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US20120195936A1 - Rna with a combination of unmodified and modified nucleotides for protein expression - Google Patents

Rna with a combination of unmodified and modified nucleotides for protein expression Download PDF

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US20120195936A1
US20120195936A1 US13/388,140 US201013388140A US2012195936A1 US 20120195936 A1 US20120195936 A1 US 20120195936A1 US 201013388140 A US201013388140 A US 201013388140A US 2012195936 A1 US2012195936 A1 US 2012195936A1
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rna
mrna
modified
triphosphate
protein
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Carsten Rudolph
Michael Kormann
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Ethris GmbH
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Ethris GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • 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
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/785—Alveolar surfactant peptides; Pulmonary surfactant peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity

Definitions

  • the invention relates to a polyribonucleotide, in particular messenger RNA, which contains a combination of unmodified and modified nucleotides, for protein expression and the use of such RNAs for the therapy of diseases and for diagnostic procedures.
  • messenger RNA which contains a combination of unmodified and modified nucleotides, for protein expression and the use of such RNAs for the therapy of diseases and for diagnostic procedures.
  • mRNA Messenger RNAs
  • mRNA are polymers which are built up of nucleoside phosphate building blocks mainly with adenosine, cytidine, uridine and guanosine as nucleosides, which as intermediate carriers bring the genetic information from the DNA in the cell nucleus into the cytoplasm, where it is translated into proteins. They are thus suitable as alternatives for gene expression.
  • mRNAs for the therapy of hereditary diseases in order to treat gene defects which lead to diseases.
  • the advantage in this is that the mRNA only has to be introduced into the cytoplasm of a cell, but does not have to be inserted into the nucleus. Insertion into the nucleus is difficult and inefficient; moreover there is a considerable risk of the chromosomal DNA being altered if the vector or parts thereof become incorporated into the genome.
  • RNA wherein one of the four ribonucleotides is replaced by a modified nucleotide.
  • mRNA behaves when the uridine is totally replaced by pseudouridine It was found that such an RNA molecule is significantly less immunogenic.
  • the biological activity of these products was not yet sufficient for successful therapy.
  • RNA sequences wherein two or more types of nucleotides are fully replaced by modifications can only be made with difficulty or not at all.
  • nucleic acid In order to be able to provide the body with necessary or beneficial proteins and/or to treat a disease due to missing or deficient proteins with nucleic acids, it is desirable to have a nucleic acid available which can transfect cells, which remains stable in the cell for long enough and provides a sufficient quantity of protein, so that excessively frequent administration is avoided. At the same time, however, this nucleic acid must not cause immunological reactions to a significant extent.
  • a purpose of the present invention was to provide an agent which is suitable for the therapy of diseases caused by deficient or defective genes or diseases caused by missing or defective proteins, or which can in vivo produce necessary or beneficial proteins, which triggers a markedly diminished or no immune response, is stable in a physiological environment, i.e. is not degraded immediately after administration and overall is suitable as an agent for therapy. Further, it was a purpose of the invention to provide an agent for the therapy of diseases which can be positively influenced by in vivo production of proteins.
  • polyribonucleotide as defined in claim 1 .
  • Particularly suitable is mRNA which encodes a protein or protein fragment, a defect or lack whereof is disadvantageous to the body, or expression whereof is of advantage to the body.
  • polyribonucleotide or mRNA is used below, unless the context states otherwise, it should always be assumed that this is a polyribonucleotide or an mRNA which encodes a protein or protein fragment which is connected with an illness or lack, as described above, or encodes a protein or protein fragment which is beneficial or supportive to the body.
  • RNA ribonucleic acid or polyribonucleotides
  • mRNA messenger RNA
  • RNA wherein two types of nucleotides are each partially replaced with modified nucleotides shows high translation and transfection efficiency, i.e. the RNA transfects more cells and produces more of the encoded protein per cell than was possible with known RNA.
  • the RNA modified according to the invention is active for longer than the RNA or unmodified RNA known from the state of the art.
  • RNA according to the invention The advantages achieved with the RNA according to the invention are obtained neither with unmodified nor with fully modified RNA. It has been found that both diminished immunogenicity and also increased stability can be achieved if the content of modified uridine and cytidine nucleotides in the mRNA is specifically set and is at least 5% and not more than 50% for each. If an mRNA with no modifications is used, this is extremely immunogenic, while when all uridine and cytidine nucleotides are present in modified form the biological activity is too low for use for therapeutic purposes to be possible. RNA in which the content of modified nucleotides is very high can be produced under very difficult conditions or not at all.
  • RNA sequences which are modified in the manner according to the invention can be produced easily with reasonable efficiency.
  • the nature of the modification is critical.
  • the mRNAs modified according to the invention show low immunogenicity and have a long lifetime.
  • the stability of the RNA according to the invention is markedly increased compared to previously used nucleic acids.
  • the mRNA according to the invention is detectable 10 days after the transfection in a quantity 10 times higher than unmodified RNA.
  • the increased lifetime above all enables the use of the mRNA according to the invention for therapeutic purposes, since the high stability and hence long lifetime makes it possible to effect administration at longer time intervals which are thus also acceptable to the patients.
  • RNA according to the invention fulfills the requirements that are placed on a product to be used in therapy: as RNA it needs only to be introduced into the cytoplasm and not into the cell nucleus to develop its activity, the danger of integration into the genome does not exist, the type of modification according to the invention largely prevents an immune reaction and in addition the modification protects the RNA from rapid degradation. Hence with the RNA according to the invention it is possible to generate or to regenerate physiological functions in tissues, e.g. to restore in vivo functions which had failed owing to a deficient or defective gene, and hence to treat diseases caused by deficient or defective genes.
  • polyribonucleotides according to the invention can favorably influence diseases in that proteins are produced in vivo which can directly or indirectly have an influence on the course of the disease.
  • polyribonucleotides can also be provided which encode factors which are beneficial and supportive to the body in general or in a specific situation, e.g. growth factors, angiogenesis factors, stimulators, inducers, enzymes or other biologically active molecules.
  • FIG. 1 shows the effect of different nucleotide modifications on the immunogenicity and stability of various mRNAs.
  • FIG. 1A is a diagram on which the TNF- ⁇ level after administration of various RNAs with differently modified nucleotides is plotted. Unmodified and up to 25% singly modified RNA leads to a high level of inflammatory markers and shows the high immunogenicity of this RNA, while for RNA doubly modified according to the invention the inflammatory markers are present in tolerable amount.
  • FIGS. 1B and 1C show the biological activity (transfection efficiency and expression) of mRNA modified in various ways in human cells and mouse cells as the percentage of the cells positive for red fluorescing protein (RFP) and the quantity of RFP per cell. The diagrams show that the proteins encoded by unmodified, singly modified and completely modified RNA can only be detected at a lower percentage content, while the RNA partly doubly modified according to the invention yields significantly higher quantities of protein owing to its greater stability.
  • RFP red fluorescing protein
  • FIG. 2 shows the higher stability and longer duration of expression for multiply modified mRNA.
  • FIGS. 2A and 2B each show diagrams on which the duration of expression of various modified and unmodified mRNAs is plotted.
  • FIG. 2C shows data for RNA immunoprecipitation for unmodified RNA, singly modified RNA and multiply modified RNA.
  • FIG. 2D shows diagrams in which the immunogenicity of various mRNAs after in vivo intravenous administration is plotted. The data show that an RNA doubly modified according to the invention displays a combination of high stability and low immunogenicity.
  • FIG. 3 shows various test results which were obtained after intratracheal aerosol application of modified SP-B mRNA in SP-B conditionally deficient mice.
  • FIG. 3A shows bioluminescence images of the lung of mice treated with unmodified RNA and multiply modified RNA. It can clearly be seen that a sufficient quantity of protein is still also expressed after 5 days only by RNA modified according to the invention, while with unmodified RNA the expression is already low after 3 hours.
  • FIG. 3B shows a diagram in which the flux is plotted against the time after transfection. It can clearly be discerned that the modification according to the invention prolongs the duration of expression.
  • FIG. 3C shows the dosing scheme for SP-B mRNA.
  • FIG. 3D shows a diagram which presents the survival rate for mice which were treated with modified mRNA compared to mice which were treated with control mRNA, the survival rate in mice treated with RNA according to the invention being markedly longer.
  • FIG. 3E shows an immunostaining in which it can be seen that with RNA according to the invention which encodes SP-B the SP-B in SP-B deficient mice could be reconstituted.
  • FIG. 3F shows as the result of a semi-quantitative Western blot analysis the distribution of proteins in cell-free BALF supernatant.
  • FIGS. 3G and H show images of lung histology preparations and bronchoalveolar lavage preparations from mice treated according to 3 C.
  • FIG. 3I shows a diagram concerning the lung tolerance over time. The lung function was retained over a longer period on treatment with RNA according to the invention, while lung damage was found in animals treated with control RNA.
  • FIG. 4 shows a diagram in which the fluorescence intensity of the RFP produced was plotted against time for unmodified and differently modified mRNAs.
  • the modified mRNA is translated later and less strongly compared to the unmodified mRNA.
  • FIG. 5 shows three diagrams in which inflammatory markers for mice treated with different mRNAs are plotted. It can clearly be discerned that RNA modified according to the invention causes no inflammatory reactions, while unmodified RNA leads to a strong immune reaction.
  • FIG. 6 shows diagrams in which different typical lung parameters are plotted for mice treated with different mRNAs according to the invention.
  • the parameters are tissue elasticity (HL), tissue damping (GL), tissue inertia, airway resistance (Rn) and lung tissue composition Eta (GL/HL).
  • HL tissue elasticity
  • GL tissue damping
  • Rn airway resistance
  • GL/HL lung tissue composition Eta
  • FIG. 7 shows the expression capacity of differently modified mRNA in a diagram in which the percentage content of RFP positive cells is plotted for mRNA with a different content of modified nucleotides. The comparison shows that only mRNA modified according to the invention leads to long-lasting expression, while mRNA modified not according to the invention expresses to a lesser extent both in human cells and also in mouse cells.
  • FIG. 8 shows the expression capacity of differently modified mRNA in a diagram in which the percentage content of RFP positive cells is plotted for mRNA with differently modified nucleotides. The comparison shows that only mRNA modified according to the invention leads to long-lasting expression, while mRNA modified not according to the invention expresses to a lesser extent both in human cells and also in mouse cells.
  • FIG. 9 shows the stability of freeze-dried RNA according to the invention.
  • FIG. 10A shows a diagram in which the transfection efficiency is plotted for various modified nucleotides. It can clearly be discerned that the highest transfection efficiency is attained with RNA wherein 10% of the uridine nucleotides and 10% of the cytidine nucleotides and optionally also 5% of further nucleotides are modified.
  • FIG. 10B shows a diagram in which the TNF- ⁇ production as a marker for the immunological reaction is plotted for RNA with differently modified nucleotides.
  • RNA wherein between 5 and 50% of the uridine nucleotides and cytidine nucleotides are modified has a markedly reduced immunogenicity compared to unmodified RNA.
  • FIG. 11 shows the results of various tests with which the stability and immunogenicity of mRNA modified according to the invention, which encodes EPO, was measured.
  • Diagram 11 ( a ) shows the content of erythropoietin which is detectable 14 days after administration of mRNA encoding EPO which is modified in different ways. It is clearly discernible that after 14 days the content of EPO in mice into which mRNA modified according to the invention was injected is 4.8 times higher than in untreated mice, but also 4.8 times higher than in mice treated with unmodified RNA and is still 2.5 times higher than in mice treated with singly modified RNA.
  • Diagram 11 ( b ) shows hematocrit values 14 days and 28 days after administration of EPO-encoding mRNA with different modifications. The diagram clearly shows that mice treated with mRNA modified according to the invention have a considerably higher hematocrit value.
  • FIG. 11( c ) the production of the factors typical for an immunological reaction is plotted. It is found that all four inflammatory markers are elevated with the administration of unmodified mRNA, while with RNA modified according to the invention an immunological reaction is hardly detectable.
  • FIG. 11( d ) show the corresponding values for IFN- ⁇ and IL-12, which are also inflammatory markers.
  • mRNA modified according to the invention causes practically no immunological reaction, in contrast to unmodified mRNA.
  • FIG. 12 shows a diagram in which the survival rate of three groups of mice which were given SP-B mRNA modified according to the invention twice in one week (B) or twice a week for 28 days (C), or in the comparison group modified EGFPLuc mRNA (A) is plotted. It is found that the mice only survive as long as they are given SP-B mRNA (B, C). Without provision of SP-B mRNA, the mice die (A).
  • FIG. 13 shows cytokine levels in the bronchoalveolar lavage of mice 8 hours after administration of unmodified SP-B mRNA, SP-B mRNA modified according to the invention or SP-B plasmid DNA.
  • the results show that in contrast to the intratracheal administration of unmodified mRNA or plasmid DNA, which each lead to a marked rise in the inflammatory markers IFN ⁇ and IL-12, on administration of SP-B mRNA modified according to the invention the inflammatory markers are practically not elevated compared to the untreated group or to the group treated with perfluorocarbon.
  • FIG. 14 shows hematocrit values as obtained after repeated administration of mEPO mRNA modified according to the invention. The results show that the repeated administration of mEPO mRNA modified according to the invention is well tolerated and results in long-persisting elevation of the hematocrit.
  • FIG. 15 shows the luciferase expression of cells which were incubated with titanium implants which were provided with coatings containing different forms of RNA modified according to the invention. It was found that RNA modified according to the invention which was contained in a coating of delayed release polymer which had been applied onto titanium plates and which was gradually released therefrom did not lose its activity.
  • FIG. 16 shows the luciferase expression for coatings applied onto titanium implants which contained modified mRNA. It was found that the protein expression for mRNA modified according to the invention was far higher than for untreated RNA, but was also higher than for plasmid DNA.
  • FIGS. 17A and 17B respectively show the relative content of RFP-positive cells and the relative RFP expression of mRNA which has micro-RNA binding sites for micro-RNA 142-3p. It was found that the content of RFP-positive cells for RNA having micro-RNA binding sites was lower and the expression of the encoded protein was considerably lower in the cells which contained the corresponding micro-RNA 142-3p.
  • FIG. 18 shows the sequence of an RNA modified by incorporation of micro-RNA binding sites, which encodes RFP.
  • the RFP sequence is shown with a gray background.
  • the fourfold tandem repetition of the micro-RNA binding site for the micro-RNA 142-3p (with light gray background) with the spacing sequences (no background) is underlined.
  • a polyribonucleotide molecule with partially multiply modified nucleotides, a partially multiply modified mRNA, an IVT mRNA, and the use of the RNA molecules for the production of a drug for the treatment of diseases due to deficient or defective genes or for the treatment of diseases which can be moderated or cured by the provision of proteins in vivo, such as factors, stimulators, inducers or enzymes, are provided.
  • the mRNA according to the invention is combined with target binding sites, targeting sequences and/or with micro-RNA binding sites, in order to allow activity of the desired mRNA only in the relevant cells.
  • the RNA according to the invention is combined with micro-RNAs or shRNAs downstream of the 3′ polyA tail.
  • RNA whose duration of action has been adjusted or extended by further specific modifications is provided.
  • RNA with increased stability and decreased immunogenicity.
  • the RNA according to the invention can be made in a manner known per se. As a rule it is made by transcription of a DNA which encodes the intact or desired protein which can influence an illness or the lack or deficient form whereof causes a disease.
  • RNA should be understood to mean any polyribonucleotide molecule which, if it comes into the cell, is suitable for the expression of a protein or fragment thereof or is translatable to a protein or fragment thereof.
  • protein here encompasses any kind of amino acid sequence, i.e. chains of two or more amino acids which are each linked via peptide bonds and also includes peptides and fusion proteins.
  • the RNA according to the invention contains a ribonucleotide sequence which encodes a protein or fragment thereof whose function in the cell or in the vicinity of the cell is needed or beneficial, e.g. a protein the lack or defective form whereof is a trigger for a disease or an illness, provision whereof can moderate or prevent a disease or an illness, or a protein which can promote a process which is beneficial for the body, in a cell or its vicinity.
  • the RNA according to the invention contains the sequence for the complete protein or a functional variant thereof.
  • the ribonucleotide sequence can encode a protein which acts as a factor, inducer, regulator, stimulator or enzyme, or a functional fragment thereof, where this protein is one whose function is necessary in order to remedy a disorder, in particular a metabolic disorder or in order to initiate processes in vivo such as the formation of new blood vessels, tissues, etc.
  • functional variant is understood to mean a fragment which in the cell can undertake the function of the protein whose function in the cell is needed or the lack or defective form whereof is pathogenic.
  • the RNA according to the invention can also have further functional regions and/or 3′ or 5′ noncoding regions. The 3′ and/or 5′ noncoding regions can be the regions naturally flanking the encoded protein or else artificial sequences which contribute to the stabilization of the RNA. Those skilled in the art can discover the sequences suitable for this in each case by routine experiments.
  • the RNA contains an m7GpppG cap, an internal ribosome entry site (IRES) and/or a polyA tail at the 3′ end in particular in order to improve translation.
  • the RNA can have further regions promoting translation.
  • Critical for the RNA according to the invention is its content of modified nucleotides.
  • RNA according to the invention with increased stability and diminished immunogenicity is obtained by using for the production thereof a nucleotide mixture wherein the content of the modified cytidine nucleotides and the modified uridine nucleotides is set.
  • the RNA according to the invention is preferably produced with a nucleotide mixture which contains both unmodified and also modified nucleotides, where 5 to 50% of the cytidine nucleotides and 5 to 50% of the uridine nucleotides are modified.
  • the adenosine- and guanosine-containing nucleotides can be unmodified.
  • a nucleotide mixture can also be used wherein some of the ATPs and/or GTPs are also modified, where their content should not exceed 20% and where their content, if present, should preferably lie in a range from 0.5 to 10%.
  • an mRNA which has 5 to 50% of modified cytidine nucleotides and 5 to 50% of uridine nucleotides and 50 to 95% of unmodified cytidine nucleotides and 50 to 95% of unmodified uridine nucleotides, and the adenosine and guanosine nucleotides can be unmodified or partially modified, and they are preferably present in unmodified form.
  • the cytidine and uridine nucleotides are modified and particularly preferably the content of the modified cytidine nucleotides lies in a range from 7.5 to 25% and the content of the modified uridine nucleotides in a range from 7.5 to 25%. It has been found that in fact a relatively low content, e.g. only 10% each, of modified cytidine and uridine nucleotides can achieve the desired properties, under the precondition that these are the modifications according to the invention.
  • RNA according to the invention either all uridine nucleotides and cytidine nucleotides can each be modified in the same form or else a mixture of modified nucleotides can be used for each.
  • the modified nucleotides can have naturally or not naturally occurring modifications.
  • a mixture of various modified nucleotides can be used.
  • one part of the modified nucleotides can have natural modifications, while another part has modifications not occurring naturally or a mixture of naturally occurring modified and/or not naturally occurring modified nucleotides can be used.
  • a part of the modified nucleotides can have a base modification and another part a sugar modification. In the same way, it is possible that all modifications are base modifications or all modifications are sugar modifications or any suitable mixture thereof.
  • the stability and/or duration of action of the RNA according to the invention can be selectively adjusted.
  • At least two different modifications are used for one type of nucleotide, where one type of the modified nucleotides has a functional group via which further groups can be attached.
  • Nucleotides with different functional groups can also be used, in order to provide binding sites for the attachment of different groups.
  • a part of the modified nucleotides can bear an azido group, an amino group, a hydroxy group, a thiol group or some other reactive group which is suitable for reaction under predefined conditions.
  • the functional group can also be such that it can under certain conditions activate a naturally present group capable of binding, so that molecules with functions can be coupled.
  • Nucleotides which are modified so that they provide binding sites can also be introduced as adenosine or guanosine modifications.
  • the selection of the particular suitable modifications and the selection of the binding sites to be made available depends on what groups are to be introduced and with what frequency these are to be present.
  • the content of the nucleotides provided with functional and/or activating groups depends on how high the content of groups to be coupled is to be and can easily be determined by those skilled in the art.
  • the content of nucleotides modified with functional and/or activating groups, if present is 1 to 25% of the modified nucleotides.
  • Those skilled in the art can if necessary determine the most suitable groups in each case and the optimal content thereof by routine experiments.
  • RNA according to the invention 2′-thiouridine as a modified uridine-containing nucleotide.
  • the RNA according to the invention contains 5′-methylcytidine as a modified cytidine nucleotide.
  • these two nucleotides are therefore preferred.
  • a combination of these two modifications are each present at a content of 10 to 30%. Nucleotides modified in another way can optionally also be present, as long as the total content of modified nucleotides does not exceed 50% of the particular nucleotide type.
  • this mRNA according to the invention additionally has a 7′-methylguanosine cap and/or a poly(A) end.
  • the mRNA is produced in its mature form, i.e. with a GppG cap, an IRES and/or a polyA tail.
  • modified uridine nucleotides and cytidine nucleotides for a specific RNA can be determined with routine experiments.
  • an mRNA whose immunogenicity is so low that the treated organism is not stressed and which has a predetermined stability and hence predetermined duration of expression is described as optimal. Methods for the testing and determination of these properties are known to those skilled in the art and are described below and in the examples.
  • the RNA according to the invention can be produced in a manner known per se.
  • a method wherein the mRNA according to the invention is produced by in vitro transcription from a mixture of ATP, CTP, GTP and UTP, wherein 5 to 50%, preferably 5 to 30% and in particular 7.5 to 25% of the cytidine nucleotides and 5 to 50%, preferably 5 to 30% and in particular 7.5 to 25% of the uridine nucleotides are modified and the rest is unmodified is for example suitable.
  • Guanosine and adenosine nucleosides, in particular adenosine can optionally also be modified.
  • the modification of UTP and CTP in the stated range is essential for the invention.
  • modified UTP and/or modified CTP are lower or higher, the advantageous properties are no longer achieved. Thus it has been found that outside the claimed ranges the mRNA is no longer so stable. Moreover, with a lower content of modification immunological reactions are to be expected.
  • the RNA is appropriately made using a nucleotide mixture, the nucleoside contents whereof are partly modified and partly unmodified in accordance with the desired ratio, where according to the invention at least 5% of the uridine nucleosides and at least 5% of the cytidine nucleosides are modified, but in total not more than 50% of uridine nucleosides and cytidine nucleosides respectively are modified.
  • Further nucleosides i.e. adenosine and guanosine, can be modified, however an upper limit of 50% modification, preferably 20%, should also not be exceeded for these nucleosides.
  • nucleosides to be modified can have modifications such as are also to be found in naturally occurring nucleosides, e.g. methylations or binding variations, but also “synthetic”, i.e. not occurring in nature, modifications or a mixture of nucleosides with natural and/or synthetic modifications can be used.
  • naturally modified nucleosides of at least one type can be combined with synthetically modified nucleosides of the same type or another type or else naturally and synthetically modified nucleosides of one type with only naturally, only synthetically or mixed naturally/synthetically modified nucleosides of another type, where “type” here refers to the type of the nucleosides, i.e. ATP, GTP, CTP or UTP.
  • modified nucleosides with functional groups, which provide binding sites, with non-functionally modified nucleosides.
  • functional groups which provide binding sites
  • non-functionally modified nucleosides particularly preferably 2-thiouridine and 5-methylcytidine are used as modified nucleosides.
  • 2′-azido and 2′-amino nucleosides are preferably considered.
  • the length of the mRNA used according to the invention depends on the gene product or protein or protein fragment which is to be provided or supplemented. Hence the mRNA can be very short, e.g. have only 20 or 30 nucleotides, or else corresponding to the length of the gene have several thousand nucleotides. Those skilled in the art can select the suitable sequence each time in the usual way.
  • 2′-Thiouridine is preferably used as the modified uridine-containing nucleotide for the production of the RNA according to the invention. Furthermore, it is preferable to use 5′-methylcytidine as the modified cytidine nucleotide.
  • a nucleotide mixture which as well as ATP and GTP respectively contains 95 to 50% of unmodified CTP and 95 to 50% of unmodified UTP and 5 to 50% of 2′-thiouridine nucleotides and 5 to 50% of methylcytidine nucleotides is preferably used.
  • a polyribonucleotide wherein 5 to 50%, preferably 5 to 30% and in particular 7.5 to 25% of the uridine nucleotides are 2′-thiouridine nucleotides and 5 to 50%, preferably 5 to 30% and in particular 7.5 to 25% of the cytidine nucleotides are 5′-methylcytidine nucleotides and the adenosine and guanosine nucleotides are unmodified nucleotides is particularly preferred.
  • Such a combination leads to the production of a partially modified RNA which is characterized by particularly high stability.
  • RNA which was produced with a nucleotide mixture which as CTP and UTP contained 5 to 50% of 2-thiouridine and 5-methylcytidine nucleotides respectively is especially stable, i.e. had a lifetime increased up to 10-fold compared to unmodified RNA or RNA modified in known manner.
  • 1 to 50%, preferably 2 to 25%, of the 5 to 50% modified uridine or cytidine nucleotides are nucleotides which have binding site-creating or activating groups as a modification, i.e. 0.5 to 20%, preferably 1 to 10% of the cytidine nucleotides and/or uridine nucleotides can have a modification which creates a binding site, such as for example azido, NH, SH or OH groups.
  • the polyribonucleotide molecule built up of unmodified and modified nucleotides has a 7′-methylguanosine cap and/or a poly(A) end.
  • the RNA can also have additional sequences, e.g. non-translated regions and functional nucleic acids, such as are well known to those skilled in the art.
  • the RNA according to the invention is preferably provided as in vitro transcribed RNA (IVT RNA).
  • IVT RNA in vitro transcribed RNA
  • the materials necessary for performing the in vitro transcription are known to those skilled in the art and available commercially, in particular buffers, enzymes and nucleotide mixtures.
  • the nature of the DNA used for the production of the RNA according to the invention is also not critical; as a rule it is cloned DNA.
  • RNA in particular mRNA, which has a predetermined content of modified uridine nucleosides and modified cytidine nucleosides is provided.
  • the optimal content of modified uridine nucleosides and cytidine nucleosides for a specific mRNA can be determined by routine experiments which are well known to those skilled in the art.
  • the RNA according to the invention is preferably used for the therapy of diseases or for the provision of proteins beneficial to the body.
  • the RNA according to the invention When used for the therapy of diseases, it preferably has the in vitro transcript for a protein or protein fragment, a defect or lack whereof leads to a disease condition or the provision whereof leads to the moderation of an illness.
  • a DNA is preferably used which encodes a protein or protein fragment, a defect or lack whereof leads to a disease or is connected with an illness.
  • the DNA of a gene, a defect or lack whereof leads to a disease or illness is used for the production of the RNA according to the invention.
  • RNA which encodes a protein the presence, perhaps temporary, whereof is beneficial or curative for an organism is used for the production of the RNA according to the invention.
  • any state wherein physical and/or mental/psychological disorders or changes are subjectively and/or objectively present, or where the abnormal course of physical, mental or psychological processes makes medical care necessary and may lead to inability to work is regarded as a disease or illness.
  • proteins or protein fragments which, without a genetic defect being present, are to be made fully or temporarily available to the body since they are missing either because of disorders of some kind or because of natural circumstances or because they can benefit the body under certain conditions, e.g. in the treatment of defects or in the context of implantation.
  • proteins or protein fragments which, without a genetic defect being present, are to be made fully or temporarily available to the body since they are missing either because of disorders of some kind or because of natural circumstances or because they can benefit the body under certain conditions, e.g. in the treatment of defects or in the context of implantation.
  • proteins or protein fragments which, without a genetic defect being present, are to be made fully or temporarily available to the body since they are missing either because of disorders of some kind or because of natural circumstances or because they can benefit the body under certain conditions, e.g. in the treatment of defects or in the context of implantation.
  • proteins or protein fragments which, without a genetic defect being present, are to be made fully or temporarily available to the body since they are missing either because of disorders of
  • the mRNA modified according to the invention can advantageously be used in order to promote the ingrowth of implanted prostheses. If it is available on the surface of prostheses to be inserted such as tooth implants, hip endoprostheses, knee endoprostheses or vertebral fusion bodies, the mRNA according to the invention can release factors which can promote the ingrowth, new formation of blood vessels and other functions which are necessary for the newly inserted prostheses.
  • the administration of biologically active substances such as growth factors such as BMP-2 or angiogenesis factors in the context of implantation of prostheses or thereafter is known. Since biological substances very often have extremely short half-lives, it was previously necessary to use very high dosages, which burdens the patient with severe side effects.
  • the desired and/or needed proteins can be used selectively and suitably dosed. This decreases or even completely spares the patient the side effects.
  • the RNA according to the invention which encodes desired and/or needed substances such as growth factors, angiogenesis factors etc. can be applied onto the implant in a coating releasing the RNA in a measured manner and then released gradually therefrom in a measured manner, so that the cells in the vicinity of the implant can continuously or intermittently produce and if necessary release the desired factors.
  • Carriers as a rule biocompatible, synthetic, natural or mixed natural-synthetic polymers, the release properties whereof can be specifically adjusted, are well known and thus need no more detailed explanation here. Polylactide or polylactide/glycolide polymers are for example used. In this way it is possible selectively to release the desired factors continuously, intermittently, over a longer or shorter time and at the desired site.
  • a deficient or defective gene or deficiency or lack are understood to mean genes which are not expressed, incorrectly expressed or not expressed in adequate quantity and as a result cause diseases or illnesses, e.g. by causing metabolic disorders.
  • RNA according to the invention can appropriately be used in any case where a protein, which would naturally be present in the body but is not present or is present in deficient form or in too small a quantity because of gene defects or diseases, is to be provided to the body. Proteins and the genes encoding them, the deficiency or defect whereof are linked with a disease, are known. Various proteins and genes in case of a lack whereof the RNA according to the invention can be used are listed below.
  • SCID Severe combined immunodeficiencies
  • X-SCID X-chromosomally inherited combined immunodeficiencies
  • ADA-SCID SCID due to lack of adenosine deaminase
  • SCID with RAG1 mutation SCID with RAG2 mutation
  • SCID with JAK3 mutation SCID with IL7R mutation
  • SCID with CD45 mutation SCID with CD3 ⁇ mutation
  • Septic granulomatoses X-chromosomal recessive CGD mutation of the gp91-phox gene CGD cytochrome b positive type 1 mutation of the p47-phox gene CGD cytochrome b positive type 2 mutation of the p67-phox gene CGD cytochrome b negative mutation of the p22-phox
  • Type Variant Clinical features Defective enzyme I-H Hurler-Pfaundler syndrome dysmorphia (gargoylism), ⁇ -L-iduronidase cognitive retardation, skeletal malformation (dysostosis), corneal clouding, decreased growth, hernias, hepatomegaly I-S Scheie's disease not mentally retarded, ⁇ -L-iduronidase skeletal malformation (dysostosis), corneal clouding, heart valve faults I-H/S Hurler/Scheie variants mentally between I-H and I-S ⁇ -L-iduronidase II Hunter's syndrome moderate cognitive retardation, iduronate sulfate skeletal malformation (dysostosis), silfatase considerable somatic changes, premature deafness III Sanfilippo type A cognitive retardation, dysmorphia, heparan sulfate syndrome corneal clouding can be lacking, sulfamidase type B frequently hearing impairment, ⁇ -N
  • the above table shows examples of genes in which a defect leads to a disease which can be treated by transcript replacement therapy with the RNA according to the invention.
  • hereditary diseases can be mentioned which for example affect the lungs, such as SPB deficiency, ABCA3 deficiency, cystic fibrosis and ⁇ 1-antitrypsin deficiency, which affect plasma proteins and cause clotting defects and complement defects, immune defects such as for example SCID, septic granulomatosis and storage diseases.
  • a protein e.g. an enzyme, is defective, which can be treated by treatment with the RNA according to the invention, which makes the protein encoded by the defective gene or a functional fragment thereof available.
  • proteins which can be encoded by the RNA according to the invention are erythropoietin (EPO), growth hormone (somatotropin, hGH), cystic fibrosis transmembrane conductance regulator (CFTR), growth factors such as GM-SCF, G-CSF, MPS, protein C, hepcidin, ABCA3 and surfactant protein B.
  • EPO erythropoietin
  • somatotropin somatotropin, hGH
  • cystic fibrosis transmembrane conductance regulator CFTR
  • growth factors such as GM-SCF, G-CSF, MPS, protein C, hepcidin, ABCA3 and surfactant protein B.
  • RNA according to the invention diseases which can be treated with the RNA according to the invention are hemophilia A/B, Fabry's disease, CGD, ADAMTS13, Hurler's disease, X chromosome-mediated A- ⁇ -globulinemia, adenosine deaminase-related immunodeficiency and respiratory distress syndrome in the newborn, which is linked with SP-B.
  • the mRNA according to the invention contains the sequence for surfactant protein B (SP-B) or for erythropoietin.
  • proteins which can be encoded by RNA modified according to the invention are growth factors such as BMP-2 or angiogenesis factors.
  • RNA is now provided for this so that the replacement of the missing protein can take place at the level of the transcript.
  • the replacement at the transcript level has the effect that the glycosylation typical in humans takes place in the body.
  • proteins that are recombinant i.e. normally produced in microorganisms
  • the glycosylation is as a rule different from that in the body where replacement is to be effected. This can lead to side effects.
  • the protein expressed from the RNA according to the invention is identical with the endogenous protein as regards structure and glycosylation, which is as a rule not the case with recombinant proteins.
  • proteins replacement or introduction whereof can be desirable are functional proteins such as erythropoietin and growth factors such as somatotropin (hGH), G-CSF, GM-CSF and thrombopoietin.
  • hGH somatotropin
  • G-CSF G-CSF
  • GM-CSF GM-CSF
  • thrombopoietin thrombopoietin
  • RNA according to the invention offers not only the advantage that the missing protein can be provided selectively and in the correct dosage but in addition it is possible to provide the protein in a time window.
  • the relevant healing factor or growth factor can be provided for a limited time by dosed administration of the RNA.
  • FGF fibroblast growth factor
  • TGF transforming growth factor
  • BMPs bone morphogenetic protein
  • BMP1 to 7, 8a & b, 10 & 15 platelet-derived growth factor
  • PDGF-A, PDGF-B, PDGF-C and PDGF-D epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF-A to F and PIGF), insulin-like growth factors, e.g. IgF1 and IgF2, hepatocyte growth factor (HGF), interleukins, e.g. interleukin-1B, IL-8 and IL-1 to 31, nerve growth factor (NGF) and other factors which stimulate the formation of erythrocytes, neutrophils, blood vessels, etc.
  • EGF epidermal growth factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • VEGF-A to F and PIGF vascular endothelial growth factor
  • insulin-like growth factors e.g. IgF1 and IgF2
  • HGF hepatocyte growth factor
  • interleukins e.g.
  • the RNA according to the invention can also be selectively used in the field of cancer diseases. Through the expression of tailor-made T cell receptors in T lymphocytes which recognize specific tumor-associated antigens, these can become still more effective. It has already been shown that in principle mRNA can be successfully used in this field. However until now its use was prevented by the immunogenic effects already described above. With the less immunogenic and highly stable RNA provided according to the invention, it is now possible to express T cell receptors appropriately.
  • RNA according to the invention can also be used to express transcription factors which ensure that somatic cells are reprogrammed into embryonic stem cells. Examples of this are O-cp3/4, Sox2, KLF4 and c-MYC. Stable RNA, especially mRNA, according to the invention which encodes these transcription factors can thus lead to the production of stem cells without creating the side effects which can occur with the previously considered gene transfer via viral or non-viral vectors.
  • RNA according to the invention is that, in contrast to the use of DNA vectors, the duration of the treatment is adjustable.
  • the transcription factors are only transiently active, in order to reprogram somatic cells into stem cells.
  • the activity is controllable over time.
  • there is the danger of integration of the genes administered which leads to complications, e.g. tumorigenesis, and moreover renders it impossible to control the duration.
  • RNA according to the invention also offers new possibilities.
  • the standard development of vaccines depends on killed or weakened pathogens. More recently, DNA which encodes a protein of the pathogen has also come under consideration. The production of these vaccines is laborious and very time-consuming. Often side effects arise and lead to vaccinations being refused.
  • the mRNA according to the invention it is possible to provide a vaccine which does not have the problems associated with pathogens or DNA. In addition, such a vaccine can be produced very quickly as soon as the antigen sequences of a pathogen are known. This is particularly advantageous under the threat of pandemics.
  • an RNA is provided which encodes an antigenic part of a disease pathogen, e.g.
  • RNA which encodes an amino acid sequence which has a combination of several epitopes, optionally linked by spacer sections.
  • a combination with immunomodulating substances is also possible, either through the RNA encoding a fusion protein or as a combination of nucleic acids.
  • RNA according to the invention can also encode proteins which as factors, stimulators, inducers, etc. have an influence on the course of disease.
  • diseases which are not directly attributable to a gene defect but wherein the disease process can be positively influenced by means of mRNA expression.
  • examples are: erythropoietin for stimulation of the formation of erythrocytes, G-CSF or GM-SCF for the formation of neutrophils, growth factors for the formation of new blood vessels, for bone and wound healing as factors for “tissue engineering”, treatment of tumors by induction of apoptosis or by formation of proteinaceous cell poisons, e.g. diphtheria toxin A, by induction of pluripotent stem cells (iPS) etc.
  • erythropoietin for stimulation of the formation of erythrocytes
  • G-CSF or GM-SCF for the formation of neutrophils
  • growth factors for the formation of new blood vessels
  • for bone and wound healing as factors for “t
  • Cytokines which are associated with inflammation such as for example TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-8, IL-6, IL-12 or other cytokines known to those skilled in the art are normally measured.
  • the expression of DC activation markers can also be used for the estimation of immunogenicity.
  • a further indication of an immunological reaction is the detection of binding to the Toll-like receptors TLR-3, TLR-7 and TLR-8 and to helicase RIG-1.
  • the immunogenicity is as a rule determined in relation to a control.
  • either the RNA according to the invention or an RNA that is unmodified or modified in another way is administered to cells and the secretion of inflammatory markers in a defined time interval as a reaction to the administration of the RNA is measured.
  • either unmodified RNA can be used, in which case the immune response should be lower, or RNA which is known to cause little or no immune response, in which case the immune response to the RNA according to the invention should then lie in the same range and not be elevated.
  • the RNA according to the invention it is possible to lower the immune response compared to unmodified RNA by at least 30%, as a rule at least 50% or even 75% or even to prevent it completely.
  • the immunogenicity can be determined by measurement of the aforesaid factors, in particular by measurement of the TNF- ⁇ and IL-8 levels and the binding capacity to TLR-3, TLR-7, TLR-8 and helicase RIG-1.
  • the quantity of one or more of the aforesaid factors after administration of the polyribonucleotide concerned can be measured.
  • a quantity of the mRNA to be tested can be administered to mice via the caudal vein or i.p. and then one or more of the aforesaid factors can be measured in the blood after a predefined period, e.g. after 7 or 14 days.
  • the quantity of factor is then related to the quantity of factor which is present in the blood of untreated animals.
  • the TNF- ⁇ levels and IL-8 levels also provide very good indications.
  • the mRNA according to the invention it is possible to lower the binding capacity to TLR-3, TLR-7, TLR-8 and RIG-1 by at least 50% compared to unmodified RNA. As a rule it is possible to lower the binding to said factors by at least 75% or even by 80%.
  • the binding capacity to TLR-3, TLR-7, TLR-8 and RIG-1 lies in the same range for the mRNA according to the invention and for animals to which no mRNA was administered.
  • the mRNA according to the invention causes practically no inflammatory or immunological reactions.
  • the RNA according to the invention has such low immunogenicity that the general condition of the patient is not affected. A slight increase in the aforesaid factors can thus be tolerated as long as the general condition does not worsen as a result.
  • Further properties of the mRNA according to the invention are its efficiency and stability. For this, transcription efficiency, transfection efficiency, translation efficiency and duration of protein expression are important and can be determined by methods known per se.
  • the transcription efficiency indicates how efficiently RNA can be produced from DNA.
  • problems can arise with the use of a high content of modified nucleotides.
  • the RNA modified according to the invention can be produced with high transcription efficiency.
  • RNA In order to obtain stable and adequate expression of the proteins encoded by the RNA, it is important that sufficient RNA reaches the desired cells. This can be determined in that after administration of labeled RNA the content of RNA which has reached the cells is determined by measurement of the labeling. Flow cytometry can be used for the determination of the labeling. When labeling is effected with a fluorescent molecule, the transfection efficiency can be calculated, for example as the percentage of the cell population wherein the fluorescence intensity is higher compared to control cells which were only treated with PBS.
  • RNA modified according to the invention can be produced effectively, in contrast to RNA wherein two or more nucleotide types have been 100% replaced by modified nucleotides, and that the transfection efficiency for RNA according to the invention, wherein only a part of the nucleotides is modified, is far higher than with RNA wherein any one type of nucleotides is 100% modified.
  • the translation efficiency designates the efficiency with which the RNA is translated into the protein.
  • the translation efficiency can be determined by comparing the proportion of translation for RNA modified according to the invention with the translation ratio for unmodified RNA. As a rule, the translation efficiency with the RNA according to the invention is somewhat lower than with unmodified RNA. This is however more than compensated by the far higher stability which is manifested in the duration of the protein expression.
  • the RNA according to the invention in particular provides for high stability, which results in long-continuing protein expression.
  • the RNA modified according to the invention is intended for the treatment of diseases due to gene defects, the longer it remains in the cell the more valuable it is. The more rapidly the RNA is degraded, the more rapidly the protein expression ends and the more often the RNA must be administered.
  • a stable RNA which remains in the cell for a long time the frequency of dosing can be greatly reduced. It has been found that RNA modified according to the invention is stably expressed for up to 4 weeks.
  • the duration of the protein expression can be adjusted by influencing the stability.
  • RNA according to the invention A further valuable property of the RNA according to the invention is thus that the duration of action can be adjusted selectively via the stability so that the duration of the protein expression can be tailored so that it takes place in a desired time window.
  • a very long-acting RNA can be used where this is necessary.
  • the RNA modified according to the invention, expression whereof can last up to 4 weeks, is thus ideally suited for the treatment of chronic diseases since here it only has to be given every 4 weeks.
  • the high stability and long-lasting protein expression is also advantageous, e.g. for the use of RNA encoding erythropoietin.
  • RNA according to the invention can also especially advantageously be used for the treatment of hemophilia.
  • the frequency of administration can be reduced, so that RNA encoding the factor now only has to be given every 2 or even every 4 weeks.
  • the stability of the mRNA according to the invention can be determined by methods known per se. Particularly suitable are methods for the determination of the viability of cells which contain RNA modified according to the invention in comparison to cells which contain unmodified or fully modified RNA, e.g. in comparison to RNA that is unmodified or modified in known manner.
  • the production of the encoded protein over time can also be monitored.
  • stability of an RNA is understood to mean that when it has been introduced into the cell, the RNA which can express the desired protein or is translatable into the protein or a functional fragment thereof, remains capable of expression over a prolonged period, is not immediately degraded and is not inactivated.
  • a method for testing the stability and the survival time of RNA in a cell thus consists in determining how long a protein encoded by the RNA is detectable in the cell or performs its function. Methods for this are described in the examples.
  • an mRNA with a sequence encoding a reporter molecule can be introduced into the cell, optionally together with an RNA encoding a desired protein and after predefined time periods the presence of reporter molecule and optionally protein are then determined.
  • Suitable reporter molecules are well known in the state of the art and those commonly used can also be used here.
  • RFP red fluorescing protein, is used as the reporter molecule.
  • the RNA according to the invention can be used for therapy so that in the cell into which the RNA is introduced a protein can be formed which is naturally not expressed to the desired extent or at all.
  • the RNA according to the invention can be used both when the protein is not formed owing to a deficiency of a gene and also in the cases when owing to a disease a protein is not formed or in cases where the introduction of the protein is advantageous for the body.
  • the RNA can also be used for supplementing a protein which is not expressed to an adequate extent.
  • the dose used in each case depends on the function which the RNA is to fulfill.
  • the duration of action of the RNA according to the invention can be deliberately adjusted. The duration of the treatment depends on the particular indication. If the RNA is used for the chronic therapy of a disease due to a deficient gene, the duration of action will be as long as possible, while with other indications it can be deliberately adjusted to a time window.
  • an IVT mRNA which encodes the surfactant protein B is used as the RNA.
  • this protein When this protein is deficient in mammals, it results in the development of the respiratory distress syndrome of the premature and newborn. In the newborn, this syndrome often leads to death owing to a lung disease.
  • the use of a multiply modified in vitro transcribed mRNA encoding SP-B wherein 5 to 50% of the uridine nucleosides and 5 to 50% of the cytidine nucleosides are modified results in the protein being formed and the disease being moderated or cured.
  • an IVT mRNA which encodes erythropoietin is used as the RNA.
  • Erythropoietin is a very important protein for the body which for example in kidney diseases is no longer available in adequate quantity and therefore must be supplied.
  • Recombinant erythropoietin which has been produced in microorganisms or animal cells and hence has a glycosylation not occurring naturally, is at present used for this. With the use of the recombinant EPO there were in rare cases severe side effects, for example erythrocyte aplasia.
  • the IVT mRNA provided according to the invention contains a ribonucleic acid which encodes erythropoietin, wherein 5 to 50% of the uridine nucleotides and 5 to 50% of the cytidine nucleotides are modified.
  • an EPO-encoding mRNA wherein 15 to 25% of the uridine nucleotides and 15 to 25% of the cytidine nucleotides are modified is provided. It has been found that this mRNA has markedly reduced immunogenicity compared to unmodified RNA. At the same time it displays a transfection efficiency of over 90% and a stability such that the hematocrit value is still elevated after 14 days.
  • the EPO produced by the RNA according to the invention in the body has the correct glycosylation, side effects are not to be expected.
  • the hematocrit value could be kept at the desired level for a prolonged period.
  • a non-immunogenic stable RNA which is usable in vivo in mammals and provides the necessary protein in a form which is very similar if not identical to the naturally present endogenous protein and in particular has the endogenous glycosylation.
  • the mRNA according to the invention can be used directly as such. However, there is also the possibility of further modifying the mRNA in order to introduce further beneficial properties. Firstly, the mRNA can be modified by attaching other coding or non-coding sequences to the coding strand. Secondly, it can also be modified by binding further molecules to functional groups provided in the modified nucleotides.
  • the mRNA according to the invention can be combined with targeting ligands which bind to surface receptors specific for the target cells, so that a receptor-mediated transfection of the target cell is possible.
  • vehicles which are suitable for the introduction of mRNA into cells, or else the mRNA itself can be modified with a ligand.
  • suitable vehicles for the introduction of mRNA into cells are cationic agents. These include cationic lipids, cationic polymers or also nanoparticles, nanocapsules, magnetic nanoparticles and nanoemulsions. Suitable vehicles are known to those skilled in the art and described in the specialist literature. Suitable ligands are also well known to those skilled in the art and described in the literature and available. As ligands for example transferrin, lactoferrin, clenbuterol, sugar, uronic acids, antibodies, aptamers, etc. can be used.
  • the mRNA itself can also be modified with a ligand.
  • mRNAs with modified nucleosides that bear a primary amino group or an azido group in the 2′ position of the ribose are preferred. Examples can be found in the table above. Such modifications are particularly preferred since they contribute to the biological activity. Via these modifications, the ligand can easily be incorporated by amide formation or “click” chemistry, e.g. by bioconjugate techniques.
  • an RNA sequence which can bind to proteins e.g. receptors, (aptamer) is introduced at the 5′ end of the mRNA.
  • the mRNA is modified by additional modification with inert polymers, e.g. polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Methods for this are well known to those skilled in the art, and processes such as are known for ligands can be used.
  • a binding site for polyethylene glycol, to which the PEG is bound after transcription can be provided in a small part of the modified nucleotides used for the mRNA according to the invention.
  • the polyethylene glycol serves for the extracellular stabilization of the mRNA, i.e. it protects the polyribonucleotide molecule until it has arrived in the cell. On entry into the cell, the PEG is cleaved off.
  • the bond between PEG and RNA is preferably designed such that the cleavage on entry into the cell is facilitated.
  • a functional group can be provided which is pH-dependently cleaved off.
  • Other molecules stabilizing the RNA can also be provided via appropriate active sites on the modified nucleotides. In this way, the mRNA can be protected by steric stabilization against enzymatic degradation and an interaction with components of biofluids prevented.
  • the mRNA thus modified can be designated as “stealth” mRNA.
  • RNA according to the invention is preferably protected by the methods described in EP 11 98 489. It has been found that firstly the RNA modified according to the invention can also advantageously be stabilized and protected by this method and secondly that the activity of RNA according to the invention thus treated is not or not significantly restricted. Hence in a preferred embodiment of the present invention, RNA modified according to the invention is treated in accordance with EP 11 98 489.
  • micro-RNA binding sites for micro-RNA 142-3p which is expressed in hematopoietic cells, but not in cells of other origin.
  • the expression is controlled such that the mRNA translation in hematopoietic cells is markedly diminished compared to other cells.
  • the expression in other cell types can be selectively controlled by incorporation of the relevant suitable micro-RNA binding sites, which are known to those skilled in the art.
  • the mRNA according to the invention is combined with a target or a binding site for at least one micro-RNA which is present only in healthy cells, but not the cells affected by the disease.
  • a target or a binding site for at least one micro-RNA which is present only in healthy cells, but not the cells affected by the disease.
  • the protein encoded by the mRNA is produced only in the cells which need the protein.
  • the selection of the suitable targets is made by routine methods which are well known to those skilled in the art. A common method which is performed at the DNA level is the cloning of a micro-RNA binding site into 3′UTR (Gu et al, Nat Struct Mol Biol. 2009 February; 16(2): 144-50, Brown et al, Nat Biotechnol. 2007 December; 25(12): 1457-67, Brown et al, Nat Med.
  • an RNA equipped with a binding site for micro-RNA is used when the RNA encodes a cytotoxin.
  • RNA according to the invention can be combined with micro-RNAs or shRNAs downstream of the 3′ polyA tail.
  • This has the advantage that the mRNA-micro-RNA/shRNA hybrid can be cleaved intracellularly by Dicer and thereby two active molecules which intervene in different pathogenic cascades can be released.
  • Such a hybrid can be provided for the treatment of diseases such as cancer or asthma.
  • the RNA according to the invention is suitable for simultaneously complementing a deficient mRNA and intervening in a defective micro-RNA cascade.
  • an RNA with advantageous properties is provided which can be tested with a screening method wherein a sequence coding for a reporter protein, e.g. red fluorescing protein (RFP), is used.
  • a sequence coding for a reporter protein e.g. red fluorescing protein (RFP)
  • RFP red fluorescing protein
  • modified multiply wherein at least 5% respectively of the uridine nucleosides and cytidine nucleosides are replaced by modified nucleosides leads to a markedly reduced immunogenicity towards human primary monocytes in the blood and at the same time can yield high transfection rates of more than 80%.
  • This can for example be tested in alveolar epithelial cells type II in humans or in the mouse.
  • the duration of the RNA expression for RNAs modified according to the invention is significantly longer than with known RNA. It has been found that mainly owing to the higher stability and lower immunogenicity of the mRNA multiply modified according to the invention the expression lasts longer than with known preparations. In a quantitative assessment, a derivative modified according to the invention showed a 10 times higher quantity of expression product 10 days after the transfection than non- or only singly modified RNA.
  • a further subject of the invention is a method for the screening of nucleotide sequences in order to test the immunogenicity and expression quality, wherein the mRNA sequence is contacted with at least one receptor selected from TLR3, TLR3, TLR8 and helicase RIG-1 and the binding capacity measured in comparison with a control sequence.
  • a control sequence a sequence is used the binding capacity whereof is known. The weaker the binding to at least one of these receptors is, the more promising is the sequence.
  • mRNA according to the invention can be tested with a screening method on an RNA expressing a reporter protein.
  • the red fluorescing protein (RFP) is preferred as the reporter protein.
  • Sequences encoding this protein which have nucleotides with different modifications can be tested for their immunogenicity and transfection efficiency.
  • uridine nucleosides can be partially replaced by 2-thiouridine nucleosides (also referred to below as s2U) and cytidine nucleosides can be partially replaced by 5-methylcytidine nucleosides (also referred to below as m5C).
  • FIGS. 1A , 1 B, 1 C, 2 A and 2 B show the results which are obtained on performing such a screening method. More detailed particulars are to be found in the examples.
  • the results shown in the figures are based on experiments which were performed for RFP RNA and show that only multiply modified mRNA wherein at least 5% of the uridine nucleosides and at least 5% of the cytidine nucleosides respectively are modified lead to markedly reduced immunogenicity towards human primary monocytes in the blood, both ex vivo and in vivo, and at the same time can yield high transfection rates of more than 80% both in alveolar epithelial cells type II in humans and also in the mouse.
  • the duration of the expression for mRNAs modified according to the invention is significantly longer than for unmodified mRNA.
  • a method for testing whether an RNA under consideration is suitable for therapy with the use of an mRNA immunoprecipitation test (RIP).
  • RIP mRNA immunoprecipitation test
  • RNA modified according to the invention for treatment in the lung
  • multiply modified mRNA which codes for a fusion protein of enhanced green fluorescent protein and luciferase (EGFPLuc) was introduced directly into the lung of a mouse and tested as to whether luciferase was expressed in comparison with unmodified EGFPLuc RNA.
  • the luciferase expression reached a maximum after three hours in the lung, although the total luminescent flux rapidly declined after 24 hours to very low proportions 5 days after the treatment. In contrast to this, high expression values were observed up to 5 days after the treatment in mice which had been treated with multiply modified EGFPLuc mRNA.
  • an RNA is provided whose therapeutic potential allows treatment of the disease attributable to SP-B deficiency, namely s2U (0.25) m5C (0.25) SP-B mRNA.
  • SP-B is a relatively small amphipathic peptide which is encoded by a single gene and through proteolytic processing creates a precursor with 381 amino acids in type II alveolar epithelial cells which coat the alveoli. It improves the distribution, adsorption and stability of the surfactant lipids which are necessary for the reduction of the surface tension in the alveoli.
  • symptoms such as thickened alveolar walls, cellular infiltration and interstitial edema occur.
  • This lung damage is accompanied by congestion, i.e. an increased number of erythrocytes and an increased number of macrophages, neutrophils and corresponding proportions of inflammatory cytokines in the broncho-alveolar fluid.
  • Congenital SP-B deficiency which arises through mutations in the SP-B gene, is critical for the replacement of the surfactant and leads to a fatal failure of the respiratory tract in the newborn during the first months of life.
  • a lung transplant is the only currently available therapeutic intervention.
  • an mRNA therapy for SP-B deficiency which is rendered possible with the RNA according to the invention, is an important alternative treatment.
  • RNA according to the invention can be used for the treatment of this disease, preferably with perfluorocarbon as vehicle.
  • a pharmaceutical preparation comprising perfluorocarbon and s2U (0.25) m5C (0.25) SP-B mRNA is provided.
  • This combination makes it possible to reconstitute SP-B in the lung of patients with SP-B deficiency, so that the chances of survival are increased.
  • administration at regular intervals e.g. 1 to 3 times weekly is sufficient for this.
  • the SP-B mRNA is administered for this intratracheally as an aerosol by spraying at high pressure. It has been found that the mRNA according to the invention can ameliorate the symptoms described above and thus improve the lung function, which can be demonstrated by testing of the lung parameters, as described in detail in the examples.
  • the mRNA according to the invention can be effectively used in therapeutic procedures and makes a treatment of diseases due to missing or defective proteins possible.
  • Systemic administration of the multiply modified mRNA is possible.
  • the mRNA translation in cells which are not affected by the gene defect is undesirable, e.g. because undesired side effects arise.
  • the corresponding vector can either be supplemented by sequences which enable addressing of the tissue affected, e.g. via ligands.
  • sequences to which endogenous micro-RNAs bind, which are not expressed in the target cell can be added to the vector which contains the mRNA, so that the mRNA are degraded in all cells which contain the relevant endogenous micro-RNAs, while they are retained in the target cells.
  • side effects can be minimized.
  • the RNA according to the invention can be administered in a manner known per se to patients who need the protein or protein fragment encoded by RNA, e.g. because they have a disease due to a deficient gene.
  • the RNA is formulated as a pharmaceutical preparation with normal pharmaceutically acceptable additives.
  • the form of the preparation depends on the location and the nature of administration. Since the RNA according to the invention is characterized by particularly high stability, it can be formulated in many ways, depending on where and in what form it is to be used. It has been found that the RNA according to the invention is so stable that it can be freeze-dried, processed in this form, e.g. crushed or milled, and stored, and can then be reconstituted when required and retains its biological activity.
  • RNA When the RNA is administered systemically, it is usually formulated as an injectable liquid with normal additives such as agents adjusting the tonicity and stabilizers, preferably as a unit dosage form.
  • stabilizers those normally known, such as for example lipids, polymers and nanosystems or liposomes, are used.
  • a composition suitable for parenteral administration which contains RNA modified according to the invention which encodes EPO.
  • the RNA according to the invention is provided in a form suitable for uptake via the lung, e.g. by inhalation.
  • Suitable formulae for this are known to those skilled in the art.
  • the preparation is in a form which can be introduced into the respiratory tract via normal nebulizers or inhalers, e.g. as a liquid for nebulizing or as a powder.
  • Devices for administration as liquid are known, and ultrasound nebulizers or nebulizers with a perforated oscillating membrane which operate with low shear forces compared to nozzle jet nebulizers are suitable.
  • powder aerosols are also suitable for mRNA complexed with cationic lipids and also bare mRNA is available after the freeze-drying with the sugar sucrose as powder that can then be crushed to a respirable size and moreover shows biological activity.
  • a pharmaceutical composition intended for pulmonary administration is combined with perfluorocarbon, which is administered previously or simultaneously with the pharmaceutical composition in order to increase the transfection efficiency.
  • RNA modified according to the invention is provided in a delayed release polymer as a carrier for the coating of implants.
  • RNA modified according to the invention can be used as such or else an RNA protected with a coating polymer and/or polymer complex.
  • a further subject of the invention are implants on the surface whereof there is a coating of a delayed release polymer which contains RNA which encodes beneficial factors for the ingrowth of the implant.
  • a coating of a delayed release polymer which contains RNA which encodes beneficial factors for the ingrowth of the implant.
  • both coatings which contain mRNA which encodes only one factor and also coatings which contain mRNAs which encode several factors, e.g. various growth factors or growth factors and angiogenesis factors or further factors promoting ingrowth, are possible here.
  • the various factors can also be provided in a form such that they are released at staggered intervals.
  • RNA which encodes one or more growth factors and one or more angiogenesis factors should be understood to mean both an RNA sequence which encodes more than one protein, singly or as a fusion protein, and also a mixture of different RNA sequences which encode different proteins, where each RNA sequence encodes one protein.
  • s2U 0.25) m5C (0.25) mRNA which encoded a fusion protein of enhanced green fluorescent protein and luciferase (EGFPLuc) which was introduced directly into the lungs of the mouse could intensify and prolong the luciferase expression in vivo in comparison to unmodified EGFPLuc mRNA.
  • a high pressure spray device for intratracheal administration known per se as described for example in (6) was used, perfluorocarbon (fluorinated FC-77) being administered beforehand in order to increase the transfection efficiency (7).
  • SP-B is a relatively small amphipathic peptide which is encoded by a single gene and in epithelial cells of the alveolar type II is converted by proteolytic processing into a precursor with 381 amino acids which coats the alveoli (8, 9). It improves the distribution, adsorption and stability of the surface-active lipids which are necessary for the reduction of the surface tension in the alveolus. If the gene for this protein is deficient, disorders in the respiratory tract occur after birth which can rapidly lead to death.
  • a knockout mouse model for SP-B deficiency was selected in order to test a gene therapy with multiply modified mRNA of SP-B according to the invention.
  • a mouse model was chosen wherein the mouse SP-B cDNA was expressed under the control of exogenous doxycycline in SP-B ⁇ / ⁇ knockout mice. Withdrawal of doxycycline in adult SP-B ⁇ / ⁇ mice resulted in a decreased content of SP-B in the lung, which resulted in respiratory failure when the SP-B concentration fell below 25% of the normal level. Conditioned transgenic mice which received doxycycline survived normally (13, 14).
  • the therapeutic strategy used comprised the following: (i) pre-treatment of the mice with perfluorocarbon before the introduction of SP-B mRNA, in order to increase expression and (ii) repeated use of SP-B mRNA twice weekly every third or fourth day for four weeks ( FIG. 3C ).
  • s2U 0.5) m5C
  • SP-B mRNA was administered intratracheally as an aerosol into conditional SP-B ⁇ / ⁇ mice using a high pressure nebulizer. This treatment saved the mice from respiratory failure and extended their average lifespan to 28.8 ⁇ 1.1 days ( FIG. 3D ), up to the defined endpoint of the study.
  • the pulmonary histology was normal in mice which had been treated for 4 weeks with s2U (0.25) m5C (0.25) SP-B mRNA, while the lungs of the mice which had received s2U (0.25) m5C (0.25) EGFPLuc control mRNA displayed thickened alveolar walls, cellular infiltration and interstitial edema after 4 days ( FIG. 3G ).
  • This lung damage was accompanied by congestion (elevated number of erythrocytes) and an elevated number of macrophages and neutrophils and an elevated level of inflammatory cytokines ( FIG. 3H and FIG. S 4 ) in the bronchoalveolar lavage fluid (HALF), while this was largely prevented in the mice treated with SP-B mRNA.
  • results show the therapeutic efficacy of the multiply modified mRNA in a mouse model for a lethal lung disease.
  • the further application of the mRNA therapy can still be improved as follows: (i) undesired mRNA translation in cells of unaffected tissue could lead to undesired effects outside the target region, (ii) if the multiply modified mRNA also reaches unaffected tissue, an adequate quantity of mRNA must be provided and (iii) repeated dosing is necessary for short-duration mRNA activity.
  • micro-RNA biology can be enlisted in order to prevent undesired mRNA translation in cells not affected by the disease.
  • mRNA degradation can be selectively caused in cells not affected by the disease, during which however the mRNA is retained in the target cells, as a result of which side effects are minimized (16, 17).
  • the targeting ligands which bind specific receptors to cell surfaces, can be combined, so that receptor-mediated transfection of the target cell is enabled. Since mRNA can be produced in large quantities nowadays (18) and efficient production processes for the production even of multiply modified mRNA on a large scale are possible, the clinical use of the mRNA according to the invention is possible and this makes it possible to develop mRNA systems specifically tailor-made for each disease (19, 20), whereby the dosing frequency and the short-duration activity can be kept to a minimum, which is not possible with the currently known therapies. In this way, according to the invention an effective molecular therapy for the treatment of disease due to a gene deficiency is provided.
  • mice Three groups of mice were set up.
  • One group of SP-B deficient mice received mRNA modified according to the invention twice in one week (B), a second group received mRNA modified according to the invention twice a week for 28 days (C), and for comparison a third group of mice received modified EGFP-Luc mRNA (A).
  • mice which received no SPB mRNA modified according to the invention died after a short time.
  • the mice which received the RNA according to the invention survived only as long as they were given the SP-B RNA according to the invention. This proves that the RNA according to the invention is biologically active and can replace necessary protein.
  • the experiment was performed as follows.
  • Kaplan-Meier survival curves were plotted and a Wilcoxon-Gehan test performed.
  • mice described in example 1 which all received doxycycline, were used, it was investigated whether the RNA according to the invention causes inflammatory reactions in an early phase after administration.
  • 5 groups were set up and cytokine levels, IFN ⁇ and IL-12 were measured in the bronchoalveolar lavage of mice 8 hours after administration of different preparations.
  • the six groups received the following preparations: a) control, untreated, i.e.
  • mRNA modified according to the invention was tested and in comparison to this an mRNA modified not according to the invention, in which two different modifications of uridine nucleotides were used and non-modified mRNA.
  • the mRNA molecules modified according to the invention were:
  • modified nucleotides each had a content of 10% and RNA molecules which in addition to 10%/10% s2U/m5C and s2U/5mC each contained a further 5% of modified nucleotides, namely once C 2 ′NH 2 and once 5% G′N 3 .
  • the results show that the mRNA modified according to the invention displays a very high transfection efficiency, while unmodified mRNA and mRNA modified not according to the invention each show far lower transfection and translation efficiency.
  • the immunogenicity was also tested for the modified mRNA previously described, by investigating the TNF- ⁇ level on human PBMCs after administration of 5 ⁇ g of each mRNA. The results are shown in FIG. 10B . As is clearly seen, the TNF- ⁇ level is markedly elevated on administration of unmodified mRNA or with mRNA wherein two types of modified uridine nucleotides were used. The TNF- ⁇ level is lower by at least 50% with the RNAs modified according to the invention than with unmodified RNA.
  • a plasmid, pCS2+DsRedT4, containing an SP6 promoter was used for the in vitro transcription of RFP cDNA (678 bp).
  • a plasmid, pCS2+DsRedT4, containing an SP6 promoter was used for the in vitro transcription of SP-B cDNA (1146 bp).
  • a pVAX1 plasmid (Invitrogen) containing a T7 promoter was used.
  • a pST1-2 ⁇ -globin-UTR-A-(120) construct containing a T7 promoter which was obtained as described in (19) was used. The constructs were cloned using standard techniques of molecular biology.
  • the pCS2+DsRed.T4, EGFPLuc and SP-B plasmids were linearized with XbaI.
  • the linearized vector DNAs were purified with the NucleoSpin Extract II kit (Macherey-Nagel) and assessed by spectrophotometry.
  • the in vitro transcription was performed with the mMESSAGE-mMACHINE SP6 or T7 Ultrakit (Ambion).
  • the SP-6 kit capped the mRNA with 7-methylGpppG, while the T7 kit created the analogous antireverse cap (ARCA; 7-methyl-(3′-O-methyl)GpppGm 7 G(5′)ppp(5′)G in a transcription reaction with ultrahigh yield.
  • the following modified ribonucleic acid triphosphates were added to the reaction system in the stated ratios: 2′-thiouridine 5′-triphosphate, 5′-methylcytidine 5′-triphosphate, pseudouridine 5′-triphosphate and N 6 -methyladenosine 5′-triphosphate (all from TriLink BioTechnologies and checked for purity with HPLC and 31 P NMR).
  • RNA from the pVAX1 SP-B plasmid was enzymatically polyadenylated using the poly(A) tail kit (Ambion).
  • the poly(A) tails were approximately 200 nt long. All capped mRNAs (RFP, EGFPLuc and SP-B) were purified using the MEGAclear kit (Ambion) and analyzed for size and purity with the Agilent RNA 6000 Nano Assay on a Bioanalysis Instrument 2100 (Agilent Technologies).
  • FCS fetal calf serum
  • FCS fetal calf serum
  • penicillin-streptomycin 1% penicillin-streptomycin
  • gentamycin 0.5% gentamycin.
  • 80 000 cells per well were plated out in 24-well plates.
  • the cells were transfected with 200 ng of mRNA with the use of Lipofectamin 2000 (Invitrogen) according to the manufacturer's instructions. After 4 hours, the cells were washed with PBS and serum-containing medium was added. For analyses of long-term expression, the cells were regularly subdivided (when the confluence was >90%).
  • a flow cytometry analysis was performed on the A549 and MLE12 cells which had been transfected with RFP mRNA, as described above.
  • the cells were removed from the plate surface with 0.25% trypsin/EDTA, washed three times with PBS and again suspended in PBS in order to measure the fluorescence using an FACSCalibur (BD Biosciences).
  • the transfection efficiency was calculated from the percentage of the cell population which exceeded the fluorescence intensity of the control cells, which had only been treated with PBS. At least 2500 cells per tube were counted. The data were analyzed with Cellquest Pro.
  • Enzyme-linked immunosorbent assays were performed using human IL-8 and TNF- ⁇ kits (RayBio), mouse IFN- ⁇ and IL-12 (P40/P70) kits (RayBio) and mouse IFN- ⁇ kit (RnD Systems).
  • C t values were obtained using the iCycler IQ software 3.1 (Bio-Rad) which automatically calculated the baseline cycles and threshold values.
  • the fixing solution and then the glycine stop-fix solution and ice-cold 1 ⁇ PBS were added to the cells and the cells were pelletized at 4° C. Then the cells were again suspended in lysis buffer to which the protease inhibitors PIC and PMSF had been added, and incubated for 30 mins on ice. After 10 minute centrifugation at 2400 rpm at 4° C., the supernatant was subjected to the capture reaction. The TLR-mRNA/RIG-mRNA complexes were captured overnight on magnetic beads in 8-well PCR strips, as described in the ChIP-IT Express manual.
  • D-luciferin substrate was dissolved in water, the pH adjusted to 7 and the final volume adjusted such that a concentration of 30 mg/ml was reached. 50 ⁇ l of this solution were applied onto the nostrils of the anesthetized mice and absorbed by snuffling (1.5 mg luciferin/mouse). After 10 mins, the bioluminescence was measured with an IVIS100 imaging system (Xenogen) as described in (21) using the following camera settings: visual field 10, f1 f-stop, high resolution and illumination times from 1 to 10 mins. The signal in the pulmonary region was quantitatively assessed and analyzed, the background being subtracted using the Living Image Software Version 2.50 (Xenogen).
  • IVIS100 imaging system Xenogen
  • mice 6 to 8 week old female BALB/C mice (Charles River Laboratories) were kept under specific pathogen-free conditions and kept in individually ventilated cages with a 12-hour light: 12-hour dark cycle and supplied with food and water ad libitum. The animals were acclimatized for at least 7 days before the start of the experiments. All animal manipulations were approved and were checked by the local ethical committee and performed according to the guidelines of the German Animal Protection Law. For all experiments except for the injection into the caudal vein, the animals were anesthetized i.p. with a mixture of medetomidine (0.5 mg/kg), midazolam (5 mg/kg) and fentanyl (50 ⁇ g/kg).
  • medetomidine 0.5 mg/kg
  • midazolam 5 mg/kg
  • fentanyl 50 ⁇ g/kg
  • mice BALB/c and SP-B ⁇ / ⁇ mice were anesthetized as described in (14) and immobilized on a plate system (Halowell EMC) such that the upper teeth were at an angle of 45°.
  • a modified cold light otoscope Beta 200 (Heine Optotechnik) was used in order to optimally illuminate the pharynx.
  • the lower jaw of the mouse was opened with a small spatula and blunt forceps were used to push the tongue aside and maximally expose the oropharynx.
  • Homozygotic SP-B ⁇ / ⁇ mice ⁇ doxycycline ⁇ modified mRNA were anesthetized as described above.
  • vecuronium bromide 0.1 mg/kg was injected intraperitoneally.
  • the pulmonary mechanical measurements were performed as described in (22).
  • a blunt steel cannula (external diameter 1 mm) was inserted in the trachea with tracheostomy.
  • the piston pump respirator served both as respirator and also as a measurement device (flexiVent, SAV).
  • Vt used was 8.4 ⁇ 1.4 ⁇ l/g in animals which were receiving doxycycline and 8.9 ⁇ 0.4 ⁇ l/g BW in animals which were receiving doxycycline and mRNA (N.S.).
  • the dynamic-mechanical properties of the respiratory system and also the pulmonary entry impedance were measured at 5 minute intervals in animals after insufflation twice at 15 ⁇ l/g for 1 sec in order to create a standard volume history. For the oscillatory measurement, the ventilation was stopped at the PEEP level.
  • Zrs In order to determine the impedance of the respiratory system (Z rs ) by forced oscillations (FOT), which consisted of a pseudorandom oscillatory signal of 8 secs, an amplitude of 3 ml/g was used. The forced signal had frequencies between 1.75 and 19.6 Hz (23, 24). The data were collected at 256 Hz and analyzed with a window of 4 secs with 66% overlap. The pulmonary impedance data were represented as resistance (real part) and reactivity (imaginary part) of the respiratory system within the frequency domain.
  • the pulmonary impedance data (Zrs) were subdivided using the constant phase model of the lung, as proposed by Hantos et al. (25). In this model, Zrs consists of a respiratory resistance (Rn), a respiratory tract inertia (inertia), a tissue elasticity (H L ) and a tissue damping (G L ) according to the equation:
  • the fit quality is represented as the coherence of the determination (COD), and the data are rejected if the COD is below 0.85.
  • the total protein content of the lavage supernatants was determined with the Bio-Rad protein assay kit (Bio-Rad). 10 ⁇ g of total protein were separated under non-reducing conditions on NuPage 10% bis-tris gels using a NOVEX Xcell II mini-cell system (Novex). After the electrophoresis, the proteins were transferred onto a PVDF membrane (ImmobilonP) with a NuPage blot module (Novex). Surfactant protein B (SP-B) was detected with polyclonal rabbit antiserum which was directed against SP-B (c329, gift from Dr W.
  • Sections fixed (3% paraformaldehyde) and embedded in paraffin wax were subjected to immunohistochemistry as recommended by the manufacturer (Abcam, www.abcam.com/technica).
  • the slides were incubated with anti-human anti-mouse SP-B antibody and with Texas red-conjugated anti-rabbit IgG antibody (both from Abcam, 1:500) and counterstained with DAPI.
  • Fluorescent images were obtained by Zeiss Axiovert 135.
  • modified mRNA was produced which contained an EPO-encoding part.
  • the expression efficiency of this mRNA was tested.
  • 5 ⁇ g of mRNA modified according to the invention or of non-modified mRNA were injected i.m. into mice. Each group of mice had four members.
  • the content of EPO in the serum was assessed quantitatively with an ELISA test.
  • the hematocrit value was assessed in whole blood from mice in the same experiment.
  • the data shown in the appended FIG. 11 each represent the mean value ⁇ SEM.
  • the scatter blot shows the individual hematocrit values. Bars show median values. *P ⁇ 0.05 compared to the untreated group at each time point; +P ⁇ 0.05 compared to the unmodified mEPO group at each time point.
  • the inflammatory markers are in the non-pathological range, while for unmodified RNA or modified RNA only with modified uridine nucleotides the inflammatory markers are markedly elevated.
  • an mRNA which encodes EPO which is very stable and at the same time causes few or no immunological reactions.
  • Such an mRNA can advantageously be used for the treatment of erythropoietin deficiency. Because of the high stability, administration is only necessary every 2 to 4 weeks.
  • the hematocrit value was then determined in the whole blood from the mice on days 0, 21, 34, 42 and 51.
  • the results are shown in FIG. 14 .
  • the data in the diagram show the mean ⁇ standard error. *P ⁇ 0.05 compared to the hematocrit value on day 0.
  • mRNA modified according to the invention is also suitable for bringing proteins promoting healing or ingrowth into the vicinity of implants in order thus to promote the healing process or the ingrowth.
  • a coating which contained mRNA which encoded luciferase was applied onto titanium plates. It was then investigated whether and for how long luciferase could be detected in the vicinity, free or in cells.
  • RNA for luciferase which is secreted from the cell expressing it as a model for proteins which are to be released into the vicinity, such as for example growth factors or angiogenesis factors.
  • RNA which encodes a luciferase which is not secreted but remains in the cell was used as a model for proteins which are to have some kind of effect in the cell.
  • RNA which encoded Metridia luciferase was used, wherein compared to the wild type 25% of the uridine units were replaced by s2U and 25% of the cytidine units were replaced by m5C.
  • a firefly luciferase-encoding mRNA was used wherein likewise 25% of the uridine units were replaced by s2U and 25% of the cytidine units were replaced by the modified m5C.
  • the mRNA modified according to the invention protected by a polymer complex, was embedded in a carrier material which was applied as a layer onto titanium plates.
  • the carrier material was polylactide (PDLLA), a well-known material for this purpose, which can selectively release the contained mRNA gradually.
  • PLLA polylactide
  • An advantage of such a coating is that the release can be specifically adjusted. The results show that the polylactide fragments released on degradation do not impair the activity of the mRNA, so that this system is very suitable.
  • the mRNA itself is stabilized by a coating polymer.
  • Metridia luciferase-encoding plasmid DNA pDNA
  • modified mRNA 9 ⁇ g respectively of Metridia luciferase pDNA or doubly modified s2U (0.25) m5C (0.25) mRNA in 200 ⁇ l of H 2 O (+if necessary 500 ⁇ g of lactose) were complexed with 9.4 ⁇ g of L-PEI (L-polyethyleneimine) in 200 ⁇ l of H 2 O.
  • L-PEI L-polyethyleneimine
  • a coating polymer solution 2.4 ⁇ l of 409.1 mM P6YE5C
  • the complexes were suspended in 72 ⁇ l of a PDLLA (poly-DL lactide)/EtOAc (50 mg/ml PDLLA) mixture on ice and dispersed by means of a micropotter. Autoclaved titanium plates
  • A549 cells in 200 ⁇ l of RPMI-1640 medium were added (5000 cells/200 ⁇ l). From the second day, 50 ⁇ l of the supernatant were taken in each case, the medium changed and the Metridia luciferase expression determined on the following days by means of 100 ⁇ l of coelenterazine solution (0.003 mM final concentration) for each.
  • the activity of the Metridia luciferase-encoding mRNA modified according to the invention was tested when this had been deposited onto calcium phosphate particles and introduced into the coating in this form.
  • 4 ⁇ g of Metridia luciferase s2U (0.25) m5C (0.25) mRNA in 600 ⁇ l of 1 ⁇ HES were mixed each time with 33 ⁇ l of 2.5M CaCl 2 .
  • A549 cells in 200 ⁇ l of RPMI-1640 medium were added (5000 cells/200 ⁇ l).
  • results can be seen in the diagram in FIG. 15 .
  • the results show clearly that mRNA modified according to the invention stays active even when it is protected with a polymer coating, introduced into a delayed release matrix and applied onto titanium implants.
  • the mRNA modified according to the invention remains biologically active and is continuously translated into the encoded protein.
  • the secretion capacity is also retained, which is seen from the fact that the Meridia luciferase can be detected in the cell culture medium (as a model for secreted bone growth factors such as for example BMP-2).
  • the results surprisingly show that the coating with modified mRNA yields higher protein expression than the coating of titanium implants with the analogous plasmid DNA.
  • modified mRNA precipitated onto calcium phosphate particles retains its activity and can exercise its advantageous properties in the titanium implant coating. The biological activity is retained. This is of particular importance since calcium phosphate can be directly incorporated into the bone.
  • firefly luciferase-encoding DNA or RNA As indicated above, a further experiment was performed with firefly luciferase-encoding DNA or RNA. For this, 9 ⁇ g of firefly luciferase pDNA or modified s2U (0.25) m5C (0.25) mRNA respectively in 200 ⁇ l of H 2 O were complexed with 9.4 ⁇ g of L-PEI in 200 ⁇ l of H 2 O. After this, the complexes were introduced into 100 ⁇ l of a coating polymer solution (2.4 ⁇ l of 409.1 mM P6YE5C) and lyophilized overnight.
  • a coating polymer solution 2.4 ⁇ l of 409.1 mM P6YE5C
  • PLLA poly-DL-lactic acid
  • EtOAc ethyl acetate
  • titanium implants can be coated with mRNA modified according to the invention during which the mRNA also further remains biologically active and translates the encoded protein.
  • the protein formed remains in the cell and can be detected intracellularly.
  • the results show that the coating with modified mRNA leads to higher protein expression than the coating of titanium implants with the analogous plasmid DNA.
  • a micro-RNA binding site was incorporated into the mRNA in order to enable cell-specific regulation of the mRNA expression.
  • HEK293 cells were cultured in MEM with 10% FCS and 1% penicillin-streptomycin. 24 hrs before the transfection, 100 000 cells/well, were sown into a 24-well plate. Directly before the transfection, the medium was replaced by 400 ⁇ l of Optimem (Invitrogen). U937 cells were cultured in RPMI-1640 medium with 10% FCS and 1% penicillin-streptomycin. Directly before the transfection 800 000 U937 cells in 400 ⁇ l of Optimem medium (Invitrogen) per well were sown into a 24-well plate.
  • Optimem Invitrogen
  • EGFP mRNA and 250 ng of RFP miRNA-BS mRNA were diluted to 50 ⁇ l with Optimem.
  • 2 ⁇ l of Lipofectamine 2000 were made up to 50 ⁇ l with Optimem and incubated for 5 mins at room temperature.
  • the mRNA solution was pipetted into the Lipofectamine 2000 solution and incubated for a further 20 mins at room temperature.
  • the resulting solution was pipetted into the wells with the cells and after 4 hrs penicillin-streptomycin (5 ⁇ l) was added and the incubation continued overnight in the incubator.
  • the HEK293 cells were washed with PBS and detached from the floor of the wells by addition of trypsin before being centrifuged for 5 mins at 300 G.
  • the U937 cells were also centrifuged for 5 mins at 300 G.
  • the supernatant was removed and the respective cells then washed twice with PBS.
  • the cells were resuspended in 500 ⁇ l of PBS for the FACS analysis.
  • the ratio of the expression of EGFP to the expression of RFP is shown as the number of positive cells ( FIG. 17 a ) and as the mean RFP fluorescence intensity ( FIG. 17 b ).
  • the results show that by the incorporation of a micro-RNA binding site into in vitro transcribed mRNA the expression can be cell-specifically regulated.
  • the untranslated sequence of a fourfold repetition of a micro-RNA binding site which are separated from one another by short spacing sequences, is situated 3′ from the RFP sequence and 5′ from the polyA tail (SEQ ID No. 1).
  • a micro-RNA binding site which binds to the micro-RNA 142-3p was used.
  • This micro-RNA is expressed in hematopoietic cells such as U937 cells, but not in cells of other origin, such as HEK-293 cells.
  • micro-RNA 142-3p binds to the RFP miRNA-BS mRNA, e.g. in the U937 cells, the degradation of the mRNA is initiated by RNA interference. As a result the formation of RFP is decreased, i.e. fewer cells express RFP at lower intensity than in cells in which micro-RNA 142-3p is not present.
  • U937 and HEK-293 cells were each co-transfected with EGFP mRNA (without micro-RNA binding site) and RFP miRNA-BS mRNA (with fourfold tandem repetition of the micro-RNA binding site for the micro-RNA 142-3p) and the expression of EGFP and RFP then measured by FACS. Since the RFP miRNA-BS mRNA is degraded because of RNA interference more rapidly in U937 cells than in HEK-293 cells, while the EGFP mRNA is equally stable in both cells, it is expected that the ratio of EGFP to RFP will be higher in HEK-293 cells than in U937 cells. This could be confirmed in the experiments performed.
  • the diagram shows clearly that the number of RFP-positive U937 cells after normalization to the number of EGFP-positive cells is markedly lower than in HEK-293 cells. The same applies for the quantity of RFP formed per cell.
  • the results thus also show clearly that the scale of the biological activity of in vitro transcribed mRNA can be controlled after transfection in cells by the incorporation of micro-RNA binding sites. The mRNA translation can thus be suppressed in cells in which the mRNA translation is undesired. Side effects can also be reduced thereby.
  • the mRNA used for the experiments in this example has the following sequence (SEQ ID No. 1).
  • the RFP sequence is shown with a gray background.
  • the underlined sequence shows the fourfold tandem repetition of the micro-RNA binding site for the micro-RNA 142-3p with spacing sequences. After synthesis, the sequence was cloned into the vector pVAX1 using BamHI-EcoRv.

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