Optimized tailing of messenger rna
AssigneeSanofi Pasteur Limited
InventorsJonathan ABYSALH, Jorel VARGAS
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Abstract
The present invention relates to a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) comprising a modified ribonucleotide, said method comprising adding the IVT mRNA to a tailing polymerase in a reaction buffer comprising 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent.
Claims101
A method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) comprising a modified ribonucleotide, said method comprising adding the IVT mRNA to a tailing polymerase in a reaction buffer comprising 30 mM or less of an alkali metal salt and 5 mM or more of a reducing agent.
The method of any one of the preceding claims, wherein the reducing agent in the reaction buffer has a concentration of 5 mM to 50 mM.
The method of any one of the preceding claims, wherein the reducing agent is selected from dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP).
The method of any one of the preceding claims, wherein the alkali metal salt is NaCl or KCl.
The method of any one of the preceding claims, wherein the modified ribonucleotide is selected from pseudouridine, N1-methylpseudouridine, 5-methylcytidine, and 5-methoxyuridine.
The method of any one of the preceding claims, wherein the modified ribonucleotide is a modified uridine.
The method of any one of the preceding claims, wherein at least 93% of the IVT mRNA is tailed.
The method of any one of the preceding claims, wherein the mRNA tail comprises about 100 to about 800 ribonucleotides.
The method of any one of the preceding claims, wherein the reaction buffer maintains a pH of about pH 7 to about pH 8.
The method of any one of the preceding claims, wherein the reaction buffer maintains the activity of the tailing polymerase by providing a divalent cation.
The method of any one of the preceding claims, wherein the IVT mRNA does not contain a modified ribonucleotide at the 3′ end.
The method of any one of the preceding claims, wherein the IVT mRNA does not contain a modified uridine at the 3′ end.
The method of any one of the preceding claims, wherein the IVT mRNA does not contain a N1-methylpseudouridine at the 3′ end.
The method of any one of the preceding claims, wherein the tailing polymerase is a polyA polymerase.
A reaction buffer for use in a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) comprising 30 mM or less of an alkali metal salt, 5 mM or more of a reducing agent.
A reaction buffer for use in a method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) comprising 300 mM or less of an alkali metal salt, 50 mM or more of a reducing agent, wherein the buffer is diluted 10-fold prior to use.
A method for generating an in vitro transcribed (IVT) messenger RNA (mRNA) comprising a modified ribonucleotide, the method comprising:
- (i) preparing a DNA template wherein the terminal 3′ residue of the DNA template does not encode a modified ribonucleotide of the IVT mRNA;
- and
- (ii) transcribing the DNA template with an RNA polymerase in an in vitro transcription (IVT) reaction comprising the modified ribonucleotide.
A method for tailing in vitro transcribed (IVT) messenger RNA (mRNA) comprising a modified ribonucleotide, comprising:
- (i) providing untailed IVT mRNA which does not comprise the modified ribonucleotide at the 3′ end;
- and
- (ii) adding a reaction buffer and a tailing polymerase.
Dependencies are inferred from the claim text.