Methods of transcriptionally modulating gene expression

Abstract

The present invention provides a method of transcriptionally modulating the expression of a gene of interest, the expression of which is associated with a defined physiological or pathological effect within a multicellular organism. The method comprises contacting a cell which is capable of expressing the gene with an amount of a molecule effective to transcriptionally modulate expression of the gene and thereby affect the level of the protein encoded by the gene which is expressed by the cell. Molecules useful in the practice of the invention are characterized as follows (a) do not naturally occur in the cell, (b) specifically transcriptionally modulate expression of the gene of interest, and (c) bind to DNA or RNA or bind to a protein through a domain of such protein which is not a ligand binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand binding domain is normally associated with the defined physiological or pathological effect.

What is claimed is:  
     
         1 . A method of transcriptionally modulating the expression of a gene of interest, the expression of which is associated with a defined physiological or pathological effect within a multicellular organism, which comprises contacting a cell, which is capable of expressing the gene, with an amount of a molecule effective to transcriptionally modulate expression of the gene and thereby affect the level of the protein encoded by the gene which is expressed by the cell, which molecule (a) does not naturally occur in the cell, (b) specifically transcriptionally modulates expression of the gene of interest, and (c) binds to DNA or RNA, or binds to a protein through a domain of such protein which is not a ligand-binding domain of a receptor which naturally occurs in the cell, the biding of a ligand to which ligand-bind domain is normally associated with the defined physiological or pathological effect.  
     
     
         2 . A meth  claim 1 , wherein the cell is a cell the multicellular organism.  
     
     
         3 . A method of  claim 1 , wherein the cell is a human cell.  
     
     
         4 . A method of  claim 1 , wherein the cell is an animal cell.  
     
     
         5 . A method of  claim 1 , wherein the cell is a plant cell.  
     
     
         6 . A method of  claim 1 , wherein the gene of interest is a human gene.  
     
     
         7 . A method of  claim 1 , wherein the gene of interest encodes a hematopoietic protein.  
     
     
         8 . A method of  claim 7 , wherein the hematopoietic protein is a colony stimulating factor.  
     
     
         9 . A method of  claim 8 , wherein the colony stimulating factor is granulocyte-macrophage colony stimulating factor (GM-CSF).  
     
     
         10 . A method of  claim 8 , wherein the colony stimulating factor is granulocyte colony stimulating factor (CSF).  
     
     
         11 . A method of  claim 8 , wherein the colony stimulating factor is macrophage colony stimulating factor (M-CSF).  
     
     
         12 . A method of  claim 7 , wherein the hematopoietic protein is erythropoietin (EPO).  
     
     
         13 . A method of  claim 1 , wherein the gene of interest encodes an interleukin (IL).  
     
     
         14 . A method of  claim 1 , wherein the gene of interest encodes a growth hormone.  
     
     
         15 . A method of  claim 14 , where the growth hormone is selected from the group consisting of human, bovine, porcine, avian, ovine, piscine, and equine growth hormone, and polypeptide analogs thereof having the biological activity of the corresponding naturally occurring growth hormone.  
     
     
         16 . A method of  claim 1  wherein the gene of interest is a viral gene.  
     
     
         17 . A method of  claim 16 , wherein the viral gene is a retroviral gene.  
     
     
         18 . A method of  claim 17 , wherein the retroviral gene is a gene from the HIV, HTLV-1, or HTLV-2 virus.  
     
     
         19 . A method of  claim 16 , wherein the viral gene is a gene from a hepatitis virus.  
     
     
         20 . A method of  claim 16 , wherein the viral gene is a gene from a herpes virus.  
     
     
         21 . A method of  claim 16 , wherein the viral gene is a gene from an animal virus.  
     
     
         22 . A method of  claim 16 , wherein the viral gene is a gene from a papilloma virus.  
     
     
         23 . A method of  claim 16 , wherein the viral gene is a gene from a cytomegalovirus.  
     
     
         24 . A method of  claim 21 , wherein the animal virus is a pseudorabies, Marek's, Newcastle's Disease, or IBR virus.  
     
     
         25 . A method of  claim 1 , wherein the gene of interest is a plant gene.  
     
     
         26 . A method of  claim 25 , wherein the plant gene encodes an agronomically important trait.  
     
     
         27 . A method of  claim 26 , wherein the agronomically important trait is selected from the group consisting of germination, sprouting, flowering, fruit ripening, salt tolerance, herbicide resistance, pesticide resistance, fungicide resistance, temperature resistance, and growth.  
     
     
         28 . A method of  claim 1 , wherein the gene of interest is a protozoan gene.  
     
     
         29 . A method of  claim 28 , wherein the protozoan is selected from the group consisting of Trypanosoma, Plasmodium, Leishmania, Giardia, Entamoeba, Toxoplasma, Babesia, and Cryptosporidiosis.  
     
     
         30 . A method of  claim 1 , wherein the gene of interest is a helminth gene.  
     
     
         31 . A method of  claim 1 , wherein the gene of interest is an oncogene.  
     
     
         32 . A method of  claim 31 , wherein the oncogene is the phl-abl oncogene.  
     
     
         33 . A method of  claim 31 , wherein the oncogene is selected from the group consisting of H-, N-, and K-ras oncogenes.  
     
     
         34 . A method of  claim 31 , wherein the oncogene is the neu oncogene.  
     
     
         35 . A method of  claim 31 , wherein the oncogene is the src oncogene.  
     
     
         36 . A method of  claim 1 , wherein the gene of interest encodes a naturally occurring receptor.  
     
     
         37 . A method of  claim 36 , wherein the receptor is the human low density lipoprotein (LDL) receptor.  
     
     
         38 . A method of  claim 36 , wherein the receptor is the receptor for a hematopoietic protein selected from the group consisting of M-CSF, G-CSF, GM-CSF, and EPO.  
     
     
         39 . A method of  claim 36 , wherein the receptor is the receptor for an interleukin (IL) selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 and IL-8.  
     
     
         40 . A method of  claim 36 , wherein the receptor is a cell surface protein which mediates infection of the cell by a virus.  
     
     
         41 . A method of  claim 40 , wherein the virus is selected from the group consisting of HIV, HTLV-1, HTLV-2, a hepatitis virus, a herpes virus, an animal virus, a papilloma virus, a cytomegalo virus, and a rhinovirus.  
     
     
         42 . A method of  claim 1 , wherein (c) the protein is not the protein encoded by the gene of interest.  
     
     
         43 . A method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of transcriptionally modulating the expression of a gene of interest which comprises contacting a sample which contains a predefined number of cells with a predetermined amount of a molecule to be tested, each such cell comprising DNA consisting essentially of (i) a modulatable transcriptional regulatory sequence of the gene of interest, (ii) a promoter of the gene of interest, and (iii) a reporter gene, which expresses a polypeptide capable of producing a detectable signal, coupled to, and under the control of, the promoter, under conditions such that the molecule, if capable of acting as a transcriptional modulator of the gene of interest, causes a measureable detectable signal to be produced by the polypeptide expressed by the reporter gene, quantitatively determining the amount of the signal produced, comparing the amount so determined with the amount of produced signal detected in the absence of any molecule being tested or upon contacting the sample with any other molecule, and thereby identifying the molecule as one which causes a change in the detectable signal produced by the polypeptide expressed by the reporter gene, and thus identifying the molecule as a molecule capable of transcriptionally modulating the expression of the gene of interest.  
     
     
         44 . A method of  claim 43 , wherein the molecule (a) does not naturally occur in the cell, (b) specifically transcriptionally modulates expression of the gene of interest, and (c) binds to DNA or RNA or binds to a protein through a domain of such protein which is not a ligand binding domain of a receptor which naturally occurs in the cell, the binding of a ligand to which ligand binding domain is normally associated with a defined physiological or pathological effect.  
     
     
         45 . A method of  claim 43 , wherein the sample comprises cells in monolayers.  
     
     
         46 . A method of  claim 43 , wherein the sample comprises cells in suspension.  
     
     
         47 . A method of  claim 43 , wherein the cells comprise human, animal, or plant cells.  
     
     
         48 . A method of  claim 43 , wherein the predefined number of cells is from about 5×10 2  to about 5×10 5  cells.  
     
     
         49 . A method of  claim 48 , wherein the predefined number of cells is from about 10 3  to about 5×10 4  cells.  
     
     
         50 . A method of  claim 43 , wherein the predetermined amount of the molecule to be tested is based upon the volume of the sample.  
     
     
         51 . A method of  claim 43 , wherein the predetermined amount is from about 0.1 nM to about 5 mM.  
     
     
         52 . A method of  claim 51 , wherein the predetermined amount is from about 0.1 μM to about 500 μM.  
     
     
         53 . A method of  claim 52 , wherein the predetermined amount is less than about 100 μM.  
     
     
         54 . A method of  claim 43 , wherein the contacting is effected from about 1 to about 24 hours.  
     
     
         55 . A method of  claim 54 , wherein the contacting is effected from about 2 to about 12 hours.  
     
     
         56 . A method of  claim 43 , wherein the contacting is effected with more than one predetermined amount of the molecule to be tested.  
     
     
         57 . A method of  claim 43 , wherein the molecule to be tested is a purified molecule.  
     
     
         58 . A method of  claim 43 , wherein the modulatable transcriptional regulatory sequence comprises a cloned genomic regulatory sequence.  
     
     
         59 . A method of  claim 43 , wherein the DNA consists essentially of more than one modulatable transcriptional regulatory sequence.  
     
     
         60 . A method of  claim 43 , wherein the modulatable transcriptional regulatory sequence comprises less than about 100 kilobases.  
     
     
         61 . A method of  claim 60 , wherein the modulatable transcriptional regulatory sequence comprises less than about 50 kilobases.  
     
     
         62 . A method of  claim 61 , wherein the modulatable transcriptional regulatory sequence comprises a sequence of less than about 10 kilobases.  
     
     
         63 . A method of  claim 52 , wherein the modulatable transcriptional regulatory sequence comprises a sequence of less than about 2 kilobases.  
     
     
         64 . A method of  claim 43 , wherein the reporter gene is inserted downstream of the promoter of the gene of interest by homologous recombination.  
     
     
         65 . A method  claim 43 , wherein the reporter gene encodes a luciferase.  
     
     
         66 . A method of  claim 43 , wherein the reporter gene encodes chloramphenicol acetyltransferase.  
     
     
         67 . A method of  claim 43 , wherein the reporter gene encodes beta glucuronidase.  
     
     
         68 . A method of  claim 43 , wherein the reporter gene encodes beta galactosidase.  
     
     
         69 . A method of  claim 43 , wherein the reporter gene encodes neomycin phosphotransferase.  
     
     
         70 . A method of  claim 43 , wherein the reporter gene encodes guanine xanthine phosphoribosyltransferase.  
     
     
         71 . A screening method according to  claim 43  which comprises separately contacting each of a plurality of substantially identical samples, each of which contains a predefined number of cells, with a predetermined amount of a different molecule to be tested.  
     
     
         72 . A screening method of  claim 71 , wherein the plurality of samples comprises more that about 10 4  samples.  
     
     
         73 . A screening method of  claim 72 , wherein the plurality of samples comprises more than about 5×10 4  samples.  
     
     
         74 . A screening method of  claim 71 , wherein more that about 10 3  samples per week are contacted with different molecules.  
     
     
         75 . A method of essentially simultaneously screening molecules to determine whether the molecules are capable of transcriptionally modulating one or more genes of interest in a panel of such genes which comprises essentially simultaneously screening the molecules against each of the genes of interest according to the method of  claim 71 .  
     
     
         76 . A method for transcriptionally modulating in a multicellular organism the expression of a gene of interest, the expression of which is associated with a defined physiological or pathological effect in the organism, which comprises administering to the organism an amount of a molecule effective to transcriptionally modulate expression of the gene and thus affect the defined physiological or pathological effect, which molecule (a) does not naturally occur in the organism, (b) specifically transcriptionally modulates expression of the gene of interest, and (c) binds to DNA or RNA or binds to a protein through a domain of such protein which is not a ligand binding domain of a receptor which naturally occurs in the organism, the binding of a ligand to which ligand binding domain is normally associated with the defined physiological or pathological effect.  
     
     
         77 . A method of  claim 76 , wherein the multicellular organism is a human being.  
     
     
         78 . A method of  claim 76 , wherein the multicellular organism is an animal.  
     
     
         79 . A method of  claim 76 , wherein the multicellular organism is a plant.  
     
     
         80 . A method of  claim 76 , wherein the defined pathological effect is a disorder and modulated expression of the gene of interest is associated with amelioration of the disorder.  
     
     
         81 . A method of  claim 77 , wherein the defined pathological effect is a disorder selected from the group consisting of cancer, a hematopoietic dysfunction, diabetes, tissue inflammation, atherosclerosis, viral infections, dysfunctions of memory or learning, and dysfunctions in a cholesterol or other metabolic pathway.  
     
     
         82 . A method of  claim 78 , wherein the defined physiological effect is growth and the organism is an animal such as a cow, a pig, a bird, a fish, a sheep or a horse.  
     
     
         83 . A method of  claim 79 , wherein the defined physiological or pathological effect is an agronomically important trait.  
     
     
         84 . A method of  claim 76 , wherein the administering comprises topical contact.  
     
     
         85 . A method of  claim 77  or  78 , wherein the administering comprises oral, transdermal, intravenous, intramuscular or subcutaneous administration.