammatory reaction, avoiding dangerous (toxic) excess. As the inflammatory reaction wanes, production of the anti-inflammatory protein will decrease, thereby avoiding prolonged immunosuppression.
[0044] In embodiments, provided is an inflammation-inducible hybrid promoter comprising one or more elements disclosed in Tables 2 or 3. A person skilled in the art will appreciate that certain modifications can be made to the inflammation-inducible hybrid promoters disclosed herein without eliminating the inflammation-inducible property of the hybrid promoter. For example, provided are inflammation-inducible hybrid promoters that comprise the elements of another inflammation-inducible hybrid promoter disclosed herein, wherein the 5’ to 3’ order of the elements has been changed. Further, the orientation of one or more of the elements of a hybrid promoter disclosed herein can be changed such that the reverse version or the reverse compliment version of a given element is used in a hybrid promoter. Further, provided are inflammation-inducible hybrid promoters that include sequence in addition (e.g., added at either end or inserted within the hybrid promoter sequence) to the elements of a hybrid promoter disclosed herein. Likewise, provided are inflammation-inducible hybrid promoters in which certain sequence has been removed from the hybrid promoters disclosed herein (e.g., as a terminal or internal deletion). As discussed herein, many ways of testing whether a promoter remain inducible by inflammation after a modification are known in the art. The inflammationinducible hybrid promoter may comprise any of the elements disclosed in Tables 2 or 3 in the 5’-> 3’ orientation indicated in Tables 2 or 3 or a reverse or a reverse compliment form.
[0045] Also provided herein are inflammation-inducible hybrid promoters that share certain percent sequence identity with an inflammation-inducible hybrid promoter disclosed herein. As used herein, the term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position. A degree identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. Methods and computer programs for determining both sequence identity and similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11 ; 12(1 Pt 1):387-95), BLASTP, BLASTN, FASTA (Altschul et al., Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403-10), and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http://www.ncbi.nlm.nih.gov/blast/). In comparing sequences, these methods account for various substitutions, deletions, and other modifications.
[0046] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising:
(a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; (h) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13;
(i) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 80% identical to SEQ ID NO:37 or SEQ ID NO: 38, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0047] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising:
(a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
[0048] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising:
(a) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; (d) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(e) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0049] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising:
(a) a sequence that is at least 90% identical to SEQ ID NO:2, a sequence that is at least 90% identical to SEQ ID NO:3, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 90% identical SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO: 15 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 90% identical to SEQ ID NO:21 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(f) a sequence that is at least 90% identical to SEQ ID NO:23 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0050] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising:
(a) a sequence that is at least 90% identical to SEQ ID NO:25, a sequence that is at least 90% identical to SEQ ID NO:26, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 90% identical to SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO:30 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to SEQ ID NO:32 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(e) a sequence that is at least 90% identical to SEQ ID NO:34 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0051] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising: (a) SEQ ID N0:2, SEQ ID N0:3, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs:9-12 and SEQ ID NO: 13;
(c) SEQ ID NO: 15 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) SEQ ID NO: 19 or SEQ ID NO:39, and any one of SEQ ID NOs:4-6, 16, or 28;
(e) SEQ ID NO:21 and any one of SEQ ID NOs:4-6, 16, or 28; or
(f) SEQ ID NO:23 and any one of SEQ ID NOs:4-6, 16, or 28.
[0052] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising:
(a) SEQ ID NO:25, SEQ ID NO:26, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs:4-6, 16, or 28, and SEQ ID NO: 13;
(c) SEQ ID NO:30 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) SEQ ID NO:32 and any one of SEQ ID NOs:4-6, 16, or 28; or
(e) SEQ ID NO:34 and any one of SEQ ID NOs:4-6, 16, or 28.
[0053] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising from 5’ to 3’ :
(a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13; (i) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 80% identical to SEQ ID NO:37 or SEQ ID NO: 38, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0054] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising from 5’ to 3’ :
(a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
[0055] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising from 5’ to 3’ :
(a) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or (e) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0056] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising from 5’ to 3’ :
(a) a sequence that is at least 90% identical to SEQ ID NO:2, a sequence that is at least 90% identical to SEQ ID NO:3, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 90% identical SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO: 15 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 90% identical to SEQ ID NO:21 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(f) a sequence that is at least 90% identical to SEQ ID NO:23 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0057] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising from 5’ to 3’ :
(a) a sequence that is at least 90% identical to SEQ ID NO:25, a sequence that is at least 90% identical to SEQ ID NO:26, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 90% identical to SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO:30 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to SEQ ID NO:32 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(e) a sequence that is at least 90% identical to SEQ ID NO:34 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
[0058] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising from 5’ to 3’ :
(a) SEQ ID NO:2, SEQ ID NO:3, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs:9-12 and SEQ ID NO: 13; (c) SEQ ID N0: 15 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) SEQ ID NO: 19 or SEQ ID NO:39, and any one of SEQ ID NOs:4-6, 16, or 28;
(e) SEQ ID NO:21 and any one of SEQ ID NOs:4-6, 16, or 28; or
(f) SEQ ID NO:23 and any one of SEQ ID NOs:4-6, 16, or 28.
[0059] Provided herein is an inflammation-inducible hybrid promoter, the promoter comprising from 5’ to 3’ :
(a) SEQ ID NO:25, SEQ ID NO:26, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs:4-6, 16, or 28, and SEQ ID NO: 13;
(c) SEQ ID NO:30 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) SEQ ID NO:32 and any one of SEQ ID NOs:4-6, 16, or 28; or
(e) SEQ ID NO:34 and any one of SEQ ID NOs:4-6, 16, or 28.
[0060] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In one embodiment, the hybrid promoter comprises SEQ ID NO: 1. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3, and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO:2, (b) SEQ ID NO:3, and (c) any one of SEQ ID NOs:4-6, 16, or 28.
[0061] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the hybrid promoter comprises SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:9- 12 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical SEQ ID NO: 13. In some embodiments, the hybrid promoter comprises (a) any one of SEQ ID NOs:9-12 and (b) SEQ ID NO: 13.
[0062] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In one embodiment, the hybrid promoter comprises SEQ ID NO: 14. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO: 15 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0063] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17 or SEQ ID NO: 18. In one embodiment, the hybrid promoter comprises SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 19, 39, or 48 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) any one of SEQ ID NOs: 19, 39, or 48 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0064] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20. In one embodiment, the hybrid promoter comprises SEQ ID NO:20. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO:21 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0065] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22. In one embodiment, the hybrid promoter comprises SEQ ID NO:22. In some embodiments, the hybrid promoter comprises (a) comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and (b) comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO:23 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0066] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24. In one embodiment, the hybrid promoter comprises SEQ ID NO:24. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25, (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:26, and (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO:25, (b) SEQ ID NO:26, and (c) any one of SEQ ID NOs:4-6, 16, or 28.
[0067] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:27. In one embodiment, the hybrid promoter comprises SEQ ID NO:27. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the hybrid promoter comprises (a) any one of SEQ ID NOs:4- 6, 16, or 28 and (b) SEQ ID NO: 13.
[0068] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:29. In one embodiment, the hybrid promoter comprises SEQ ID NO:29. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO:30 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0069] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31. In one embodiment, the hybrid promoter comprises SEQ ID NO:31. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:32 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO:32 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0070] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33. In one embodiment, the hybrid promoter comprises SEQ ID NO:33. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:34 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4- 6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) SEQ ID NO:34 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0071] In some embodiments, the hybrid promoter comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:35 or SEQ ID NO:36. In one embodiment, the hybrid promoter comprises SEQ ID NO:35 or SEQ ID NO:36. In some embodiments, the hybrid promoter comprises (a) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:37, 38, or 49 and (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-6, 16, or 28. In some embodiments, the hybrid promoter comprises (a) any one of SEQ ID NOs:37, 38, or 49 and (b) any one of SEQ ID NOs:4-6, 16, or 28.
[0072] In some embodiments, the hybrid promoter comprises one or more sequences sequence that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences in Tables 2 or 3. In some embodiments, the hybrid promoter comprises one or more sequences disclosed in Tables 2 or 3.
[0073] Nucleic Acid Constructs and Vectors
[0074] In one aspect, provided are nucleic acid constructs and their use for the expression of a transgene in a cell. Also provided are recombinant vectors and their use for the introduction of a transgene or an expression construct into a cell.
[0075] Nucleic acid constructs include expression constructs including plasmids. The term “expression construct” refers to a recombinant polynucleotide construct that includes a nucleic acid coding for an RNA capable of being transcribed in a cell. Methods for constructing expression constructs and plasmids through standard recombinant techniques are known in the art.
[0076] In some embodiments, the nucleic acid constructs or vectors disclosed herein comprise recombinant nucleic acid constructs that include additional DNA elements, including DNA segments that provide for the replication of the DNA in a host cell and expression of the target gene in target cells at appropriate levels. “Vector,” as used herein, means a vehicle that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. Nonlimiting examples of vectors include a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini-circle, or a virus (including virus derived sequences). A vector may also refer to a virion comprising a nucleic acid to be delivered into a host cell, either in vitro or in vivo. In some embodiments, a vector refers to a virion comprising a recombinant viral genome, wherein the viral genome comprises one or more ITRs and a transgene. In one embodiment, the recombinant vector is a viral vector or a combination of multiple viral vectors. In one aspect, provided is a vector comprising any of the expression constructs disclosed herein.
[0077] Provided herein are nucleic acid constructs and vectors comprising an inflammationinducible hybrid promoter disclosed herein operatively linked to a transgene. In embodiments, the nucleic acid constructs or vectors disclosed herein comprise additional regulatory elements, including, but not limited to, promoters, enhancers, translation initiation signals, introns, and/ or splicing enhancers. In embodiments, the nucleic acid constructs or vectors disclosed herein comprise a polyadenylation sequence, which may include splice donor and acceptor sites. In embodiments, the nucleic acid constructs or vectors disclosed herein comprise an internal ribosome entry site (IRES). An IRES sequence may be used to produce more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to produce a protein that contains more than one polypeptide chain or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in photoreceptors, RPE and ganglion cells. In one embodiment, the IRES is located 3' of the transgene.
[0078] The term “transgene” is used to refer to a polynucleotide that can be introduced into a cell or organism to express a gene product of interest. The transgene can encode a “therapeutic polypeptide” or “therapeutic protein,” which are to be understood herein as a polypeptide or protein that can have a beneficial effect on a subject. The individual may be a human. In embodiment, the subject suffers from a disease. Such therapeutic polypeptide may be selected from, but is not limited to, the group consisting of an enzyme, a co-factor, a cytokine, an antibody, a growth factor, a hormone and an anti-inflammatory protein. In embodiments, the gene product of interest treats, prevents or suppresses symptoms associated with an arthritic disease. In embodiments, the gene product of interest treats, prevents or suppresses symptoms associated with an arthritic disease, wherein a subject experiences these symptoms in the joints. It is understood herein that the gene product of interest may include biosimilars of said gene product of interest. [0079] In one embodiment, the gene product of interest is a nucleic acid. In some embodiments, the transgene encodes an antisense oligonucleotide, an siRNA, or an RNAi.
[0080] In one embodiment, the gene product of interest is an immunosuppressant. In some embodiments, the gene product of interest is selected from the group consisting of ANP32A, Nrf2, IL-6 inhibitor, soluble IL-6 receptor, IL-6 receptor antagonist, humanized anti-IL-6 monoclonal antibody, chimeric anti-IL-6 monoclonal antibody, humanized rabbit anti-IL-6 monoclonal antibody, interleukin 1 (IL-1) inhibitor, tumor necrosis factor alpha (TNFa) inhibitor, IL-1 receptor antagonist, soluble IL-1 receptor, IL- 17 inhibitor, IL-12/IL-23 inhibitor, T-cell costimulation inhibitor, B cell depleting and inhibiting agents, IL-15 inhibitor, IL-22 inhibitor, inhibitor of GM-CSF, insulin-like growth factor (IGF-1), fibroblast-growth factor (FGF), receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor, complement 5a inhibitor, bone morphogenetic protein family member (BMPs), transforming growth factor-beta (TGF-P), growth differentiation factor family member (GDFs), interleukin- 18 inhibitor, IL-2 inhibitor, soluble TNFa (sTNFa) receptor p55, sTNFa receptor p75, sTNFa receptors fused with an IgG, inhibitors of TNFa receptor p55, inhibitors of sTNFa receptor p75, dominant negative IxB-kinase (dn-IKK-P), interleukin-4 (IL-4), interleukin- 10 (IL-10), interleukin- 13 (IL-13), IL-33 inhibitor, CCL17-inhibitor, interferon beta (IFN-P), tissue inhibitor of MMP family member (TIMPs), plasminogen-activator inhibitor (PAIs), serine protease inhibitor (serpins), signaling molecules/transcription factors, extracellular matrix components, vasoactive intestinal peptide (VIP), Cluster of Differentiation 39 (CD39), Cluster of Differentiation 73 (CD73) and Superoxide dismutase (SOD).
[0081] In some embodiments, the gene product of interest is selected from the group consisting of tocilizumab, sarilumab, olokizumab, sirukumab, Siltucimab, Clazakizumab, anakinra, canakinumab, rilonacept, gevokizumab and lutikizumab, etanercept, infliximab, adalimumab, certilizumab pegol, golimumab, secukinumab, brodalumab, ixekizumab, ustekinumab, risankizumab, guselkumab, tildrakizumab, abatacept, rituximab, belimumab, ianalumab, tabalumab, AMG-714, Fezakunimab, lenzilumab, namilumab, rhFGF- 18/sprifermin, denosumab, C5aR-151, Tadekinig alfa/ IL-18 binding protein, basiliximab, daclizumab, F8IL10/Dekavil, SMAD, Sox9, IkB, collagen, cartilage oligomeric matrix protein (COMP), proteoglycans and elastin.
[0082] In one embodiment, the gene product of interest is an interleukin 6 (IL-6) inhibitor. In one embodiment, the gene product of interest is a soluble IL-6 receptor. In one embodiment, the gene product of interest is an IL-6 receptor antagonist. In some embodiments, the IL-6 receptor antagonist is at least one of tocilizumab and sarilumab. In one embodiment, the gene product of interest is a humanized or a chimeric anti-IL-6 monoclonal antibody. In some embodiments, the anti-IL-6 antibody is olokizumab or sirukumab. In one embodiment, the chimeric anti-IL-6 monoclonal antibody is siltucimab. In one embodiment, the humanized rabbit anti-IL-6 monoclonal antibody is clazakizumab.
[0083] In one embodiment, the gene product of interest is an interleukin 1 (IL-1) inhibitor. In some embodiments, the IL-1 inhibitor is selected from the group consisting of anakinra, canakinumab, rilonacept, gevokizumab and lutikizumab. In embodiments, the IL-1 inhibitor is selected from the group consisting of (i) an IL-1 receptor antagonist (ii) an anti-IL-1 monoclonal antibody, (iii) a human dimeric anti-IL-1 fusion protein, and (iv) a human dual variable domain anti-IL-1 immunoglobulin.
[0084] In one embodiment, the IL-1 receptor antagonist is a human IL-1 receptor antagonist. In one embodiment, the human IL-1 receptor antagonist anakinra.
[0085] In one embodiment, the anti-IL-1 monoclonal antibody is a human anti-IL-1 monoclonal antibody. In one embodiment, the human IL-1 monoclonal antibody canakinumab or gevokizumab.
[0086] In one embodiment, the human dimeric anti-IL-1 fusion protein is the human dimeric anti-IL-1 fusion protein rilonacept.
[0087] In one embodiment, the human dual variable domain anti-IL-1 immunoglobulin, is the human dual variable domain anti-IL-1 immunoglobulin lutikizumab.
[0088] In one embodiment, the gene product of interest is a tumor necrosis factor alpha (TNFa) inhibitor. In embodiments, the TNFa inhibitor is selected from the group consisting of etanercept, infliximab, adalimumab, certilizumab pegol and golimumab. In one embodiment, the TNFa inhibitor is etanercept.
[0089] In one embodiment, the gene product of interest is an IL-1 receptor antagonist. In one embodiment, the gene product of interest is a soluble IL-1 receptor.
[0090] In one embodiment, the gene product of interest is an IL- 17 inhibitor. In embodiments, the IL- 17 inhibitor is selected from the group consisting of secukinumab, brodalumab and ixekizumab.
[0091] In one embodiment, the gene product of interest is an IL-12/IL-23 inhibitor. In embodiments, the IL-12/IL-23 inhibitor is selected from the group consisting of ustekinumab, risankizumab, guselkumab and tildrakizumab.
[0092] In one embodiment, the gene product of interest is a T-cell costimulation inhibitor. In one embodiment, the T-cell costimulation inhibitor is abatacept. [0093] In one embodiment, the gene product of interest is a B cell depleting and inhibiting agent. In one embodiment, the B cell depleting and inhibiting agent is selected from the group consisting of rituximab, belimumab, ianalumab and tabalumab.
[0094] In one embodiment, the gene product of interest is an IL- 15 inhibitor. In one embodiment, the IL-15 inhibitor is AMG-714.
[0095] In one embodiment, the gene product of interest is an IL-22 inhibitor. In one embodiment, the IL-22 inhibitor is fezakunimab.
[0096] In one embodiment, the gene product of interest is an inhibitor of GM-CSF. In embodiments, the inhibitor of GM-CSF is lenzilumab or namilumab.
[0097] In one embodiment, the gene product of interest is an insulin-like growth factor (IGF-1).
[0098] In one embodiment, the gene product of interest is a fibroblast-growth factor (FGF). In one embodiment, the fibroblast-growth factor (FGF) is rhFGF-18/sprifermin.
[0099] In one embodiment, the gene product of interest is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor. In one embodiment, the RANKL inhibitor is denosumab.
[0100] In one embodiment, the gene product of interest is a complement 5a inhibitor. In one embodiment, the complement 5a inhibitor is C5aR-151.
[0101] In one embodiment, the gene product of interest is a bone morphogenetic protein family member (BMPs).
[0102] In one embodiment, the gene product of interest is a transforming growth factor-beta (TGF-p).
[0103] In one embodiment, the gene product of interest is a growth differentiation factor family member (GDFs).
[0104] In one embodiment, the gene product of interest is an interleukin- 18 inhibitor. In one embodiment, the interleukin- 18 inhibitor is Tadekinig alfa/ IL-18 binding protein.
[0105] In one embodiment, the gene product of interest is an IL-2 inhibitor. In embodiments, the IL-2 inhibitor is basiliximab or daclizumab.
[0106] In one embodiment, the gene product of interest is a soluble TNFa (sTNFa) receptor p55.
[0107] In one embodiment, the gene product of interest is a sTNFa receptor p75. In one embodiment, the gene product of interest is a sTNFa receptor fused with an IgG. In one embodiment, the gene product of interest is an inhibitor of TNFa receptor p55. In one embodiment, the gene product of interest is an inhibitor of sTNFa receptor p75. [0108] In one embodiment, the gene product of interest is a dominant negative IxE-kinase (dn-IKK-p).
[0109] In one embodiment, the gene product of interest is an interleukin-4 (IL-4).
[0110] In one embodiment, the gene product of interest is an interleukin- 10 (IL- 10). In one embodiment, the IL- 10 is F8IL10/Dekavil.
[OHl] In one embodiment, the gene product of interest is an interleukin- 13 (IL-13).
[0112] In one embodiment, the gene product of interest is an IL-33 inhibitor.
[0113] In one embodiment, the gene product of interest is a CCL17-inhibitor,
[0114] In one embodiment, the gene product of interest is an interferon beta (IFN-P).
[0115] In one embodiment, the gene product of interest is a tissue inhibitor of MMP family member (TIMPs),
[0116] In one embodiment, the gene product of interest is a plasminogen-activator inhibitor (PAIs).
[0117] In one embodiment, the gene product of interest is a serine protease inhibitor (serpins).
[0118] In one embodiment, the gene product of interest is a signaling molecule/transcription factor. In embodiments, the signaling molecule/transcription factor is selected from the group consisting of SMAD, Sox9 and IkB.
[0119] In one embodiment, the gene product of interest is an extracellular matrix component. In embodiments, the extracellular matrix component is selected from the group consisting of collagen, cartilage oligomeric matrix protein (COMP), proteoglycans and elastin. [0120] In one embodiment, the gene product of interest is a vasoactive intestinal peptide (VIP).
[0121] In one embodiment, the gene product of interest is a Cluster of Differentiation 39 (CD39).
[0122] In one embodiment, the gene product of interest is a Cluster of Differentiation 73 (CD73).
[0123] In one embodiment, the gene product of interest is a superoxide dismutase (SOD).
[0124] In one embodiment, the gene product of interest is a sequence-specific nuclease
(complex) for functional genome editing. Functional genome editing systems for use in all embodiments of the invention are known to the person skilled in the art and include: Transcription Activator-Like Effector Nucleases (TALENs, see, e.g., Gaj et al., ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013 Jul;31(7):397-405), zinc-finger nucleases (ZFNs, see., e.g., Gaj et al. (2013) supra), meganucleases such as I-Scel (Arnould et al., Engineered I-Crel derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol. 2007 Aug 3 ;371(l):49-65; Takeuchi et al., Tapping natural reservoirs of homing endonucleases for targeted gene modification. Proc Natl Acad Sci U S A. 2011 Aug 9;108(32): 13077-82.), RNA-guided endonuclease systems like CRISPR/Cas (see, e.g., Mali et al., Cas9 as a versatile tool for engineering biology. Nat Methods. 2013 Oct;10(10):957-63.; Mali et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013 Sep;31(9):833-8; Cong et al., Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15;339(6121): 819-23) and CRISPR/Cpfl (see, e.g., Zetsche et al., Cpfl is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22;163(3):759-71), triplexforming molecules, synthetic polyamides and designer zinc-finger proteins (see, e.g., Uil et al., Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities. Nucleic Acids Res. 2003 Nov l;31(21):6064-78). Functional genome editing systems employ nucleases which create site-specific double-strand breaks at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous endjoining or homologous recombination. As a result, targeted mutations are obtained. It is advantageous to replace a defective gene (causing a disease or disorder) with a normal allele at its natural location by any of these methods, because it does not require that the full coding sequences and regulatory sequences are included in the rAAV virion when only a small portion of the gene needs to be altered. Expression of the partially replaced gene is also thought to be more consistent with normal cell biology than full genes that are carried by virions. A preferred gene editing system may be CRISPR (comprising CRISPR/Cpfl and CRISPR-Cas), because it is quicker and cheaper than other methods. A major advantage is also that CRISPR can be easily repurposed to target different DNA sequences using the CRISPR single guide RNAs. Thus, alternatively, or in combination with another embodiment, provided is a nucleic acid construct that comprises at least one of (i) a polynucleotide comprising a sequence encoding at least one guide RNA (gRNA); wherein the guide RNA is substantially complementary - in some embodiments complementary - to a target polynucleotide sequence(s) in a genome; and (ii) a polynucleotide comprising a sequence encoding a nuclease; wherein the nuclease forms a ribonuclease complex with the guide RNA, and wherein the ribonuclease complex makes site-specific double-stranded DNA breaks in the genome.
[0125] In embodiments, the gene product of interest is an antibody, antigen-binding fragment thereof, or a chimeric antigen receptor (CAR). [0126] Viral Vectors
[0127] Viral vectors for the expression of a target gene in a target cell, tissue, or organism are known in the art and include, for example, an AAV vector, adenovirus vector, lentivirus vector, retrovirus vector, poxvirus vector, baculovirus vector, herpes simplex virus vector, vaccinia virus vector, or a synthetic virus vector (e.g., a chimeric virus, mosaic virus, or pseudotyped virus, and/or a virus that contains a foreign protein, synthetic polymer, nanoparticle, or small molecule).
[0128] AA V vectors
[0129] AAV are small, single-stranded DNA viruses which require helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITR) and two open reading frames which encode the Rep proteins and Cap proteins, respectively. The Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD, and 40 kD. These proteins function mainly in regulating AAV replication and rescue and integration of the AAV into a host cell's chromosomes. The Cap reading frame encodes three structural proteins of molecular weight 85 kD (VP 1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid. More than 80% of total proteins in AAV virion comprise VP3. Flanking the rep and cap open reading frames at the 5' and 3' ends are about 145 bp long inverted terminal repeats (ITRs). The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene.
[0130] Recombinant adeno-associated virus “rAAV” vectors include any vector derived from any AAV serotype. rAAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, in some embodiments, the Rep and/or Cap genes, but retain functional flanking ITR sequences.
[0131] In some embodiments, the viral vector is an rAAV virion, which comprises an rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome comprises an expression construct disclosed herein.
[0132] In some embodiments, the viral vector disclosed herein comprises a nucleic acid comprising an AAV 5' ITR and 3' ITR located 5' and 3' of the transgene, respectively. However, in certain embodiments, it may be desirable for the nucleic acid to contain the 5 ' ITR and 3' ITR sequences arranged in tandem, e.g., 5' to 3' or a head-to-tail, or in another alternative configuration. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs or to have 5' ITRs (or conversely, 3' ITRs) located both 5' and 3' to the transgene. The ITRs sequences may be located immediately upstream and/or downstream of the heterologous molecule, or there may be intervening sequences. The ITRs need not be the wild-type nucleotide sequences, and may be altered (e.g., by the insertion, deletion, or substitution of nucleotides) so long as the sequences provide for functional rescue, replication, and packaging. The ITRs may be selected from AAV2, or from among the other AAV serotypes, as described herein.
[0133] In some embodiments, provided is a vector comprising a nucleic acid sequence comprising (i) an expression construct disclosed herein and (ii) one or more inverted terminal repeats (ITR). In one embodiment, the nucleic acid sequence comprises a 5’ ITR and a 3’ ITR. In one embodiment, the 5’ ITR and a 3’ ITR are derived from AAV serotype AAV2. In one embodiment, the 5’ ITR and a 3’ ITR are derived from AAV serotype AAV5.
[0134] In some embodiments, the viral vector is an AAV vector, such as an AAV1 (i.e., an AAV containing AAV1 ITRs and AAV1 capsid proteins), AAV2 (z.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (z.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (i.e., an AAV containing AAV9 ITRs and AAV9 capsid proteins), AAVrh74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid proteins), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid proteins), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid proteins).
[0135] In some embodiments, the viral vector is a pseudotyped AAV vector, containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2/9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2/10 (i.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2/7m8 (i.e., an AAV containing AAV2 ITRs and AAV7m8 capsid proteins).
[0136] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more of capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In embodiments, the capsid protein is a variant AAV capsid protein. In one embodiment, the capsid protein is AAV2 variant rAAV2-retro (SEQ ID NO:44 from WO 2017/218842, incorporated herein by reference in its entirety). In embodiments, the capsid protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence disclosed in WO 2017/218842. In embodiments, an AAV with tropism for joint tissues is used, including, but not limited to an AAV comprising a viral capsid protein disclosed in International Patent Publication No. WO2019/141765, entitled “A modified raav capsid protein for gene therapy” or International Patent Publication No. WO202 1/009684, entitled “Modified aav capsid proteins for treatment of arthritic disease”, both incorporated herein by reference in their entireties). In embodiments, the capsid protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence disclosed in International Patent Publication Nos. WO2019/141765 or WO202 1/009684.
[0137] In embodiments, the AAV particle comprises a capsid protein comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence in Table 1.
Table 1. Exemplary AAV capsid protein sequences. * SEQ ID NO:44 from International Patent Publication No. WO2017/218842. Remaining sequences corresponds to SEQ ID NOS: 1-7 from International Patent Publication Nos. WO2019/141765 and W02021/009684. SIN = SEQ ID NO.
[0138] In some embodiments, the AAV vector contains two or more capsid proteins selected from different serotypes. In some embodiments, the AAV vector contains a capsid from a first serotype and a capsid protein from a second serotype. In some embodiments, the AAV vector contains capsid proteins from a first and a second AAV serotype, respectively, in aratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some embodiments, the AAV vector contains capsid proteins from a first and a second AAV serotype, respectively, in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.
[0139] In some embodiments, a mixture of (1) AAV vectors comprising capsid proteins from a first serotype and (2) AAV vectors comprising capsid proteins from a second serotype is used. In some embodiments, a ratio of the (1) AAV vectors comprising capsid proteins from a first serotype and (2) AAV vectors comprising capsid proteins from a second serotype, respectively, of 1 : 1, 1 :2, 1:3, 1 :4, 1 :5, 1 :6, 1 :7, 1:8, 1 :9, 1 : 10, 1 : 15, 1 :20, 1:25, 1 :30, 1 :35, 1 :40, 1 :45, or 1 :50 is used. In some embodiments, a ratio of the (1) AAV vectors comprising capsid proteins from a first serotype and (2) AAV vectors comprising capsid proteins from a second serotype, respectively, of 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1:6, 1:7, 1 :8, 1 :9, 1 : 10, 1 : 15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, or 1 :50 is used.
[0140] Other viral vectors
[0141] Other viral vectors include adenoviral (AV) vectors, for example, those based on human adenovirus type 2 and human adenovirus type 5 that have been made replication defective through deletions in the El and E3 regions. The transcriptional cassette can be inserted into the El region, yielding a recombinant E1ZE3 -deleted AV vector. Adenoviral vectors also include helper-dependent high-capacity adenoviral vectors (also known as high- capacity, “gutless” or “gutted” vectors), which do not contain viral coding sequences. These vectors contain the cis-acting elements needed for viral DNA replication and packaging, mainly the inverted terminal repeat sequences (ITR) and the packaging signal (CY). These helperdependent AV vector genomes have the potential to carry from a few hundred base pairs up to approximately 36 kb of foreign DNA.
[0142] Alternatively, other systems such as lentiviral vectors can be used. Lentiviral-based systems can transduce nondividing as well as dividing cells making them useful for applications targeting, for examples, the nondividing cells of the CNS. Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome providing the potential for very long-term gene expression.
[0143] Polynucleotides, including plasmids, YACs, minichromosomes and minicircles, carrying the target gene containing the expression cassette can also be introduced into a cell or organism by nonviral vector systems using, for example, cationic lipids, polymers, or both as carriers. Conjugated poly-L-lysine (PLL) polymer and polyethylenimine (PEI) polymer systems can also be used to deliver the vector to cells. Other methods for delivering the vector to cells includes hydrodynamic injection and electroporation and use of ultrasound, both for cell culture and for organisms. For a review of viral and non-viral delivery systems for gene delivery see Nayerossadat, N. et al., Viral and nonviral delivery systems for gene delivery. Adv Biomed Res. 2012; 1 :27.
[0144] rAAV virion production
[0145] The rAAV virions disclosed herein may be constructed and produced using the materials and methods described herein, as well as those known to those of skill in the art. Such engineering methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). Further, methods suitable for producing a rAAV cassette in an adenoviral capsid have been described in U.S. Pat. Nos. 5,856,152 and 5,871,982.
[0146] Briefly, in order to package the rAAV genome into a rAAV virion, a host cell is used that contains sequences necessary to express AAV rep and AAV cap or functional fragments thereof as well as helper genes essential for AAV production. The AAV rep and cap sequences are obtained from an AAV source as identified herein. The AAV rep and cap sequences may be introduced into the host cell in any manner known to one in the art, including, without limitation, transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences may be transfected into the host cell by one or more nucleic acid molecules and exist stably in the cell as an episome. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. Another embodiment has the rep and cap sequences transiently expressed in the host cell. For example, a useful nucleic acid molecule for such transfection comprises, from 5' to 3', a promoter, an optional spacer interposed between the promoter and the start site of the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.
[0147] The rep and cap sequences, along with their expression control sequences, may be supplied on a single vector, or each sequence may be supplied on its own vector. The rep and cap sequences are supplied on the same vector. Alternatively, the rep and cap sequences may be supplied on a vector that contains other DNA sequences that are to be introduced into the host cells. The promoter used in this construct may be any suitable constitutive, inducible or native promoters known to one of skill in the art. The molecule providing the rep and cap proteins may be in any form which transfers these components to the host cell. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as those for marker genes. This molecule does not contain the AAV ITRs and generally does not contain the AAV packaging sequences. To avoid the occurrence of homologous recombination, other virus sequences, particularly those of adenovirus, are avoided in this plasmid. This plasmid is desirably constructed so that it may be stably transfected into a cell. [0148] Although the molecule providing rep and cap may be transiently transfected into the host cell, it may be preferred that the host cell be stably transformed with sequences necessary to express functional rep/cap proteins in the host cell, e.g., as an episome or by integration into the chromosome of the host cell. Depending upon the promoter controlling expression of such stably transfected host cell, the rep/cap proteins may be transiently expressed (e.g., through use of an inducible promoter).
[0149] The methods employed for constructing embodiments of this disclosure are conventional genetic engineering or recombinant engineering techniques such as those described in the references above. For example, the rAAV may be produced utilizing a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif), according to manufacturer’s instructions. See also Herzog et al., Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat Med. 1999 Jan;5(l):56-63, employing the plasmid with the transgene, a helper plasmid containing AAV rep and cap, and a plasmid supplying adenovirus helper functions of E2A, E40rf6 and VA. In embodiments, a two- plasmid system is used, e.g., such as the system described in Grimm et al., Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6. Molecular therapy: the journal of the American Society of Gene Therapy. 2003;7(6):839-50. While this specification provides illustrative examples of specific constructs, using the information provided herein, one of skill in the art may select and design other suitable constructs, using a choice of spacers, promoters, and other elements, including at least one translational start and stop signal, and the optional addition of polyadenylation sites.
[0150] The rAAV virions are then produced by culturing a host cell containing a rAAV virus as described herein which contains a rAAV genome to be packaged into a rAAV virion, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof. Suitable viral helper genes, e.g., adenovirus E2A, E40rf6 and VA, among other possible helper genes, may be provided to the culture in a variety of ways known to the art, including on a separate plasmid. Thereafter, the recombinant AAV virion which directs expression of the transgene is isolated from the cell or cell culture in the absence of contaminating helper virus or wildtype AAV.
[0151] Expression of the transgenes may be measured in ways known in the art. For example, a target cell may be infected in vitro, and the number of copies of the transgene in the cell monitored by Southern blotting or quantitative polymerase chain reaction (PCR). The level of RNA expression may be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and the level of protein expression may be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or by the specific methods detailed below in the Examples.
[0152] Cells
[0153] In embodiments, the nucleic acid constructs and vectors disclosed herein are used to deliver an inflammation-inducible promoter operatively linked to a transgene to a cell. Provided herein are cells comprising a promoter, a nucleic acid construct, or a vector disclosed herein. In embodiments, the cell is a fibroblast-like synoviocyte (FLS), cartilage cell, chondrocyte, chondroblast, adipocyte, osteoclast, T-lymphocyte, macrophage, monocyte, neutrophil, osteoblast, dendritic cell, plasma cell, mast cell, (skeletal) muscle cell, cardiomyocyte, or B lymphocyte. In one embodiment, the cell is a synovial cell. The “synovium” or “synovial tissue” or “synovial cells” as used herein refers to the cellular lining covering the non-cartilaginous surfaces of the synovial joints, as further described in Tak (2000, Examination of the synovium and synovial fluid. In: Firestein GS, Panyani GS, Wollheim FA editors. Rheumatoid Arthritis. New York: Oxford Univ. Press, Inc. 55-68) and incorporated herein by reference. The synovium consists of the intimal lining layer (or synovial lining layer) and the synovial sublining (subsynovium), which merges with the joint capsule. The intimal lining layer comprises intimal macrophages (or macrophage-like synoviocytes or type A synoviocytes) and FLS (or type B synoviocytes). “Synovium” may therefore be replaced by, or is synonymous with, “synovial tissue”. A synovial cell can include any cell present in the synovium including FLS and macrophage-like synoviocytes. A synoviocyte cell may also be a neutrophil, T, B cells and/or connective tissue cells, which may all be present in the synovium. “Fibroblast-like synoviocytes” (FLS) are cells of mesenchymal origin that display many characteristics that are in common with fibroblasts, such as expression of specific proteins, such as for example several types of collagens. However, FLS also secrete proteins that are normally absent in other fibroblast lineages, such as for example lubricin. In addition, FLS express molecules that are important for the mediation of cell adhesion, such as cadherin- 11, VCAM-1, several integrins and their receptors. Specific for FLS is the expression of CD55 and this protein is therefore typically used to identify FLS in the synovium by immunohistochemistry. FLS represent a specialized cell type located inside joints in the synovium, whose cells play a crucial role in the pathogenesis of chronic inflammatory diseases, such as rheumatoid arthritis (RA). The term “rheumatoid synovium” or “rheumatoid synovial cells” or “rheumatoid synovial tissue” refers to the inflamed synovium of the joints of an individual suffering from RA. The rheumatoid synovium is characterized by intimal lining hyperplasia and by accumulation of FLS, T-cells, plasma cells, macrophages, B-cells, natural killer cells and dendritic cells in the synovial sublining. These accumulated cells are comprised in the definition of rheumatoid synovial cells. During the progression of RA, the synovial tissue becomes a place where constant inflammatory processes take place, which can eventually lead to cartilage damage and joint destruction and deformation. FLS that are present in the synovium during RA have been reported to display an altered phenotype compared to the FLS present in normal tissues. For example, the FLS in rheumatoid synovium lose “contact inhibition”, z.e., they lose the property to arrest their growth when more cells come into contact with each other. In addition, they lose the dependency to grow on adhesive surfaces. As a result, the number of FLS in the diseased synovium increases. The inflammation is further enhanced by the production of several pro-inflammatory signaling molecules, particularly interleukins IL-6 and IL-8, prostanoids and matrix metalloproteinases (MMPs). The cells may be isolated.
[0154] Pharmaceutical Compositions
[0155] Provided herein are pharmaceutical compositions comprising a nucleic acid construct or vector disclosed herein and a pharmaceutically acceptable excipient.
[0156] The nucleic acid construct or vector disclosed herein may be assessed for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for storage and/or administration to a patient.
[0157] Formulations of the nucleic acid constructs or vectors disclosed herein disclosed herein involve the use of a pharmaceutically and/or physiologically acceptable vehicle or carrier, such as buffered saline or other buffers, e.g., PBS or HEPES, to maintain pH at appropriate physiological levels. The nucleic acid constructs or vectors disclosed herein can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to the vector, a pharmaceutically and/or physiologically acceptable excipient, carrier, buffer, stabilizer, antioxidants, preservative, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Additional carriers are provided in International Patent Publication No. W02000/015822, entitled “Methods for treatment of degenerative retinal diseases,” incorporated herein by reference. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used. In some cases, Ringer's Injection, Lactated Ringer's Injection, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
[0158] Pharmaceutical compositions comprising a nucleic acid construct or vector disclosed herein may formulated with one or more pharmaceutically-acceptable excipients, which can be a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and/or a preservative. Some examples of materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
[0159] A bulking agent is a compound which adds mass to a pharmaceutical formulation and contributes to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol and sorbitol.
[0160] The use of a surfactant can reduce aggregation of the reconstituted protein and/or reduce the formation of particulates in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution. Suitable surfactants according to the present invention include polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl- sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol e.g. Pluronics, PF68, etc.).
[0161] Preservatives may be used in formulations of invention. Suitable preservatives for use in the formulation of the invention include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3 -pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, e.g., in "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995).
[0162] For delayed release, nucleic acid construct or vector disclosed herein may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.
[0163] If a vector is to be stored long-term, it may be frozen in the presence of glycerol.
[0164] Methods
[0165] Provided herein are methods of regulating the expression of a transgene in response to inflammatory stimuli. Provided herein is a method of inducing the expression of transgene, the method comprising providing a cell comprising (i) a nucleic acid construct comprising an inflammation-inducible hybrid promoter disclosed herein operatively linked to a transgene or (ii) a vector comprising an inflammation-inducible hybrid promoter disclosed herein operatively linked to a transgene, and contacting the promoter with one or more inflammatory stimuli.
[0166] Depending on the application, a person skilled in the art may perform a method disclosed herein in vitro, in vivo, or ex vivo.
[0167] In embodiments, gene expression is induced by delivering an inflammationinducible hybrid promoter disclosed herein operatively linked to a transgene to a cell in a subject, wherein the subject produces inflammatory stimuli (including, but not limited to, cytokines or chemokines) that induce the expression of the transgene. In embodiments, gene expression is induced by delivering an inflammation-inducible hybrid promoter disclosed herein operatively linked to a transgene to a cell in a subject, wherein the cell in the subject is exposed to exogenous inflammatory stimuli (including, but not limited to, LPS) that induce the expression of the transgene.
[0168] In some embodiments, the inflammatory stimulus is a cytokine, a chemokine, a growth factor, a pathogen associated molecular pattern (PAMP), or a damage associated molecular pattern (DAMP). In embodiments, the inflammation-inducible hybrid promoter responds to one or more of IL-1, IL-alpha, IL-1 beta, IL-2, IL-5, IL-4, IL-6, IL-7, IL-8, IL- 10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-25, tumor necrosis factor alpha (TNF-a), interferon-alpha (IFN-a), interferon-P (IFN-beta), interferon gamma (IFNy), granulate colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB), macrophage inflammatory protein- la (MIP-la = C-X-C motif chemokine 2 (CCL2)), CCL3, CCL4, CCL5, RANTES, stromal cell-derived factor 1 (SDF-1 = C-X-C motif chemokine 12 (CXCL12)), Neutrophil-activating protein 3 (NAP-3 (CXCL1)), monocyte chemoattractant protein (MCP-1), CXCL8, IFN-y-induced protein (IP-10 (CXCL-10)), lymphotactin, fractalkine, transforming growth factor beta (TGF-beta), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), or cyclooxygenase-2 (COX-2), nitric oxide (NO). In one embodiment, the inflammation-inducible hybrid promoter responds to lipopolysaccharides (LPS). In embodiments, the inflammation-inducible hybrid promoter responds to PAMPs (including, but not limited to, dsRNA, ssRNA, CpG DNA, peptidoglycan, flagellin, lipoteichoic acid (LT A), and diacyl lipopeptide). In embodiments, the inflammation-inducible hybrid promoter responds to DAMPs (including, but not limited to, DNA, RNA, ATP, ECM components (glycoproteins, proteoglycans, glycosaminoglycans), defensins, heat shock proteins (HSP), HMGB-1, S-100 proteins, uric acid and uric acid crystals, mtDNA, and fatty acids).
[0169] All other referenced patents and applications are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0170] To facilitate a better understanding of the present invention, the following Examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the invention. EXAMPLES
[0171] Example 1: Identification of Inflammation-Inducible Promoters
[0172] To identify inflammation-inducible promoters or cis-regulatory elements to drive gene-expression under inflammatory conditions, the Survey of Regulatory Elements, (SuRE) methodology was used (van Arensbergen et al., Genome-wide mapping of autonomous promoter activity in human cells. Nat Biotechnol. 2017 Feb;35(2): 145-153).
[0173] In brief, a barcoded library containing around 300 million human genome DNA elements with an average insert size of about 300 base pairs was screened in HT1080, a human fibrosarcoma cell line. Elements that showed increased expression in the presence of TNF alpha (TNFa) and IL-1 beta (IL-1 (3) were subsequently used to create hybrid combinations, either by rationale design and fusing them to the NFkB-responsive CMV promoter by cloning, or randomly by creating a new library of hybrids of 96 elements and the NFkB-responsive CMV promoter, containing over 40,000 hybrid combinations on total which were then again screening using SuRE. HT1080, a human fibrosarcoma cell line was used to identify hybrid promoters, comprising one or more DNA elements, that showed increased expression in presence of TNF alpha (TNFa) and IL-1 beta (IL-lp). The NFkB-responsive CMV promoter, herein referred to as NFkB-CMV promoter (Khoury et al., Inflammation-inducible anti-TNF gene expression mediated by intra-articular injection of serotype 5 adeno-associated virus reduces arthritis. J Gene Med. 2007 Jul;9(7)), was used as a control. The most promising hybrid promoter candidates were subjected to further analysis (Table 2).
Table 2. Selected inflammation-inducible hybrid promoters. SEQ ID NO: 1 includes part of bacterial plasmid backbone. SEQ ID NO: 10 is missing a 3’ “G” as compared to SEQ ID NO:9. SEQ ID NO: 16 is missing 5’ “CTAGCA” as compared to SEQ ID NO:4. SEQ ID NO:24 includes part of bacterial plasmid backbone. SEQ ID NO:28 is missing 5’ “GT” as compared to SEQ ID NO:4. SEQ ID NO:38 comprises a point mutation as compared to SEQ ID NO:37. SEQ ID NO:39 comprises three point mutations as compared to SEQ ID NO: 19. Constructs marked with * were used in the Examples. SEQ ID NOs:4-6, 16, and 28 may be replaced a different sequence selected from a group comprising SEQ ID NOs:4-6, 16, and 28. SEQ ID NOs:9-12 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:9-12. SEQ ID NOs: 19, 39, and 48 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:19, 39, and 48. SEQ ID NOs:37, 38, and 49 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:37, 38, and 49. SIN = SEQ ID NO. [0174] Example 2: Induction of Selected Promoters by Inflammatory Cytokines (Plasmid Transfections)
[0175] To assess the induction of selected hybrid promoters by inflammatory cytokines, the hybrid promoters were cloned into plasmids containing a single stranded AAV2 genome with luciferase as a reporter gene. Subsequently, the plasmids were transfected into HEK293T/17 cells (human embryonic kidney cells stably expressing the adenoviral El A and ElB-55k proteins and the SV40 large T antigen), HT1080 cells (human fibrosarcoma cell line), C2C12 cells (mouse myoblast cell line) cells, and primary fibroblast-like synoviocytes from rheumatoid arthritis patients (RA-FLS cells), respectively. The cells were stimulated with cytokines and luciferase expression was determined.
[0176] Cloning of selected elements into plasmids containing the AAV2 ITRs and the Luciferase reporter gene.
[0177] To clone hybrid promoters g201, g202, g210, g211, g221, and g229 into plasmids containing a single stranded AAV2 genome with Luciferase as a reporter gene, the hybrid promoters were PCR amplified using primers flanking Mlul and Hindlll restriction sites. After PCR amplification, the DNA was loaded and run on a 0.6% agarose gel to isolate fragments with the right size, and after gel extraction and DNA isolation the DNA was digested with Mlul and Hindlll restriction enzymes. Digested elements were then put again on DNA gel electrophoresis, 0.6% agarose, and bands were successfully isolated (-500 bp). Plasmid pART548, containing the AAV2 ITRs and Luciferase as a reporter gene, was digested with Mlul and Hindlll, and upon gel electrophoresis, the band at 5248 bp was isolated. The hybrids were ligated into cut pART548 backbone. The ligation mixture was transformed to E. coli XL 10 Gold cells and the constructs were checked by restriction digestions and sequencing. The ITRs and expression cassette were sequenced confirming the presence of the ITRs and to confirm the sequence of the hybrid promotes. The final recombinant AAV plasmids contained AAV2 ITRs, a hybrid promoter of interest, a LUC2P reporter gene and the human growth hormone polyadenylation signal (hGHpA) (Fig. 1).
[0178] Hybrid promoters gl87, gl90, g231, ng007, ng008 and ng012 were cloned in the EcoRV site of plasmid pUC57. Subsequently, these plasmids were digested with Mlul and Hindlll restriction enzymes and ligated in pART548 digested with the same enzymes, as described above. The ITRs and expression cassette were sequenced confirming the presence of the ITRs and the sequence of the hybrid promoters.
[0179] Transfection of plasmids into HEK293T/17, HT1080, and C2C12 cells and stimulation of cells with cytokines [0180] HEK293T/17, HT1080, and C2C12 cells were seeded 24 h prior to transfection.
HEK293T/17 and HT1080 were seeded at a density of 50,000 cells per well in 200 pl total volume in 96 well plates, whereas C2C12 cells were seeded at 10,000 cells per well in 300 pl total volume in 48 well plates. The cells were incubated at 37 °C, 5% CO2. For all cell lines, DMEM + GlutaMAX-I (Gibco) supplemented 10% (v/v) Fetal Bovine Serum (FBS), and 1% (v/v) Gibco® Antibiotic- Antimycotic was used. After 24 h, HEK293T/17 and HT1080 cells were transfected using the Invitrogen Lipofectamine™ 2000 Transfection Reagent, and C2C12 cells using the Invitrogen Lipofectamine™ 3000 Transfection Reagent by adding the Lipofectamine/plasmid DNA/Opti-MEM® I medium (Gibco) mixture, according to the manufacturer’s instructions. After 48 h of incubation with the Lipofectamine/plasmid DNA complexes at 37 °C, 5% CO2, cells were stimulated with cytokines (10 ng/mL TNFa and 1 ng/mL IL-ip). For transfection of plasmids with hybrid promoters gl90, g201, g202, g210, g211, g221, g231 and g229, the medium of the cells containing the plasmid and lipofection reagent, was exchanged for fresh DMEM + GlutaMAX-I medium supplemented 10% (v/v) Fetal Bovine Serum (FBS), and 1% (v/v) Gibco® Antibiotic- Antimycotic with or without the cytokines. For transfection of plasmids with hybrid promoters gl87, ng007, ng008, and ng012, the medium composition for cytokine stimulation was DMEM + GlutaMAX-I (Gibco) supplemented 1% (v/v) Fetal Bovine Serum (FBS), and 0.5% (v/v) Gibco® Antibiotic- Antimycotic, with or without the cytokines. Cells were incubated for another 24 h and were either directly harvested and lysed to measure luciferase activity, or first assessed for cell death and toxicity using the Cell Counting kit-8 (CCK8) from Dojindo. To this end, 30 pL of CCK8 reagent was added to each well, the plates were incubated for 2 h at 37 °C, and absorbance of the plate measured at 450 nm. After measurement of cell toxicity/cell viability, the media of the cells was removed, and the cells were all washed with PBS buffer, except HEK293T/17 cells since they are prone to detachment. After washing, cells were subsequently harvested and lysed using passive lysis buffer from Promega. Finally, luciferase activity was measured in the lysates using the One gio substrate from Promega. The luciferase signal was measured with the Omega fluostar machine.
[0181] A separate transfection experiment was performed with C2C12 cells and plasmids containing the gl90, g202, g210, g211 and g221 hybrid promoters. To mimic cytokine levels in different inflammatory murine models, the amount and type of cytokines was varied. In addition, Lipopolysaccharide (LPS) was used as an inflammatory component. C2C12 cells were seeded at a density of 5,000 cells per well in 200 pl total volume in 96 well plates, 24 h prior to transfection. Transfection was performed using Invitrogen Lipofectamine™ 3000 Transfection Reagent according to manufacturer’s instructions. Upon 48 h incubation of the cells with the Lipofectamine/plasmid DNA complexes at 37 °C, 5% CO2, cells were stimulated with cytokines, using three different stimulation conditions, namely 10 pg/mL IL-ip, 5 pg/mL TNFa, and 50 pg/mL IL-6; or 50 pg/mL IL-ip, 100 pg/mL TNFa, and 50,000 pg/mL IL-6; or 5 pg/mL LPS. Cells were stimulated for 24 h and the medium composition for stimulation was DMEM + GlutaMAX-I (Gibco) supplemented 10% (v/v) Fetal Bovine Serum (FBS), and 1% (v/v) Gibco® Antibiotic-Antimycotic, with or without the stimulatory agents. After stimulation, cells were harvested and lysed using passive lysis buffer from Promega. Finally, luciferase activity was measured in the lysates using the One gio™ substrate from Promega, similar to prior experiments. The Luciferase signal was measured with the Omega fluostar machine.
[0182] Transfection of plasmid into RA-FLS from different donors, and stimulation of cells with cytokines
[0183] RA-FLS cells were cultured in T175 flasks in DMEM+GlutaMAX-I (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) IM Hepes (Gibco) and 1% (v/v) Gibco® Antibiotic- Antimycotic at 37 °C, 5% CO2 prior to nucleofection experiments in order to reach proper cell densities for nucleofections. Once the cells had reached proper cell densities for nucleofection experiments, they were trypsinized and counted. Subsequently, the cells were centrifuged, the supernatant was removed, and the cells were resuspended.
[0184] For transfection of plasmids with hybrid promoters gl90, g201, g202, g210, g211, g221, g229, and g231 resuspension of the cells and addition of the plasmid DNA was performed using the Basic Nucleofector™ Kit Primary Fibroblasts. Cells were nucleofected (electroporated) in aluminum cuvets using the Amaxa biosystems Nucleofector® II device. Per nucleofection reaction, 600,000 cells were transfected. After nucleofections, cells were plated in 24 well plates at densities of 30,000, 60,000 or 40,000 cells per well in the in DMEM+ GlutaMAX-I (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) IM Hepes (Gibco) and 1% (v/v) Gibco® Antibiotic- Antimycotic and incubated at 37 °C, 5% CO2. The next day, the medium of the cells was refreshed. 48 h after transfection, cells were stimulated with cytokines. To this end, the medium of the cells was refreshed with new medium with or without the cytokines (10 ng/mL TNFa and 1 ng/mL IL-ip).
[0185] For transfection of plasmids with hybrid promoters gl87, ng007, ng008 and ng012, the Amaxa P2 Primary Cell 4D-Nucleofector X Kit S (V4XP-2032) was used. The nucleofection procedure was done according to manufacturer’s protocol. Lonza’s 4D- Nucleofector system was used, and cells were nucleofected using the 16-well cuvette system. Per nucleofection reaction, approximately 70,000 cells were transfected. After nucleofections, cells were plated in 48 well plates, at 20,000 cells per well in DMEM+GlutaMAX-I (Gibco) supplemented with 10% (v/v) FBS, 1% (v/v) IM Hepes (Gibco) and 1% (v/v) Gibco® Antibiotic-Antimycotic and incubated at 37 °C, 5% CO2. 48 h after transfection, cells were stimulated with cytokines. To this end, the medium of the cells was refreshed with new DMEM+GlutaMAX-I (Gibco) medium supplemented 1% (v/v) Fetal Bovine Serum (FBS), and 0.5% (v/v) Gibco® Antibiotic- Antimycotic with or without the cytokines (10 ng/mL TNFa and 1 ng/mL IL-ip).
[0186] For hybrid promoters, gl90, g201, g202, g210, g211, g221, g229, and g231, cells were incubated for another 48 hand subsequently they were lysed using passive lysis buffer from Promega. Finally, luciferase activity was measured in the lysates using the One gio™ substrate from Promega. The Luciferase signal was measured with the Omega fluostar machine. For hybrid promoters, gl87, ng007, ng008 and ng012, cells were incubated for 48 h, following a cell death and toxicity assessment, using the Cell Counting kit-8 (CCK8) from Dojindo. To this end, 30 pL of CCK8 reagent was added to each well, the plates were incubated for 2 h at 37 °C, and absorbance of the plate measured at 450 nm. After measurement of cell toxicity/cell viability, the media of the cells was removed, and the cells were all washed with PBS buffer. After washing, cells were lysed and Luciferase signal was measured similar to previous group of hybrid promoters.
[0187] Results
[0188] Inflammation-inducible promoters show increased reporter gene expression in the presence of inflammatory stimulants in a variety of cell types. Results for HEK293T/17 cells are shown in Figs. 3A, 4A, 5A, 6A, 7A, and 9A. Results for HT1080 cells are shown in Figs. 2A, 8A, 10A, 11A, 12A, and 13A. Results for C2C12 cells are shown in Figs. 2B, 2E, 3D, 4B, 4E, 5D, 6D, 8B, 10B, 11B, and 12B. Results for primary RA-FLS are shown in Figs. 2D, 3C, 4D, 5C, 6C, 7C, 8C, 9B, 10C, 11C, 12C, and 13B.
[0189] Inflammation-inducible promoters show increased reporter gene expression in the presence of different inflammatory stimulants (see, e.g., Figs. 2E, 3D, 4E, 5D, and 6D).
[0190] Example 3: Induction of Selected Promoters by Inflammatory Cytokines (AAV Vector Transduction)
[0191] To assess the induction of selected hybrid promoters by inflammatory cytokines, the AAV2 genomes containing a s luciferase reporter gene under the control of selected hybrid promoters were packed into AAV5 capsids. The AAV2/5 viral particles were used to transduce HEK293T/17, mFLS AXR7, or RA-FLS, respectively.
[0192] AA V productions
[0193] Recombinant AAV5 viruses containing AAV2 genomes with the luciferase gene driven by different hybrid promoters g201, g202, g210, g211, g221, and g229, gl87, gl90, g231, ng007, ng008 and ng012, as well as the NFkB-CMV promoter, were produced in adherent HEK293T/17 cells. Cells were cultured in DMEM + GlutaMAX-I (Gibco) supplemented 10% (v/v) FBS, and 1% (v/v) Gibco® Antibiotic- Antimycotic in 8 layer cell flasks at 37 °C, 5% CO2, and transfected with the AAV2 vector plasmids and pDP5-Kan3 helper/ AAV5 packaging plasmid, a derivative of pDP5 with the ampicillin resistance gene replaced by the kanamycin resistance gene (references: Grimm D, Kay MA, Kleinschmidt JA. Helper virus-free, optically controllable, and two-plasmid-based production of adeno- associated virus vectors of serotypes 1 to 6. Molecular therapy: the journal of the American Society of Gene Therapy. 2003;7(6):839-50); Grimm D, Kern A, Rittner K, Kleinschmidt JA. Novel tools for production and purification of recombinant adenoassociated virus vectors. Human gene therapy. 1998;9(18):2745-60).) Transfections were performed with Polyethylenimine “Max” (PEI MAX®) transfection reagent from Polysciences. The medium was removed from the HEK293T/17 cells and replaced by the plasmid/PEI MAX mixture diluted in prewarmed DMEM, high glucose, GlutaMAX™. Cells were incubated for 96 hours at 37 °C, 5% CO2. Subsequently, cells were lysed under high salt conditions, and the crude viral lysate was purified by centrifugation, filtration, and POROS CaptureSelect AAVX resin (AAVX). Viruses were formulated in PBS formulation buffer pH 7.3 with 0.001% Pluronic®F- 68. Titers (Vector Genomes/mL) was determined by qPCR.
[0194] A A V transductions
[0195] AAV transduction experiments were done in a 48 well plate format. The following cell lines with their specific seeding cell densities were used: HEK293T/17 cells were seeded at 20,000 cells per well, in 300 pL total volume; mouse FLS AXR7 and RA-FLS were seeded at 8,000 cells per well in 300 pL total volume. After 24 hours, cells were transduced with the rAAV vectors (diluted in DMEM + GlutaMAX-I (Gibco), without FBS and without Gibco® Antibiotic- Antimycotic ) at a multiplicity of infection in terms of VG/cell (MOI) of 200,000. Four hours post transduction an equal amount of DMEM + GlutaMAX-I (Gibco) supplemented with 2% (v/v) FBS, and 1% (v/v) Gibco® Antibiotic-Antimycotic, and 0.8 pM doxorubicin, was added to each well resulting in a final concentration of 1% FBS, 0.5% (v/v) Gibco® Antibiotic-Antimycotic and 0.4 pM doxorubicin. Cells were stimulated with cytokines 24 h after AAV transductions. The cytokines were either human or mouse IL-ip and TNFa, depending on the cell type (1 ng/mL IL-ip and 10 ng/mL TNFa in DMEM + GlutaMAX-I (Gibco) supplemented with 1% (v/v) Fetal Bovine Serum, and 0.5% (v/v) Gibco® Antibiotic- Antimycotic). If applicable, cells were incubated with or without these cytokines for 72 h and cell toxicity was assessed using the Cell Counting kit-8 (CCK8) from Dojindo. To this end, 30 pL of CCK8 reagent was added to each well, the plates were incubated for 2 h at 37 °C, and absorbance of the plate measured at 450 nm. After measurement of cell toxicity/cell viability, the media of the cells was removed, and the cells were all washed with PBS buffer except HEK293T/17 cells since they are prone to detachment. After washing, cells were lysed using 60 pL passive lysis buffer from Promega, at each well. Then samples were used for One gio™ luciferase measurement, using One gio™ substrate from Promega. The Luciferase signal was measured with the Omega fluostar machine. The measurement was done at different machine gain values due to the differences of the signal found in different cell lines and donors.
[0196] Results
[0197] Inflammation-inducible promoters show increased reporter gene expression in the presence of inflammatory stimulants in a variety of cell types. Results for HEK293T/17 cells are shown in Figs. 2A, 3A, 4A, 5A, 6A, 7A, and 13A. Results for murine FLS are shown in Figs. 2C, 3B, 4C, 5B, 6B, and 7B. Results for primary RA-FLS are shown in Figs. 2D, 3C, 4D, 5C, 6C, 7C, and 13B
[0198] Selected hybrid promoter sequences are shown in Table 3.
Table 3. Hybrid promoter sequences. Some sequences are split into several components merely for illustrative purposes. Constructs marked with * were used in the Examples. SIN = SEQ ID NO. Underlined (i.e., (1) simply underlined or (2) bold and underlined) sequence may be replaced fully or partially by an alternative sequence or may be omitted. SEQ ID NOs:4-6, 16, and 28 may be replaced a different sequence selected from a group comprising SEQ ID NOs:4-6, 16, and 28. SEQ ID NOs:9-12 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:9-12. SEQ ID NOs:19, 39, and 48 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs: 19, 39, and 48. SEQ ID NOs:37, 38, and 49 may be replaced may be replaced a different sequence selected from a group comprising SEQ ID NOs:37, 38, and 49,

Claims

We claim:
1. An inflammation-inducible promoter, the promoter comprising:
(a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13;
(i) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 80% identical to SEQ ID NO:37 or SEQ ID NO: 38, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
2. The inflammation-inducible promoter of claim 1, the promoter comprising from 5’ to 3’ :
(a) a sequence that is at least 80% identical to SEQ ID NO:2, a sequence that is at least 80% identical to SEQ ID NO:3, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; (b) a sequence that is at least 80% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 80% identical SEQ ID NO: 13;
(c) a sequence that is at least 80% identical to SEQ ID NO: 15 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 80% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 80% identical to SEQ ID NO:21 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 80% identical to SEQ ID NO:23 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 80% identical to SEQ ID NO:25, a sequence that is at least 80% identical to SEQ ID NO:26, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 80% identical to SEQ ID NO: 13;
(i) a sequence that is at least 80% identical to SEQ ID NO:30 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 80% identical to SEQ ID NO:32 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 80% identical to SEQ ID NO:34 and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 80% identical to SEQ ID NO:37 or SEQ ID NO: 38, and a sequence that is at least 80% identical to any one of SEQ ID NOs:4-6, 16, or 28.
3. The inflammation-inducible promoter of claim 1, the promoter comprising:
(a) a sequence that is at least 90% identical to SEQ ID NO:2, a sequence that is at least 90% identical to SEQ ID NO:3, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 90% identical SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO: 15 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; (e) a sequence that is at least 90% identical to SEQ ID NO:21 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 90% identical to SEQ ID NO:23 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(g) a sequence that is at least 90% identical to SEQ ID NO:25, a sequence that is at least 90% identical to SEQ ID NO:26, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 90% identical to SEQ ID NO: 13;
(i) a sequence that is at least 90% identical to SEQ ID NO:30 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 90% identical to SEQ ID NO:32 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 90% identical to SEQ ID NO:34 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 90% identical to SEQ ID NO:37 or SEQ ID NO: 38, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
4. The inflammation-inducible promoter of claim 3, the promoter comprising from 5’ to 3’ :
(a) a sequence that is at least 90% identical to SEQ ID NO:2, a sequence that is at least 90% identical to SEQ ID NO:3, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(b) a sequence that is at least 90% identical to any one of SEQ ID NOs:9-12 and a sequence that is at least 90% identical SEQ ID NO: 13;
(c) a sequence that is at least 90% identical to SEQ ID NO: 15 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(d) a sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO:39, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(e) a sequence that is at least 90% identical to SEQ ID NO:21 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(f) a sequence that is at least 90% identical to SEQ ID NO:23 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; (g) a sequence that is at least 90% identical to SEQ ID NO:25, a sequence that is at least 90% identical to SEQ ID NO:26, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(h) a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28, and a sequence that is at least 90% identical to SEQ ID NO: 13;
(i) a sequence that is at least 90% identical to SEQ ID NO:30 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(j) a sequence that is at least 90% identical to SEQ ID NO:32 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28;
(k) a sequence that is at least 90% identical to SEQ ID NO:34 and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28; or
(l) a sequence that is at least 90% identical to SEQ ID NO:37 or SEQ ID NO: 38, and a sequence that is at least 90% identical to any one of SEQ ID NOs:4-6, 16, or 28.
5. The inflammation-inducible promoter of claim 3, the promoter comprising:
(a) SEQ ID NO:2, SEQ ID NO:3, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs:9-12 and SEQ ID NO: 13;
(c) SEQ ID NO: 15 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) any one of SEQ ID NOs: 19, 39, or 48, and any one of SEQ ID NOs:4-6, 16, or 28;
(e) SEQ ID NO:21 and any one of SEQ ID NOs:4-6, 16, or 28;
(f) SEQ ID NO:23 and any one of SEQ ID NOs:4-6, 16, or 28;
(g) SEQ ID NO:25, SEQ ID NO:26, and any one of SEQ ID NOs:4-6, 16, or 28;
(h) any one of SEQ ID NOs:4-6, 16, or 28, and SEQ ID NO: 13;
(i) SEQ ID NO:30 and any one of SEQ ID NOs:4-6, 16, or 28;
(j) SEQ ID NO:32 and any one of SEQ ID NOs:4-6, 16, or 28;
(k) SEQ ID NO:34 and any one of SEQ ID NOs:4-6, 16, or 28; or
(l) any one of SEQ ID NOs:37, 38, or 49, and any one of SEQ ID NOs:4-6, 16, or 28.
6. The inflammation-inducible promoter of claim 5, the promoter comprising from 5’ to 3’ :
(a) SEQ ID NO:2, SEQ ID NO:3, and any one of SEQ ID NOs:4-6, 16, or 28;
(b) any one of SEQ ID NOs: 9- 12 and SEQ ID NO: 13;
(c) SEQ ID NO: 15 and any one of SEQ ID NOs:4-6, 16, or 28;
(d) any one of SEQ ID NOs: 19, 39, or 48, and any one of SEQ ID NOs:4-6, 16, or 28;
(e) SEQ ID NO:21 and any one of SEQ ID NOs:4-6, 16, or 28; (f) SEQ ID NO:23 and any one of SEQ ID NOs:4-6, 16, or 28;
(g) SEQ ID NO:25, SEQ ID NO:26, and any one of SEQ ID NOs:4-6, 16, or 28;
(h) any one of SEQ ID NOs:4-6, 16, or 28, and SEQ ID NO: 13;
(i) SEQ ID NO:30 and any one of SEQ ID NOs:4-6, 16, or 28;
(j) SEQ ID NO:32 and any one of SEQ ID NOs:4-6, 16, or 28;
(k) SEQ ID NO:34 and any one of SEQ ID NOs:4-6, 16, or 28; or
(l) any one of SEQ ID NOs:37, 38, or 49, and any one of SEQ ID NOs:4-6, 16, or 28.
7. The inflammation-inducible promoter of one of claims 1-6, the promoter comprising:
(a) a sequence that is at least 80% identical to SEQ ID NO: 1;
(b) a sequence that is at least 80% identical to SEQ ID NO:7 or SEQ ID NO:8;
(c) a sequence that is at least 80% identical to SEQ ID NO: 14;
(d) a sequence that is at least 80% identical to SEQ ID NO: 17 or SEQ ID NO: 18;
(e) a sequence that is at least 80% identical to SEQ ID NO:20;
(f) a sequence that is at least 80% identical to SEQ ID NO:22;
(g) a sequence that is at least 80% identical to SEQ ID NO:24;
(h) a sequence that is at least 80% identical to SEQ ID NO:27;
(i) a sequence that is at least 80% identical to SEQ ID NO:29;
(j) a sequence that is at least 80% identical to SEQ ID NO:31;
(k) a sequence that is at least 80% identical to SEQ ID NO:33; or
(l) a sequence that is at least 80% identical to SEQ ID NO:35 or SEQ ID NO:36.
8. The inflammation-inducible promoter of claim 7, the promoter comprising:
(a) a sequence that is at least 90% identical to SEQ ID NO: 1;
(b) a sequence that is at least 90% identical to SEQ ID NO:7 or SEQ ID NO:8;
(c) a sequence that is at least 90% identical to SEQ ID NO: 14;
(d) a sequence that is at least 90% identical to SEQ ID NO: 17 or SEQ ID NO: 18;
(e) a sequence that is at least 90% identical to SEQ ID NO:20;
(f) a sequence that is at least 90% identical to SEQ ID NO:22;
(g) a sequence that is at least 90% identical to SEQ ID NO:24;
(h) a sequence that is at least 90% identical to SEQ ID NO:27;
(i) a sequence that is at least 90% identical to SEQ ID NO:29;
(j) a sequence that is at least 90% identical to SEQ ID NO:31;
(k) a sequence that is at least 90% identical to SEQ ID NO:33; or (1) a sequence that is at least 90% identical to SEQ ID NO:35 or SEQ ID NO:36.
9. The inflammation-inducible promoter of claim 8, the promoter comprising any one of SEQ ID NOs: l, 7, 8, 14, 17, 18, 20, 22, 24, 27, 29, 31, 33, 35, or 36.
10. An expression construct comprising:
(a) the inflammation-inducible promoter of any one of claims 1-9; and
(b) an operatively linked transgene.
11. The expression construct of claim 10, wherein the transgene encodes a therapeutic protein.
12. A recombinant nucleic acid comprising the inflammation-inducible promoter of any one of claims 1-9 or the expression construct of claim 10 or 11.
13. A vector comprising the inflammation-inducible promoter of any one of claims 1-9 or the expression construct of claim 10 or 11.
14. The vector of claim 13, wherein the vector is a viral vector.
15. The vector of claim 14, wherein the viral vector is selected from the group consisting of adenoviral vector, adeno-associated virus (AAV) vector, and lentiviral vector.
16. The vector of claim 15, wherein the vector comprises an AAV2 genome.
17. The vector of claim 15 or 16, wherein the vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV.rh8, AAV.rhlO, AAV.rh39, AAV.43, AAV2/2-66, AAV2/2-84, or AAV2/2-125 capsid.
18. The vector of claim 15 or 16, wherein the vector comprises an capsid protein comprising any one of SEQ ID NOs:40-47.
19. The vector of claim 13, wherein the vector is a non-viral vector.
20. The vector of claim 19, wherein the vector is a plasmid, bacmid, cosmid, or, closed-ended linear DNA (ceDNA).
21. A composition comprising the vector of any one of claims 13-20 and a pharmaceutically acceptable excipient.
22. A cell comprising the inflammation-inducible promoter of any one of claims 1-9, the expression construct of claim 10 or 11, or the vector of any one of claims 13.
23. The cell of claim 22, wherein the cell is an isolated cell.
24. The cell of claim 22 or 23, wherein the cell is a fibroblast-like synoviocyte, cartilage cell, chondrocyte, or chondroblast.
25. A method of inducing the expression of a transgene, the method comprising
(a) providing the cell of claim 22 or 23;
(b) contacting the promoter with one or more inflammatory stimuli.
26. The method of claim 25, wherein the one or more inflammatory stimuli are selected from the group consisting of inflammatory cytokines, inflammatory chemokines, growth factors, pathogen associated molecular patterns (PAMP), or a damage associated molecular patterns (DAMP).
27. The method of claim 26, wherein the one or more inflammatory stimuli are one or more inflammatory cytokines or chemokines.
28. The method of claim 27, wherein the one or more inflammatory cytokines or chemokines are one or more of IL-1, IL-2, IL-5, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL- 17, IL-18, IL-21, IL-22, IL-23, IL-25, tumor necrosis factor alpha (TNF-a), interferonalpha (IFN-a), interferon-P (IFN-P), interferon gamma (IFNy), granulate colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB), macrophage inflammatory protein-la (MIP-la), CCL3, CCL4, CCL5, RANTES, stromal cell-derived factor 1 (SDF-1), Neutrophil-activating protein 3 (NAP-3), monocyte chemoattractant protein (MCP-1), CXCL8, IFN-y-induced protein (IP- 10), lymphotactin, fractalkine, or combinations thereof.
29. The method of claim 28, wherein the one or more inflammatory cytokines or chemokines are one or more of IL-1, IL-6, IL-8, IL- 17, IL-23, TNF-a, (IP- 10), or combinations thereof.
30. The method of claim 25, wherein the one or more inflammatory stimuli are lipopolysaccharides.
EP24718226.4A 2023-04-13 2024-04-11 Inflammation-inducible promoters Pending EP4694906A1 (en)

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