Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

• US 10,064,959 B2
• Filed: 04/21/2017
• Issued: 09/04/2018
• Est. Priority Date: 10/01/2010
• Status: Active Grant

• Current Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Original Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Inventors: Schrum, Jason P., Siddiqi, Suhaib, Ejebe, Kenechi
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/493,829
• Publication Number:
  US 20170239374A1
• Time in Patent Office: 501 Days
• Field of Search: 435 912, 435 61
• CPC Class Codes: A61K 48/0066 Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: G01N 33/559 through a gel, e.g. Ouchterlony technique

Abstract

The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using thereof.

Claims

1. A synthetic messenger ribonucleic acid (mRNA) that is synthesized according to a method comprising the steps of:

a) providing a complementary deoxyribonucleic acid (cDNA) that encodes a pharmaceutical protein of interest;

b) selecting a nucleotide that disrupts a binding of a major groove binding partner with the mRNA, wherein the nucleotide has decreased binding affinity to the major groove binding partner selected from the group consisting of toll-like receptor (TLR) 3, TLR7, TLR8, retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and wherein the nucleotide comprises a modification on the major groove face of the nucleobase where an atom of the major groove face of the nucleobase is replaced or substituted with an alkyl group; and

c) contacting the provided cDNA and the selected nucleotide with an RNA polymerase under conditions such that an mRNA transcript is synthesized.

2. The synthetic mRNA of claim 1, wherein the method further comprises after c), the step of:

d) 5′

-capping the mRNA transcript concomitantly or post-transcriptionally such that the mRNA is synthesized.

3. The synthetic mRNA of claim 1, wherein the mRNA is at least 300 nucleotides in length.

4. The synthetic mRNA of claim 1, wherein the nucleotide comprises a modification on the major groove face of a pyrimidine nucleobase.

5. The synthetic mRNA of claim 4, wherein the pyrimidine nucleobase is selected from cytosine and uracil.

6. The synthetic mRNA of claim 5, wherein the pyrimidine nucleobase is uracil.

7. The synthetic mRNA of claim 5, wherein the pyrimidine nucleobase is cytosine.

8. The synthetic mRNA of claim 5, wherein the nucleotide comprises 1-methyl-pseudouridine or 5-methyl-uridine.

9. The synthetic mRNA of claim 8, wherein the nucleotide comprises 1-methyl-pseudouridine.

10. The synthetic mRNA of claim 7, wherein the nucleotide comprises 5-methyl-cytidine.

11. The synthetic mRNA of claim 4, wherein the major groove binding partner is TLR3, TLR7, or TLR8.

12. The synthetic mRNA of claim 4, wherein the major groove binding partner is RIG-I, MDA5, or LGP2.

13. A pharmaceutical composition comprising the synthetic mRNA of claim 1 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition comprising the synthetic mRNA of claim 3 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising the synthetic mRNA of claim 4 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising the synthetic mRNA of claim 6 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprising the synthetic mRNA of claim 7 and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising the synthetic mRNA of claim 8 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising the synthetic mRNA of claim 9 and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising the synthetic mRNA of claim 10 and a pharmaceutically acceptable carrier.

21. The synthetic mRNA of claim 2, wherein the 5′

-capping is performed concomitantly.

22. The synthetic mRNA of claim 2, wherein the mRNA is at least 300 nucleotides in length.

23. The synthetic mRNA of claim 2, wherein the nucleotide comprises a modification on the major groove face of a pyrimidine nucleobase.

24. The synthetic mRNA of claim 23, wherein the pyrimidine nucleobase is selected from cytosine and uracil.

25. The synthetic mRNA of claim 24, wherein the nucleotide comprises 1-methyl-pseudouridine or 5-methyl-uridine.

26. The synthetic mRNA of claim 23, wherein the major groove binding partner is TLR3, TLR7, or TLR8.

27. The synthetic mRNA of claim 23, wherein the major groove binding partner is RIG-I, MDA5, or LGP2.

28. A pharmaceutical composition comprising the synthetic mRNA of claim 2 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition comprising the synthetic mRNA of claim 22 and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising the synthetic mRNA of claim 23 and a pharmaceutically acceptable carrier.