RNA preparations comprising purified modified RNA for reprogramming cells

Patent metadata

Field	Value
Publication number	US-9012219-B2
Application number	US-96246810-A
Country	US
Kind code	B2
Filing date	Dec 7, 2010
Priority date	Aug 23, 2005
Publication date	Apr 21, 2015
Grant date	Apr 21, 2015

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.

First claim

We claim:

1. A method for generating a reprogrammed cell colony comprising reprogrammed cells that exhibit a second differentiated state, comprising:
introducing into human or mammalian cells that exhibit a first differentiated state a purified RNA preparation comprising in vitro-synthesized modified mRNA molecules that:
(i) contain at least one pseudouridine that is not further modified and/or 1-methylpseudouridine in place of at least one unmodified uridine nucleoside, (ii) exhibit a 5′ cap, (iii) exhibit a 3′ poly(A) tail comprising 50-200 or greater than 150 nucleotides, and (iv) encode at least one reprogramming factor,
wherein said purified RNA preparation is free of an amount of RNA contaminant molecules, including double-stranded RNA molecules, that would activate an immune response in said cells sufficient to prevent survival of said cells for at least 20 days in culture and the generation of a reprogrammed cell colony; and
repeating said introducing on multiple days, under conditions such that at least one reprogrammed cell colony is generated that comprises reprogrammed cells that exhibit a second differentiated state.

2. The method of claim 1 , wherein the at least one pseudouridine that is not further modified or said 1-methypseudouridine is present in place of substantially all of the corresponding unmodified uridine nucleosides.

3. The method of claim 1 , wherein said reprogrammed cells are dedifferentiated cells or induced pluripotent stem cells (iPS cells).

4. The method of claim 1 , wherein said at least one reprogramming factor is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, L-MYC, N-MYC, and SOX2.

5. The method of claim 1 , wherein all of the uridine nucleosides in said modified mRNA molecules are replaced by said pseudouridine that is not further modified or 1-methylpseudouridine.

6. The method of claim 5 , wherein all of the cytidine nucleosides in said modified mRNA molecules are replaced by 5-methylcytidine nucleosides.

7. The method of claim 1 , wherein said at least 20 days in culture is at least 40 days in culture.

8. The method of claim 1 , wherein said reprogrammed cells are able to form a reprogrammed cell line, survive and replicate in culture for at least 40 days, and exhibit a morphology and express markers which are characteristic of an iPS cell.

9. The method of claim 1 , wherein said reprogrammed cells express one or more markers characteristic of said second differentiated state selected from NANOG, KLF4, LIN28, SSEA4, CRIPTO, REX1 and TRA-1-60.

10. The method of claim 1 , wherein said at least one reprogramming factor comprises a plurality of protein reprogramming factors including: OCT4, SOX2, KLF4, and at least one MYC family protein.

11. The method of claim 10 , wherein said plurality of reprogramming factors additionally encode at least one protein reprogramming factor selected from LIN28 and NANOG.

12. The method of claim 1 , wherein greater than 98% of said modified mRNA molecules are capped.

13. The method of claim 1 , wherein said modified mRNA molecules exhibit 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.

14. The method of claim 1 , wherein said modified mRNA molecules exhibit at least one particular sequence selected from the group consisting of: a heterologous 5′ UTR sequence, a Kozak sequence, an IRES sequence, and a 3′ UTR sequence; wherein said particular sequence results in greater translation of the modified mRNA molecules compared to the same mRNA molecules that do not exhibit said particular sequence.

15. The method of claim 14 , wherein said 5′ UTR sequence or 3′ UTR sequence is from a Xenopus or human alpha globin or beta globin mRNA, or wherein said 5′ UTR sequence is a sequence from a tobacco etch virus (TEV) RNA.

16. The method of claim 1 , wherein said purified RNA preparation is generated using at least one method selected from the group consisting of:
a) a process comprising treating a composition comprising said modified mRNA molecules with one or more enzymes that specifically digest one or more RNA contaminant molecules or contaminant DNA molecules such that said purified RNA preparation is generated;
b) a process comprising subjecting a composition comprising said modified mRNA molecule to column purification such that said purified RNA preparation is generated; and
c) a process comprising treating a composition comprising said modified mRNA molecules with a ribonuclease III (RNase III) enzyme such that short RNase III digestion products are generated, and purifying said short RNase III digestion products away from said modified mRNA molecules such that said purified RNA preparation is generated.

17. The method of claim 1 , further comprising, after said introducing, contacting said cell with at least one growth factor and/or cytokine selected from the group consisting of: IL-12; IFN-α; TNF-α; RANTES; MIP-1α or β; IL-6; IFN-β; IL-8; α-melanocyte-stimulating hormone (α-MSH); transforming growth factor-β1 (TGF-β1); insulin-like growth factor-I (IGF-I); TGF; PDGF-BB; stem cell factor (SCF); FMS-like tyrosine kinase 3 ligand (Flt3L); granulocyte-colony stimulating factor (G-CSF); IL-3; IL-6; erythropoietin; basic fibroblast growth factor (bFGF); insulin-like growth factor 2 (IGFII); and bone morphogenetic protein 4 (BMP-4).

18. The method of claim 1 , further comprising introducing into said cell modified mRNA encoding at least one cytokine or growth factor selected from the group consisting of: TGF; PDGF-BB; stem cell factor (SCF); FMS-like tyrosine kinase 3 ligand (Flt3L); granulocyte-colony stimulating factor (G-CSF); IL-3; IL-6; erythropoietin; basic fibroblast growth factor (bFGF); insulin-like growth factor 2 (IGFII); bone morphogenetic protein 4 (BMP-4); heat shock protein; HSP70; platelet-derived growth factor (PDGF); vascular endothelial growth factor (V-EGF); insulin-like growth factor (IGF); and a protein that down-regulates or antagonizes growth factor signaling.

19. The method of claim 1 , wherein said repeating said introducing on multiple days comprises repeating said introducing daily for 10-16 days.

20. A method for generating a reprogrammed cell colony comprising reprogrammed cells that exhibit a second differentiated state, comprising:
introducing into human or mammalian cells that exhibit a first differentiated state a purified RNA preparation comprising in vitro-synthesized modified mRNA molecules that:
(i) contain at least one modified nucleoside selected from the group consisting of: pseudouridine that is not further modified, 1-methylpseudouridine, and 5-methylcytidine in place of at least one canonical nucleoside, (ii) exhibit a 5′ cap, (iii) exhibit a 3′ poly(A) tail comprising 50-200 or greater than 150 nucleotides, and (iv) encode at least one reprogramming factor,
wherein said purified RNA preparation is free of an amount of RNA contaminant molecules, including double-stranded RNA molecules, that would activate an immune response in said cells sufficient to prevent survival of said cells for at least 20 days in culture and the generation of a reprogrammed cell colony; and
repeating said introducing on multiple days, under conditions such that at least one reprogrammed cell colony is generated that comprises reprogrammed cells that exhibit a second differentiated state.

21. The method of claim 20 , wherein said at least one modified nucleoside is said pseudouridine that is not furthe

Assignees

• Kariko Katalin
• Weissman Drew
• Dahl Gary
• Person Anthony
• Meis Judith
• Jendrisak Jerome
• Univ Pennsylvania

Inventors

• Kariko Katalin
• Weissman Drew
• Dahl Gary
• Person Anthony
• Meis Judith
• Jendrisak Jerome

Classifications

• A61K48/005 Primary

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title

• C12N2310/3341

  5-Methylcytosine · CPC title

• C07K14/505

  Erythropoietin [EPO] · CPC title

• C07K14/47

  from mammals · CPC title

• A61K48/0041 Primary

  the non-active part being polymeric · CPC title