RNA POLYMERASE VARIANTS

• US 20200131550A1
• Filed: 10/18/2019
• Published: 04/30/2020
• Est. Priority Date: 08/18/2017
• Status: Active Grant

• Current Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Original Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Inventors: Rabideau, Amy E., Dousis, Athanasios, Ravichandran, Kanchana, Hobert, Elissa
• Granted Patent: US 11,066,686 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/1247 DNA-directed RNA polymerase (2.7.7.6)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Y 207/11023 [RNA-polymerase-subunit] kinase (2.7.11.23)

Abstract

The present disclosure provides, in some aspects, variant RNA polymerases, the use of which increases transcription efficiency while reducing the number of double-stranded RNA contaminates and run-on transcripts produced during an in vitro transcription reaction.

Claims

1. A ribonucleic acid (RNA) polymerase variant comprising an amino acid substitution, relative to wild-type RNA polymerase, that causes a loop structure of the RNA polymerase variant to undergo a conformational change to a helix structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex.

30. The RNA polymerase variant of claim 1, wherein the amino acid substitution is a high-helix propensity amino acid at a position selected from E42, S43, Y44, E45, M46, G47, R257, A258, G259, A260, L261, and A262 of a wild-type RNA polymerase, optionally wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:

1 or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO;

1.

33. A method of producing a ribonucleic acid (RNA) comprising contacting a DNA template with the RNA polymerase of claim 1 under conditions that result in the production of RNA transcript.

34. A method of performing an in vitro transcription (IVT) reaction, comprising contacting a DNA template with the RNA polymerase of claim 1 in the presence of nucleoside triphosphates and buffer under conditions that result in the production of RNA transcript.

35. The method of claim 33, wherein the RNA transcript produced, when delivered to cells, optionally in unpurified form, stimulates a cytokine response that is at least 50% lower relative to RNA produced using wild-type RNA polymerase.

36. The method of claim 33, wherein the concentration of double-stranded RNA (dsRNA) transcript produced is at least 50% lower relative to dsRNA transcript produced using wild-type polymerase.

37. The method of claim 33, wherein less than 20% of the RNA transcripts produced exhibit 3′

heterogeneity.

38. The method of claim 33, wherein less than 50% of the RNA transcript produced is double-stranded contaminant.

39. The method of claim 33, wherein less than 50% of the RNA transcript produced is run-on RNA transcript.

40. The method of claim 33, wherein the amount of full-length RNA transcript produced is at least 15 times greater than the amount of the DNA template.

41. The method of claim 33, wherein the ratio of double-stranded contaminant:

full-length RNA transcript produced is less than 1;

1.

42. The method of claim 33, wherein the RNA transcript produced has less than 1 mutation per 100 nucleotides relative to the DNA template.

43. A nucleic acid encoding the RNA polymerase variant of claim 1.

44. A vector comprising the nucleic acid of claim 43.

45. A host cell comprising the nucleic acid of claim 43.

46. A composition or kit comprising the RNA polymerase variant of claim 1 and an in vitro transcription (IVT) reagent.

47. A ribonucleic acid (RNA), optionally a messenger RNA (mRNA), produced by the method of claim 1.

2-27. -27. (canceled)

28. A RNA polymerase variant, optionally a T7 RNA polymerase variant, comprising a C terminus that comprises a XAFAX_n motif, a FXFAX_n motif, a FAXAX_n motif, or a FAFXX_n motif, wherein each X is any amino acid, and n is any integer greater than zero.

29.

31-32. -32. (canceled)

48. (canceled)

49. A co-transcriptional capping method for ribonucleic acid (RNA) synthesis, the method comprising reacting a polynucleotide template with a RNA polymerase variant, optionally a T7 RNA polymerase variant, nucleoside triphosphates, and a cap analog under in vitro transcription reaction conditions to produce RNA transcript.

50-74. -74. (canceled)

75. A T7 ribonucleic acid (RNA) polymerase comprising an amino acid sequence that has at least 90% identity with a wild-type T7 RNA polymerase and is modified to comprise (a) an alanine at an amino acid position corresponding to amino acid position 43 of SEQ ID NO:

1 and (b) an additional C-terminal glycine.

76-77. -77.