Making and using in vitro-synthesized ssRNA for introducing into mammalian cells to induce a biological or biochemical effect
The present invention relates to compositions, kits and methods for making and using RNA compositions comprising in vitro-synthesized ssRNA inducing a biological or biochemical effect in a mammalian cell or organism into which the RNA composition is repeatedly or continuously introduced. In certain embodiments, the invention provides compositions and methods for changing the state of differentiation or phenotype of a human or other vertebrate cell. For example, the present invention provides mRNA and methods for reprogramming cells that exhibit a first differentiated state or phenotype to cells that exhibit a second differentiated state or phenotype, such as to reprogram human somatic cells to pluripotent stem cells.
1. A method of generating a purified RNA preparation comprising:
a) performing in vitro transcription (IVT) such that an IVT RNA composition is generated that comprises single-strand RNA (ssRNA) and double-stranded RNA (dsRNA) contaminants, wherein said ssRNA encodes a protein;
b) contacting said IVT RNA composition with a prokaryotic endoRNase III to generate a reaction mixture, wherein magnesium cations are present in said reaction mixture at a concentration of about 1-4 mM, and wherein said prokaryotic endoRNase III digests all, or practically all, of said dsRNA contaminants to generate dsRNA digestion products; and
c) purifying said ssRNA to generate a purified composition comprising said ssRNA, and wherein 0.05% 0.01% or less of the mass of RNA in said purified composition is dsRNA of 30 basepairs or longer.
2. The method of claim 1 , wherein said magnesium cations are present in said reaction mixture at a concentration of about 1-2 mM.
3. The method of claim 1 , wherein said ssRNA comprises mRNA.
4. The method of claim 3 , wherein said mRNA encodes a human cell reprogramming factor protein or a human therapeutic protein.
5. The method of claim 1 , wherein said ssRNA comprises one or more modified nucleosides.
6. The method of claim 5 , wherein said one or more modified nucleosides are selected from the group consisting of: pseudouridine (ψ), 1-methyl-pseudouridine (m 1 Ψ), 5-methylcytidine (m 5 C), 5-methyluridine (m 5 U), 2′-O-methyluridine (Um or m 2′-O U), 2-thiouridine (s 2 U), and N6-methyladenosine.
7. The method of claim 1 , further comprising: d) repeatedly transfecting isolated human or animal cells with said purified composition, without inducing significant cytotoxicity or cell death in said cells.
8. The method of claim 7 , wherein said ssRNA comprises mRNA encoding reprogramming factors.
9. The method of claim 7 , wherein said ssRNA comprises mRNA that encodes at least one protein selected from the group consisting of: OCT4, SOX2; KLF4; LIN28; NANOG; MYC; c-MYC; c-MYC(T58A); and L-MYC.
10. The method of claim 1 , wherein 0.001% or less of the mass of RNA in said purified composition is dsRNA of 30 basepairs or longer.
11. The method of claim 1 , wherein 0.0002% or less of the mass of RNA in said purified composition is dsRNA of 30 basepairs or longer.
12. The method of claim 1 , wherein said ssRNA comprises a cap structure.
13. The method of claim 1 , wherein said ssRNA comprises a Cap I structure where the 5′ penultimate nucleoside comprises a 2′-O-methyl-ribosyl group.
14. The method of claim 1 , wherein said ssRNA comprises a poly A tail.
15. The method of claim 1 , wherein said prokaryotic endoRNase III is from E. coli.
16. The method of claim 1 , wherein all, or practically all, of said dsRNA contaminants are digested by said prokaryotic endoRNase III to a size of about 12 to 15 bp in length.