IP Library › Granted Patent US 12,649,939
Granted Patent B2
US 12,649,939 · App. 16/771,362 · Granted Jun 9, 2026

Processes for the production of oligonucleotides

Inventors: Andreas Crameri (Stevenage, GB); David Graham Tew (Stevenage, GB)
Assignee: GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED
C12P19/34C12N9/1264C12N9/22C12N9/93C12N15/1031C12N15/113C12P19/30C12N2310/11C12N2310/314C12N2310/315C12N2310/321C12N2310/322C12N2310/3231C12N2310/332C12N2310/341C12Y605/01003
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Quick Facts
Patent No.
US 12,649,939
App. No.
16/771,362
Granted
Jun 9, 2026
Kind
B2
Abstract

The invention relates to novel processes using enzymes for the production of oligonucleotides, wherein said processes are suitable for use in the production of chemically modified oligonucleotides, such as those for use in therapy.

Claims (52)

1 . A process for producing a single-stranded oligonucleotide product, wherein the single-stranded oligonucleotide product comprises at least one modified nucleotide residue selected from the group consisting of modification at the 2′ position of the sugar moiety, modification of the nucleobase, and modification of the backbone of the nucleotide residue, wherein the process comprises:

a) providing a pool of oligonucleotides comprising segments of the single-stranded oligonucleotide product, wherein at least one segment comprises the at least one modified nucleotide residue, and wherein at least one segment is produced by enzymatic synthesis using a single-stranded ligase (ssLigase);

b) providing a template oligonucleotide having a sequence complementary to the sequence of the single-stranded oligonucleotide product, wherein the template oligonucleotide has a property that allows it to be separated from the single-stranded oligonucleotide product;

c) contacting the pool of oligonucleotides with the template oligonucleotide in conditions to allow annealing of the segments to the template oligonucleotide;

d) joining the segments by enzymatic ligation with a ligase to form the single-stranded oligonucleotide product annealed to the template oligonucleotide and impurity oligonucleotide strands annealed to the template oligonucleotide;

e) changing the conditions to denature the annealed template oligonucleotide and any impurity oligonucleotide strands, and separating the impurity oligonucleotide strands from the annealed template oligonucleotide and single-stranded oligonucleotide product;

f) changing the conditions to denature the annealed template oligonucleotide and the single-stranded oligonucleotide product, and separating the single-stranded oligonucleotide product; and

g) recycling the template oligonucleotide,

wherein a process for producing the at least one segment using a ssLigase comprises:

(i) adding a 3′,5′ nucleotide bisphosphate, having at least one phosphate oxygens substituted by sulphur, to the 3′-OH of a single-stranded oligonucleotide primer by using the ssLigase;

(ii) removing the 3′-phosphate, 3′-thiophosphate or 3′-dithiophosphate by using a phosphatase;

(iii) repeating steps (i) and (ii) to produce the segment attached to the single-stranded oligonucleotide primer;

(iv) releasing the segment from the single-stranded oligonucleotide primer by using a sequence specific endonuclease.

2 . The process according to claim 1 , wherein each segment is produced by enzymatic synthesis.

3 . The process according to claim 1 , wherein the 3′,5′ nucleotide bisphosphate is selected from the group consisting of a 3′,5′ bisthiophosphate, a 3′-phosphate-5′-thiophosphate, a 3′-thiophosphate-5′-phosphate, a 3′,5′ bisdithiophosphate, a 3′-phosphate-5′-dithiophosphate, and a 3′-dithiophosphate-5′-phosphate.

4 . The process according to claim 1 , wherein at least one segment is produced using a transferase.

5 . The process according to claim 4 , wherein the process for producing the segment using a transferase comprises:

(i) adding a nucleotide-5′-triphosphate, alpha-thiotriphosphate or alphadithiotriphosphate, which has a protecting group on its 3′-OH, to the 3′-OH of a single-stranded oligonucleotide primer, by using a transferase;

(ii) deprotecting the 3′-position to regenerate the 3′-OH;

(iii) repeating steps (i) and (ii) to produce the segment attached to the single-stranded oligonucleotide primer; and

(iv) releasing the segment from the single-stranded oligonucleotide primer by using a sequence specific endonuclease.

6 . The process according to claim 1 , wherein each segment is produced by enzymatic synthesis using a ssLigase.

7 . The process according to claim 1 , wherein the process is semi-continuous or continuous.

8 . The process according to claim 1 , wherein the single-stranded oligonucleotide product is produced at gram or kilogram scale and/or the process is carried out in a 1 L or larger reactor.

9 . The process according to claim 1 , whereby the denaturing results from a temperature increase, a change in pH, or a change in salt concentration in a buffering solution.

10 . The process according to claim 9 , wherein the process includes two steps of increasing the temperature: i) to denature annealed template oligonucleotide and impurity oligonucleotide strands and ii) to denature annealed template oligonucleotide and single stranded oligonucleotide product.

11 . The process according to claim 1 , wherein each segment is independently 3 to 15 nucleotides long.

12 . The process according to claim 1 , wherein the single-stranded oligonucleotide product is 10 to 200 nucleotides long.

13 . The process according to claim 12 , wherein the single-stranded oligonucleotide product is 20 nucleotides long and comprises three segments comprising:

(i) a 5′ segment that is 7 nucleotides long, a central segment that is 6 nucleotides long and a 3′ segment that is 7 nucleotides long;

(ii) a 5′ segment that is 6 nucleotides long, a central segment that is 8 nucleotides long and a 3′ segment that is 6 nucleotides long;

(iii) a 5′ segment that is 5 nucleotides long, a central segment that is 10 nucleotides long and a 3′ segment that is 5 nucleotides long;

(iv) a 5′ segment that is 4 nucleotides long, a central segment that is 12 nucleotides long and a 3′ segment that is 4 nucleotides long; or

(v) a 5′ segment that is 3 nucleotides long, a central segment that is 14 nucleotides long and a 3′ segment that is 3 nucleotides long.

14 . The process according to claim 12 , wherein the single-stranded oligonucleotide product is 20-30 nucleotides long.

15 . The process according to claim 1 , wherein the template oligonucleotide is attached to a support material.

16 . The process according to claim 15 , wherein multiple, repeated copies of the template oligonucleotide are attached via a single attachment point to the support material.

17 . The process according to claim 15 , wherein the template oligonucleotide is attached to the support material at multiple attachment points.

18 . The process according to claim 15 , wherein the support material is selected from the group consisting of polyethylene glycol, an organic polymer, DNA, a protein, a dendrimer, a polysaccharide, an oligosaccharide, and a carbohydrate.

19 . The process according to claim 18 , wherein the support material is polyethylene glycol.

20 . The process according to claim 15 , wherein the support material is selected from the group consisting of a glass bead, a polymeric bead, a fibrous support, a membrane, a streptavidin coated bead, and cellulose, or the support material is part of the reaction vessel itself.

21 . The process according to claim 1 , wherein the modification is at the 2′ position of the sugar moiety and the modification is selected from the group consisting of 2′-F, 2′-OMe, 2′-MOE, and 2′-amino, or wherein the oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), or a bridged nucleic acid (BNA).

22 . The process according to claim 1 , wherein the modification is in the nucleobase and the modification is selected from the group consisting of a 5-methyl pyrimidine, a 7-deazaguanosine and an abasic nucleotide.

23 . The process according to claim 1 , wherein the modification is in the backbone and the modification is selected from the group consisting of phosphorothioate, phosphoramidate and phosphorodiamidate.

24 . The process according to claim 1 , wherein the resulting single-stranded oligonucleotide product is at least 90% pure.

25 . The process according to claim 24 , wherein the resulting single-stranded oligonucleotide product is at least 98% pure.

26 . The process according to claim 1 , wherein the single-stranded oligonucleotide product is a gapmer.

27 . A process for producing a double-stranded oligonucleotide product, wherein two (2) complementary single-stranded oligonucleotides are each produced by the process according to claim 1 and are mixed under conditions to allow annealing.

28 . The process according to claim 1 , wherein the single-stranded oligonucleotide product is a therapeutic oligonucleotide.

29 . The process according to claim 1 , wherein the ssLigase is an RNA ligase.

30 . The process according to claim 1 , wherein the property that allows the template oligonucleotide to be separated from the single-stranded oligonucleotide product is molecular weight of the template oligonucleotide.

31 . The process according to claim 30 , wherein repeated copies of the template oligonucleotide are joined together to form a single oligonucleotide, with or without a linker between each copy of template oligonucleotide.

Assignments (2)
CHANGE OF ADDRESS Recorded Oct 8, 2025
From: GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED
To: GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED
Reel/Frame 073032/0390 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 10, 2020
From: CRAMERI, ANDREAS; TEW, DAVID GRAHAM
To: GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED
Reel/Frame 052892/0806 →
Priority Claims (1)
GB 1721307 · Dec 19, 2017 · national
Continuity (1)
Related Publication 20240287566A1 · Aug 29, 2024
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