Methods and Compositions for Regulating Gene Expression

Abstract

In certain embodiments, the disclosure relates to compositions and methods relating to a translation-based gene regulation system that functions in mammalian cells. In certain specific embodiments, the disclosure relates to methods of regulating gene expression via modulating translational termination.

1 . A method for inducing expression of a target protein in a host cell, comprising contacting the host cell with an effective amount of an agent, wherein the host cell comprises a nucleic acid encoding a target protein, wherein the nucleic acid is operably linked to a promoter and has been modified to contain at least one nonsense mutation downstream to and in close proximity of the start codon that initiates translation of the target protein, and wherein the agent suppresses the nonsense mutation. 
   
   
       2 . The method of  claim 1 , wherein the host cell is a mammalian cell. 
   
   
       3 . The method of  claim 1 , wherein the agent is an aminoglycoside antibiotic or an analog thereof. 
   
   
       4 . The method of  claim 3 , wherein the aminoglycoside antibiotic is G418. 
   
   
       5 . The method of  claim 1 , wherein the agent is an acetylamino benzoic acid compound or a derivative thereof. 
   
   
       6 . The method of  claim 5 , wherein the agent is selected from 3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid and 3-{2-[4-(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoic acid. 
   
   
       7 . The method of  claim 4 , wherein the number of codons between the nonsense mutation and the start codon is selected from: 0, 1, 2, 3, 4, and 5. 
   
   
       8 . The method of  claim 1 , wherein the target protein is a secreted protein. 
   
   
       9 . The method of  claim 8 , wherein the nonsense mutation is introduced in a region of the nucleic acid which encodes a signal peptide. 
   
   
       10 - 12 . (canceled) 
   
   
       13 . A recombinant polynucleotide molecule comprising a nucleic acid encoding a target protein, wherein the nucleic acid is operably linked to a promoter and has been modified to contain at least one nonsense mutation downstream to and in close proximity of the start codon that initiates translation of the target protein. 
   
   
       14 - 19 . (canceled) 
   
   
       20 . A host cell comprising the polynucleotide molecule of  claim 13 . 
   
   
       21 . (canceled) 
   
   
       22 . The host cell of  claim 20 , wherein the cell is a mammalian cell and further comprises an agent which suppresses the nonsense mutation. 
   
   
       23 . (canceled) 
   
   
       24 . A recombinant vector comprising:
 a) a promoter;   b) a nucleic acid encoding a linker peptide, wherein the nucleic acid is operably linked to the promoter, and wherein the nucleic acid has been modified to contain at least one nonsense mutation located downstream to and in close proximity of the start codon that initiates translation of the linker peptide; and   c) at least one cloning site downstream to the nucleic acid for introducing a target nucleic acid sequence which encodes a target protein to be fused in frame to the carboxyl terminus of the linker peptide.   
   
   
       25 - 26 . (canceled) 
   
   
       27 . A method for identifying a nucleic acid construct having a desired biological activity from a population of nucleic acid constructs, comprising:
 a) producing a population of candidate nucleic acid constructs, wherein each of the candidate nucleic acid construct is cloned into the multiple cloning sites of the vector of  claim 24 ;   b) expressing the population of candidate nucleic acid constructs in a test cell; and   c) identifying a nucleic acid construct that, when expressed in the test cell in the presence of an agent that suppresses the nonsense mutation, produces a desired biological activity.   
   
   
       28 - 40 . (canceled) 
   
   
       41 . A method for identifying an agent that suppresses a nonsense mutation, comprising:
 (a) providing a test host cell which contains a polynucleotide molecule comprising a nucleic acid encoding a target protein, wherein the nucleic acid is operably linked to a promoter and has been modified to contain at least one nonsense mutation downstream to and in close proximity of the start codon that initiates translation of the target protein;   (b) providing a control host cell comprising a wild type nucleic acid encoding the target protein expressed under an appropriate promoter and the expression of the target protein is at wild type level;   (c) contacting the test host cell and the control host cell with a candidate agent; and   (d) assaying for the level of the target protein in the test host cell and in control host cell;   wherein if the candidate agent induces translation of the target protein in the test host cell to a significant level, the candidate agent is an agent that suppresses the nonsense mutation.   
   
   
       42 - 51 . (canceled) 
   
   
       52 . A method of inducing expression of a target gene in an individual, comprising:
 (a) obtaining cells from the individual;   (b) maintaining the cells under conditions appropriate for cell growth and cell division;   (c) introducing into the cells of step (b) a DNA construct comprising a nucleic acid encoding a target protein, wherein the nucleic acid is operably linked to a promoter and has been modified to contain at least one nonsense mutation downstream to and in close proximity of the start codon that initiates translation of the target protein;   (d) returning the cells produced in step (c) to the individual; and   (e) administering to the individual an agent that suppresses the nonsense mutation.   
   
   
       53 . A kit for regulating gene expression, comprising a vector comprising: (a) a promoter; (b) an nucleic acid encoding a linker peptide, wherein the nucleic acid is operably linked to the promoter, and wherein the nucleic acid has been modified to contain at least one nonsense mutation located downstream to and in close proximity of the start codon that initiates translation of the linker peptide; and (c) at least one cloning site downstream to the nucleic acid for introducing a target nucleic acid sequence which encodes a target protein to be fused in frame to the carboxyl terminus of the linker peptide. 
   
   
       54 - 55 . (canceled) 
   
   
       56 . The kit of  claim 53 , further comprising an agent that suppresses a nonsense mutation. 
   
   
       57 - 60 . (canceled) 
   
   
       61 . A method of inducing expression of a target gene in an individual, comprising:
 (a) administering to the individual a DNA construct comprising a nucleic acid encoding a target protein, wherein the nucleic acid is operably linked to a promoter and has been modified to contain at least one nonsense mutation downstream to and in close proximity of the start codon that initiates translation of the target protein; and   (b) administering to the individual an agent that suppresses the nonsense mutation.   
   
   
       62 . (canceled) 
   
   
       63 . The method of  claim 61 , wherein the number of codons is selected from: 0, 1, 2, 3, 4 and 5.