RNA WITH A COMBINATION OF UNMODIFIED AND MODIFIED NUCLEOTIDES FOR PROTEIN EXPRESSION

• US 20120195936A1
• Filed: 07/30/2010
• Published: 08/02/2012
• Est. Priority Date: 07/31/2009
• Status: Abandoned Application

• Current Assignee: Ethris Gmbh
• Original Assignee: Ethris Gmbh
• Inventors: Rudolph, Carsten, Kormann, Michael
• Application Number:
  US13/388,140
• Publication Number:
  US 20120195936A1
• Time in Patent Office: Days
• US Class Current: 424/400
• CPC Class Codes: A61K 38/1816 Erythropoietin [EPO]
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0066 Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• CPC Class Codes: A61L 27/227 Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/785 Alveolar surfactant peptides; Pulmonary surfactant peptides
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 2310/334 Modified C
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity

Abstract

The invention relates to a polyribonucleotide with a sequence that codes a protein or protein fragment, wherein the polyribonucleotide comprises a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidin nucleotides are modified uridine nucleotides or modified cytidin nucleotides.

Claims

1-30. -30. (canceled)

31. A polyribonucleotide comprising, a sequence which encodes a protein or protein fragment, wherein the polyribonucleotide contains a combination of unmodified and modified nucleotides, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are respectively modified uridine nucleotides and modified cytidine nucleotides.

32. The polyribonucleotide of claim 31, wherein the polyribonucleotide is mRNA, wherein optionally the mRNA is in vitro transcribed mRNA (IVT mRNA).

33. The polyribonucleotide of claim 31, wherein the RNA encodes a protein or protein fragment, a defect or lack whereof can trigger a disease, which can moderate, prevent or cure an illness or which can contribute a beneficial or necessary function, and/or wherein 15 to 30%, preferably 7.5 to 25%, of the uridine nucleosides and 15 to 30%, preferably 7.5 to 25%, of the cytidine nucleosides are modified, and/or wherein it contains at least two types of modified uridine nucleosides and/or at least two types of modified cytidine nucleosides, wherein optionally at least one type of the modified uridine nucleosides and/or cytidine nucleosides has as a modification a functional group for attachment of one or more function bearers, wherein preferably the function bearer is a target sequence, a PEG group and/or a targeting ligand.

34. The polyribonucleotide of claim 31, wherein modified uridines are selected from 2-thiouridine, 5-methyluridine, pseudouridine, 5-methyluridine 5′

-triphosphate (m5U), 5-idouridine 5′

-triphosphate (15U), 4-thiouridine 5′

-triphosphate (S4U), 5-bromouridine 5′

-triphosphate (Br5U), 2′

-methyl-2′

-deoxyuridine 5′

-triphosphate (U2′

m), 2′

-amino-2′

-deoxyuridine 5′

-triphosphate (U2′

NH2), 2′

-azido-2′

-deoxyuridine 5′

-triphosphate (U2′

N3) and 2′

-fluoro-2′

-deoxyuridine 5′

-triphosphate (U2′

F), and/or wherein the modified cytidines are selected from 5-methylcytidine, 3-methylcytidine, 2-thio-cytidine, 2′

-methyl-2′

-deoxycytidine 5′

-triphosphate (C2′

m), 2′

-amino-2′

-deoxycytidine 5′

-triphosphate (C2′

NH2), 2′

-fluoro-2′

-deoxycytidine 5′

-triphosphate (C2′

F), 5-iodocytidine 5′

-triphosphate (15U), 5-bromocytidine 5′

-triphosphate (Br5U) and 2′

-azido-2′

-deoxycytidine 5′

-triphosphate (C2′

N3), and/or wherein it has an m7GpppG cap and/or at least one IRES and/or a polyA tail at the 5′

end.

35. The polyribonucleotide of claim 31, for use for transcript replacement therapy.

36. The polyribonucleotide of claim 31, wherein the polyribonucleotide contains an mRNA sequence which encodes at least one factor which is beneficial and supportive for the body in general or in a specific situation.

37. The polyribonucleotide of claim 31, wherein the polyribonucleotide contains an RNA sequence which encodes a growth factor, angiogenesis factor, stimulator, inducer, an enzyme or another biologically active molecule, and/or wherein it contains an mRNA sequence which encodes surfactant protein B (SP-B), EPO, ABCA3, BMP-2 or a fragment thereof, and/or further containing at least one target sequence or a targeted sequence for endogenous micro-RNAs which are not expressed in the target cell.

38. The polyribonucleotide of claim 37, further comprising a sequence encoding SP-B, for use for the treatment of respiratory distress syndrome in the newborn, or further comprising a sequence encoding EPO, for use for the treatment of EPO deficiency, or which contains at least one sequence encoding a growth factor, angiogenesis factor, stimulator, inducer or an enzyme, for use for the coating of an implant.

45. A pharmaceutical composition comprising, at least one RNA of claim 31, together with pharmaceutically acceptable additives.

46. The pharmaceutical composition of claim 45, in a form for intratracheal and/or pulmonary administration or in the form of a layer for application onto an implant, which optionally additionally comprises at least one perfluorocarbon for administration before or during the administration of the RNA-containing composition, which preferably contains perfluorocarbon and s2U_(0.25)m5C_(0.25) SP-B mRNA.

47. An implant, comprising, a coating of modified RNA of claim 31 in a delayed release polymer as carrier.

48. The implant of claim 47, which is a dental implant, a hip endoprosthesis, knee endoprosthesis or a vertebral fusion body.

49. The implant of claim 47, wherein the carrier polymer contains at least one type of modified RNA, and/or wherein the carrier polymer contains RNA which encodes at least one protein beneficial in connection with an implantation, and/or wherein the carrier polymer contains RNA which encodes one or more growth factors and one or more angiogenesis factors.

50. The implant of claim 48, wherein the carrier polymer contains at least one type of modified RNA, and/or wherein the carrier polymer contains RNA which encodes at least one protein beneficial in connection with an implantation, and/or wherein the carrier polymer contains RNA which encodes one or more growth factors and one or more angiogenesis factors.

39. A polyribonucleotide comprising, a sequence which encodes a protein or protein fragment, obtainable from a nucleotide mixture of the nucleotides ATP, GTP, CTP and UTP, wherein 5 to 50% of the cytidine nucleotides and 5 to 50% of the uridine nucleotides are modified.

40. The polyribonucleotide of claim 39, wherein the polyribonucleotide is mRNA, wherein optionally the mRNA is in vitro transcribed mRNA (IVT mRNA).

41. The polyribonucleotide of claim 39, wherein the RNA encodes a protein or protein fragment, a defect or lack whereof can trigger a disease, which can moderate, prevent or cure an illness or which can contribute a beneficial or necessary function, and/or wherein 15 to 30%, preferably 7.5 to 25%, of the uridine nucleosides and 15 to 30%, preferably 7.5 to 25%, of the cytidine nucleosides are modified, and/or wherein it contains at least two types of modified uridine nucleosides and/or at least two types of modified cytidine nucleosides, wherein optionally at least one type of the modified uridine nucleosides and/or cytidine nucleosides has as a modification a functional group for attachment of one or more function bearers, wherein preferably the function bearer is a target sequence, a PEG group and/or a targeting ligand.

42. The polyribonucleotide of claim 39, wherein modified uridines are selected from 2-thiouridine, 5-methyluridine, pseudouridine, 5-methyluridine 5′

-triphosphate (m5U), 5-idouridine 5′

-triphosphate (15U), 4-thiouridine 5′

-triphosphate (S4U), 5-bromouridine 5′

-triphosphate (Br5U), 2′

-methyl-2′

-deoxyuridine 5′

-triphosphate (U2′

m), 2′

-amino-2′

-deoxyuridine 5′

-triphosphate (U2′

NH2), 2′

-azido-2′

-deoxyuridine 5′

-triphosphate (U2′

N3) and 2′

-fluoro-2′

-deoxyuridine 5′

-triphosphate (U2′

F), and/or wherein the modified cytidines are selected from 5-methylcytidine, 3-methylcytidine, 2-thio-cytidine, 2′

-methyl-2′

-deoxycytidine 5′

-triphosphate (C2′

m), 2′

-amino-2′

-deoxycytidine 5′

-triphosphate (C2′

NH2), 2′

-fluoro-2′

-deoxycytidine 5′

-triphosphate (C2′

F), 5-iodocytidine 5′

-triphosphate (15U), 5-bromocytidine 5′

-triphosphate (Br5U) and 2′

-azido-2′

-deoxycytidine 5′

-triphosphate (C2′

N3), and/or wherein it has an m7GpppG cap and/or at least one IRES and/or a polyA tail at the 5′

end.

43. The polyribonucleotide of claim 39, for use for transcript replacement therapy.

44. The polyribonucleotide of claim 39, characterized in that it contains an RNA sequence which encodes a growth factor, angiogenesis factor, stimulator, inducer, an enzyme or another biologically active molecule, and/or wherein it contains an mRNA sequence which encodes surfactant protein B (SP-B), EPO, ABCA3, BMP-2 or a fragment thereof, and/or further containing at least one target sequence or a targeted sequence for endogenous micro-RNAs which are not expressed in the target cell.

51. A method for the screening of nucleotide sequences in order to test the immunogenicity and expression quality, comprising,

a) contacting an RNA sequence with at least one receptor selected from TLR3, TLR7, TLR8 and helicase RIG-1;

and

b) measuring the binding capacity.