Synthesis and use of anti-reverse phosphorothioate analogs of the messenger RNA cap

• US 8,153,773 B2
• Filed: 06/19/2008
• Issued: 04/10/2012
• Est. Priority Date: 06/19/2007
• Status: Active Grant

• Current Assignee: Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System), University Of Warsaw
• Original Assignee: Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System), University Of Warsaw
• Inventors: Jemielity, Jacek, Grudzien-Nogalska, Ewa M., Kowalska, Joanna, Darzynkiewicz, Edward, Rhoads, Robert E.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/280,282
• Publication Number:
  US 20100233757A1
• Time in Patent Office: 1,391 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 530/300
• US Class Current: 536/23.1
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

New RNA cap analogs are disclosed containing one or more phosphorothioates groups. The analogs also contain modifications at the 2′-O position of 7-methylguanosine that prevent them from being incorporated in the reverse orientation during in vitro synthesis of mRNA and that hence are “anti-reverse cap analogs” (ARCAs). The ARCA modification ensures that the S atom is precisely positioned within the active sites of cap-binding proteins in both the translational and decapping machinery. The new S-ARCA analogs are resistant to in vivo decapping enzymes. Some S-ARCAs have a higher affinity for eIF4E than the corresponding analogs not containing a phosphorothioate group. When mRNAs containing the various S-ARCAs are introduced into cultured cells, some are translated as much as five-fold more efficiently than mRNAs synthesized with the conventional analog m⁷GpppG.

Claims

1. A composition comprising

2. A composition as recited in claim 1, wherein said composition consists essentially of a single stereoisomer.

3. A composition as recited in claim 1, wherein said composition comprises a mixture of at least two diastereomers, a first diastereomer and a second diastereomer;

wherein said first and second diastereomers are otherwise identical, except that said first and second diastereomers have different stereochemical configurations at a chiral phosphorus atom;

wherein said chiral phosphorus atom is a phosphorus atom that is bound to a sulfur atom.

4. An RNA molecule whose 5′

end incorporates a composition as recited in claim 1.

5. A method for synthesizing an RNA molecule as recited in claim 4 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence of an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

6. A method for synthesizing a protein or peptide in vitro, said method comprising translating an RNA molecule as recited in claim 4 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

7. A method for synthesizing a protein or peptide in vivo, said method comprising introducing an RNA molecule as recited in claim 4 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

8. A composition as recited in claim 1;

wherein n is 3 and said composition contains a β

-phosphorothioate group, or wherein n is 4 and said composition contains a γ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by Dcp2 under physiological conditions.

9. A composition as recited in claim 1;

wherein n is 3 and said composition contains a γ

-phosphorothioate group;

or wherein n is 4 and said composition contains a δ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by DcpS under physiological conditions, and said composition inhibits cap-dependent translation.

10. A method for synthesizing a protein or peptide in vivo;

said method comprising introducing into cells an RNA molecule whose 5′

end incorporates a composition as recited in claim 8, wherein the RNA molecule comprises an open reading frame;

under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame;

wherein the in vivo protein expression is higher than the in vivo protein expression that would be obtained from an otherwise-identical method in which each Y is an oxygen atom, and in which no Y is a sulfur atom.

11. A composition comprising

12. A composition as recited in claim 11, wherein said composition consists essentially of a single stereoisomer.

13. A composition as recited in claim 11, wherein said composition comprises a mixture of at least two diastereomers, a first diastereomer and a second diastereomer;

wherein said first and second diastereomers are otherwise identical, except that said first and second diastereomers have different stereochemical configurations at a chiral phosphorus atom;

wherein said chiral phosphorus atom is a phosphorus atom that is bound to a sulfur atom.

14. An RNA molecule whose 5′

end incorporates a composition as recited in claim 11.

15. A method for synthesizing an RNA molecule as recited in claim 14 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence of an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

16. A method for synthesizing a protein or peptide in vitro, said method comprisng translating an RNA molecule as recited in claim 14 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

17. A method for synthesizing a protein or peptide in vivo, said method comprising introducing an RNA molecule as recited in claim 14 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

18. A composition as recited in claim 11;

wherein n is 3 and said composition contains a β

-phosphorothioate group, or wherein n is 4 and said composition contains a γ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by Dcp2under physiological conditions.

19. A composition as recited in claim 11;

wherein n is 3 and said composition contains a γ

-phosphorothioate group;

or wherein n is 4 and said composition contains a δ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by DcpS under physiological conditions, and said composition inhibits cap-dependent translation.

20. A method for synthesizing a protein or peptide in vivo;

said method comprising introducing into cells an RNA molecule whose 5′

end incorporates a composition as recited in claim 17, wherein the RNA molecule comprises an open reading frame;

under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame;

wherein the in vivo protein expression is higher than the in vivo protein expression that would be obtained from an otherwise-identical method in which each Y is an oxygen atom, and in which no Y is a sulfur atom.

21. A composition comprising

22. A composition as recited in claim 21, wherein said composition consists essentially of a single stereoisomer.

23. A composition as recited in claim 21, wherein said composition comprises a mixture of at least two diastereomers, a first diastereomer and a second diastereomer;

wherein said first and second diastereomers are otherwise identical, except that said first and second diastereomers have different stereochemical configurations at a chiral phosphorus atom;

wherein said chiral phosphorus atom is a phosphorus atom that is bound to a sulfur atom.

24. An RNA molecule whose 5′

end incorporates a composition as recited in claim 21.

25. A method for synthesizing an RNA molecule as recited in claim 24 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence of an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

26. A method for synthesizing a protein or peptide in vitro, said method comprising translating an RNA molecule as recited in claim 24 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

27. A method for synthesizing a protein or peptide in vivo, said method comprising introducing an RNA molecule as recited in claim 24 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

28. A composition as recited in claim 21;

wherein n is 3 and said composition contains a β

-phosphorothioate group, or wherein n is 4 and said composition contains a γ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by Dcp2 under physiological conditions.

29. A composition as recited in claim 21;

wherein n is 3 and said composition contains a γ

-phosphorothioate group;

or wherein n is 4 and said composition contains a δ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by DcpS under physiological conditions, and said composition inhibits cap-dependent translation.

30. A method for synthesizing a protein or peptide in vivo;

said method comprising introducing into cells an RNA molecule whose 5′

end incorporates a composition as recited in claim 28, wherein the RNA molecule comprises an open reading frame;

under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame;

wherein the in vivo protein expression is higher than the in vivo protein expression that would be obtained from an otherwise-identical method in which each Y is an oxygen atom, and in which no Y is a sulfur atom.

31. A composition comprising

32. A composition as recited in claim 31, wherein said composition consists essentially of a single stereoisomer.

33. A composition as recited in claim 31, wherein said composition comprises a mixture of at least two diastereomers, a first diastereomer and a second diastereomer;

wherein said first and second diastereomers are otherwise identical, except that said first and second diastereomers have different stereochemical configurations at a chiral phosphorus atom;

wherein said chiral phosphorus atom is a phosphorus atom that is bound to a sulfur atom.

34. An RNA molecule whose 5′

end incorporates a composition as recited in claim 31.

35. A method for synthesizing an RNA molecule as recited in claim 34 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence of an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

36. A method for synthesizing a protein or peptide in vitro, said method comprising translating an RNA molecule as recited in claim 34 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

37. A method for synthesizing a protein or peptide in vivo, said method comprising introducing an RNA molecule as recited in claim 34 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

38. A composition as recited in claim 31;

wherein n is 3 and said composition contains a β

-phosphorothioate group, or wherein n is 4 and said composition contains a γ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by Dcp2 under physiological conditions.

39. A composition as recited in claim 31;

wherein n is 3 and said composition contains a γ

-phosphorothioate group;

or wherein n is 4 and said composition contains a δ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by DcpS under physiological conditions, and said composition inhibits cap-dependent translation.

40. A method for synthesizing a protein or peptide in vivo;

said method comprising introducing into cells an RNA molecule whose 5′

end incorporates a composition as recited in claim 38, wherein the RNA molecule comprises an open reading frame;

under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame;

wherein the in vivo protein expression is higher than the in vivo protein expression that would be obtained from an otherwise-identical method in which each Y is an oxygen atom, and in which no Y is a sulfur atom.

41. A composition comprising

42. A composition as recited in claim 41, wherein said composition consists essentially of a single stereoisomer.

43. A composition as recited in claim 41, wherein said composition comprises a mixture of at least two diastereomers, a first diastereomer and a second diastereomer;

wherein said first and second diastereomers are otherwise identical, except that said first and second diastereomers have different stereochemical configurations at a chiral phosphorus atom;

wherein said chiral phosphorus atom is a phosphorus atom that is bound to a sulfur atom.

44. An RNA molecule whose 5′

end incorporates a composition as recited in claim 41.

45. A method for synthesizing an RNA molecule as recited in claim 44 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence of an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

46. A method for synthesizing a protein or peptide in vitro, said method comprising translating an RNA molecule as recited in claim 44 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

47. A method for synthesizing a protein or peptide in vivo, said method comprising introducing an RNA molecule as recited in claim 44 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

48. A composition as recited in claim 41;

wherein n is 3 and said composition contains a β

-phosphorothioate group, or wherein n is 4 and said composition contains a γ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by Dcp2 under physiological conditions.

49. A composition as recited in claim 41;

wherein n is 3 and said composition contains a γ

-phosphorothioate group;

or wherein n is 4 and said composition contains a δ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by DcpS under physiological conditions, and said composition inhibits cap-dependent translation.

50. A method for synthesizing a protein or peptide in vivo;

said method comprising introducing into cells an RNA molecule whose 5′

end incorporates a composition as recited in claim 48, wherein the RNA molecule comprises an open reading frame;

under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame;

wherein the in vivo protein expression is higher than the in vivo protein expression that would be obtained from an otherwise-identical method in which each Y is an oxygen atom, and in which no Y is a sulfur atom.

51. A composition comprising

52. A composition as recited in claim 51, wherein said composition consists essentially of a single stereoisomer.

53. A composition as recited in claim 51, wherein said composition comprises a mixture of at least two diastereomers, a first diastereomer and a second diastereomer;

wherein said first and second diastereomers are otherwise identical, except that said first and second diastereomers have different stereochemical configurations at a chiral phosphorus atom;

wherein said chiral phosphorus atom is a phosphorus atom that is bound to a sulfur atom.

54. An RNA molecule whose 5′

end incorporates a composition as recited in claim 51.

55. A method for synthesizing an RNA molecule as recited in claim 54 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence of an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

56. A method for synthesizing a protein or peptide in vitro, said method comprising translating an RNA molecule as recited in claim 54 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

57. A method for synthesizing a protein or peptide in vivo, said method comprising introducing an RNA molecule as recited in claim 54 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

58. A composition as recited in claim 51;

wherein n is 3 and said composition contains a β

-phosphorothioate group, or wherein n is 4 and said composition contains a γ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by Dcp2 under physiological conditions.

59. A composition as recited in claim 51;

wherein n is 3 and said composition contains a γ

-phosphorothioate group;

or wherein n is 4 and said composition contains a δ

-phosphorothioate group; and

wherein said composition is not hydrolyzed by DcpS under physiological conditions, and said composition inhibits cap-dependent translation.

60. A method for synthesizing a protein or peptide in vivo;

said method comprising introducing into cells an RNA molecule whose 5′

end incorporates a composition as recited in claim 58, wherein the RNA molecule comprises an open reading frame;

under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame;

wherein the in vivo protein expression is higher than the in vivo protein expression that would be obtained from an otherwise-identical method in which each Y is an oxygen atom, and in which no Y is a sulfur atom.

61. A composition comprising one or more compounds selected from the group consisting of

m₂^7,2′

-OGpp_spG;

m₂<sup>7,3′

-O</sup>Gpp_spG;

m₂<sup>7,2′

-O</sup>Gppp_spG;

m₂<sup>7,3′

-O</sup>Gppp_spG;

m₂<sup>7,2′

-O</sup>Gpp_sppG;

m₂<sup>7,3′

-O</sup>Gpp_sppG;

m₂<sup>7,2′

-O</sup>Gp_sp_spG;

m₂<sup>7,3′

-O</sup>Gp_sp_spG;

m₂<sup>7,2′

-O</sup>Gpp_sp_sG;

m₂<sup>7,3′

-O</sup>Gpp_sp_sG;

bn⁷m<sup>2′

-O</sup>Gpp_spG;

bn⁷m<sup>3′

-O</sup>Gpp_spG;

bn⁷m<sup>2′

-O</sup>Gppp_spG;

bn⁷m<sup>3′

-O</sup>Gppp_spG;

bn⁷m<sup>2′

-O</sup>Gpp_sppG;

bn⁷m<sup>3′

-O</sup>Gpp_sppG;

bn⁷m<sup>2′

-O</sup>Gp_sp_spG;

bn⁷m<sup>3′

-O</sup>Gp_sp_spG;

bn⁷m<sup>2′

-O</sup>Gpp_sp_sG; and

bn⁷m<sup>3′

-O</sup>Gpp_sp_sG.

62. A composition as recited in claim 61, wherein said composition consists essentially of a single stereoisomer of one of said compounds.

63. A composition as recited in claim 61, wherein said composition comprises a mixture of at least two diastereomers of one of said compounds, a first diastereomer and a second diastereomer;

wherein said first and second diastereomers are otherwise identical, except that said first and second diastereomers have different stereochemical configurations at a chiral phosphorus atom;

wherein said chiral phosphorus atom is a phosphorus atom that is bound to a sulfur atom.

64. An RNA molecule whose 5′

end incorporates a composition as recited in claim 61.

65. A method for synthesizing an RNA molecule as recited in claim 64 in vitro;

said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template;

in the presence of an RNA polymerase;

under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy;

whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

66. A method for synthesizing a protein or peptide in vitro, said method comprising translating an RNA molecule as recited in claim 64 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

67. A method for synthesizing a protein or peptide in vivo, said method comprising introducing an RNA molecule as recited in claim 64 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.