Regulation of endogenous gene expression in cells using zinc finger proteins

Abstract

The present invention provides methods for modulating expression of endogenous cellular genes using recombinant zinc finger proteins.

1 . A method of inhibiting expression of an endogenous cellular gene in a cell, the method comprising the step of: 
 administering to the cell a nucleic acid molecule comprising an RNA sequence which encodes a first engineered zinc finger protein, wherein the nucleic acid molecule expresses the zinc finger protein in the cell;    such that the zinc finger protein contacts a first target site in the endogenous cellular gene, wherein the K d  of the zinc finger protein is less than about 25 nM;    thereby inhibiting expression of the endogenous cellular gene.    
     
     
         2 . The method of  claim 1 , wherein the step of administering further comprises administering a second zinc finger protein-encoding RNA sequence that expresses a second zinc finger protein in the cell, and wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with the second zinc finger protein.  
     
     
         3 . The method of  claim 2 , wherein the first and second target sites are adjacent.  
     
     
         4 . The method of  claim 3 , wherein the first and second zinc finger proteins are covalently linked, forming a fusion protein.  
     
     
         5 . The method of  claim 1 , wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.  
     
     
         6 . The method of  claim 5 , wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.  
     
     
         7 . The method of  claim 2 , wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.  
     
     
         8 . The method of  claim 7 , wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.  
     
     
         9 . A method of inhibiting expression of an endogenous cellular gene in a cell, the method comprising the step of: 
 administering to the cell a nucleic acid molecule comprising an RNA sequence which encodes an engineered fusion zinc finger protein, wherein the nucleic acid molecule expresses the fusion zinc finger protein in the cell, and wherein the fusion zinc finger protein comprises six fingers and a regulatory domain;    such that the fusion zinc finger protein contacts a target site in the endogenous cellular gene, wherein the K d  of the fusion zinc finger protein is less than about 25 nM;    thereby inhibiting expression of the endogenous cellular gene.    
     
     
         10 . The method of  claim 1 , wherein the cell is selected from the group consisting of animal cell, a plant cell, a bacterial cell, a protozoal cell, or a fungal cell.  
     
     
         11 . The method of  claim 10 , wherein the cell is a mammalian cell.  
     
     
         12 . The method of  claim 11 , wherein the cell is a human cell.  
     
     
         13 . The method of  claim 1 , wherein expression of the endogenous cellular gene is inhibited by at least about 75%-100%.  
     
     
         14 . The method of  claim 1 , wherein the endogenous cellular gene is selected from the group consisting of VEGF, ERα, IGF-I, c-myc, c-myb, ICAM, and Her2/Neu.  
     
     
         15 . The method of  claim 1 , wherein the endogenous cellular gene is VEGF.  
     
     
         16 . The method of  claim 1 , wherein the inhibition of gene expression prevents gene activation.  
     
     
         17 . The method of  claim 5  or  claim 7 , wherein the regulatory domain is selected from the group consisting of a transcriptional repressor, an endonuclease, a methyl transferase, and a histone deacetylase.  
     
     
         18 . The method of  claim 1 , wherein the step of administering the nucleic acid molecule to the cell comprises administering the nucleic acid molecule in a lipid:nucleic acid complex or as naked nucleic acid.  
     
     
         19 . The method of  claim 1 , wherein the cell comprises less than about 1.5×10 6  copies of the zinc finger protein.  
     
     
         20 . The method of  claim 1 , wherein the target site is upstream of a transcription initiation site of the endogenous cellular gene.  
     
     
         21 . The method of  claim 1 , wherein the target site is adjacent to a transcription initiation site of the endogenous cellular gene.  
     
     
         22 . The method of  claim 1 , wherein the target site is adjacent to an RNA polymerase pause site, wherein the RNA polymerase pause site is downstream of a transcription initiation site of the endogenous cellular gene.  
     
     
         23 . The method of  claim 1 , wherein the zinc finger protein comprises an SP-1 backbone.  
     
     
         24 . The method of  claim 23 , wherein the zinc finger protein comprises a regulatory domain and is humanized.  
     
     
         25 . A method of activating expression of an endogenous cellular gene in a cell, the method comprising the step of: 
 administering to the cell a nucleic acid molecule comprising an RNA sequence which encodes an engineered zinc finger protein, wherein the nucleic acid molecule expresses the zinc finger protein in the cell; and    contacting a first target site in the endogenous cellular gene with the zinc finger protein, wherein the K d  of the zinc finger protein is less than about 25 nM;    thereby activating expression of the endogenous cellular gene.    
     
     
         26 . The method of  claim 25 , wherein the step of administering further comprises administering a second zinc finger protein-encoding nucleic acid molecule that expresses a second zinc finger protein in the cell, and wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with the second zinc finger protein.  
     
     
         27 . The method of  claim 26 , wherein the first and second target sites are adjacent.  
     
     
         28 . The method of  claim 27 , wherein the first and second zinc finger proteins are covalently linked, forming a fusion protein.  
     
     
         29 . The method of  claim 25 , wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.  
     
     
         30 . The method of  claim 29 , wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.  
     
     
         31 . The method of  claim 26 , wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.  
     
     
         32 . The method of  claim 31 , wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.  
     
     
         33 . A method of activating expression of an endogenous cellular gene in a cell, the method comprising the step of: 
 administering to the cell a nucleic acid molecule comprising an RNA sequence which encodes an engineered fusion zinc finger protein, wherein the nucleic acid molecule expresses a fusion zinc finger protein in the cell, and wherein the fusion zinc finger protein comprises six fingers and a regulatory domain; and    contacting a target site in the endogenous cellular gene with the fusion zinc finger protein, wherein the K d  of the fusion zinc finger protein is less than about 25 nM;    thereby activating expression of the endogenous cellular gene.    
     
     
         34 . The method of  claim 25 , wherein the cell is selected from the group consisting of animal cell, a plant cell, a bacterial cell, a protozoal cell, or a fungal cell.  
     
     
         35 . The method of  claim 34 , wherein the cell is a mammalian cell.  
     
     
         36 . The method of  claim 35 , wherein the cell is a human cell.  
     
     
         37 . The method of  claim 25 , wherein the endogenous cellular gene is activated to at least about 200-500%.  
     
     
         38 . The method of  claim 25 , wherein the endogenous cellular gene is selected from the group consisting of FAD2-1, EPO, GM-CSF, GDNF, VEGF, and LDL-R.  
     
     
         39 . The method of  claim 25 , wherein the endogenous cellular gene is VEGF.  
     
     
         40 . The method of  claim 25 , wherein the activation of gene expression prevents repression of gene expression.  
     
     
         41 . The method of  claim 30  or 32, wherein the regulatory domain is selected from the group consisting of a transcriptional activator, or a histone acetyltransferase.  
     
     
         42 . The method of  claim 25 , wherein the step of administering the nucleic acid molecule to the cell comprises administering the nucleic acid molecule in a lipid:nucleic acid complex or as naked nucleic acid.  
     
     
         43 . The method of  claim 25 , wherein the cell comprises less than about 1.5×10 6  copies of the zinc finger protein.  
     
     
         44 . The method of  claim 25 , wherein the target site is upstream of a transcription initiation site of the endogenous cellular gene.  
     
     
         45 . The method of  claim 25 , wherein the target site is adjacent to a transcription initiation site of the endogenous cellular gene.  
     
     
         46 . The method of  claim 25 , wherein the target site is adjacent to an RNA polymerase pause site, wherein the RNA polymerase pause site is downstream of a transcription initiation site of the endogenous cellular gene.  
     
     
         47 . The method of  claim 25 , wherein the zinc finger protein comprises an SP-1 backbone.  
     
     
         48 . The method of  claim 47 , wherein the zinc finger protein comprises a regulatory domain and is humanized.  
     
     
         49 . A method of modulating expression of an endogenous cellular gene in a cell, the method comprising the step of: 
 administering to the cell a nucleic acid molecule comprising an RNA sequence which encodes an engineered zinc finger protein, wherein the nucleic acid molecule expresses the zinc finger protein in the cell; and    contacting a first target site in the endogenous cellular gene with the zinc finger protein,    thereby modulating expression of the endogenous cellular gene.    
     
     
         50 . The method of  claim 49 , wherein the step of administering further comprises administering a second zinc finger protein-encoding nucleic acid molecule comprising an RNA sequence that expresses a second zinc finger protein in the cell, and wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with the second zinc finger protein.  
     
     
         51 . The method of  claim 50 , wherein the first and second target sites are adjacent.  
     
     
         52 . The method of  claim 51 , wherein the first and second zinc finger proteins are covalently linked, forming a fusion protein.  
     
     
         53 . The method of  claim 49 , wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.  
     
     
         54 . The method of  claim 53 , wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.  
     
     
         55 . The method of  claim 50 , wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.  
     
     
         56 . The method of  claim 55 , wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.  
     
     
         57 . A method of modulating expression of an endogenous cellular gene in a cell, the method comprising the step of: 
 administering to the cell a nucleic acid molecule comprising an RNA sequence which encodes an engineered fusion zinc finger protein, wherein the nucleic acid molecule expresses the fusion zinc finger protein in the cell, and wherein the fusion zinc finger protein comprises six fingers and a regulatory domain;    such that the fusion zinc finger protein contacts a target site in the endogenous cellular gene;    thereby modulating expression of the endogenous cellular gene.    
     
     
         58 . The method of  claim 49 , wherein the cell is selected from the group consisting of animal cell, a plant cell, a bacterial cell, a protozoal cell, or a fungal cell.  
     
     
         59 . The method of  claim 58 , wherein the cell is a mammalian cell.  
     
     
         60 . The method of  claim 59 , wherein the cell is a human cell.  
     
     
         61 . The method of  claim 49 , wherein the endogenous cellular gene is selected from the group consisting of VEGF, ERα, IGF-I, c-myc, c-myb, ICAM, Her2/Neu, FAD2-1, EPO, GM-CSF, GDNF, and LDL-R.  
     
     
         62 . The method of  claim 61 , wherein the endogenous cellular gene is VEGF.  
     
     
         63 . The method of  claim 52  or  claim 54 , wherein the regulatory domain is selected from the group consisting of a transcriptional activator, a transcriptional repressor and an endonuclease.  
     
     
         64 . The method of  claim 49 , wherein the step of administering the nucleic acid molecule to the cell comprises administering the nucleic acid molecule in a lipid:nucleic acid complex or as naked nucleic acid.  
     
     
         65 . The method of  claim 49 , wherein the target site is upstream of a transcription initiation site of the endogenous cellular gene.  
     
     
         66 . The method of  claim 49 , wherein the target site is adjacent to a transcription initiation site of the endogenous cellular gene.  
     
     
         67 . The method of  claim 49 , wherein the target site is adjacent to an RNA polymerase pause site, wherein the RNA polymerase pause site is downstream of a transcription initiation site of the endogenous cellular gene.  
     
     
         68 . The method of  claim 49 , wherein the zinc finger protein comprises an SP-1 backbone.  
     
     
         69 . The method of  claim 68 , wherein the zinc finger protein comprises a regulatory domain and is humanized.