Compositions and methods for reprogramming eukaryotic cells

Abstract

The present invention relates to methods for changing the state of differentiation of a eukaryotic cell, the methods comprising introducing mRNA encoding one or more reprogramming factors into a cell and maintaining the cell under conditions wherein the cell is viable and the mRNA that is introduced into the cell is expressed in sufficient amount and for sufficient time to generate a cell that exhibits a changed state of differentiation compared to the cell into which the mRNA was introduced, and compositions therefor. For example, the present invention provides mRNA molecules and methods for their use to reprogram human somatic cells into pluripotent stem cells.

1 - 24 . (canceled) 
     
     
         25 . A composition comprising in vitro-synthesized RNA encoding a plurality of iPS cell induction factors selected from the group consisting of KLF1, KLF2, KLF4, KLF5, LIN28, c-MYC, L-MYC, N-MYC, NANOG, OCT3, OCT4, and SOX1, SOX2, SOX3 and SOX15,
 wherein said in vitro-synthesized RNA: (a) has been treated with ribonuclease III; (b) exhibits a poly(A) tail that is 50-200 nucleotides or greater than 150 nucleotides in length; and (c) comprises a population of mRNA molecules of which greater than 95% are capped.   
     
     
         26 . The composition of  claim 25 , wherein said plurality comprises 3, 4, 5 or 6 of the iPS cell induction factors selected from the group consisting of KLF4, LIN28, c-MYC, L-MYC, N-MYC, NANOG, OCT4 and SOX2. 
     
     
         27 . The composition of  claim 25 , wherein said plurality of iPS cell induction factors comprises each of OCT4, SOX2, KLF4, and a MYC family protein. 
     
     
         28 . The composition of  claim 27 , wherein the MYC family protein is selected from the group consisting of c-MYC, L-MYC, and N-MYC. 
     
     
         29 . The composition of  claim 27 , wherein said plurality of iPS cell induction factors further comprises at least one iPS cell induction factor selected from among LIN28 and NANOG. 
     
     
         30 . The composition of  claim 25 , wherein greater than 98% of said in vitro-synthesized modified mRNA molecules are capped. 
     
     
         31 . The composition of  claim 25 , wherein said in vitro-synthesized RNA exhibits a cap with a cap1 structure, wherein the 2′ hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is methylated. 
     
     
         32 . The composition of  claim 25 , wherein said in vitro-synthesized RNA exhibits at least one particular sequence selected from the group consisting of: a heterologous 5′ UTR sequence, a Kozak sequence, an IRES sequence, and 3′ UTR sequence. 
     
     
         33 . The composition of  claim 25 , wherein said 5′ UTR sequence or 3′ UTR sequence is from a  Xenopus  or human alpha globin or beta globin mRNA. 
     
     
         34 . The composition of  claim 25 , wherein said in vitro-synthesized RNA is synthesized by amplification of mRNA molecules derived from one or more cells using an RNA amplification reaction method that results in synthesis of sense RNA molecules. 
     
     
         35 . The composition of  claim 25 , wherein said in vitro-synthesized RNA contains at least one pseudouridine nucleoside that is not further modified (Ψ). 
     
     
         36 . The composition of  claim 35 , wherein said pseudouridine nucleoside is present in place of all of the corresponding unmodified uridine nucleosides. 
     
     
         37 . The composition of  claim 25 , wherein, except for the cap, said in vitro-synthesized RNA comprises only unmodified guanosine, adenosine, cytidine and uridine nucleosides. 
     
     
         38 . A kit comprising the composition of  claim 25 . 
     
     
         39 . The kit of  claim 38 , further comprising at least one component selected from the group consisting of a transfection reagent, a culture medium, cells, a container, a box, a buffer, an inhibitor, a label, a positive control molecule, a negative control molecule, a refrigerant, instructions for use, cell culture equipment, and detection/analysis equipment. 
     
     
         40 . A mammalian somatic cell comprising the composition of  claim 25 . 
     
     
         41 . The cell of  claim 40 , wherein said cell is selected from a fibroblast, a keratinocyte, an adipocyte, a lymphocyte, a T cell, a B cells, a mononuclear cord blood cell, a buccal mucosa cell, an hepatic cell, and a cancer cell. 
     
     
         42 . The cell of  claim 40 , wherein said cell exhibits a disease state or a known pathology. 
     
     
         43 . The cell of  claim 40 , wherein said cell is free of a known disease or pathology. 
     
     
         44 . The cell of  claim 40 , wherein said cell is in vitro or in vivo.