RNA polymerase variants for co-transcriptional capping

Patent metadata

Field	Value
Publication number	US-11485960-B2
Application number	US-202117325883-A
Country	US
Kind code	B2
Filing date	May 20, 2021
Priority date	Feb 20, 2019
Publication date	Nov 1, 2022
Grant date	Nov 1, 2022

Abstract

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

First claim

What is claimed is:

1. A ribonucleic acid (RNA) polymerase variant comprising an RNA polymerase that comprises an amino acid substitution at position D351 relative to an RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, wherein the RNA polymerase variant has at least 95% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the amino acid substitution at position D351 is selected from D351C, D351I, D351K, D351L, D351M, D351P, D351Q, D351R, D351S, D351T, D351V, and D351W.

2. A ribonucleic acid (RNA) polymerase variant comprising an RNA polymerase that comprises an amino acid substitution at positions E350 and D351, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1, wherein the RNA polymerase variant has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

3. A method comprising producing a mRNA in an in vitro transcription reaction that comprises a deoxyribonucleic acid (DNA), nucleoside triphosphates, and the RNA polymerase variant of claim 2 .

4. A composition or kit comprising the RNA polymerase variant of claim 2 and an in vitro transcription (IVT) reaction component.

5. A nucleic acid encoding the RNA polymerase variant of claim 2 .

6. The RNA polymerase variant of claim 2 , wherein the amino acid substitution at position E350 is selected from E350W, E350A, E350K, and E350N.

7. The RNA polymerase variant of claim 6 , wherein the amino acid substitution at position E350 is E350W.

8. The RNA polymerase variant of claim 2 , wherein the amino acid substitution at position D351 is D351V.

9. The RNA polymerase variant of claim 7 , wherein the amino acid substitution at position D351 is D351V.

10. The RNA polymerase variant of claim 2 , further comprising a C-terminal G.

11. The RNA polymerase variant of claim 9 , further comprising a C-terminal G.

12. The RNA polymerase variant of claim 2 , further comprising an amino acid substitution at position G47, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

13. The RNA polymerase variant of claim 12 , wherein the amino acid substitution at position G47 is G47A.

14. The RNA polymerase variant of claim 9 , further comprising an amino acid substitution at position G47, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

15. The RNA polymerase variant of claim 14 , wherein the amino acid substitution at position G47 is G47A.

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

17. A ribonucleic acid (RNA) polymerase variant comprising an RNA polymerase that comprises a C-terminal G and an amino acid substitution at positions G47, E350, and D351, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

18. The RNA polymerase variant of claim 17 , 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.

19. A ribonucleic acid (RNA) polymerase variant comprising an RNA polymerase that comprises the amino acid sequence of SEQ ID NO: 126.

20. The RNA polymerase variant of claim 6 , wherein the amino acid substitution at position E350 is E350A.

21. The RNA polymerase variant of claim 1 , wherein the amino acid substitution at position D351 is D351V.

22. The RNA polymerase variant of claim 1 , further comprising an amino acid substitution at position K387.

23. The RNA polymerase variant of claim 22 , wherein the amino acid substitution at position K387 is selected from K387H, K387N, and K387S.

24. The RNA polymerase variant of claim 1 , comprising amino acid substitutions G47A, E350A, D351V, and K387H, and further comprising a C-terminal G.

25. The RNA polymerase variant of claim 24 , comprising the amino acid sequence of SEQ ID NO: 116.

26. The RNA polymerase variant of claim 1 , comprising amino acid substitutions G47A, E350A, D351V, and K387N, and further comprising a C-terminal G.

27. The RNA polymerase variant of claim 26 , comprising the amino acid sequence of SEQ ID NO: 117.

28. The RNA polymerase variant of claim 1 , comprising amino acid substitutions G47A, E350A, D351V, and K387S, and further comprising a C-terminal G.

29. The RNA polymerase variant of claim 28 , comprising the amino acid sequence of SEQ ID NO: 115.

30. The RNA polymerase variant of claim 2 , wherein the RNA polymerase variant has at least 98% identity to the amino acid sequence of SEQ ID NO: 1.

31. The RNA polymerase variant of claim 17 , wherein the RNA polymerase variant has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

Assignees

• Modernatx Inc

Inventors

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

Classifications

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12N9/1247 Primary

  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