GENERATION OF HUMAN iPS CELLS BY A SYNTHETIC SELF-REPLICATIVE RNA

Abstract

The disclosure provides methods and compositions useful for obtaining induced stem cells, methods of making and use thereof.

1 - 18 . (canceled) 
     
     
         19 . An RNA replicon comprising:
 a plurality of non-structural replicase domain from an alphavirus and at least two heterologous polynucleotide sequences that encode reprogramming factors (RFs) for inducing the generation of pluripotent stem cells when expressed in a somatic cell;   wherein the RNA replicon comprises from 5′ to 3′: (the plurality of non-structural replicase domains from an alphavirus)-(a promoter)-(a first RF)-(a first RF separating region)-(a second RF)-(a second RF separating region)-(optional additional RFs-optional additional separating regions)-(optional selectable marker)-(alphavirus 3′UTR and polyA tail)-(optional selectable marker)-(optional promoter);   wherein the RFs are heterologous polynucleotide sequences which encode reprogramming factors that induce de-differentiation of a somatic cell to a pluripotent cells;   wherein the RFs are polynucleotides encoding RFs selected from the group consisting of Oct polypeptides, Klf polypeptides, Sox polypeptides, Myc polypeptides, Nanog, Lin28 and Glis1;   wherein at least one of the RFs is an Oct polypeptide; and   wherein at least one of the RFs is a Sox polypeptide.   
     
     
         20 . The RNA replicon of  claim 19 , wherein the replicon comprises sequences obtained from an alphavirus selected from the group consisting of Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus Western Equine Encephalitis virus (WEE), Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus and Buggy Creek virus. 
     
     
         21 . The RNA replicon of  claim 20 , wherein the RF separating regions each consist of one or more of the following: an IRES, a small promoter and a self-cleaving peptide. 
     
     
         22 . The RNA replicon of  claim 21 , wherein the polynucleotide encoding the Sox polypeptide encodes a SOX-2 polypeptide having at least 95% identity to a sequence of SEQ ID NO:6. 
     
     
         23 . The RNA replicon of  claim 21 , wherein the polynucleotide encoding the Sox polypeptide encodes a SOX-2 polypeptide having a sequence of SEQ ID NO:6. 
     
     
         24 . The RNA replicon of  claim 21 , wherein the polynucleotide encoding the Sox polypeptide comprises a sequence as set forth in SEQ ID NO:5, and where the thymidine residues are replaced with uracil residues. 
     
     
         25 . The RNA replicon of  claim 21 , wherein the polynucleotide encoding the Oct polypeptide encodes an Oct polypeptide having at least 95% identity to a sequence of SEQ ID NO:4. 
     
     
         26 . The RNA replicon of  claim 21 , wherein the polynucleotide encoding the Oct polypeptide encodes an Oct polypeptide having a sequence of SEQ ID NO:4. 
     
     
         27 . The RNA replicon of  claim 21 , wherein the polynucleotide encoding the Oct polypeptide comprises a sequence as set forth in SEQ ID NO:3, and where the thymidine residues are replaced with uracil residues. 
     
     
         28 . The RNA replicon of  claim 21 , wherein the replicon comprises from 5′ to 3′: (VEE RNA replicases)-(promoter)-(a first RF)-(a first RF separating region)-(a second RF)-(a second RF separating region)-(optional additional RFs)-(optional additional separating regions)-(optional selectable marker)-(VEE 3′UTR and polyA tail)-(optional selectable marker)-(optional promoter);
 wherein the RFs are heterologous polynucleotide sequences which encode reprogramming factors that induce de-differentiation of a somatic cell to a pluripotent cells; 
 wherein the RFs are polynucleotides encoding RFs selected from the group consisting of Oct polypeptides, Klf polypeptides, Sox polypeptides, Myc polypeptides, Nanog, Lin28 and Glis1; 
 wherein at least one of the RFs is an Oct polypeptide; and 
 wherein at least one of the RFs is a Sox polypeptide. 
 
     
     
         29 . A composition comprising human cells transformed with a replicon of  claim 21 . 
     
     
         30 . The composition of  claim 29 , further comprising B18R conditioned media. 
     
     
         31 . The composition of  claim 29 , wherein the human cells are somatic cells. 
     
     
         32 . The composition of  claim 31 , wherein the human cells are fibroblasts. 
     
     
         33 . A method of making stem cells comprising culturing the composition of  claim 29 , for at least 30 days under conditions to express the coding sequences of the replicon and isolating stem cells. 
     
     
         34 . An in vitro method for making stem cells, comprising:
 transforming somatic cells in vitro with a replicon of  claim 28 , culturing the somatic cells in vitro under conditions to promote expression of the replicon and isolating stem cells.   
     
     
         35 . The in vitro method of  claim 34 , wherein the culturing comprises culturing the cells in media supplemented with B18R. 
     
     
         36 . The in vitro method of  claim 35 , wherein the B18R supplemented media is produced by transfection of B18R mRNA into primary human fibroblasts. 
     
     
         37 . An in vitro method to obtain a population of induced stem cells from human somatic cells, comprising:
 contacting a human somatic cell in vitro with the RNA replicon of  claim 21  or  claim 28 ,   culturing the somatic cell in vitro to express the de-differentiation factors;   selecting cells that display a stem cell morphology and/or stem cell markers; and   subculturing the cells in vitro to obtain a population of induced stem cells.   
     
     
         38 . The in vitro method of  claim 37 , wherein the cells are selected by detecting expression of a Tumor Rejection Antigen 1-60 and/or 1-81. 
     
     
         39 . An isolated human somatic cell comprising the RNA replicon of  claim 21 . 
     
     
         40 . The human somatic cell of  claim 39 , wherein the somatic cell de-differentiates when cultured under conditions to express the de-differentiation polynucleotides in the RNA replicon. 
     
     
         41 . A cell population comprising the cells of  claim 40 . 
     
     
         42 . The method of  claim 33  or  claim 34 , wherein the cells are subcultured in a medium that inhibits the degradation of the RNA replicon.