Alternative rna purification strategies
AssigneeModernaTX, Inc.
InventorsChristopher Ladd Effio, Tahir Kapoor, Joseph Elich, Amy E. Rabideau
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Abstract
Provided herein are methods of purifying nucleic acids (e.g., mRNAs) from mixtures, including protease digestion of residual proteins, RNase III digestion of double-stranded RNAs, salt precipitation of mRNA, continuous removal of transcribed RNA, and improvement of column chromatography using high salt concentrations.
Claims100
A method of purifying in vitro transcribed mRNA, the method comprising:
- (a) adding a high-salt buffer to a composition comprising mRNA to produce a high-salt mRNA composition comprising a salt concentration of at least 100 mM;
- (b) contacting a stationary phase with the composition produced in (a);
- and
- (c) eluting mRNA from the stationary phase of (b) to obtain eluted mRNA.
A method of purifying in vitro transcribed mRNA, the method comprising:
- (i) contacting a mixture comprising the mRNA with a protease;
- (ii) incubating the mixture for a period of time sufficient for the protease to cleave one or more proteins in the mixture to produce peptide fragments;
- and
- (iii) isolating the mRNA from the mixture to obtain an isolated mRNA composition.
A method of removing in vitro transcribed mRNA from an in vitro transcription reaction, the method comprising:
- (i) in a reaction vessel comprising a mixture comprising a DNA molecule, nucleotide triphosphates (NTPs) including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), and an RNA polymerase, in vitro transcribing a DNA molecule, whereby the RNA polymerase transcribes the DNA molecule to produce an mRNA, wherein the mRNA is removed from the reaction vessel by the steps of:
- (1) transferring a portion of the mixture from the reaction vessel to a column comprising a stationary phase;
- (2) passing the portion of the mixture through the column, whereby the stationary phase retains mRNA from the mixture;
- and
- (3) re-introducing a flowthrough from the column into the reaction vessel, wherein the concentration of mRNA in the flowthrough of step (3) is lower than the concentration of mRNA in the portion of the mixture of step (1);
- and
- (ii) isolating the mRNA from the mixture to obtain an isolated mRNA composition.
A method of reducing double-stranded RNA in an mRNA composition, the method comprising:
- (i) in a reaction vessel comprising a mixture comprising a DNA molecule and an RNA polymerase, in vitro transcribing a DNA molecule, whereby the RNA polymerase transcribes the DNA molecule to produce an mRNA;
- (ii) contacting the mixture with an RNase III;
- (iii) incubating the RNase III to cleave one or more double-stranded RNAs (dsRNAs) in the mixture;
- and
- (iv) isolating the mRNA from the mixture to obtain an isolated mRNA composition.
A composition comprising mRNA formulated in a lipid nanoparticle, wherein a concentration of proteins in the mRNA prior to formulation in the lipid nanoparticle is 0.8% (% w/w) or less, 0.6% or less, 0.4% or less, or 0.2% or less, and wherein at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the mRNA molecules of the mRNA composition comprise a poly(A) tail.
A composition comprising:
- (i) an mRNA;
- (ii) a DNA;
- (iii) one or more nucleotide triphosphates;
- (iv) one or more proteins or peptide fragments thereof;
- (v) a protease in an amount sufficient to cleave one or more proteins in the mixture into peptide fragments.
Dependencies are inferred from the claim text.