Controllable Transcription

Abstract

The present invention relates to a stable method for introducing at least one inducible cassette into a cell, and permitting controllable transcription from within that inducible cassette. The method may be used for any cell type, from any eukaryotic organism, but has a particular application in the introduction of inducible cassettes into pluripotent stem cells, such as animal or human pluripotent stem cells (hPSCs). The inducible cassette is controllably inserted in such a way to ensure that the genetic material it contains is not silenced or subject to negative influences from the insertion site, and transcription of the genetic material is controlled.

1 . A method for the production of oligodendrocytes from pluripotent stem cells, comprising:
 a) inserting a gene encoding a transcriptional regulator protein into a first genomic safe harbour site; and   b) inserting two or more of SOX-10, OLIG2, NKX2.2 and NKX6.2 genes operably linked to an inducible promoter into a second genomic safe harbour site,   wherein said inducible promoter is regulated by the transcriptional regulator protein; and   wherein said first and second genomic safe harbour sites are different.   
     
     
         2 . The method of  claim 1 , wherein activity of said transcriptional regulator protein is controlled by an exogenously supplied substance. 
     
     
         3 . The method of  claim 1 , wherein said transcriptional regulator protein is constitutively expressed. 
     
     
         4 . The method of  claim 1 , wherein said transcriptional regulator protein is selected from the group consisting of: a tetracycline—responsive transcriptional activator protein (rtTa), a Tetracycline repressor (TetR), a VgEcR synthetic receptor, a hybrid transcriptional regulator protein comprising a DNA binding domain from a yeast GAL4 protein, a truncated ligand binding domain from a human progesterone receptor, and an activation domain from the human NF-κB. 
     
     
         5 . The method of  claim 4 , wherein activity of rtTA is controlled by tetracycline or a derivative thereof. 
     
     
         6 . The method of  claim 4 , wherein activity of rtTA is controlled by doxycycline. 
     
     
         7 . The method of  claim 1 , wherein the inducible promoter comprises a Tet Responsive Element (TRE). 
     
     
         8 . The method of  claim 1 , wherein said first and second genomic safe harbour sites are selected from any two of a hROSA26 locus, a AAVS1 locus, a CLYBL gene, and a CCR5 gene. 
     
     
         9 . The method of  claim 1 , wherein insertion of said gene encoding a transcriptional regulator protein into a first genomic safe harbour site occurs on both chromosomes of the cell and/or insertion of said inducible cassette into a second genomic safe harbour site occurs on both chromosomes of the cell. 
     
     
         10 . The method of  claim 1 , wherein said method is performed ex vivo. 
     
     
         11 . The method of  claim 1 , further comprising inserting an inducible cassette or transgene into a further genomic safe harbour site different from the first and second genomic safe harbour sites. 
     
     
         12 . The method of  claim 1 , wherein said pluripotent stem cell is an induced pluripotent stem cell (iPSC). 
     
     
         13 . The method of  claim 1 , wherein said oligodendrocytes are human. 
     
     
         14 . An oligodendrocyte made by the method of  claim 1 . 
     
     
         15 . A method for therapy, comprising the oligodendrocyte of  claim 14 . 
     
     
         16 . A method for in vitro diagnostics, comprising the oligodendrocyte of  claim 14 . 
     
     
         17 . A method for tissue engineering, comprising the oligodendrocyte of  claim 14 . 
     
     
         18 . A cell, comprising: a modified genome comprising an inserted gene encoding a transcriptional regulator protein into a first genetic safe harbour site; and an inserted inducible cassette comprising a genetic sequence operably linked to an inducible promoter into a second genetic safe harbour site, wherein said inducible promoter is regulated by the transcriptional regulator protein and said first and second sites are different. 
     
     
         19 . The cell of  claim 18 , wherein the cell is an animal cell. 
     
     
         20 . An optimized tetR sequence, comprising: a sequence selected from the group consisting of a sequence with 80% homology to SEQ ID NO. 6, a sequence with 85% homology to SEQ ID NO. 6, a sequence with 90% homology to SEQ ID NO. 6, a sequence with 95% homology to SEQ ID NO. 6, and a sequence with 99% homology to SEQ ID NO. 6.