RNA preparations comprising purified modified RNA for reprogramming cells

Patent metadata

Field	Value
Publication number	US-11739300-B2
Application number	US-202016827098-A
Country	US
Kind code	B2
Filing date	Mar 23, 2020
Priority date	Dec 7, 2009
Publication date	Aug 29, 2023
Grant date	Aug 29, 2023

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 comprising:
administering a single dose of a purified RNA preparation to mammalian cells or a mammalian subject without triggering a detectable innate immune response,
wherein said purified RNA preparation was made by a process comprising purifying a preparation of in vitro-synthesized RNA molecules obtained by a process comprising in vitro transcription (IVT),
wherein said in vitro-synthesized RNA molecules encode at least one recombinant protein and comprise a modified nucleoside selected from ψ, m 1 ψ, m 5 U, mo 5 U, and s 2 U in place of at least a portion of uridine nucleosides in said in vitro-synthesized RNA molecules,
wherein said purifying uses a purification process that removes RNA contaminant molecules comprising double-stranded RNA (dsRNA) molecules that are toxic to mammalian cells by inducing an innate immune response,
wherein said purified RNA preparation is free of said RNA contaminant molecules such that less than 0.01% of the total RNA in said purified RNA preparation consists of said RNA contaminant molecules based on dsRNA dot blotting assays that use a dsRNA-specific monoclonal antibody (mAb) selected from J2 mAb and K1 mAb to quantify the amount of said RNA contaminant molecules in said purified RNA preparation that is spotted on a membrane.

2. The method of claim 1 , wherein said in vitro-synthesized RNA molecules comprise a modified nucleoside selected from ψ, m 1 ψ, m 5 U, mo 5 U, and mo 5 U in place of all or nearly all uridine nucleosides in said in vitro-synthesized RNA molecules.

3. The method of claim 1 , wherein said mammalian subject is a human, and wherein said mammalian cell is a human cell.

4. The method of claim 1 , wherein said mammalian cells comprise toll-like receptor 3 (TLR3) which can be activated by said RNA contaminant molecules.

5. The method of claim 1 , wherein said purifying comprises treating the in vitro-synthesized RNA with a ribonuclease III (RNase III) enzyme and then purifying it away from the RNase III digestion products.

6. The method of claim 1 , wherein said purifying comprises the use of HPLC.

7. A method comprising:
administering a single dose of a purified RNA preparation to a mammalian cell or a mammalian subject,
wherein said purified RNA preparation was made by a process comprising purifying a preparation of in vitro-synthesized RNA molecules obtained by a process comprising in vitro transcription (IVT),
wherein said in vitro-synthesized RNA molecules encode at least one recombinant protein and comprise a modified nucleoside selected from ψ, m 1 ψ, m 5 U, mo 5 U, and s 2 U in place of at least a portion of uridine nucleosides in said in vitro-synthesized RNA molecules,
wherein said purifying generates a purified RNA preparation that exhibits reduced immunogenicity that is detectable using an in vitro MDDC immunogenicity assay by measuring secretion of less IFN-α or TNF-α cytokine secreted by human or murine monocyte-derived dendritic cells (MDDCs) transfected with said purified RNA preparation than is secreted from MDDCs transfected with said preparation of in vitro-synthesized RNA molecules that have not been subjected to said purifying.

8. The method of claim 7 , wherein said in vitro-synthesized RNA molecules comprise a modified nucleoside selected from ψ, m 1 ψ, m 5 U, mo 5 U, and mo 5 U in place of all or nearly all uridine nucleosides in said in vitro-synthesized RNA molecules.

9. The method of claim 7 , wherein said mammalian subject is a human, and wherein said mammalian cell is a human cell.

10. The method of claim 7 , wherein said mammalian cell comprises toll-like receptor 3 (TLR3) which can be activated by said RNA contaminant molecules.

11. The method of claim 7 , wherein said purified RNA preparation is free of double-stranded RNA contaminant molecules such that less than 0.01% of the total RNA in said purified RNA preparation consists of said RNA contaminant molecules.

12. The method of claim 11 , wherein said less than 0.01% of the total RNA in said purified RNA preparation consists of said RNA contaminant molecules is based on dsRNA dot blotting assays that use a dsRNA-specific monoclonal antibody (mAb) selected from J2 mAb and K1 mAb to quantify the amount of said RNA contaminant molecules in said purified RNA preparation that is spotted on a membrane.

13. The method of claim 7 , wherein said purifying comprises treating the in vitro-synthesized RNA with a ribonuclease III (RNase III) enzyme and then purifying it away from the RNase III digestion products.

14. The method of claim 7 , wherein said purifying comprises the use of HPLC.

Assignees

• Univ Pennsylvania

Inventors

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

Classifications

• C12N2310/335

  Modified T or U · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• A61K48/0091

  Purification or manufacturing processes for gene therapy compositions · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/10 Primary

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title