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: contacting a population of mammalian somatic cells with a synthetic transcription factor that activates expression of a pluripotency factor gene, under conditions and for a period of time sufficient to: (i) reprogram the mammalian somatic cell to an induced pluripotent stem cell (iPSC), and/or (ii) transdifferentiate the mammalian somatic cell to a target cell different in cell type from said mammalian somatic cell. 
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein said mammalian somatic cells are human cells. 
     
     
         4 . (canceled) 
     
     
         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 . The method of  claim 1 , wherein the synthetic transcription factor comprises:
 a. at least one guide RNA comprising a DNA-binding segment and a polypeptide-binding segment, wherein the DNA-binding segment binds the promoter region of the pluripotency factor gene; and   b. at least one transcriptional modulator, which binds said polypeptide-binding segment of said guide RNA.   
     
     
         8 . The method of  claim 7 , 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 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 7 , wherein said transcriptional modulator comprises an enzymatically inactive Cas9 polypeptide (dCas9). 
     
     
         12 . The method of  claim 11 , 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 according to  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 , p16 Ink4a , 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 - 22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the transcriptional modulator is a transcriptional modulator polypeptide and wherein said population of mammalian somatic cells is further contacted with isolated gRNA nucleic acid. 
     
     
         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 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . A method of nuclear reprogramming a mammalian primary somatic cell, the method comprising: contacting a population of mammalian primary somatic cells with:
 (a) at least one guide RNA comprising (i) a DNA-binding segment complementary to a portion of a promoter region of a pluripotency factor gene, and (ii) a polypeptide-binding segment; and   (b) at least one transcriptional modulator comprising:
 (i) dCas9 capable of binding to said polypeptide-binding segment of said guide RNA; and 
 (ii) a functional domain selected from a transcriptional activation domain and a repressor domain, 
   under conditions and for a period of time sufficient to reprogram the mammalian somatic primary cell to an induced pluripotent stem cell (iPSC).   
     
     
         29 - 34 . (canceled) 
     
     
         35 . A population of iPSCs produced by a method according to  claim 1 . 
     
     
         36 . The population of iPSCs of  claim 35  being substantially free of expression vector components. 
     
     
         37 . A pharmaceutical composition comprising the iPSCs of  claim 35  and a pharmaceutically acceptable carrier. 
     
     
         38 . A method of treating a disease amenable to stem cell therapy in a patient, the method comprising administering to the patient in need thereof a therapeutically effective amount of a pharmaceutical composition of  claim 37 . 
     
     
         39 . (canceled) 
     
     
         40 . A kit for practicing the method of  claim 1 . 
     
     
         41 . A screening method for identifying a candidate pluripotency factor gene, the method comprising:
 (a) contacting a population of mammalian somatic cells with:
 (i) at least one candidate guide RNA comprising a DNA-binding segment complementary to a portion of a promoter region of the candidate pluripotency factor gene; and a polypeptide-binding segment; and 
 (ii) at least one transcriptional modulator, which binds said polypeptide-binding segment of said candidate RNA, 
 for a period of time and under conditions sufficient to reprogram said mammalian somatic cells to induced pluripotent stem cells (iPSCs), thereby forming a population of test cells; and 
   (b) culturing said test cells,   
       wherein said candidate pluripotency factor gene is not selected from oct3/4, sox2, klf4, c-myc, lin28, and nanog. 
     
     
         42 - 46 . (canceled)