Granted Patent
B2
US 12,655,395 · App. 18/453,897 · Granted Jun 16, 2026
RNA preparations comprising purified modified RNA for reprogramming cells
Inventors:
Katalin Kariko (Rydal, PA); Drew Weissman (Wynnewood, PA); Gary Dahl (Madison, WI); Anthony Person (Madison, WI); Judith Meis (Fitchburg, WI); Jerome Jendrisak (Madison, WI)
C12N5/0696A61K38/1709A61K38/1816A61K38/44A61K38/465A61K38/50A61K48/005A61K48/0075C07K14/47C07K14/4712C07K14/505C12N9/0075C12N9/22C12N9/78C12N15/117C12N15/85C12N15/87C12N2310/17C12N2310/335C12N2320/30C12N2501/602C12N2501/603C12N2501/604C12N2501/605C12N2501/606C12N2501/608C12N2506/02C12Y114/13039C12Y301/04012C12Y305/04004
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Abstract
The present invention provides compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-strand mRNA encoding an iPS cell induction factor. The purified RNA preparations are preferably substantially free of RNA contaminant molecules that: i) would activate an immune response in the somatic cells, ii) would decrease expression of the single-stranded mRNA in the somatic cells, and/or iii) active RNA sensors in the somatic cells. In certain embodiments, the purified RNA preparations are substantially free of partial mRNAs, double-stranded RNAs, un-capped RNA molecules, and/or single-stranded run-on mRNAs.
Claims (14)
1 . A method for reprogramming a human or animal cell that exhibits a first differentiated state or phenotype to a cell that exhibits a second differentiated state or phenotype comprising: introducing into the cell that exhibits a first differentiated state a purified RNA preparation comprising modified mRNA molecules that contain at least one modified nucleoside selected from the group consisting of: pseudouridine (Ψ) and 5-methylcytosine (m 5 C) and that encode at least one reprogramming factor and culturing the cell under conditions wherein the cell exhibits a second differentiated state.
2 . The method of claim 1 , wherein the at least one modified nucleoside is present in place of substantially all of the corresponding unmodified canonical nucleoside.
3 . The method of claim 1 , wherein said purified RNA preparation:
i) comprises at least one single-stranded mRNA that encodes an iPS cell induction factor, wherein substantially all of said first single-stranded mRNAs comprise at least one pseudouridine residue and/or at least one 5-methylcytidine residue, and
ii) is substantially free of RNA contaminant molecules which are able to activate RNA sensors in said somatic cell.
4 . The method of claim 3 , wherein said RNA contaminant molecules are selected from the group consisting of: partial mRNAs encoding only a portion of said reprogramming factor, RNA molecules that are smaller than the full-length mRNA, RNA molecules that are larger than the full-length mRNA, double-stranded mRNA molecules, and un-capped mRNA molecules.
5 . The method of claim 1 , wherein said cell that exhibits a second differentiated state is a transdifferentiated cell.
6 . The method of claim 1 , wherein said reprogrammed cell that exhibits a second differentiated state is a redifferentiated cell.
7 . The method of claim 1 , wherein said modified mRNA molecules that encode at least one reprogramming factor is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
8 . The method of claim 1 , wherein the method further comprises contacting said cell that exhibits a first differentiated state or phenotype with at least one growth factor and/or cytokine.
9 . The method of claim 1 , wherein all of the uridine nucleosides in said modified mRNA are replaced by pseudouridine nucleosides.
10 . The method of claim 1 , wherein all of the cytidine nucleosides in said modified mRNA are replaced by 5-methylcytidine nucleosides.
11 . The method of claim 1 , wherein said purified RNA preparation is free of an amount of RNA contaminant molecules that would activate an immune response in said cell that prohibits said cell from surviving at least 10 days in culture.
12 . The method of claim 1 , wherein said cell that exhibits a second differentiated state is able to form a cell line.
Continuity (8)
Provisional Application
61267312
· Dec 7, 2009
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