Transcriptional regulation of target genes

Abstract

The present invention describes a method of identifying inducible genetic regulatory sequences that can control the transcription of specific gene transcripts. Methods of using inducible genetic regulatory sequences are also discussed. In particular, the genetic regulatory sequences of the present invention can modulate the transcription of a nucleic acid transcript in vivo.

What is claimed is:  
     
         1 . A method of identifying a genetic regulatory sequence responsive to a pulsatile electromagnetic stimulus, comprising: 
 (a) inserting a stimulator into a tissue of an animal subject;    (b) applying a pulsatile electromagnetic signal with the stimulator; wherein at least a part of the tissue is stimulated;    (c) identifying a gene that has either enhanced or diminished transcription in the part of the tissue stimulated; and    (d) selecting a genetic regulatory sequence involved in the enhanced or diminished transcription of the gene; wherein the genetic regulatory sequence is identified as being responsive to the pulsatile electromagnetic stimulus.    
     
     
         2 . The method of  claim 1 , wherein the pulsatile electromagnetic signal is provided by a bipolar stimulator.  
     
     
         3 . The method of  claim 1 , wherein the pulsatile electromagnetic signal is provided by a monopolar stimulator.  
     
     
         4 . The method of  claim 1 , wherein at least a part of the tissue is not stimulated by the stimulator; and wherein the identifying of step (c) is performed by comparing the transcription of the genes in the part of the tissue stimulated with that of a part of the tissue that is not stimulated.  
     
     
         5 . The method of  claim 4 , wherein a second stimulator is placed into the part of the tissue that is not stimulated; and wherein no pulsatile signal is applied by the second stimulator.  
     
     
         6 . The method of  claim 1 , wherein the tissue is neural tissue.  
     
     
         7 . The method of  claim 6 , wherein the neural tissue is brain tissue.  
     
     
         8 . The method of  claim 1 , wherein the pulsatile electromagnetic signal is a magnetic stimulation.  
     
     
         9 . An isolated genetic regulatory sequence identified by performing the method of  claim 1 .  
     
     
         10 . A vector for in vivo expression of a gene of interest in a mammalian host cell, wherein the vector comprises the gene of interest and a transcriptional control region comprising a genetic regulatory sequence, and wherein the gene of interest is operatively linked to the transcriptional control region, and the genetic regulatory sequence is responsive to a pulsatile electromagnetic stimulus.  
     
     
         11 . The vector of  claim 10 , wherein the transcriptional control region exhibits tissue specificity and is from a gene encoding a protein expressed by the host cell.  
     
     
         12 . The vector of  claim 10 , that is a replication defective virus.  
     
     
         13 . The vector of  claim 10 , wherein the host cell is a neural tissue cell.  
     
     
         14 . The vector of  claim 13 , wherein the neural tissue cell is a brain cell.  
     
     
         15 . A non-human mammalian host transformed with the vector of  claim 10 , wherein the expression of the gene of interest in the non-human mammalian host can be modulated by applying a pulsatile signal with a stimulator.  
     
     
         16 . A cell transformed with the vector of  claim 10 , wherein the expression of the gene of interest in the transformed cell can be modulated by applying a pulsatile signal with a stimulator.  
     
     
         17 . The cell of  claim 16 , wherein the transformed cell may be transplanted into a recipient mammalian host, and wherein the expression of the gene of interest in the recipient mammalian host can be modulated by applying a pulsatile signal with a stimulator.  
     
     
         18 . The replication defective viral vector of  claim 10  selected from the group consisting of a replication defective herpes simplex virus (HSV), a replication defective papillomavirus, a replication defective Epstein Barr virus (EBV), a replication defective adenovirus, a gutless adenovirus, a replication defective adeno-associated virus (AAV), and a replication defective lentivirus.  
     
     
         19 . A method of delivering the vector of  claim 10 , to a target tissue of an animal subject comprising administering the vector to the tissue of the animal subject, wherein the expression of the gene of interest in the target tissue can be modulated by applying a pulsatile signal with a stimulator.  
     
     
         20 . The method of  claim 19 , wherein the transcription of the gene of interest in the target tissue is stimulated by a stimulator that is present in the target tissue.  
     
     
         21 . A method of regulating the expression of a gene of interest in a tissue of an animal subject in which the vector of  claim 10  has been administered, comprising applying a pulsatile signal with a stimulator which modulates the transcription of the gene of interest in the tissue.  
     
     
         22 . The method of  claim 21 , wherein the response to the pulsatile signal by the genetic regulatory sequence stimulates the transcription of the gene of interest.  
     
     
         23 . The method of  claim 21 , wherein the response to the pulsatile signal by said genetic regulatory sequence hinders the transcription of the gene of interest.  
     
     
         25 . A method of identifying a genetic regulatory sequence that is responsive to a peptide comprising: 
 (a) contacting a peptide with a cell;    (b) identifying a gene that exhibits altered transcription relative to a cell not contacted with the peptide; and    (c) selecting a genetic regulatory sequence involved in the altered transcription of the gene; wherein the genetic regulatory sequence is identified as being responsive to the peptide.    
     
     
         26 . The method of  claim 25 , wherein the altered transcription is an enhancement or diminishment of transcription.  
     
     
         27 . An isolated genetic regulatory sequence identified by performing the method of  claim 25 .  
     
     
         28 . The method of  claim 25 , wherein identifying a gene exhibiting altered transcription is by 2-dimensional gel electrophoresis or Northern blot analysis.  
     
     
         29 . A vector for the in vivo expression of a gene of interest in a mammalian host cell, wherein the vector comprises the gene of interest and a transcriptional control region comprising the genetic regulatory sequence of  claim 27 , and wherein the gene of interest is operatively linked to said transcriptional control region and the genetic regulatory sequence is responsive to the peptide.  
     
     
         30 . A method of ameliorating symptoms due to Parkinson's disease comprising: 
 (a) administering a vector to the subthalamic nucleus of a patient having a symptom of Parkinson's disease; wherein the vector comprises a nucleic acid encoding glutamic acid decarboxylase operatively under the control of a genetic regulatory sequence that is stimulated by a pulsatile electromagnetic signal;    (b) inserting a stimulator into the subthalamic nucleus of the patient; and    (c) applying a pulsatile electromagnetic signal with the stimulator, wherein the glutamic acid decarboxylase is expressed and leads to the amelioration of a symptom of Parkinson's disease.    
     
     
         31 . The method of  claim 30 , wherein the vector is a replication defective viral vector.  
     
     
         32 . The method of  claim 30 , wherein the vector is a CBA-GAD65 viral vector.  
     
     
         33 . A method of modulating the release of a stored compound by a cell comprising: 
 (a) administering a vector to the subthalamic nucleus of an animal subject; wherein the vector comprises a nucleic acid encoding a protein operatively under the control of a genetic regulatory sequence that is stimulated by a pulsatile electromagnetic signal, and wherein the expression of the protein stimulates the production of a compound that is stored by a cell of the animal subject;    (b) inserting a stimulator into the subthalamic nucleus of the animal subject;    (c) applying a pulsatile electromagnetic signal with the stimulator, wherein the protein is expressed and the compound is produced; and wherein the cell stores the compound; and    (d) applying a second pulsatile electromagnetic signal with the stimulator, wherein the release of the stored compound from the cell is modulated.    
     
     
         34 . The method of  claim 33 , wherein the compound is GABA.  
     
     
         35 . The method of  claim 34 , wherein applying of a second pulsatile electromagnetic signal is at a low frequency and the release of GABA from the cell is increased.  
     
     
         36 . The method of  claim 34 , wherein applying of a second pulsatile electromagnetic signal is at a high frequency and the release of GABA from the cell is decreased.