NOVEL PROCESSES FOR THE PRODUCTION OF OLIGONUCLEOTIDES

• US 20180023122A1
• Filed: 07/07/2017
• Published: 01/25/2018
• Est. Priority Date: 07/11/2016
• Status: Active Grant

• Current Assignee: GlaxoSmithKline Intellectual Property Development Limited (Glaxosmithkline Holdings Ltd.)
• Original Assignee: GlaxoSmithKline Intellectual Property Development Limited (Glaxosmithkline Holdings Ltd.)
• Inventors: CRAMERI, Andreas, Hill, Malcolm Leithhead, Lovelock, Sarah Louise, Schober, Markus, Tew, David Graham, Thomas, Pamela Joan
• Granted Patent: US 10,640,812 B2
• Time in Patent Office: Days
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Y 605/01001 DNA ligase (ATP) (6.5.1.1)
• CPC Class Codes: G01N 2030/027 Liquid chromatography

Abstract

Disclosed herein are novel processes for the production of oligonucleotides that are suitable for use in the production of chemically modified oligonucleotides, such as those for use in therapy.

Claims

1. A process for producing a single stranded oligonucleotide product having at least one modified nucleotide residue, comprising:

a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product;

b) providing a pool of oligonucleotides (II);

c) contacting (I) and (II) in conditions to allow annealing;

d) changing the conditions to separate any impurities, comprising denaturing the annealed template and impurity oligonucleotide strands and separating the impurities; and

e) changing the conditions to separate the product, comprising denaturing the annealed template and product oligonucleotide strands and separating the product.

2. A process according to claim 1, comprising:

a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product;

b) providing a pool of oligonucleotides (II) containing oligonucleotides that are segments of the product sequence, wherein at least one segment contains at least one modified nucleotide residue;

c) contacting (I) and (II) in conditions to allow annealing;

d) joining the segment oligonucleotides to form the product;

e) changing the conditions to separate any impurities, comprising denaturing the annealed template and impurity oligonucleotide strands and separating the impurities; and

f) changing the conditions to separate the product, comprising denaturing the annealed template and product oligonucleotide strands and separating the product.

3. A process according to claim 1 or claim 2, whereby the denaturing results from a temperature increase, changing the pH, or changing the salt concentration in a buffering solution.

4. A process according to any one of claims 1 to 3, including two steps of increasing the temperature:

i) to denature any annealed impurities and ii) to denature annealed product.

5. A process according to any one of claims 2-4, wherein the segment oligonucleotides are joined by enzymatic ligation, optionally wherein the enzyme is a ligase.

6. A process according to any one of claims 2-5, wherein the segments are 3 to 15 nucleotides long.

7. A process according to any preceding claim, wherein the product is 10 to 200 nucleotides long, optionally 20 to 30 nucleotides long, optionally 20 to 25 nucleotides long.

8. A process according to claim 7, wherein said product is 20 nucleotides long, said product comprising three segment oligonucleotides:

(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;

or

(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.

9. A process according to any preceding claim, wherein the property that allows the template to be separated from the product is that the template is attached to a support material.

10. The process according to claim 9, wherein the support material is a soluble support material, optionally wherein the support material is selected from the group consisting of polyethylene glycol, a soluble organic polymer, DNA, a protein, a dendrimer, a polysaccharide, an oligosaccharide, and a carbohydrate.

11. The process according to claim 9, wherein the support material is an insoluble support material, optionally 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, cellulose or is part of the reaction vessel itself, e.g. a reaction wall.

12. A process according to any one of claims 9-11, wherein multiple, repeated copies of the template are attached in a continuous manner via a single attachment point to the support material.

13. A process according to any one of claims 1-12, wherein the property that allows the template to be separated from the product is the molecular weight of the template.

14. A process according to any preceding claim, wherein the template, or the template and support material, are recycled for use in future reactions.

15. A process according to any preceding claim, wherein the reaction is carried out using a continuous or semi-continuous flow process.

16. The process according to any preceding claim, wherein the modification is at the 2′

position of the sugar moiety, optionally selected from the group consisting of 2′

-F, 2′

-OMe, 2′

-MOE, and 2′

-amino, or wherein the oligonucleotide comprises a PMO, a LNA, a PNA, a BNA, or a SPIEGELMER.

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

18. The process according to any preceding claim, wherein the modification is in the backbone, optionally wherein the modification is selected from the group consisting of phosphorothioate, phosphoramidate and phosphorodiamidate.

19. A process according to any preceding claim, wherein the resulting product is at least 90% pure, optionally wherein the product is at least 95% pure, optionally wherein the product is at least 98% pure.

20. A process for producing a double stranded oligonucleotide product, wherein 2 complimentary single stranded oligonucleotides are produced by the method of any preceding claim and then mixed under conditions to allow annealing.

21. A process as claimed in any preceding claim, wherein the process is for producing a therapeutic oligonucleotide.

22. A process as claimed in any preceding claim, wherein the product is produced at gram or kilogram scale and/or the process is carried out in a 1 L or larger reactor.

23. An oligonucleotide produced by the process of any one of claims 1 to 22, optionally wherein the oligonucleotide is a gapmer.

24. A ligase comprising the amino acid sequence as set out in SEQ ID NO:

3, wherein the amino acid at position 368 is replaced with R or K; and

/or the amino acid at position 371 is replaced with L, K, Q, V, P, or R.

25. A ligase comprising the amino acid sequence as set forth in any one of the following SEQ ID NOs:

10-28.

26. Use of a ligase comprising the amino acid sequence as set forth in SEQ ID NO:

6 or SEQ ID NO;

8 for ligating a 5′

segment, containing one or more modified sugar moieties, to a 3′

segment, wherein all of the sugar moieties within the 3′

segment are unmodified, optionally wherein the 5′

segment, contains one or more sugar moieties with a 2′

-OMe modification.