| abstract |
A reprogramming method of a human somatic cell or of another mammal that resides in vitro and has a first state or differentiated phenotype in an induced pluripotent stem cell (iPSC), comprising: a) introducing into said cell having a first state or differentiated phenotype a purified RNA preparation comprising modified mRNA molecules synthesized in vitro that - encodes a plurality of 4 or more, 5 or more or 6 iPSC induction factors selected from the group consisting of OCT4, SOX2, KLF4, MYC , NANOG and LIN28, - comprise at least one modified nucleoside selected from the group consisting of 1- methylpseudouridine (m1Ψ) instead of at least a portion of the homologous unmodified uridine nucleoside, pseudouridine that is not additionally modified (Ψ ) instead of at least a portion of the homologous unmodified uridine nucleoside, 5-methyluridine (m5U) instead of at least a portion of the nucleoside homologous unmodified uridine, 5-methoxyuridine (mo5U) instead of at least one portion of the homologous unmodified uridine nucleoside, 2-thiouridine (s2U) instead of at least one homologous unmodified uridine nucleoside portion and 5 -methylcitidine (m5C) instead of at least a portion of the homologous unmodified uridine nucleoside, and - has a 5 'cap and a 3' poly (A) tail; wherein said modified mRNA molecules that make up said purified RNA preparation are purified using a method that eliminates: RNA pollutant molecules that are immunogenic and toxic to the cell by induction of an innate immune response, as can be detected by measuring the decrease in secretion of IFN-α or TNF-α cytokines from dendritic cells transfected with said purified modified mRNA molecules compared to the secretion of said cytokines from dendritic cells transfected with unpurified modified mRNA molecules; such that said purified RNA preparation is free of RNA contaminant molecules that, if present, would activate an immune response in said cell, sufficient to prevent the survival of said cell and the generation of a reprogrammed cell; and b) repeating said introduction of said purified RNA preparation in multiple days and culturing the cell, in which the cell is reprogrammed in an iPSC. |