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EP-4735007-A2 - IN VITRO TRANSCRIPTION METHOD

EP4735007A2EP 4735007 A2EP4735007 A2EP 4735007A2EP-4735007-A2

Abstract

The present disclosure relates generally to methods of producing RNA from a DNA template. More particularly, the method of the present disclosure comprises an in vitro transcription method for producing RNA from close-ended DNA (ceDNA) templates.

Inventors

  • CHANG, CHENG

Assignees

  • Seqirus Inc.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A method of producing an RNA molecule by in vitro transcription, said method including the steps of: (a) providing a close-ended DNA template encoding the RNA molecule; and (b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8.
  2. 2. The method of claim 1, wherein the reaction mixture has a pH of about 6.5 to about 7.5.
  3. 3. The method of claim 1, wherein the reaction mixture has a pH of about 7.0.
  4. 4. The method of claim 1, wherein the close-ended DNA template is formulated in a solution having a pH of about 6 to about 8.
  5. 5. The method of claim 4, wherein the solution has a pH of about 6.5 to about 7.5.
  6. 6. The method of claim 4, wherein the solution has a pH of about 7.0.
  7. 7. The method of claim 1, wherein the solution comprises a buffer.
  8. 8. The method of claim 7, wherein the solution comprises a Tris buffer.
  9. 9. The method of claim 8, wherein the solution comprises the Tris buffer at a concentration of about 8 mM to about 12 mM.
  10. 10. The method of claim 8, wherein the solution comprises the Tris buffer at a concentration of about 10 mM.
  11. 11. The method of claim 1, wherein the close-ended DNA template is or comprises doggybone DNA (dbDNA).
  12. 12. The method of claim 1, wherein the close-ended DNA template has been linearized.
  13. 13. The method of claim 1, wherein the method further includes the earlier step of linearizing the close-ended DNA template.
  14. 14. The method of claim 1, wherein the RNA molecule is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA molecule.
  15. 15. The method of claim 1, wherein the RNA molecule encodes an immunogenic protein.
  16. 16. An isolated RNA molecule produced by a method of in vitro transcription, said method including the steps of: (a) providing a close-ended DNA template encoding the RNA molecule; and (b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8..
  17. 17. A pharmaceutical composition comprising the isolated RNA molecule of claim 16 and optionally a pharmaceutically acceptable carrier, diluent or excipient.
  18. 18. The pharmaceutical composition of claim 17, wherein the isolated RNA molecule is contained in or otherwise associated with a lipid-based carrier.
  19. 19. The pharmaceutical composition of claim 18, wherein the lipid-based carrier is or comprises a lipid nanoparticle.
  20. 20. The isolated RNA molecule of claim 16 for use in: (a) eliciting an immune response; and/or (b) treating a disease, disorder or condition in a subject.

Description

"IN VITRO TRANSCRIPTION METHOD" CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority from United States Provisional Patent Application No. 63/510,969 filed on 29 June 2023, the contents of which is incorporated herein by reference in its entirety. TECHNICAL FIELD The present disclosure relates generally to methods of producing RNA from a DNA template. More particularly, the method of the present disclosure comprises an in vitro transcription method for producing RNA from close-ended DNA (ceDNA) templates. BACKGROUND In vitro transcription (IVT) is a biochemical process of mRNA synthesis in a cell-free transcription system in which an RNA polymerase assembles ribonucleotides using DNA as a template. The template typically contains an open reading frame (ORF) that is often, but not exclusively, comprised of the complete coding sequence of a gene, along with 5' and 3' regulatory elements and a poly(A) tail. Therefore, a quality template DNA is a prerequisite for efficient IVT of standard mRNAs or those mRNAs synthesized with modified nucleosides. Plasmid DNA is commonly used in IVT methods to produce an RNA of interest. However, conventional bacterial fermentation techniques used to produce plasmid DNA are slow, expensive, limited by a lack of manufacturing capacity and may produce poor DNA quality owing to unstable or difficult sequences. As such, the production of template DNA of sufficient quality and quantity imposes a bottleneck in the process of mRNA synthesis. Accordingly, there is a need for improved approaches for producing RNA by IVT to address one or more of these limitations. SUMMARY In vitro transcription (IVT) is typically performed using a plasmid DNA template. However, the disadvantages associated with the use of plasmid DNA in such reactions has driven the search for improved DNA templates. Close-ended DNA (ceDNA) templates are generated through a cell-free and enzymatically-based process which avoids the selective pressure often associated with plasmid instability, while also delivering rapid and reliable GMP DNA. IVT reactions also generally require less ceDNA material as compared to when using plasmid DNA templates. ceDNA templates can further support the production of difficult sequences, such as those encompassing long polyA tails or those that comprise large (>20kb) genes of interest, thereby allowing greater flexibility in supporting a range of genetic medicines. The inventors have surprisingly found that certain parameters, and in particular pH, can play an important role in the yield or efficiency of RNA production from IVT based on a close-ended DNA template. In a first aspect, the present disclosure provides a method of producing an RNA molecule by in vitro transcription, said method including the steps of: (a) providing a close-ended DNA template encoding the RNA molecule; and (b) transcribing the RNA molecule from the close-ended DNA template in a reaction mixture having a pH of between about 6 to about 8. Suitably, the reaction mixture has a pH of about 6.5 to about 7.5. In one example, the reaction mixture has a pH of about 7.0. In certain examples, the close-ended DNA template is formulated in a solution having a pH of about 6 to about 8. In other examples, the solution has a pH of about 6.5 to about 7.5. According to some examples, the solution has a pH of about 7.0. Suitably, the solution comprises a buffer. In certain examples, the solution comprises a Tris buffer. In other examples, the solution comprises the Tris buffer at a concentration of about 8 mM to about 12 mM. Referring to some examples, the solution comprises the Tris buffer at a concentration of about 10 mM. In some examples, the close-ended DNA template is or comprises doggybone DNA (dbDNA). In certain examples, the close-ended DNA template has been linearized. Suitably, the method further includes the earlier step of linearizing the close-ended DNA template. In another example, the RNA molecule is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA molecule. Suitably, the RNA molecule encodes an immunogenic protein. In a second aspect, the present disclosure provides an isolated RNA molecule produced by the method described herein. In a third aspect, the present disclosure provides a pharmaceutical composition comprising the isolated RNA molecule described herein and optionally a pharmaceutically acceptable carrier, diluent or excipient. Accordingly, the isolated RNA molecule is contained in or otherwise associated with a lipid-based carrier. In some examples, the lipid-based carrier is or comprises a lipid nanoparticle. In other examples, the present disclosure provides an isolated RNA molecule or a pharmaceutical composition as described herein for use in: (a) eliciting an immune response; and/or (b) treating a disease, disorder or condition in a subject. In a fourth aspect, the present disclosure provides a method of eliciting an immune response in a subject