Methods for nuclear reprogramming using synthetic transcription factors

Abstract

The current disclosure provides methods for reprogramming mammalian somatic cells by regulating the expression of endogenous cellular genes. Cellular reprogramming of somatic cells can be induced by activating the transcription of embryonic stem cell-associated genes (e.g., oct3/4) and suppressing the transcription of somatic cell-specific and/or cell death-associated genes. The endogenous transcription machinery can be modulated using synthetic transcription factors (activators and suppressors), to allow for faster, and more efficient nuclear reprogramming under conditions amenable for clinical and commercial applications. The current disclosure further provides cells obtained from such methods, along with therapeutic methods for using such cells for the treatment of diseases amendable to stem cell therapy, as well as kits for such uses.

1 . A method of nuclear reprogramming a mammalian somatic cell, the method comprising:
 (a) contacting a population of mammalian somatic cells with:
 (i) a first nucleic acid encoding at least one guide RNA, wherein the at least one guide RNA comprises a DNA-binding segment and a polypeptide-binding segment, the DNA-binding segment binds a promoter region of an endogenous pluripotency factor gene of the mammalian somatic cells; and 
 (ii) a second nucleic acid encoding at least one transcriptional modulator, wherein the at least one transcriptional modulator binds the polypeptide-binding segment of the at least one guide RNA, and the at least one transcriptional modulator comprises an enzymatically inactive Cas9 polypeptide (dCas9); and 
   (b) culturing the mammalian somatic cells for a period under conditions sufficient to (i) reprogram the mammalian somatic cell to an induced pluripotent stem cell (iPSC), and/or transdifferentiate the mammalian somatic cell to a target cell different in cell type from said mammalian somatic cell.   
     
     
         2 . The method of  claim 1 , wherein the period is from about 2 to about 14 days. 
     
     
         3 . The method of  claim 1 , wherein said mammalian somatic cells are human cells. 
     
     
         4 . The method of  claim 3 , wherein said mammalian somatic cells are fibroblasts. 
     
     
         5 . The method of  claim 3 , wherein said mammalian somatic cells are primary blood cells. 
     
     
         6 . The method of  claim 5 , wherein said blood cells are peripheral blood mononuclear cells (PBMCs) or cord blood mononuclear cells. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein said pluripotency factor gene is selected from the group consisting of oct3/4, sox2, klf4, c-myc, lin28, nanog, glis-1, bcl2, and bclx. 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein said dCas9 is fused to a transcriptional activation domain. 
     
     
         13 . The method of  claim 12 , wherein the transcriptional activation domain is VP64 or p65. 
     
     
         14 . The method of  claim 1 , wherein the method further comprises contacting the population of mammalian somatic cells with a second synthetic transcription factor that represses expression of a second pluripotency factor gene. 
     
     
         15 . The method according to  claim 14 , wherein the second pluripotency factor gene being repressed is selected from p19 Arf , p16Ink4a, ROCK, a PKA/PKG/PKC family kinase gene, and genes that when repressed inhibit the mTOR pathway. 
     
     
         16 . The method of  claim 14 , wherein said second synthetic transcription factor comprises a second transcriptional modulator comprising dCas9 fused to a transcriptional suppressor domain. 
     
     
         17 . The method of  claim 16 , wherein the transcriptional suppressor domain is KRAB. 
     
     
         18 . The method of  claim 1 , wherein said population of mammalian somatic cells is contacted with at least one expression vector encoding said synthetic transcription factor. 
     
     
         19 . The method of  claim 18 , wherein said expression vector is a non-viral, episomal vector. 
     
     
         20 . The method of  claim 18 , wherein said population of mammalian somatic cells is contacted with an expression vector encoding both said guide RNA and said transcriptional modulator. 
     
     
         21 . The method of  claim 18 , wherein said population of mammalian somatic cells is contacted with a first expression vector encoding said guide RNA and a second expression vector encoding said transcriptional modulator. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein said population of mammalian somatic cells is contacted with at least three synthetic transcription factors, each targeting a different gene. 
     
     
         25 . The method of  claim 24 , wherein said population of mammalian somatic cells is contacted with:
 (i) dCas9 fused to a transcriptional activation domain;   (ii) a gRNA comprising a DNA-binding segment complementary to at least a portion of the promoter region of a mammalian oct3/4 gene;   (iii) a gRNA comprising a DNA-binding segment complementary to at least a portion of the promoter region of a mammalian sox2 gene; and   (iv) a gRNA comprising a DNA-binding segment complementary to at least a portion of the promoter region of a mammalian klf4 gene.   
     
     
         26 . The method of  claim 1 , wherein said population of mammalian somatic cells is contacted with at least four synthetic transcription factors activating expression of four different pluripotency factor genes. 
     
     
         27 - 46 . (canceled)