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

• US 9,334,328 B2
• Filed: 01/11/2013
• Issued: 05/10/2016
• Est. Priority Date: 10/01/2010
• Status: Active Grant

• Current Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Original Assignee: Moderna Therapeutics, Inc. (Moderna, Inc.)
• Inventors: Schrum, Jason P., Siddiqi, Suhaib, Ejebe, Kenechi
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/739,212
• Publication Number:
  US 20130203115A1
• Time in Patent Office: 1,215 Days
• Field of Search: 435/91.21
• US Class Current: 1/1
• 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 method of synthesizing a ribonucleic acid (RNA), comprising the steps of:

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

b) selecting a nucleotide that is known to disrupt a binding of a major groove binding partner with a RNA, 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 the RNA is synthesized.

2. The method of claim 1, wherein the ribonucleic acid (RNA) is a messenger RNA (mRNA).

3. The method of claim 1, wherein the major groove binding partner is TLR3.

4. The method of claim 1, wherein the major groove binding partner is TLR7.

5. The method of claim 1, wherein the major groove binding partner is TLR8.

6. The method of claim 1, wherein the major groove binding partner is RIG-I.

7. The method of claim 1, wherein the major groove binding partner is MDA5.

8. The method of claim 1, wherein the major groove binding partner is LGP2.

9. The method of claim 1, wherein the nucleobase comprising a modification on the major groove face is a pyrimidine nucleobase.

10. The method of claim 2, wherein the nucleobase comprising a modification on the major groove face is a pyrimidine nucleobase.