RNA polymerase variants for co-transcriptional capping

Patent metadata

Field	Value
Publication number	US-12180518-B2
Application number	US-202217816696-A
Country	US
Kind code	B2
Filing date	Aug 1, 2022
Priority date	Feb 20, 2019
Publication date	Dec 31, 2024
Grant date	Dec 31, 2024

Abstract

The present disclosure provides RNA polymerase variants for high efficiency transcription.

First claim

What is claimed is:

1. A method comprising:
incubating a deoxyribonucleic acid (DNA) in an in vitro transcription reaction that comprises nucleoside triphosphates, buffer, and a ribonucleic acid (RNA) polymerase variant, wherein the RNA polymerase variant (i) comprises an amino acid substitution at position D351 relative to an RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1 and (ii) has at least 95% identity to the amino acid sequence of SEQ ID NO: 1, thereby producing a messenger RNA (mRNA).

2. The method of claim 1 , wherein the amino acid substitution at position D351 is selected from D351C, D351I, D351K, D351L, D351M, D351P, D351Q, D351R, D351S, D351T, D351V, and D351W.

3. The method of claim 2 , wherein the amino acid substitution at position D351 is D351V.

4. The method of claim 1 , wherein the RNA polymerase variant further comprises an amino acid substitution at position E350, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

5. The method of claim 4 , wherein the RNA polymerase variant further comprises an amino acid substitution at position E350, and the amino acid substitution at position E350 is selected from E350W, E350A, E350K, and E350N.

6. The method of claim 5 , wherein the amino acid substitution at position E350 is E350W.

7. The method of claim 1 , wherein the RNA polymerase variant further comprises a C-terminal G and an amino acid substitution at positions G47 and E350, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

8. The method of claim 7 , wherein the amino acid substitution at position G47 is G47A, the amino acid substitution at position E350 is E350W, and the amino acid substitution at position D351 is D351V.

9. The method of claim 1 , wherein the RNA polymerase variant has at least 98% identity to the amino acid sequence of SEQ ID NO: 1.

10. The method of claim 9 , wherein the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 126.

11. The method of claim 1 , wherein the in vitro transcription reaction further comprises a cap analog.

12. The method of claim 11 , wherein the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

13. The method of claim 11 , wherein the DNA includes a 2′-deoxythymidine residue or 2′-deoxycytidine residue at template position +1.

14. The method of claim 11 , wherein the cap analog and nucleoside triphosphates are present in the reaction at equimolar concentrations.

15. The method of claim 11 , wherein the molar ratio of cap analog to nucleosides in the reaction is greater than 1:1.

16. The method of claim 11 , wherein the in vitro transcription reaction comprises a concentration of the cap analog that is at least 5-fold lower than a concentration of the cap analog required to produce an equivalent amount of mRNA using a control T7 RNA polymerase.

17. The method of claim 1 , wherein the buffer comprises dithiothreitol (DTT), magnesium ions, and tris.

18. The method of claim 1 , wherein the nucleoside triphosphates are naturally-occurring, synthetic, or modified.

19. The method of claim 1 , wherein greater than 50% of the mRNA produced is homogeneous at the 3′ end.

20. The method of claim 1 , wherein less than 5 ng of double-stranded RNA is produced per 25 μg of mRNA produced.

Assignees

• Modernatx Inc

Inventors

• Dousis Athanasios
• Ravichandran Kanchana
• Rabideau Amy E
• Franklin Margaret
• Smith Kevin
• Hall Michelle Lynn

Classifications

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C07H21/00

  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 title

• C07H21/02

  with ribosyl as saccharide radical · CPC title