Novel processes for the production of oligonucleotides
AssigneeGlaxoSmithKline Intellectual Property Development Limited
InventorsAndreas CRAMERI, Malcolm Leithhead Hill, Sarah Louise Lovelock, Markus Schober, David Graham Tew, Pamela Joan Thomas
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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.
Claims26
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.
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.
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.
A process according to any preceding claim, wherein the template, or the template and support material, are recycled for use in future reactions.
A process according to any preceding claim, wherein the reaction is carried out using a continuous or semi-continuous flow process.
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.
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.
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.
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.
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.
A process as claimed in any preceding claim, wherein the process is for producing a therapeutic oligonucleotide.
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.
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.
A ligase comprising the amino acid sequence as set forth in any one of the following SEQ ID NOs:10-28.
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.
Dependencies are inferred from the claim text.