Methods for producing microRNAs

Abstract

The invention relates to recombinant vectors for inducible and/or tissue specific expression of double-stranded RNA molecules that interfere with the expression of a target gene. In certain embodiments, the invention relates to the use of Tet (tetracycline)-responsive RNA Polymerase II (Pol II) promoters (e.g., TetON or TetOFF) to direct inducible knockdown in certain cells of an integrated or an endogenous gene, such as p53. The invention also relates to a method for producing transgenic animals (e.g., mice) expressing inducible (such as tetracycline-regulated), reversible, and/or tissue-specific double-stranded RNA molecules that interfere with the expression of a target gene.

1 . An artificial nucleic acid construct comprising an RNA Polymerase II (Pol II) promoter operably linked to a coding sequence for expressing a precursor molecule for an siRNA, said siRNA inhibiting the expression of a target gene, wherein the nucleic acid construct directs the expression of the precursor molecule and/or the siRNA, and substantially inhibits the expression of the target gene when stably integrated into a host cell genome.  
   
   
       2 . The nucleic acid construct of  claim 1 , wherein said Pol II promoter is an inducible promoter, a tissue-specific promoter, and/or a developmental stage-specific promoter.  
   
   
       3 . The nucleic acid of  claim 2 , wherein the inducible promoter is a tetracyclin-responsive promoter.  
   
   
       4 . The nucleic acid construct of  claim 3 , wherein the tetracyclin-responsive promoter is a TetON promoter, the transcription from which promoter is activated at the presence of tetracyclin (tet), doxycycline (Dox), or a tet analog.  
   
   
       5 . The nucleic acid construct of  claim 3 , wherein the tetracyclin-responsive promoter is a TetOFF promoter, the transcription from which promoter is turned off at the presence of tetracyclin (tet), doxycycline (Dox), or a tet analog.  
   
   
       6 . The nucleic acid construct of  claim 2 , wherein the Pol II promoter is an LTR promoter or a CMV promoter.  
   
   
       7 . The nucleic acid construct of  claim 2 , wherein the precursor molecule is a precursor microRNA.  
   
   
       8 . The nucleic acid construct of  claim 7 , wherein the precursor microRNA (miR) is an artificial miR comprising coding sequence for said siRNA for said target gene.  
   
   
       9 . The nucleic acid construct of  claim 8 , wherein the miR comprises a backbone design of microRNA-30 (miR-30).  
   
   
       10 . The nucleic acid construct of  claim 8 , wherein the miR comprises a backbone design of miR-15a, -16, -19b, -20, -23a, -27b, -29a, -30b, -30c, -104, -132s, -181, -191, -223.  
   
   
       11 . The nucleic acid construct of  claim 2 , wherein the precursor molecule is a short hairpin RNA (shRNA).  
   
   
       12 . The nucleic acid construct of  claim 1 , wherein a single integrated copy of the nucleic acid construct is sufficient for substantially inhibiting the expression of the target gene.  
   
   
       13 . The nucleic acid construct of  claim 1 , further comprising an enhancer for the Pol II promoter.  
   
   
       14 . The nucleic acid construct of  claim 1 , further comprising a reporter gene under the control of a second promoter.  
   
   
       15 . The nucleic acid construct of  claim 14 , wherein the second promoter and the reporter gene is downstream of (3′-to) the coding sequence for the precursor molecule.  
   
   
       16 . The nucleic acid construct of  claim 15 , wherein the reporter gene is translated from an internal ribosomal entry site (IRES) between a second promoter and the reporter gene.  
   
   
       17 . The nucleic acid construct of  claim 1 , further comprising at least one selectable marker.  
   
   
       18 . The nucleic acid construct of  claim 1 , further comprising a reporter gene, wherein the coding sequence for expressing the precursor molecule is embeded or inserted into the 5′-UTR (untranslated region), 3′-UTR, or an intron of the reporter gene.  
   
   
       19 . The nucleic acid construct of  claim 1 , further comprising a Pol III promoter upstream of the coding sequence for expressing the precursor molecule.  
   
   
       20 . The nucleic acid construct of  claim 1 , wherein the target gene is associated with a disease condition selected from cancer or infectious disease.  
   
   
       21 . The nucleic acid construct of  claim 20 , wherein the target gene is over-expressed or abnormally active in the disease.  
   
   
       22 . The nucleic acid construct of  claim 20 , wherein the target gene is an oncogene or an antagonist/inhibitor or dominant negative mutation of a tumor suppressor gene.  
   
   
       23 . A cell comprising the nucleic acid construct of  claim 1 .  
   
   
       24 . The cell of  claim 23 , which is a mammalian cell.  
   
   
       25 . The cell of  claim 23 , wherein the Pol II promoter is an inducible promoter, and wherein the cell further comprises an additional construct for expressing an activator or an inhibitor of the inducible promoter.  
   
   
       26 . The cell of  claim 25 , wherein the inducible promoter is a tet-responsive promoter, and wherein the additional construct encodes tTA or rtTA.  
   
   
       27 . A non-human mammal comprising the cell according to  claim 23 .  
   
   
       28 . The non-human mammal of  claim 27 , which is a chimeric mammal.  
   
   
       29 . The non-human mammal of  claim 27 , which is a transgenic mammal.  
   
   
       30 . A method for inhibiting the expression of a target gene of interest in a cell, comprising introducing a construct according to  claim 1  into the cell, wherein the siRNA molecule derived from the precursor molecule is specific for the target gene.  
   
   
       31 . The method of  claim 30 , further comprising inhibiting at least one additional target gene(s) of interest in the cell by introducing at least one additional constructs according to  claim 1  into the cell, wherein each of the siRNA molecules derived from the precursor molecules are specific for the additional target genes, respectively.  
   
   
       32 . A method for treating a gene-mediated disease, comprising introducing into an individual having the disease a construct according to  claim 1 , where the siRNA derived from the precursor molecule is specific for the gene mediating the disease.  
   
   
       33 . A method of validating a gene as a potential target for treating a disease, comprising: 
 (1) introducing a construct according to  claim 1  into a cell associated with the disease, wherein the siRNA molecule derived from the precursor molecule is specific for the gene;    (2) assessing the effect of inhibiting the expression of the gene on one or more disease-associated phenotype;    wherein a positive effect on at least one disease-associated phenotype is indicative that the gene is a potential target for treating the disease.