RNA preparations comprising purified modified RNA for reprogramming cells

A purified RNA preparation comprising a plurality of in vitro-synthesized single-stranded mRNA molecules that encode 2 or more, 3 or more, 4 or more, 5 or more, or 6 iPSC induction factors selected from the group consisting of OCT4, SOX2, KLF4, LIN28, NANOG, and a MYC protein selected from c-MYC, L-MYC and N-MYC;

• wherein said in vitro-synthesized mRNA molecules:
• a) comprise a modified nucleotide comprising a modified nucleoside in place of uridine that is selected from the group consisting of pseudouridine that is not further modified (Ψ), 1-methyl-pseudouridine (m1Ψ), 5-methyluridine (m5U), 5-methoxyuridine (mo5U) and 2-thiouridine (s2U);
• and
• b) have been purified using a purification process that removes RNA contaminant molecules that are immunogenic and toxic to a mammalian cell by inducing an innate immune response, such that said purified in vitro-synthesized mRNA lacks immunogenicity, as can be determined using an in vitro monocyte-derived dendritic cell (MDDC) assay by measuring an amount of IFN-α or TNF-α secreted by MDDCs transfected with said purified in vitro-synthesized mRNA molecules that is no greater than the mean+the standard error of the mean (SEM) amount of IFN-α or TNF-α secreted by MDDCs transfected with negative controls that do not contain RNA.

A composition comprising a mammalian somatic cell containing in vitro-synthesized mRNA molecules,

• wherein said in vitro-synthesized mRNA molecules encode the iPSC induction factors:
• A) OCT4, SOX2, KLF4 and MYC, or
• B) OCT4, SOX2, KLF4, MYC and LIN28, or
• C) OCT4, SOX2, KLF4, MYC and NANOG, or
• D) OCT4, SOX2, KLF4, MYC, LIN28 and NANOG,
• wherein said MYC in A)-D) is selected from c-MYC, L-MYC and N-MYC;
• and
• wherein said in-vitro synthesized mRNA molecules comprise:
• (i) guanosine,
• (ii) adenosine,
• (iii) cytidine, and
• (iv) pseudouridine in place of uridine.