Expression system

Abstract

The present invention provides a polynucleotide comprising a RNA polymerase III promoter, a region encoding a siRNA, and a transcriptional termination element comprising five consecutive thymine residues. The invention also provides for vectors, cells and non-human transgenic animal comprising the polynucleotides of the invention as well as their use in medicaments for various conditions.

1 . A polynucleotide comprising: 
 a RNA polymerase III promoter;    a region encoding a siRNA; and    a transcriptional termination element comprising five consecutive thymine residues in the sense strand.    
     
     
         2 . A polynucleotide according to  claim 1 , wherein the promoter is the RNA polymerase III H1-RNA gene promoter or a functional derivative thereof.  
     
     
         3 . A polynucleotide according to  claim 1 , wherein the promoter is the RNA polymerase III 5S, U6, adenovirus VA1, Vault, telomerase RNA, or tRNA gene promoter or a functional derivative thereof.  
     
     
         4 . A polynucleotide according to  claim 1 ,  2  or  3 , wherein the region encoding the siRNA comprises: 
 a region complementary to a target gene and a second region complementary to the first region; and  
 a spacer separating the two complementary regions.  
 
     
     
         5 . A polynucleotide according to  claim 4 , wherein: 
 the spacer region in the polynucleotide comprises two consecutive thymine residues in the sense strand immediately 3′ of the region complementary to the target gene; and    immediately 3′ of the region complementary to the first region are two thymine residues in the sense strand    wherein in the RNA transcript generated from the polynucleotide the complementary region and spacer are capable of forming a stem loop structure.    
     
     
         6 . A polynucleotide according to  claim 4  or  5 , wherein the spacer region is from seven to fifteen nucleotides in length and/or the region complementary to the target gene is from nineteen to twenty-one bases in length.  
     
     
         7 . A polynucleotide according to  claim 5  or  claim 6 , wherein the spacer region has the sequence 5′ TTCAAGAGA 3′.  
     
     
         8 . A polynucleotide according to  claim 5 ,  6  or  7 , wherein the stem loop structure can be cleaved by an enzyme to generate a siRNA molecule which 3′ overhangs at each of its termini each comprising two uridine residues.  
     
     
         9 . A polynucleotide according to any one of  claims 4  to  8 , wherein the target gene is Cdh1, p53 or CDC20.  
     
     
         10 . A polynucleotide according to any one of the preceding claims, wherein the siRNA is capable of discriminating between different alleles of the same gene.  
     
     
         11 . A vector comprising a polynucleotide according to any one of the preceding claims.  
     
     
         12 . A cell comprising a polynucleotide according to any one of  claims 1  to  10 , or a vector according to  claim 9 .  
     
     
         13 . A cell according to  claim 12 , wherein the polynucleotide, or vector is integrated into the host genome.  
     
     
         14 . A non-human transgenic animal comprising a polynucleotide according to any one of  claims 1  to  10 , a vector according to  claim 11 , or a cell according to  claim 12  or  13 .  
     
     
         15 . Use of a polynucleotide according to any one of  claims 1  to  10  or a vector according to  claim 11  to inhibit or reduce the expression of a gene.  
     
     
         16 . A method of identifying an agent capable of modulating the phenotype of a cell according to  claim 12  or  13  or a non-human transgenic animal according to  claim 14 , in a desired manner comprising determining whether a test agent can modulate the phenotype of the cell or transgenic organism in the desired manner.  
     
     
         17 . A method for identifying: 
 (i) a modulator of transcription and/or translation of a target gene; and/or    (ii) a modulator of the activity of a target polypeptide,    in a cell according to  claim 12  or  13  or a non-human transgenic animal according to  claim 14  which method comprises determining whether a test agent can modulate transcription and/or translation of the target gene or the activity of the target polypeptide.    
     
     
         18 . A method according to  claim 16  or  17 , wherein the target gene is a disease gene or the desired modulation in phenotype is the prevention or treatment of a disease or infection.  
     
     
         19 . A method of producing a pharmaceutical composition suitable for preventing or treating a specific disorder, comprising performing the method of any one of  claims 16  to  18  and formulating the agent or modulator identified by the method with a pharmaceutically acceptable carrier or diluent.  
     
     
         20 . An agent or modulator identified by a method according to any one of  claims 16  to  18 , or a pharmaceutical composition identified by a method according to  claim 19 .  
     
     
         21 . A kit comprising a polynucleotide according to any one of  claims 1  to  10 , a vector according to  claim 11 , or a cell according to  claim 12  or  13  and a means for detecting and/or quantifying the expression of the target gene.  
     
     
         22 . A pharmaceutical composition comprising a polynucleotide according to any one of  claims 1  to  10 , a vector according to  claim 11 , a cell according to  claim 12  or  13 , an agent or a modulator according to  claim 20  and a pharmaceutically acceptable excipient.  
     
     
         23 . A polynucleotide according to any one of  claims 1  to  10 , a vector according to  claim 11 , a cell according to  claim 12  or  13 , an agent or a modulator according to  claim 20  for use in a method of treatment of the human or animal body by therapy or diagnosis.  
     
     
         24 . Use of a polynucleotide according to any one of  claims 1  to  10 , a vector according to  claim 11 , a cell according to  claim 12  or  13 , or an agent or a modulator according to  claim 20  in the manufacture of a medicament for the treatment or prevention of cancer or an autosomal dominant disorder.