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WO2010088927A1 - Utilisation de pei pour l'amélioration de la libération endosomale et de l'expression d'acides nucléiques transfectés, complexés par des composés cationiques ou polycationiques - Google Patents

Utilisation de pei pour l'amélioration de la libération endosomale et de l'expression d'acides nucléiques transfectés, complexés par des composés cationiques ou polycationiques Download PDF

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WO2010088927A1
WO2010088927A1 PCT/EP2009/000886 EP2009000886W WO2010088927A1 WO 2010088927 A1 WO2010088927 A1 WO 2010088927A1 EP 2009000886 W EP2009000886 W EP 2009000886W WO 2010088927 A1 WO2010088927 A1 WO 2010088927A1
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nucleic acid
precomplexed
complexed
molecule
cationic
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Patrick Baumhof
Thomas Schlake
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Curevac SE
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Curevac AG
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • A61K48/0008—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric

Definitions

  • PEI for the improvement of endosomal release and expression of transfected nucleic acids, complexed with cationic or polycationic compounds
  • the present invention is directed to complexed precomplexed nucleic acids, preferably mRNAs, which have been precomplexed in a first step with PEI in an N/P ratio between 0.05 and 2, preferably in an N/P ratio between 0.1 and 1 , and which have been further complexed in a second step with a cationic compound.
  • the present invention is furthermore directed to the use of such complexed precomplexed nucleic acids for the improvement of the endosomal release of nucleic acids and optionally the improvement of expression of an encoded protein or peptide.
  • compositions comprising such complexed precomplexed nucleic acids and to the use of such complexed precomplexed nucleic acids or compositions thereof for gene therapy and/or the treatment of various diseases as mentioned herein, e.g. by vaccination.
  • present invention is also directed to methods for preparing and administering these complexed precomplexed nucleic acids or compositions thereof and to kits, comprising these complexed precomplexed nucleic acids or compositions thereof.
  • Transfection of nucleic acids into cells or tissue is, however, not simple and typically dependent on many factors.
  • successful delivery e.g., delivery of nucleic acids or genes into cells or tissue
  • many barriers must be overcome.
  • the vector must be able 1 ) to tightly compact and to protect the nucleic acid, e.g., to stabilize the nucleic acid and prevent it from early degradation, 2) to target specific cell- surface receptors, 3) to disrupt the endosomal membrane, and 4) to deliver the cargo, e.g. the nucleic acid or gene, into the nucleus or the cytoplasm.
  • the vector must be able 1 ) to tightly compact and to protect the nucleic acid, e.g., to stabilize the nucleic acid and prevent it from early degradation, 2) to target specific cell- surface receptors, 3) to disrupt the endosomal membrane, and 4) to deliver the cargo, e.g. the nucleic acid or gene, into the nucleus or the cytoplasm.
  • transfection e.g. of nucleic acids
  • viral or non-viral vectors For successful delivery, these viral or non-viral vectors must be able to overcome the above mentioned barriers.
  • the most successful gene therapy strategies available today rely on the use of viral vectors, such as adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses.
  • viral vectors such as adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses.
  • viruses related to immunogenicity, cytotoxicity, and insertional mutagenesis A solution to this problem may be found in the use of non-viral vectors.
  • non-viral vectors are not as efficient as viral vectors, many non-viral vectors have been developed to provide a safer alternative in gene therapy.
  • non-viral vectors include (positively charged) polymers for transfection, calcium phosphate, lipids, proteins or peptides, dendrimers, or other polymers.
  • Particularly cationic compounds such as positively charged cationic polymers, cationic lipids, etc., glycerol based cationic lipids, allow an efficient transfection of nucleic acids, due to their superior condensing properties.
  • cationic polymers may include, e.g., DEAE-dextran, poly-L-lysine, modified polyaminoacids, such as ⁇ -aminoacid-polymers, or reversed polyamides, modified polyethylenes, such as poly(N-ethyl-4-vinylpyridinium bromide) (PVP), modified acrylates, such as poly(dimethylaminoethyl methylacrylate) (pDMAEMA), modified amidoamines, such as poly(amidoamine) (pAMAM), modified polybetaaminoesters (PBAE), such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-i -pentanol polymers, dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, polyimine, such as poly(ethyleneimine) (PEI), or poly(propyleneimine), polyallyl
  • Cationic polymers may furthermore include cationic lipids, e.g., glycerol-based cationic lipids, non-glycerol-based cationic lipids, or cholesterol-based cationic lipids, etc.
  • cationic lipids e.g., glycerol-based cationic lipids, non-glycerol-based cationic lipids, or cholesterol-based cationic lipids, etc.
  • Cationic lipids may include, e.g., DOPC, DODAP, Dioleyl phosphatidylethanol-amine (DOPE), Dioleoxypropyltrimethylammonium chloride (DOTMA), DC-Choi, DOSPA, DODAB, DOIC, DMEPC, Dioctadecylamidoglicylspermin (DOGS), Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide (DIMRI), DOTAP: dioleoyloxy-3- (trimethylammonio)propane, O,O-ditetradecanoyl-N-( ⁇ -trimethylammonioacetyl) diethanolamine chloride (DC-6-14), rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]- dimethylammonium chloride (CLIPI ), rac-[2(2,3-dihexadecyloxypropyl-
  • Glycerol-based cationic lipids may include, e.g., DOTMA: [1 -(2,3- sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DOTAP, DMRIE, SOSPA, non- glycerol based cationic lipids may include, e.g., DOGS, di-C14-amidine, DOTIM, SAINT, and cholesterol-based cationic lipids may include, e.g., DC-Choi, BGTC, and CTAP, etc.
  • peptide-based vectors are particularly advantageous over other non-viral strategies in that they are more or less capable to achieve all of the above mentioned four goals.
  • cationic peptides rich in basic residues such as lysine and/or arginine are able to efficiently condense nucleic acids into small, compact particles that can be stabilized in serum.
  • a peptide ligand may be attached to the polyplex structure, which allows targeting of the complex to specific receptors and/or specific cell types.
  • Peptide sequences derived from protein transduction domains (PTDs) are in general capable to selectively lyse the endosomal membrane in its acidic environment leading to cytoplasmic release of the polyplex.
  • short peptide sequences taken from longer viral proteins can provide nuclear localization of condensates once they are in the cytoplasm (see Martin and Rice, The AAPS Journal 2007; 9 (1 ) Article 3).
  • CPPs cell penetrating peptides
  • CPPs are typically derived from viral, insect or mammalia proteins endowed with membrane translocation properties and include, e.g. penetratin; peptides derived from the family of HIV-1 TAT proteins or peptides; pVEC; VP22 (HSV VP22 (Herpes simplex)); MAP; KALA; PpTG20; Proline-rich peptides; chimeric peptides, including transportan or proteins or peptides from the MPG familiy, e.g.
  • MPG- peptides such as P ⁇ or Pa
  • peptides of the penetratin family such as Antennapedia (Drosophila antennapedia) derived peptides, e.g. pAntp or plsl
  • Pep-1 peptides of the penetratin family
  • L-oligomers peptides of the penetratin family
  • arginine-rich peptides e.g.
  • oligoarginines typically represent short peptide sequences of 10 to about 30 amino acids which can cross the plasma membrane of mammalian cells and may thus offer unprecedented opportunities for cellular drug delivery.
  • a complex is formed between the nucleic acid or gene to be transfected and the cationic compound.
  • the complex is usually a very strong binding complex formed between the positively charged cationic compound and the negatively charged nucleic acid or gene.
  • a tight binding is advantageous since it allows a good condensation of the nucleic acid or gene prior to transfection, a compact and strong binding does not necessarily allow a good endosomal escape of the complexed nucleic acid or gene and, in some cases, may even lead to a poor or diminished release of the nucleic acid or gene in the cell and thus to a poor expression.
  • the DNA in these lipoplexes is well protected from nuclease degradation. Lipoplexes are furthermore able to trigger cellular uptake and facilitate the release of DNA from the intracellular vesicles before reaching destructive lysosomal compartments.
  • cationic polymer-mediated gene transfer involves cationic polymers such as polyethylenimine (PEI), polyamdidoamine and polypropylamine dendrimers, polyallylamine, cationic dextran, chitosan, cationic proteins (polylysine, protamine, histones) and cationic peptides.
  • PEI is regarded as the most active and most studied polymer for gene delivery. According to Gao et a/. (2007, supra), DNA-to-PEI ratios, the molecular weight and configuration of PEI, as well as the concentration of DNA and polymer and the ionic strength of the solvent for preparation are important factors for PEI-mediated transfection that determine the physical properties of the DNA/PEI complexes (polyplexes) and their transfection activity.
  • poly(ethylene imine) PEI takes a prominent position, due to its potential for endosomal escape.
  • PEI has a high cationic charge density due to its nitrogen atoms which are capable of protonation.
  • PEI becomes protonated and thereby can buffer endosomal pH and thereby induces the rupture of the endosomal membrane by osmotic swelling (Sonawane et a/. 2003, J. Biol. Chem. 2003, Nov 7, 278 (45): 44826-31 ). This hypothesis is called proton sponge.
  • PEI is available in a broad range of molecular weights, from ⁇ 1 ,000 Da to 1 ,600 kDa.
  • an N/P ratio of about 2-3 is typically used in the art since approximately 90% of its charged groups must be neutralized to condense DNA (see Neu et a/., 2005, supra). Such an N/P ratio is therefore believed to be necessary to achieve stable complexes using branched or linear PEI.
  • an N/P ratio is a specific determinant indicating the ratio of nucleic acid to peptide contained in such a complex on the basis of the nitrogen/phosphate ratio of all atoms involved.
  • PEI transfection reagent
  • N/P ratio of 5 induces apoptosis in Hek293 cells
  • toxicity and transfection activity of PEI is dependent on its molecular weight.
  • the most active PEI from a commercial source is 25 kDa for BPEI (branched PEI) and 22 kDa for LPEI (linear PEI).
  • PEI with a molecular weight larger than 25 kDa is also active but exhibits greater toxicity. Such increased cytotoxicity is presumably due to aggregation of huge clusters of the cationic polymer PEI on the outer cell membrane, which thereby induces necrosis.
  • BPEI of 5 to 10 kDa appears to be more active in transfection and less toxic when compared with a standard "benchmark" of 25 kDa BPEI.
  • PEI of 2 kDa or smaller is relatively nontoxic but not active in transfection. Treatment of low-molecular weight PEI with several bifunctional cross-linking reagents generates PEI oligomers that are transfectionally active.
  • PEI may be also combined with cationic polymers.
  • Hattori eta/. (Hattori eta/. Biol. Pharm. Bull 30(9) 1773-1 778 (2007)) disclosed that both PEI but also cationic nanoparticles have been widely used for non-viral DNA transfection.
  • pDNA plasmid DNA
  • low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex with NP-OH and release pDNA from the endosome to cytoplasm by an effect termed proton sponge effect. Accordingly, a combination of cationic cholesterol-based nanoparticles and low-molecular PEI was assumed to have potential as a non-viral DNA vector for gene delivery.
  • the PEI used in these experiments was a very low molecular PEI having a molecular weight of about 600 and 1800 Da.
  • the N/P ratios (nitrogen/phosphate ratio) used in these experiments were at least 1 , typically at least 1 to about 3.
  • PEI provided a better control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA. PEI furthermore enhanced gene transfection efficiency of the microspheres up to 50-fold without exhibiting a significant cytotoxicity. Physically blending PEI with POE was therefore assumed to be a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Therefore PEI was used in the appreciate amount known in the art (N/P of 7.7) to increase the gene transfer of a plasmid DNA containing uncharged degradable POE microsphere.
  • cationic compounds known in the art particularly cationic polymers and peptides, which are suitable to efficiently condense the DNA or gene to be transfected and to allow high transfection rates.
  • Addition of PEI or use of PEI to this formulation furthermore allows to a certain extent a modification and in part improvement of gene transfection efficiency and DNA release kinetics.
  • the object underlying the present invention is therefore to provide a composition, particularly a nucleic acid composition, and tools or methods, which both lead to an efficient gene transfection and an efficient DNA release kinetics, and additionally to a good expression of the encoded protein in the cell, preferably in vivo and in vitro at low toxicities.
  • a first aspect of the present invention solves the above object by at least one "complexed precomplexed nucleic acid", preferably at least one complexed precomplexed mRNA, wherein a nucleic acid (molecule) has been precomplexed in a first step with PEI in an N/P ratio between 0.05 and 2, and the precomplexed nucleic acid (molecule) has been further complexed in a second step with a cationic or polycationic compound.
  • the term "complexed precomplexed nucleic acid” refers to a nucleic acid (molecule), preferably to an mRNA, which is precomplexed according to a first step of the present invention with PEI in the above mentioned ratio and then complexed according to a second step of the present invention with a cationic or polycationic compound, preferably in a ratio as defined herein.
  • the "complexed precomplexed nucleic acid” according to the present invention is particularly suitable for both the improvement of the endosomal release of nucleic acids and improvement of expression of the encoded proteins.
  • PEI has a high cationic charge density due to its nitrogen atoms which are capable of protonation. In the endosome PEI becomes protonated and thereby can buffer endosomal pH. Thereby, PEI induces the rupture of the endosomal membrane by osmotic swelling (Sonawane et a/. 2003, supra).
  • PEI alone or mixed or blended with a cationic polymer is not suitable to improve expression of a nucleic acid encoded protein.
  • both the endosomal escape of nucleic acids and the expression of nucleic acid encoded proteins is significantly improved by the inventive stepwise treatment of the nucleic acid, i.e. precomplexing the nucleic acid in a defined ratio with (a high-molecular) PEI, in which PEI is not able to act as an transfection agent, and complexation of the already precomplexed nucleic acid with a cationic or polycationic compound in a second step. Only this "complexed precomplexed nucleic acid”, leads to both an efficient endosomal escape and an efficient release of the nucleic acid from the cationic or polycationic compound within the cell.
  • the nucleic acid (molecule) used according is precomplexed with PEI as defined above, preferably in an N/P ratio between 0.05 and 2. More preferably, the nucleic acid (molecule) used according is precomplexed with PEI as defined above in an N/P ratio between 0.05 and 1 , even more preferably in an N/P ratio between 0.1 and 1.
  • an N/P ratio is a specific determinant indicating the ratio of a nucleic acid to a peptide or a further polymer on the basis of the nitrogen/phosphate ratio of all atoms involved.
  • the N/P ratio is determined as the ratio between the nucleic acid and the cationic polymer PEI used for precomplexation on the basis of the nitrogen/phosphate ratio of all atoms involved.
  • the nucleic acid (molecule) is precomplexed with PEI.
  • PEI is polyethyleneimine, a polymer consisting of monomeric ethyleneimine units, wherein the polymer may be linear or branched.
  • PEI is available in a broad molar range of molecular weights, from ⁇ 1 ,000 Da to 1 ,600 kDa.
  • a PEI suitable for the inventive purpose is preferably selected from a linear polyethyleneimine (LPEI) or a branched polyethleneimine (BPEI).
  • PEI suitable for the inventive purpose is preferably selected from a high-molecular weight polyethyleneimine (high- molecular PEI).
  • a high-molecular PEI is preferably selected from a high-molecular linear polyethyleneimine (LPEI) or a high-molecular branched polyethleneimine (BPEI), having an average molecular weight of about 1 to about 1 ,600 kDa, more preferably selected from a high-molecular LPEI or BPEI having an average molecular weight of about 5 to about 1 .500 kDa, even more preferably selected from a high-molecular LPEI or BPEI having an average molecular weight of about 10 to about 1.000 kDa, e.g.
  • a high-molecular LPEI or BPEI having an average molecular weight of about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or about 1 .000 kDa, or may be selected from a high-molecular LPEI or BPEI of a molecular range formed by two of any of the values as mentioned above.
  • the high- molecular PEI used according to the present invention is used in an extremely small amount, which does not induce necrosis or exhibits any toxicity in the transfected cells. Additionally, a high-molecular PEI exhibits a better transfection efficiency than a low- molecular PEI.
  • the at least one precomplexed nucleic acid (molecule), prepared according to a first step as described above is further complexed with a cationic or polycationic compound, preferably a cationic peptide.
  • the at least one precomplexed nucleic acid (molecule), prepared according to a first step as described above, is further complexed with the cationic or polycationic compound, preferably a cationic peptide, in an N/P ratio of about about 0.1 -50, preferably in a range of about 0.5-50, even more preferably in the range of about 10-50 and most preferably in the range of about 25-50 of RNAxationic or polycationic polymer, alternatively in a range of about 0.75-25 or 1 -25.
  • the cationic or polycationic compound preferably a cationic peptide
  • a cationic or polycationic compound as used in the second step of the preparation of the at least one "complexed precomplexed nucleic acid” may be selected from any cationic or polycationic compound.
  • a cationic or polycationic compound is preferably selected from any cationic or polycationic compound, suitable for complexing and thereby stabilizing the above defined at least one precomplexed nucleic acid, particularly an mRNA, e.g. by associating the at least one precomplexed nucleic acid with the cationic or polycationic compound.
  • cationic or polycationic compounds are selected from cationic or polycationic peptides or proteins, including protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophil), PLL
  • Particularly preferred peptides of this formula are oligoarginines e.g. Arg 7 , Arg ⁇ , Arg 9/ Arg 7 , H 3 R 9 , R 9 H 3 , H 3 R 9 H 3 , YSSR 9 SSY, (RKH) 4 , Y(RKH) 2 R, etc.
  • Further preferred cationic or polycationic compounds, which can be used for complexing the at least one precomplexed nucleic acid (molecule) as defined above may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, cationic lipids, e.g.
  • modified polyaminoacids such as ⁇ -aminoacid- polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl- 4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified amidoamines such as pAMAM (poly(amidoamine)), etc.
  • modified polybetaaminoester (PBAE) such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 -pentanol polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc., poly(propyleneimine), polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based
  • association or complexing the at least one precomplexed nucleic acid with a cationic or polycationic compound preferably confers a stabilizing effect to the at least one precomplexed nucleic acid additional to enhanced transfection properties of the resulting complexed precomplexed nucleic acid.
  • cationic or polycationic compounds are compounds selected from the group consisting of protamine, nucleoline, spermine, spermidine, oligoarginines as defined above, such as Arg 7 , Arg ⁇ , Arg 9 , Arg 7 , H 3 R 9 , R 9 H 3 , H 3 R 9 H 3 , YSSR 9 SSY, (RKH) 4 , Y(RKH) 2 R, etc.
  • the nucleic acid (molecule) used for preparation of the at least one "complexed precomplexed nucleic acid" according to the present invention is preferably selected from any nucleic acid, more preferably selected from any nucleic acid suitable to encode a(t least one) peptide or protein, i.e. a coding nucleic acid, e.g. a coding DNA, selected e.g. from genomic DNA, cDNA, DNA oligonucleotides, or a coding RNA, selected e.g. from (short) RNA oligonucleotides, messenger RNA (mRNA), etc.
  • a coding nucleic acid e.g. a coding DNA, selected e.g. from genomic DNA, cDNA, DNA oligonucleotides, or a coding RNA, selected e.g. from (short) RNA oligonucleotides, messenger RNA (mRNA), etc.
  • an mRNA is typically an RNA, which is composed of several structural elements, e.g. an optional 5'-UTR region, an upstream positioned ribosomal binding site followed by a coding region, an optional 3'-UTR region, which may be followed by a poly-A tail (and/or a poly-C-tail).
  • An mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides.
  • Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein.
  • the at least one "complexed precomplexed nucleic acid” according to the present invention may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), or a viral RNA (vRNA). Furthermore, the at least one "complexed precomplexed nucleic acid” according to the present invention may be a circular or linear nucleic acid, preferably a linear nucleic acid.
  • the at least one "complexed precomplexed nucleic acid" may be a single- or a double-stranded nucleic acid (which may also be regarded as a nucleic acid within the above meaning due to non-covalent association of two single-stranded nucleic acids) or a partially double-stranded or partially single stranded nucleic acid, which are at least partially self complementary (both of these partially double-stranded or partially single stranded nucleic acids are typically formed by a longer and a shorter single-stranded nucleic acid or by two single stranded nucleic acids, which are about equal in length, wherein one single-stranded nucleic acid is in part complementary to the other single- stranded nucleic acid and both thus form a double-stranded nucleic acid in this region, i.e.
  • the at least one "complexed precomplexed nucleic acid" may be a RNA, more preferably an mRNA, even more preferably a (linear) single stranded mRNA.
  • the nucleic acid (molecule) used for preparation of the at least one "complexed precomplexed nucleic acid" according to the present invention comprises a length of about 5 to about 20000 nucleotides, or 100 to about 20000 nucleotides, preferably of about 250 to about 20000 nucleotides, more preferably of about 500 to about 10000 nucleotides, even more preferably of about 500 to about 5000 nucleotides.
  • the nucleic acid (molecule) used for preparation of the at least one "complexed precomplexed nucleic acid" according to the present invention may be a coding nucleic acid, preferably any nucleic acid as defined above, more preferably a coding RNA.
  • a coding nucleic acid, particularly such a coding RNA may be a single- or a double-stranded RNA, a single- stranded RNA, and/or a circular or linear RNA, and most preferably a linear RNA, such as a (linear) single-stranded RNA, or a ((linear) single-stranded) messenger RNA (mRNA).
  • the coding nucleic acid particularly the coding RNA or mRNA, used for preparation of the at least one "complexed precomplexed nucleic acid" according to the present invention may further encode a protein or a peptide, which may be selected, without being restricted thereto, e.g. from therapeutically active proteins or peptides, from antigens, e.g. tumor antigens, pathogenic antigens (e.g.
  • coding nucleic acid particularly the coding RNA or mRNA, used for the preparation of the at least one "complexed precomplexed nucleic acid" according to the present invention may be transported into a cell, a tissue or an organism and the protein may be expressed subsequently in this cell, tissue or organism.
  • Therapeutically active proteins are selected from pathogenic proteins as defined above or from animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens), autoimmune antigens, or further antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other proteins or peptides suitable for a specific (therapeutic) application, wherein the coding nucleic acid, particularly the coding RNA or mRNA, used for the preparation of the at least one "complexed precomplexed nucleic acid" according to the present invention may be transported into a cell, a tissue or an organism and the protein may
  • therapeutically active proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may be selected from any naturally occurring recombinant or isolated protein known to a skilled person from the prior art, particularly from any naturally occurring recombinant or isolated protein known to exhibit a therpeutic effect.
  • therapeutically active proteins may comprise proteins, capable of stimulating or inhibiting the signal transduction in the cell, e.g. cytokines, antibodies, etc.
  • Therapeutically active proteins may thus comprise cytokines of class I of the family of cytokines, having 4 positionally conserved cysteine residues (CCCC) and comprising a conserved sequence motif Trp-Ser- X-Trp-Ser (WSXWS), wherein X is a non-conserved amino acid.
  • Cytokines of class I of the family of cytokines comprise the GM-CSF subfamily, e.g. IL-3, IL-5, GM-CSF, the IL- 6-subfamily, e.g. IL-6, IL-1 1 , IL-12, or the I L-2 -subfamily, e.g.
  • Therapeutically active proteins may also comprise cytokines of class Il of the family of cytokines, which also comprise 4 positionally conserved cystein residues (CCCC), but no conserved sequence motif Trp- Ser-X-Trp-Ser (WSXWS). Cytokines of class Il of the family of cytokines comprise e.g. IFN-alpha, IFN-beta, IFN-gamma, etc. Therapeutically active proteins may additionally comprise cytokines of the family of tumor necrose factors, e.g.
  • TNF-alpha TNF-beta, etc.
  • cytokines of the family of chemokines which comprise 7 transmembrane helices and interact with G-protein, e.g. IL-8, MIP-I , RANTES, CCR5, CXR4, etc., or cytokine specific receptors, such as TNF-RI, TNF-RII, CD40, OX40 (CD134), Fas, etc.
  • Therapeutically active proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, may furthermore be selected from proteins associated with genetic diseases as mentioned herein, particularly proteins, which are not or not sufficiently expressed in the context of these genetic diseases.
  • Therapeutically active proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may also be selected from any of the proteins given in the following: 0ATL3, 0FC3, 0PA3, 0PD2, 4-1 BBL, 5T4, 6Ckine, 707-AP, 9D7, A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1, ABCA4, ABCB1, ABCB11, ABCB2, ABCB4, ABCB7, ABCC2, ABCC6, ABCC8, ABCD1, ABCD3, ABCG5, ABCG8, ABL1, ABO, ABR ACAA1, ACACA, ACADL, ACADM, ACADS, ACADVL, ACATI, ACCPN, ACE, ACHE, ACHM3, ACHM1, ACLS,
  • BDMR BEST1, beta-Catenin/m, BF, BFHD, BFIC, BFLS, BFSP2, BGLAP,BGN, BHD, BHR1, BING-4, BIRC5, BJS, BLM, BLMH, BLNK, BMPR2, BPGM, BRAF, BRCAI, BRCA1/m, BRCA2, BRCA2/m, BRCD2, BRCD1, BRDT, BSCL, BSCL2, BTAA, BTD, BTK, BUB1, BWS, BZX, C0L2A1, C0L6A1, ClNH, C1QA, C1QB, C1QG, C1S, C2, C3, C4A, C4B, C5, C6, C7, C7orf2, C8A, C8B, C9, CA125, CA15-3/CA 27-29, CA195, CA19-9,
  • CA72-4 CA2, CA242, CA50, CABYR, CACD, CACNA2D1, CACNA1A, CACNA1F, CACNA1S, CACNB2, CACNB4, CAGE, CA1, CALB3, CALCA, CALCR, CALM, CALR, CAM43, CAMEL, CAP-1, CAPN3, CARD15, CASP-5/m, CASP-8, CASP-8/m, CASR, CAT, CATM, CAV3, CB1, CBBM, CBS, CCA1, CCAL2, CCALl, CCAT, CCL-1, CCL-11, CCL- 12, CCL-13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-2, CCL-20, CCL-
  • IGAD1 IGER, IGF-1R, IGF2R, IGFI, IGH, IGHC, IGHG2, IGHGI, IGHM, IGHR, IGKC, IHG1, IHH, IKBKG, ILI, IL-I RA, ILIO, IL-11, IL12, IL12RB1, IL13, IL-13R ⁇ 2, IL-15, IL-16,
  • IMMP2L INDX
  • INFGR1 INFGR2
  • INF ⁇ IFN INF ⁇
  • INS INSR
  • IPl IRF6, IRSI, ISCW, ITGA2, ITGA2B, ITGA6, ITGA7, ITGB2, ITGB3, ITGB4, ITIHI, ITM2B, IV, IVD, JAG1, JAK3, JBS, JBTS1, JMS, JPD, KAL1, KAL2, KALI, KLK2, KLK4,
  • NKX2E NM, NME1, NMP22, NMTC, NODAL, NOG, NOS3, NOTCH3, NOTCH1, NP, NPC2, NPC1, NPHL2, NPHP1, NPHS2, NPHS1, NPM/ALK, NPPA, NQO1, NR2E3, NR3C1, NR3C2, NRAS, NRAS/m, NRL, NROB1, NRTN, NSE, NSX, NTRK1, NUMA1, NXF2, NY-CO1, NY-ESO1, NY-ESO-B, NY-LU-12, ALDOA, NYS2, NYS4, NY-SAR-35, NYS1 , NYX, OA3, OAI , OAP, OASD, OAT, OCA1 , OCA2, OCD1 , OCRL, OCRL1 , OCT,
  • ODDD ODT1, OFC1, OFDI, OGDH, OGT, OGT/m, OPA2, OPA1, OPD1, OPEM, OPG, OPN, OPN 1LW, OPN 1MW, OPN 1SW, OPPG, OPTB 1, TTD, ORM1, ORP1, OS- 9, OS-9/m, OSM LIF, OTC, OTOF, OTSC1, OXCT1, OYTES1, P15, P190 MINOR BCR- ABL, P2RY12, P3, P16, P40, P4HB, P-501, P53, P53/m, P97, PABPN1, PAFAH1B1, PAFAHI PI, PAGE-4, PAGE-5, PAH, PAI-1, PAI-2, PAK3, PAP, PAPPA, PARK2, PART-1,
  • SERPINF2 SERPING1, SERPINI1, SFTPA1, SFTPB, SFTPC, SFTPD, SGCA, SGCB, SGCD, SGCE, SGM1, SGSH, SGY-1, SH2D1A, SHBG, SHFM2, SHFM3, SHFM1, SHH, SHOX, Sl, SIAL, SIALYL LEWISX , SIASD, S11, SIM1, SIRT2/m, SIX3, SJS1, SKP2, SLC10A2, SLC12A1, SLC12A3, SLC17A5, SLC19A2, SLC22A1 L, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC25A4, SLC25A5, SLC25A6, SLC26A2, SLC26A3, SLC26A4, SLC2A1,
  • Therapeutically active proteins which may be encoded by the the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may further be selected from apoptotic factors or apoptosis related proteins including AIF, Apaf e.g. Apaf-1 , Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x L , Bcl-x s , bik, CAD, Calpain, Caspase e.g.
  • Apoptotic factors or apoptosis related proteins including AIF, Apaf e.g. Apaf-1 , Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x L , Bcl-x s , bik,
  • a therapeutically active protein which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, can also be an adjuvant protein.
  • an adjuvant protein is preferably to be understood as any protein, which is capable to elicit an innate immune response as defined herein.
  • such an innate immune response comprises activation of a pattern recognition receptor, such as e.g. a receptor selected from the ToII- like receptor (TLR) familiy, including e.g. a Toll like receptor selected from human TLR1 to TLR10 or from murine Toll like receptors TLR1 to TLR13.
  • TLR ToII- like receptor
  • an innate immune response is elicited in a mammal as defined above.
  • the adjuvant protein is selected from human adjuvant proteins or from pathogenic adjuvant proteins, in particular from bacterial adjuvant proteins.
  • mRNA encoding huma proteins involved in adjuvant effects may be used as well.
  • Human adjuvant proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, typically comprise any huma protein, which is capable of eliciting an innate immune response (in a mammal), e.g. as a reaction of the binding of an exogenous TLR ligand to a
  • human adjuvants which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, are selected from the group consisting of, without being limited thereto, cytokines which induce or enhance an innate immune response, including IL-2, IL-12, IL-15, IL-18, IL-21 CCL21 , GM-CSF and TNF-alpha; cytokines which are released from macrophages, including IL-I, IL-6, IL-8, IL-12 and TNF-alpha; from components of the complement system including C1 q, MBL, C1 r, C1 s, C2b, Bb, D, MASP-1 , MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1 , CR2, CR3, CR4, C1 qR, C1
  • TICAM2 etc.
  • activated transcription factors including e.g. NF- ⁇ B, c-Fos, c-Jun, c-Myc
  • induced target genes including e.g. IL-I alpha, IL-I beta, Beta-Defensin, IL-6, IFN gamma, IFN alpha and IFN beta
  • costimulatory molecules including CD28 or CD40-ligand or PD1
  • protein domains, including LAMP cell surface proteins
  • human adjuvant proteins including CD80, CD81 , CD86, trif, flt-3 ligand, thymopentin, Gp96 or fibronectin, etc., or any species homolog of any of the above human adjuvant proteins.
  • Pathogenic adjuvant proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, typically comprise any pathogenic (adjuvant) protein, which is capable of eliciting an innate immune response (in a mammal), more preferably selected from pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc., and even more preferably from pathogenic adjuvant proteins selected from the group consisting of, without being limited thereto, bacterial proteins, protozoa proteins (e.g. profilin - like protein of Toxoplasma gondii), viral proteins, or fungal proteins, animal proteins, etc.
  • pathogenic (adjuvant) protein which is capable of eliciting an innate immune response (in a mammal)
  • pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc. and even more
  • bacterial (adjuvant) proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, may comprise any bacterial protein, which is capable of eliciting an innate immune response (preferably in a mammal) or shows an adjuvant character.
  • such bacterial (adjuvant) proteins are selected from the group consisting of bacterial heat shock proteins or chaperons, including Hsp60, Hsp70, Hsp90, Hspl OO; OmpA (Outer membrane protein) from gram-negative bacteria; bacterial porins, including OmpF; bacterial toxins, including pertussis toxin (PT) from Bordetel Ia pertussis, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, PT-9K/129G mutant from pertussis toxin, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin, cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from cholera toxin, CTE1 12K mutant from CT, Escherichia coli heat-l
  • CT
  • Bacterial (adjuvant) proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may also be selected from bacterial adjuvant proteins, even more preferably selected from the group consisting of, without being limited thereto, bacterial flagellins, including flagellins from organisms including Agrobacterium, Aquifex, Azospirillum, Bacillus, Bartonella, Bordetella, Borrelia, Burkholderia, Campylobacter, Caulobacte,
  • Bordetella bronchiseptica Borrelia burgdorferi, Burkholderia cepacia, Campylobacter jejuni, Cau/obacter crescentus, Clostridium botulinum strain Bennett clone 1, Escherichia coli, Helicobacter pylori, Lachnospiraceae bacterium, Legionella pneumophila, Listeria monocytogenes, Proteus mirabilis, Pseudomonas aeroguinosa, Pseudomonas syringae, Rhizobium meliloti, Rhodobacter sphaeroides, Roseburia prevalencecola, Roseburis hominis,
  • Bacterial flagellins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, even more preferably comprise a sequence selected from the group comprising any of the following sequences as referred to their accession numbers: I I enterocolitica
  • Protozoa proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may be selected from any protozoa protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Tc52 from Trypanosoma cruzi,
  • Viral proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, may be selected from any viral protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Respiratory Syncytial Virus fusion glycoprotein (F-protein), envelope protein from MMT virus, mouse leukemia virus protein, Hemagglutinin protein of wild-type measles virus, etc.
  • F-protein Respiratory Syncytial Virus fusion glycoprotein
  • Fungal proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, may be selected from any fungal protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, fungal immunomodulatory protein (FIP; LZ-8), etc.
  • FIP fungal immunomodulatory protein
  • pathogenic adjuvant proteins which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, may be selected from any further pathogenic protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto,
  • KLH Keyhole limpet hemocyanin
  • OspA OspA
  • the nucleic acid (molecule), preferably an mRNA, of the at least one "complexed precomplexed nucleic acid” according to the present invention may alternatively encode an antigen.
  • the term "antigen” refers to a substance which is recognized by the immune system and is capable of triggering an antigen- specific immune response, e.g. by formation of antibodies as part of an adaptive immune response.
  • the first step of an adaptive immune response is the activation of native antigen-specific T cells by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which native T cells are constantly passing.
  • the three cell types that can serve as antigen-presenting cells are dendritic cells, macrophages, and B cells.
  • Tissue dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class Il molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. Presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells.
  • effector T cells The most important function of effector T cells is the killing of infected cells by CD8 + cytotoxic T cells and the activation of macrophages by TH1 cells which together make up cell- mediated immunity, and the activation of B cells by both TH2 and TH1 cells to produce different classes of antibody, thus driving the humoral immune response.
  • T cells recognize an antigen by their T cell receptors which does not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogens' protein antigens, which are bound to MHC molecules on the surfaces of other cells.
  • T cells fall into two major classes that have different effector functions.
  • the two classes are distinguished by the expression of the cell-surface proteins CD4 and CD8. These two types of T cells differ in the class of MHC molecule that they recognize.
  • MHC molecule- MHC class I and MHC class II- which differ in their structure and expression pattern on tissues of the body.
  • CD4 + T cells bind to the MHC class Il molecule and CD8 + T cells to the MHC class I molecule.
  • MHC class I and MHC class Il have distinct distributions among cells that reflect the different effector functions of the T cells that recognize them.
  • MHC class I molecules present peptides from pathogens, commonly viruses to CD8 + T cells, which differentiate into cytotoxic T cells that are specialized to kill any cell that they specifically recognize.
  • MHC class I molecules Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. But not only pathogenic peptides from viruses are presented by MHC class I molecules, also self-antigens like tumour antigens are presented by them. MHC class I molecules bind peptides from proteins degraded in the cytosol and transported in the endoplasmic reticulum. Thereby MHC class I molecules on the surface of cells infected with viruses or other cytosolic pathogens display peptides from these pathogen.
  • the CD8 + T cells that recognize MHC class hpeptide complexes are specialized to kill any cells displaying foreign peptides and so rid the body of cells infected with viruses and other cytosolic pathogens.
  • CD4 + T cells CD4 + helper T cells
  • MHC class Il molecules are normally found on B lymphocytes, dendritic cells, and macrophages, cells that participate in immune responses, but not on other tissue cells.
  • Macrophages for example, are activated to kill the intravesicular pathogens they harbour, and B cells to secrete immunoglobulins against foreign molecules.
  • MHC class Il molecules are prevented from binding to peptides in the endoplasmic reticulum and thus MHC class Il molecules bind peptides from proteins which are degraded in endosomes. They can capture peptides from pathogens that have entered the vesicular system of macrophages, or from antigens internalized by immature dendritic cells or the immunoglobulin receptors of B cells.
  • TH1 cells activate the microbicidal properties of macrophages and induce B cells to make IgG antibodies that are very effective of opsonising extracellular pathogens for ingestion by phagocytic cells
  • TH2 cells initiate the humoral response by activating native B cells to secrete IgM, and induce the production of weakly opsonising antibodes such as IgGl and lgG3 (mouse) and lgG2 and lgG4 (human) as well as IgA and IgE (mouse and human).
  • antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, typically comprise any antigen, falling under the above definition, more preferably protein and peptide antigens, e.g. antigen, including tumor antigens, pathogenic antigens or pathogens, animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergy antigens, or autoimmune (self-)antigens, etc.
  • antigens including tumor antigens, pathogenic antigens or pathogens, animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergy antigens, or autoimmune (self-)antigens, etc.
  • antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may be antigens generated outside the cell, more typically antigens not derived from the host organism (e.g. a human) itself (i.e. non-self antigens) but rather derived from host cells outside the host organism, e.g. viral antigens, bacterial antigens, fungal antigens, protozoological antigens, animal antigens (preferably selected from animals or organisms as disclosed herein), allergy antigens, etc.
  • the host organism e.g. a human
  • non-self antigens i.e. non-self antigens
  • host cells outside the host organism e.g. viral antigens, bacterial antigens, fungal antigens, protozoological antigens, animal antigens (preferably selected from animals or organisms as disclosed herein), allergy antigens, etc.
  • Allergy antigens are typically antigens, which cause an allergy in a human and may be derived from either a human or other sources.
  • Antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may be furthermore antigens generated inside the cell, the tissue or the body, e.g. by secretion of proteins, their degradation, metabolism, etc.
  • antigens include antigens derived from the host organism (e.g. a human) itself, e.g.
  • tumor antigens self-antigens or auto-antigens, such as auto-immune self-antigens, etc., but also (non-self) antigens as defined above, which have been originally been derived from host cells outside the host organism, but which are fragmented or degraded inside the body, tissue or cell, e.g. by (protease) degradation, metabolism, etc.
  • Antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, may furthermore comprise fragments or variants of such antigens as mentioned herein, particularly of protein or peptide antigens. Fragments of such antigens in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 1 1 , or 12 amino acids), or fragments as processed and presented by
  • MHC class Il molecules preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 1 7, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence.
  • These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form.
  • Fragments of antigens as defined herein may also comprise epitopes of those antigens.
  • Epitopes also called “antigen determinants” are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
  • tumor antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, comprises tumor antigens.
  • Tumor antigens are preferably located on the surface of the (tumor) cell. Tumor antigens may also be selected from proteins, which are overexpressed in tumor cells compared to a normal cell. Furthermore, tumor antigens also includes antigens expressed in cells which are (were) not themselves (or originally not themselves) degenerate but are associated with the supposed tumor. Antigens which are connected with tumor-supplying vessels or (re)formation thereof, in particular those antigens which are associated with neovascularization, e.g.
  • Antigens connected with a tumor furthermore include antigens from cells or tissues, typically embedding the tumor. Further, some substances (usually proteins or peptides) are expressed in patients suffering (knowingly or not-knowingly) from a cancer disease and they occur in increased concentrations in the body fluids of said patients. These substances are also referred to as "tumor antigens", however they are not antigens in the stringent meaning of an immune response inducing substance.
  • the class of tumor antigens can be divided further into tumor-specific antigens
  • TSAs tumor-associated-antigens
  • TAAs tumor-associated-antigens
  • TAAs tumor-associated-antigens
  • TSAs can only be presented by tumor cells and never by normal "healthy" cells. They typically result from a tumor specific mutation. TAAs, which are more common, are usually presented by both tumor and healthy cells. These antigens are recognized and the antigen-presenting cell can be destroyed by cytotoxic T cells. Additionally, tumor antigens can also occur on the surface of the tumor in the form of, e.g., a mutated receptor. In this case, they can be recognized by antibodies.
  • tumor antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, are shown in Tables 1 and 2 below. These tables illustrate specific (protein) antigens (i.e.
  • cancer antigens with respect to the cancer disease, they are associated with.
  • Table 2 Mutant anti ens ex ressed in cancer diseases [TGFbRIl IT GFbeta receptor Il colorectal carcinoma l ⁇ Pl/m lttrriosephosphate isomerase Melanoma
  • the tumor antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, are selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AIbZIP HPGl , alpha-5-beta-1 -integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-eaten in/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP- 8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, C
  • PSM PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1 , RU2, S-100, SAGE, SART-1 , SART-2, SART-3, SCC, SIRT2/m, SpI 7, SSX-1 , SSX-2/HOM-MEL-40, SSX-4, STAMP-I , STEAP, survivin, survivin-2B, SYT-SSX-1 , SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1 , TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1 , and WT1.
  • the tumor antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, are selected from the group consisting of MAGE-A1 (e.g. MAGE-Al according to accession number M77481 ), MAGE-A2, MAGE-A3, MAGE- A6 (e.g. MAGE-A6 according to accession number NM_005363), MAGE-C1 , MAGE-C2, melan-A (e.g. melan-A according to accession number NM_00551 1 ), GP100 (e.g.
  • GP100 according to accession number M77348), tyrosinase (e.g. tyrosinase according to accession number NM_000372), survivin (e.g. survivin according to accession number AF077350), CEA (e.g. CEA according to accession number NM_004363), Her-2/neu (e.g. Her-2/neu according to accession number M1 1730), WT1 (e.g. WT1 according to accession number NM_000378), PRAME (e.g. PRAME according to accession number
  • EGFRI epidermal growth factor receptor 1
  • EGFRI epidermal growth factor receptor 1
  • MUC1 MUC1
  • mucin-1 e.g. mucin-1 according to accession number NM_002456
  • SEC61 G e.g. SEC61 G according to accession number NM_014302
  • hTERT e.g. hTERT accession number NM_198253
  • 5T4 e.g. 5T4 according to accession number NM_006670
  • NY-Eso-1 e.g. NY-Eso1 according to accession number NM_001327)
  • TRP-2 e.g. TRP-2 according to accession number NM_001922
  • STEAP PCA, PSA, PSMA, etc.
  • One further class of antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, comprises allergy antigens.
  • allergy antigens may be selected from antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g. grasses, pollens, molds, drugs, or numerous environmental triggers, etc. Allergy antigens typically belong to different classes of compounds, such as nucleic acids and their fragments, proteins or peptides and their fragments, carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc.
  • antigens which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, i.e. protein or peptide antigens and their fragments or epitopes, or nucleic acids and their fragments, particularly nucleic acids and their fragments, encoding such i.e. protein or peptide antigens and their fragments or epitopes.
  • antigens derived from animals which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may include antigens derived from, without being limited thereto, insects, such as mite (e.g. house dust mites), mosquito, bee (e.g. honey bee, bumble bee), cockroache, tick, moth (e.g.
  • mite e.g. house dust mites
  • mosquito e.g. honey bee, bumble bee
  • cockroache e.g.
  • Antigens derived from plants which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may include antigens derived from, without being limited thereto, fruits, such as kiwi, pineapple, jackfruit,papaya, lemon, orange, mandarin, melon, sharon fruit, strawberry, lychee, apple, cherryados apple, mango, passion fruit, plum, apricot, nectarine, pear, passion fruit, raspberry, grape, from vegetables, such as garlic, onion, leek, soya bean, celery, cauliflower, turnip, paprika, chickpea, fennel, zucchini, cucumber, carrot, yam, bean, pea, olive, tomato, potato, lentil, lettuce, avocado, parsley, horseradish, chirimoya, beet, pumkin, spinach, from spices, such as mustard, coriander, saffron, pepper, ani
  • Antigens derived from fungi which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may include antigens derived from, without being limited thereto, e.g.
  • Altemia sp. Aspergillus sp., Beauveria sp., Candida sp., Cladosporium sp., Endothia sp., Curcularia sp., Embellisia sp., Epicoccum sp., Fusarium sp., Malassezia sp., Penicillum sp., Pleospora sp., Saccharomyces sp., etc.
  • Antigens derived from bacteria which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, may include antigens derived from, without being limited thereto, e.g. Bacillus tetani, Staphylococcus aureus, Streptomyces griseus, etc.
  • the nucleic acid (molecule), preferably an mRNA, of the at least one "complexed precomplexed nucleic acid” according to the present invention may encode an antibody.
  • an antibody may be selected from any antibody, e.g. any recombinantly produced or naturally occurring antibodies, known in the art, in particular antibodies suitable for therapeutic, diagnostic or scientific purposes, or antibodies which have been identified in relation to specific cancer diseases.
  • antibody is used in its broadest sense and specifically covers monoclonal and polyclonal antibodies (including agonist, antagonist, and blocking or neutralizing antibodies) and antibody species with polyepitopic specificity.
  • antibody typically comprises any antibody known in the art (e.g. IgM, IgD, IgG, IgA and IgE antibodies), such as naturally occurring antibodies, antibodies generated by immunization in a host organism, antibodies which were isolated and identified from naturally occurring antibodies or antibodies generated by immunization in a host organism and recombinantly produced by biomolecular methods known in the art, as well as chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies, i.e. antibodies expressed in cells and optionally localized in specific cell compartments, and fragments and variants of the aforementioned antibodies.
  • an antibody consists of a light chain and a heavy chain both having variable and constant domains.
  • the light chain consists of an N-terminal variable domain, V L , and a C-terminal constant domain, Q.
  • the heavy chain of the IgG antibody for example, is comprised of an N-terminal variable domain, V H , and three constant domains, C H 1 , C H 2 und C H 3.
  • Single chain antibodies may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention as well, preferably by a single-stranded RNA, more preferably by an mRNA.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may encode a polyclonal antibody.
  • polyclonal antibody typically means mixtures of antibodies directed to specific antigens or immunogens or epitopes of a protein which were generated by immunization of a host organism, such as a mammal, e.g. including goat, cattle, swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse, hamster and rabbit.
  • Polyclonal antibodies are generally not identical, and thus usually recognize different epitopes or regions from the same antigen.
  • nucleic acid typically a mixture (a composition) of different nucleic acids, preferably mRNAs, will be used as the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, each nucleic acid encoding a specific (monoclonal) antibody being directed to specific antigens or immunogens or epitopes of a protein.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may encode a monoclonal antibody.
  • the term "monoclonal antibody” herein typically refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed to a single antigenic site.
  • each monoclonal antibody is directed to a single determinant on the antigen.
  • monoclonal antibodies as defined above may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods, e.g. as described in U.S. Pat. No. 4,816,567.
  • “Monoclonal antibodies” may also be isolated from phage libraries generated using the techniques described in McCafferty et a/., Nature, 348:552-554 (1990), for example.
  • an immunogen (antigen) of interest is injected into a host such as a mouse and B-cell lymphocytes produced in response to the immunogen are harvested after a period of time.
  • the B-cells are combined with myeloma cells obtained from mouse and introduced into a medium which permits the B-cells to fuse with the myeloma cells, producing hybridomas.
  • These fused cells are then placed into separate wells of microtiter plates and grown to produce monoclonal antibodies.
  • the monoclonal antibodies are tested to determine which of them are suitable for detecting the antigen of interest. After being selected, the monoclonal antibodies can be grown in cell cultures or by injecting the hybridomas into mice.
  • the peptide sequences of these monoclonal antibodies have to be sequenced and the nucleic acid sequences encoding these antibodies can be used as the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention, which can be prepared according to procedures well known in the art.
  • non-human monoclonal or polyclonal antibodies such as murine antibodies may also be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • chimeric, humanized non- human and human antibodies are also envisaged as antibodies encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention.
  • “Chimeric” antibodies which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, are preferably antibodies in which the constant domains of an antibody described above are replaced by sequences of antibodies from other organisms, preferably human sequences.
  • “Humanized” (non-human) antibodies which may be also encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, are antibodies in which the constant and variable domains (except for the hypervariable domains) described above of an antibody are replaced by human sequences.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may encode human antibodies, i.e. antibodies having only human sequences.
  • human antibodies can be isolated from human tissues or from immunized non-human host organisms which are transgene for the human IgG gene locus, and RNA sequences may be prepared according to procedures well known in the art. Additionally, human antibodies can be provided by the use of a phage display.
  • nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” may encode bispecific antibodies.
  • Bispecific antibodies in context of the invention are preferably antibodies which act as an adaptor between an effector and a respective target by two different F ⁇ -domains, e.g. for the purposes of recruiting effector molecules such as toxins, drugs, cytokines etc., targeting effector cells such as CTL, NK cells, makrophages, granulocytes, etc. (see for review: Kontermann R. E., Acta Pharmacol. Sin, 2005, 26(1 ): 1 -9).
  • Bispecific antibodies as described herein are, in general, configured to recognize by two different F ⁇ -domains, e.g. two different antigens, immunogens, epitopes, drugs, cells (or receptors on cells), or other molecules (or structures) as described above. Bispecificity means herewith that the antigen-binding regions of the antibodies are specific for two different epitopes. Thus, different antigens, immunogens or epitopes, etc. can be brought close together, what, optionally, allows a direct interaction of the two components. For example, different cells such as effector cells and target cells can be connected via a bispecific antibody.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may also encode intrabodies, wherein these intrabodies may be antibodies as defined above. Since these antibodies are intracellular expressed antibodies, i.e. antibodies which are encoded by nucleic acid (molecules) localized in specific areas of the cell and also expressed there, such antibodies may be termed intrabodies.
  • Antibodies as encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may preferably comprise full-length antibodies, i.e. antibodies composed of the full heavy and full light chains, as described above. However, derivatives of antibodies such as antibody fragments, variants or adducts, may also be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” may also encode antibody fragments selected from Fab, Fab', F(ab') 2/ Fc, Facb, pFc', Fd and Fv fragments of the aforementioned (full-length) antibodies.
  • antibody fragments are known in the art.
  • an Fab fragment, antigen binding
  • fragment is composed of one constant and one variable domain of each of the heavy and the light chain. The two variable domains bind the epitope on specific antigens.
  • the two chains are connected via a disulfide linkage.
  • an scFv (“single chain variable fragment”) fragment typically consists of the variable domains of the light and heavy chains.
  • the domains are linked by an artificial linkage, in general a polypeptide linkage such as a peptide composed of 15-25 glycine, proline and/or serine residues.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may be an siRNA.
  • An siRNA is of interest particularly in connection with the phenomenon of RNA interference. Attention was drawn to the phenomenon of RNA interference in the course of immunological research. In recent years, an RNA-based defence mechanism has been discovered, which occurs both in the kingdom of the fungi and in the plant and animal kingdom and acts as an "immune system of the genome". The system was originally described in various species independently of one another, first in C.
  • RNA- mediated virus resistance in plants RNA-mediated virus resistance in plants
  • PTGS posttranscriptional gene silencing
  • RNA interference in eukaryotes are accordingly based on a common procedure.
  • the in vitro technique of RNA interference (RNAi) is based on double-stranded RNA molecules (dsRNA), which trigger the sequence-specific suppression of gene expression (Zamore (2001 ) Nat. Struct. Biol. 9: 746-750; Sharp (2001 ) Genes Dev. 5:485-490: Hannon (2002) Nature 41 : 244-251 ).
  • dsRNA molecules have also been used in vivo (McCaffrey et a/. (2002), Nature 418: 38-39; Xia et a/. (2002), Nature Biotech. 20: 1006-1010; Brummelkamp eta/. (2002), Cancer Cell 2: 243-247).
  • an siRNA used for the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” may comprise a (single- or) double stranded, preferably a double-stranded, RNA sequence with about 8 to 30 nucleotides, preferably 1 7 to 25 nucleotides, even more preferably from 20 to 25 and most preferably from 21 to 23 nucleotides.
  • all the sections having a length of from 17 to 29, preferably from 19 to 25, most preferably from 21 to 23 base pairs that occur in the coding region of an RNA sequence as mentioned above can serve as target sequence for such an si RNA.
  • siRNAs can also be directed against nucleotide sequences of a protein, particularly of regulatory proteins, which negatively regulate induction of an (innate or adaptive) immune response, that do not lie in the coding region, in particular in the 5' non-coding region of the RNA, for example, therefore, against non-coding regions of an RNA having a regulatory function.
  • the target sequence of the siRNA, used as the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention can therefore lie in the translated and/or untranslated region of the nucleotide sequence of such a protein as defined above and/or in the region of its control elements.
  • the target sequence of an siRNA as defined above can also lie in the overlapping region of untranslated and translated sequence; in particular, the target sequence can comprise at least one nucleotide upstream of the start triplet of the coding region of the RNA.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may be an antisense RNA.
  • an antisense RNA is preferably a (single-stranded) RNA molecule transcribed off the coding, rather than the template, strand of a DNA, so that (preferably) the (entire) anti-sense mRNA sequence is complementary to the sense (messenger) RNA.
  • An antisense RNA as defined herein typically forms a duplex between the sense and antisense RNA molecules and is thus capable to block transcription of the coding strand.
  • An antisense RNA used as the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention can be directed against the nucleotide sequences, e.g. a (naturally occurring) mRNA or genomic sequence encoding a protein or peptide, which may be selected from any protein or peptide sequence suitable for that purpose.
  • the antisense RNA used herein as the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention comprises a length as defined above in general for RNA molecules, more preferably a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention as defined above, encoding a protein as defined above, e.g. a therapeutically active protein, antibody and/or antigen, may also encode fragments and/or variants of the aforementioned proteins, wherein the fragments and/or variants may have a sequence identity to one of the aforementioned proteins of at least 70%, 80% or 85%, preferably at least 90%, more preferably at least 95% and most preferably at least 99% over the whole length of the (coding) nucleic acid sequences encoding these proteins.
  • a fragment of such a protein, encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may preferably comprise a sequence of a protein as defined above, which is, with regard to its amino acid sequence (or its encoded nucleic acid sequence), N- terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid sequence). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
  • a sequence homology as defined above may therefore either refer to the entire (protein or nucleic acid) sequence or to the coding (nucleic acid) sequence of a protein as defined herein, encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • Fragments of a protein, encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may furthermore comprise a sequence of a protein as defined above, which has a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class Il molecules, preferably having a length of about 13 or more amino acids, e.g.
  • these fragments may be selected from any part of the amino acid sequence of such a protein.
  • These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form.
  • Fragments of a protein, encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may also comprise epitopes of those proteins.
  • Epitopes also called “antigen determinants” in the context of the present invention are typically fragments located on the outer surface of (native) proteins as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
  • Such epitopes of proteins, encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may furthermore be selected from any of the herein mentioned variants of such proteins.
  • “Variants” of proteins, encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention preferably comprises a sequence, wherein nucleic acids of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, are exchanged.
  • a protein may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s).
  • these fragments and/or variants have the same or an improved biological function or specific activity compared to the full-length native protein as defined above.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” may also encode a protein as defined above, wherein the encoded amino acid sequence of the protein comprises a conservative amino acid substitution(s) compared to its physiological sequence.
  • Those encoded amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined above.
  • Substitutions in which amino acids which originate from the same class are exchanged for one another are called conservative substitutions.
  • these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function.
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain.
  • Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g.
  • CD spectra circular dichroism spectra
  • Preferred conservative amino acid substitutions of preferred groups of synonymous amino acid residues within the above meaning include, without being limited thereto:
  • variants of proteins as defined above which may be encoded by the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, may also comprise those sequences, wherein nucleic acids of this nucleic acid (molecule) are exchanged according to the degeneration of the genetic code, without leading to an alteration of respective amino acid sequence of the antigen or antigenic protein, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.
  • nucleic acid sequences e.g. (m)RNA (RNA or mRNA) sequences as defined herein, or amino acid sequences, preferably their encoded amino acid sequences, e.g. the amino acid sequences of the adjuvant proteins as defined above
  • the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. gaps can be inserted into the sequence of the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position.
  • the percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions.
  • the percentage to which two sequences are identical can be determined using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et a/. (1993), PNAS USA, 90:5873-5877 or Altschul et a/. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” may occur as a mono-, di-, or even multicistronic nucleic acid (molecule), preferably an mRNA, i.e. a nucleic acid (molecule) which carries the coding sequences of at least one protein or peptide, e.g. of one, two or more proteins or peptides, as disclosed herein.
  • the term "encoding at least one protein” may mean, without being limited thereto, that the at least one "complexed precomplexed nucleic acid”, encodes at least one protein or peptide, preferably one, two, three or even more adjuvant proteins or peptides.
  • the at least one protein is encoded by a monocistronic nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid", e.g. a monocistronic (m)RNA
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” may include various types of monocistronic nucleic acid(s) (molecules) coding for different proteins as defined herein, wherein each of the at least one nucleic acid molecules preferably encodes a (preferably different) protein as defined herein.
  • a composition may contain several monocistronic nucleic acid(s) (molecules), wherein each of these monocistronic nucleic acid(s) (molecules) encodes for one (preferably different) protein, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more monocistronic nucleic acid(s) (molecules), wherein each of these monocistronic nucleic acid(s) (molecules) encodes for one (preferably different) protein.
  • the at least one protein encoded thereby may be selected independently from any one of the proteins as defined above.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may include at least one bi- or even multicistronic nucleic acid (molecule), which carries two or even more of the coding sequences of the at least one protein as defined herein.
  • Such coding sequences of the at least one (preferably different) protein of the at least one bi- or even multicistronic nucleic acid (molecule) may be separated by at least one IRES (internal ribosomal entry site) sequence, as defined below.
  • the term "encoding at least one (preferably different) protein” may mean, without being limited thereto, that the (bi- or even multicistronic) nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” may encode e.g. at least one, two, three, four, five, six, seven, eight, nine, ten, etc. (preferably different) proteins of the above mentioned group of proteins. In any case, each of these proteins may be selected independently from one of the proteins as defined above.
  • IRES internal ribosomal entry site
  • IRES sequences which can be used according to the invention are those from picornaviruses (e.g.
  • FMDV pestiviruses
  • CFFV pestiviruses
  • PV polioviruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot and mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV mouse leukoma virus
  • SIV simian immunodeficiency viruses
  • CrPV cricket paralysis viruses
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may furthermore include a mixture of at least one monocistronic nucleic acid (molecule) as defined above, and at least one bi- or even multicistronic nucleic acid (molecules) as defined above.
  • the at least one monocistronic nucleic acid (molecule) and/or the at least one bi- or even multicistronic nucleic acid (molecule) preferably encode different proteins selected from the proteins as defined herein.
  • the at least one monocistronic nucleic acid (molecule) and the at least one bi- or even multicistronic nucleic acid (molecule) may preferably also encode (in part) identical proteins.
  • Use of a mixture of at least one monocistronic nucleic acid (molecule) as defined above, and at least one bi- or even multicistronic nucleic acid (molecule) or of a mixture of several at least one monocistronic nucleic acid(s) (molecules), wherein the at least one nucleic acid (molecule) encodes at least one (preferably different) protein as defined herein may be advantageous e.g. for a staggered, e.g.
  • the different nucleic acid(s) (molecules) "complexed precomplexed nucleic acid” according to the present invention may be e.g. contained in (different parts of) a kit of parts composition and may be administered together or separately.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may be in the form of a modified nucleic acid (molecule), preferably a modified mRNA, wherein any modification, as defined herein, may be introduced into the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • Modifications as defined herein preferably lead to a stabilized nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • nucleic acid (molecule) may be, dependent on the type of nucleic acid (molecule), more suitable for a nucleic acid moleucla, e.g. a DNA or RNA in general, or, e.g. in the case of GC-modified (m)RNA or mRNA sequences, be more suitable for a coding RNA, preferably an (m)RNA and/or mRNA as defined above.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may thus be provided as a "stabilized nucleic acid (molecule)", that is to say as a nucleic acid molecule, preferably an mRNA, that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease).
  • a stabilized nucleic acid (molecule) that is to say as a nucleic acid molecule, preferably an mRNA, that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease).
  • stabilization can be effected, for example, by a modified phosphate backbone of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • a backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the nucleic acid (molecule) are chemically modified.
  • Nucleotides that may be preferably used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, preferably at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom.
  • Stabilized nucleic acid(s) may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form.
  • non-ionic phosphate analogues such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group
  • phosphodiesters and alkylphosphotriesters in which the charged oxygen residue is present in alkylated form.
  • backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-O-(1 - thiophosphate)).
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may additionally or alternatively also contain sugar modifications.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention and typically includes, without implying any limitation, sugar modifications selected from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine-5'- tri phosphate, 2 ' -f I uoro-2 ' -deoxyu rid i ne-5 ' -tri phosphate), 2 ' -deoxy-2 ' -deam i ne oligoribonucleotide (2 '-amino-2'-deoxycytidi ne-5 '-
  • Significant in this case means an increase in the expression of the protein by at least 20%, preferably at least 30%, 40%, 50% or 60%, more preferably by at least 70%, 80%, 90% or even 100% and most preferably by at least 150%, 200% or even 300% or more.
  • a nucleotide having such a base modification is preferably selected from the group of the base-modified nucleotides consisting of 2-amino-6-chloropurineriboside-5'-triphosphate, 2- aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'- triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5- aminoallyluridine-5 '-triphosphate, 5-bromocytidine-5 '-triphosphate, 5-bromouridine-5'- triphosphate, 5-iodocytidine-5 '-triphosphate, 5-iodouridi ne-5 '-triphosphate, 5- methylcytidi ne-5 '-triphosphate, 5-methyluridine-5'-triphosphate, 6-azacytidine-5'- triphosphate, 6-azauridine-5'
  • nucleotides for base modifications selected from the group of base- modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'- triphosphate, 5-bromocytidine-5 '-triphosphate, and pseudouridine-5'-triphosphate.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention can likewise be modified (and preferably stabilized) by introducing modified nucleotides containing modifications of their ribose or base moieties.
  • nucleotide analogues are defined as non-natively occurring variants of naturally occurring nucleotides. Accordingly, analogues are chemically derivatized nucleotides with non-natively occurring functional groups, which are preferably added to or deleted from the naturally occurring nucleotide or which substitute the naturally occurring functional groups of a nucleotide. Accordingly, each component of the naturally occurring nucleotide may be modified, namely the base component, the sugar (ribose) component and/or the phosphate component forming the oligonucleotide's backbone (see above). Modifications of the, preferably of the base component, may comprise e.g.
  • analogues of guanosine, uracil, adenosine, and cytosine which include, without implying any limitation, any naturally occurring or non-naturally occurring guanosine, uracil, adenosine, thymidine or cytosine that has been altered chemically, for example by acetylation, methylation, hydroxylation, etc., including 1 -methyl-adenosine, 1 -methyl-guanosine, 1 -methyl-inosine, 2,2-dimethyl- guanosine, 2,6-diaminopurine, 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine, 2'- amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-amino-6-chloropurineriboside, 2- aminopurine-riboside, 2'-araadenosine, 2'-
  • sugar component may comprise analogs such as e.g. 2'-Deoxy-2'-fluoro- oligoribonucleotide (e.g. 2'-fluoro-2'-deoxycytidine-triphosphate, 2'-fluoro-2'-deoxyuridine- triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (e.g.
  • analogue as described above, particular preference is given according to the invention to those analogues that do not interfere with a further modification that has been introduced into the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may comprise between 0.1 % and 100% (modified) nucleotides selected from (modified) nucleotides as defined above with respect to the non-modified nucleic acid (molecule), wherein preferably between 0.1 % and 100% of each natively occurring non-modified ATP, GTP, CTP, UTP (and/or TTP) nucleotide of a nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention, or of its corresponding DNA- template, may be modified using a corresponding modified nucleotide as defined above, more preferably between 0,1 % and 20%, between 10% and 30%, between 20% and 40%, between 30% and 50%, between 40% and 60%, between 50% and 70%, between 60% and 80%, between 70% and 90%, or between 80% and 100% or at least 10%, more preferably
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention can contain a lipid modification, i.e. a lipid-modified nucleic acid (molecule).
  • a lipid-modified nucleic acid (molecule) typically comprises a nucleic acid (molecule) as defined herein for the at least one "complexed precomplexed nucleic acid", at least one linker covalently linked with that nucleic acid (molecule), and at least one lipid covalently linked with the respective linker.
  • such a lipid-modified nucleic acid (molecule) comprises a (at least one) nucleic acid (molecule) as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) with that nucleic acid (molecule).
  • the lipid-modified nucleic acid (molecule) comprises a nucleic acid (molecule) as defined herein, at least one linker covalently linked with that nucleic acid (molecule), and at least one lipid covalently linked with the respective linker, and also at least one (bifunctional) lipid covalently linked (without a linker) with that nucleic acid (molecule).
  • the lipid of a lipid-modified nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention is typically a lipid or a lipophilic residue that preferably is itself biologically active.
  • Such lipids preferably include natural substances or compounds such as, for example, vitamins, e.g. alpha-tocopherol (vitamin E), including RRR-alpha-tocopherol (formerly D-alpha-tocopherol), L-alpha-tocopherol, the racemate D,L-alpha-tocopherol, vitamin E succinate (VES), or vitamin A and its derivatives, e.g.
  • retinoic acid retinol
  • vitamin D and its derivatives e.g. vitamin D and also the ergosterol precursors thereof
  • vitamin E and its derivatives vitamin K and its derivatives, e.g. vitamin K and related quinone or phytol compounds, or steroids, such as bile acids, for example cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxygenin, testosterone, cholesterol or thiocholesterol.
  • bile acids for example cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxygenin, testosterone, cholesterol or thiocholesterol.
  • Further lipids or lipophilic residues used within the scope of the present invention include, without implying any limitation, polyalkylene glycols (Oberhauser et a/., Nucl.
  • aliphatic groups such as, for example, C1 -C20-alkanes, C1 -C20-alkenes or C1 -C20-alkanol compounds, etc., such as, for example, dodecanediol, hexadecanol or undecyl residues (Saison-Behmoaras et a/, EMBO J, 1991 , 10, 1 1 1 ; Kabanov et a/., FEBS Lett., 1990, 259, 327; Svinarchuk et a/., Biochimie, 1993, 75, 49), phospholipids such as, for example, phosphatidylglycerol, diacylphosphatidylglycerol, phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylserine, phosphatidy
  • polyamines or polyalkylene glycols such as, for example, polyethylene glycol (PEG) (Manoharan et a/, Nucleosides & Nucleotides, 1995, 14, 969), hexaethylene glycol (HEG), palmitin or palmityl residues (Mishra et a/, Biochim. Biophys. Acta, 1995, 1264, 229), octadecylamines or hexylamino-carbonyl-oxycholesterol residues (Crooke eta/, J. Pharmacol. Exp. Ther., 1996, 277, 923), and also waxes, terpenes, alicyclic hydrocarbons, saturated and mono- or poly-unsaturated fatty acid residues, etc.
  • PEG polyethylene glycol
  • HEG hexaethylene glycol
  • HOG hexaethylene glycol
  • palmitin or palmityl residues Mishra e
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may likewise be stabilized in order to prevent degradation of the nucleic acid molecule in vivo by various approaches.
  • RNA is considerably less stable in solution, which is substantially due to RNA-degrading enzymes, so-called RNases (ribonucleases).
  • RNases ribonucleases
  • Even the smallest ribonuclease contaminations are sufficient to degrade RNA completely in solution.
  • RNase contaminations can generally be removed only by special treatment, in particular with diethyl pyrocarbonate (DEPC).
  • the natural degradation of mRNA in the cytoplasm of cells is very finely regulated.
  • a number of mechanisms are known in this connection in the prior art.
  • the terminal structure is typically of critical importance for an mRNA in vivo.
  • cap structure a modified guanosine nucleotide
  • poly-A tail a sequence of up to 200 adenosine nucleotides
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention can therefore be stabilized against degradation by RNases by the addition of a so-called "5 1 cap” structure.
  • a m7G(5')ppp 5'(A,G(5')ppp(5')A or G(5')ppp(5')G as the 5' cap" structure.
  • a modification is introduced only if a modification, for example a lipid modification, has not already been introduced at the 5' end of the nucleic acid (molecule) or if the modification does not interfere with the desired immunogenic properties of the (unmodified or chemically modified) nucleic acid (molecule).
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may contain, according to a further preferred embodiment of the present invention, a poly-A tail on the 3' terminus of typically about 10 to 200 adenosine nucleotides, preferably about 10 to 100 adenosine nucleotides, more preferably about 20 to 100 adenosine nucleotides or even more preferably about 40 to 80 adenosine nucleotides, e.g. 70 nucleotides.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may additionally or alternatively contain a poly-C tail on the 3' terminus of typically about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 20 to 70 cytosine nucleotides or even more preferably about 20 to 60 or even 10 or 20 to 40 cytosine nucleotides, e.g. 30 nucleotides.
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention may be modified, and thus stabilized, by modifying the G/C content of the nucleic acid (molecule), preferably of the coding region of the nucleic acid (molecule).
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention may be stabilized by modifying the G/C content of the coding region of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • the G/C content of the coding region of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention is altered, preferably increased, compared to the G/C content of the coding region of the corresponding non-modified nucleic acid (molecule) (in the following "native" nucleic acid).
  • the encoded amino acid sequence of this G/C-increased nucleic acid (molecule) is preferably not altered compared to the corresponding native nucleic acid (molecule).
  • Such alteration of the GC-sequence may be termed in the following "GC-stabilization".
  • This G/C-stabilization of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention is based on the fact that the sequence of any nucleic acid region to be translated is important for efficient translation of that nucleic acid (molecule).
  • the sequence of various nucleotides is important.
  • sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content.
  • the codons the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention are therefore varied compared to its native nucleic acid (molecule) sequence, while retaining the translated amino acid sequence, such that they include an increased amount of G/C nucleotides.
  • the most favorable codons for the stability can be determined (so-called alternative codon usage).
  • the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" there are various possibilities for G/C- modification of the nucleic acid (molecule) sequence, compared to its native sequence.
  • amino acids which are encoded by codons which contain exclusively G or C nucleotides no G/C-modification of the codon is necessary.
  • the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and GIy (GGC or GGG) require no G/C- modification, since no A or U is present.
  • codons which contain A and/or U nucleotides can be G/C-modified by substitution of other codons which code for the same amino acids but contain no A and/or
  • the codons for Pro can be G/C-modified from CCU or CCA to CCC or CCG; the codons for Arg can be G/C-modified from CGU or CGA or AGA or AGG to CGC or
  • the codons for Ala can be G/C-modified from GCU or GCA to GCC or GCG; the codons for GIy can be G/C-modified from GGU or GGA to GGC or GGG.
  • the codons for Phe can be G/C-modified from UUU to UUC; the codons for Leu can be G/C-modified from UUA, UUG, CUU or CUA to CUC or CUG; the codons for Ser can be G/C-modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be G/C-modified from UAU to UAC; the codon for Cys can be G/C-modified from UGU to UGC; the codon for His can be G/C-modified from CAU to CAC; the codon for GIn can be G/C-modified from CAA to CAG; the codons for Me can be G/C-modified from AUU or AUA to AUC; the codons for Thr can be G/C-modified from ACU or ACA to ACC or ACG; the codon for Asn can be G/C-modified from AAU to AAC; the codon for Lys can
  • substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention, particularly if provided as an RNA or mRNA, compared to its particular native nucleic acid (molecule) sequence.
  • all codons for Thr occurring in the native sequence can be G/C-modified to ACC (or ACG).
  • combinations of the above substitution possibilities are used:
  • the G/C content of the coding region of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" according to the present invention is increased by at least 7%, more preferably by at least 15%, particularly preferably by at least 20%, compared to the G/C content of the coding region of the native nucleic acid (molecule) which codes for a protein as defined herein.
  • At least 60%, more preferably at least 70 %, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the region coding for a protein as defined herein or the whole sequence of the native nucleic acid (molecule) sequence are substituted, thereby increasing the GC/content of said sequence.
  • the G/C content of the native nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” according to the present invention particularly if provided as an RNA or mRNA, to the maximum (i.e. 100% of the substitutable codons of the native nucleic acid (molecule) sequence), in particular in the region coding for a protein as defined herein.
  • a further preferred modification of the nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid” according to the present invention is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells.
  • the corresponding G/C-stabilized or native RNA sequence may be translated to a significantly poorer degree than in the case, where codons coding for relatively "frequent" tRNAs are present.
  • the region in the nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention is preferably GC-stabilized compared to the corresponding region of the respective native nucleic acid (molecule) such that at least one codon of the native sequence which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA.
  • the native nucleic acid (molecule) sequences are GC-stabilized such that codons for which frequently occurring tRNAs are available are inserted.
  • all codons of the native sequence which code for a tRNA which is relatively rare in the cell can in each case be exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA.
  • the sequential G/C content which is increased, in particular maximized, in the GC-stabilized nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” according to the present invention particularly if provided as an RNA or mRNA, with the "frequent" codons without modifying the amino acid sequence of the protein encoded by the coding region of the native nucleic acid (molecule).
  • This preferred embodiment allows provision of a particularly efficiently translated and GC-stabilized nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” according to the present invention.
  • the determination of the necessary (and/or possible) GC modification of the nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention can be carried out using the computer program as explained in WO 02/098443 - the disclosure content of which is included in its full scope in the present invention.
  • the nucleotide sequence of any desired (coding) nucleic acid (molecule) used for the at least one "complexed precomplexed nucleic acid” according to the present invention can be GC-stabilized with the aid of the genetic code or the degenerative nature thereof such that a maximum G/C content results.
  • the amino acid sequence coded by the GC-stabilized nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” according to the present invention is preferably not further modified compared to their native RNA sequence.
  • the A/U content in the environment of the ribosome binding site of an (optionally already GC-stabilized) nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention is increased compared to the A/U content in the environment of the ribosome binding site of its particular native nucleic acid (molecule) sequence.
  • This modification an increased A/U content around the ribosome binding site increases the efficiency of ribosome binding to the nucleic acid (molecule) sequence.
  • nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention may be further modified with respect to potentially destabilizing sequence elements.
  • the coding region and/or the 5' and/or 3' untranslated region of the nucleic acid (molecule) sequence as defined herein may be further modified compared to the particular native nucleic acid (molecule) sequence such that is contains no destabilizing sequence elements, the coded amino acid sequence of the nucleic acid (molecule) sequence as defined herein preferably not being modified compared to its particular native nucleic acid (molecule) sequence.
  • DSE destabilizing sequence elements
  • DSEs present in the untranslated regions (3'- and/or 5'-UTR) can also be eliminated from a nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention by such further modifications.
  • Such destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3'-UTR sections of numerous unstable RNAs (Caput et a/., Proc. Natl. Acad. Sci. USA 1986, 83:
  • AURES AU-rich sequences
  • a nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” as defined herein, is therefore preferably (further) modified compared to the native nucleic acid (molecule) sequence such that the modified nucleic acid (molecule) sequence contains no such destabilizing sequences.
  • This also applies to those sequence motifs which are recognized by possible endonucleases, e.g. the sequence
  • GAACAAG which is contained in the 3'-UTR segment of the gene which codes for the transferrin receptor (Binder et a/., EMBO J. 1994, 13: 1969 to 1980).
  • sequence motifs are also preferably removed according to the invention in the nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention.
  • the nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid" according to the present invention preferably if provided as an mRNA, has, in a further modified form at least one IRES as defined above and/or at least one 5' and/or 3' stabilizing sequence, e.g. to enhance ribosome binding or to allow expression of different encoded proteins, e.g., antibodies, therapeutically active proteins or antigens, as defined above, located on an at least one (bi- or even multicistronic) nucleic acid (molecule) as defined above.
  • IRES as defined above
  • 5' and/or 3' stabilizing sequence e.g. to enhance ribosome binding or to allow expression of different encoded proteins, e.g., antibodies, therapeutically active proteins or antigens, as defined above, located on an at least one (bi- or even multicistronic) nucleic acid (molecule) as defined above.
  • the nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” according to the present invention may furthermore have at least one (additional) 5' and/or 3' stabilizing sequence.
  • These stabilizing sequences in the 5' and/or 3' untranslated regions have the effect of increasing the half-life of the nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” as defined herein in the cytosol.
  • These stabilizing sequences can have 100% sequence homology to naturally occurring sequences which occur in viruses, bacteria and eukaryotes, but can also be partly or completely synthetic.
  • the untranslated sequences (UTR) of the globin gene e.g.
  • stabilizing sequences which can be used in the present invention for a further stabilized nucleic acid (molecule) sequence as defined herein.
  • Another example of a stabilizing sequence has the general formula (C/U)CCAN x CCC(U/A)Py x UC(C/U)CC (SEQ ID NO: 9), which is contained in the 3'UTR of the very stable RNA which codes for globin, (l)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik et a/., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414).
  • a nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” according to the present invention is therefore preferably present as globin UTR (untranslated regions)-stabilized nucleic acid (molecule).
  • any of the above modifications may be applied to a nucleic acid (molecule) sequence of the at least one "complexed precomplexed nucleic acid” according to the present invention, and may be, if suitable or necessary, be combined with each other in any combination, provided, these combinations of modifications do not interfere with each other in the respective modified nucleic acid (molecule).
  • a person skilled in the art will be able to take his choice accordingly.
  • a nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" as defined herein may be prepared using any naturally or synthetic DNA or RNA sequence available in the art as a template, i.e. any suitable (desoxy)ribonucleic acid.
  • Such naturally or synthetic DNA or RNA sequences may be obtained from any synthetic or naturally occurring source, which is available to a skilled person, e.g. may be derived from a protein or peptide library or may be transcribed from a nucleic acid library, such as a cDNA library, or may be obtained from any living or dead tissue, from a sample obtained from e.g. a human, animal or bacterial source.
  • nucleic acid (molecule) as defined herein may be prepared synthetically by methods known to a person skilled in the art, e.g., by solid phase synthesis or any other suitable method for preparing nucleic acid sequences, particularly methods suitable for preparing mRNA sequences.
  • substitutions, additions or eliminations of nucleotides or bases in these sequences are preferably carried out using a DNA matrix for preparation of a RNA or mRNA as defined herein or by techniques of the well known site directed mutagenesis or with an oligonucleotide ligation strategy (see e.g.
  • nucleic acid (molecule) of the at least one "complexed precomplexed nucleic acid" as defined herein can also be introduced into the nucleic acid (molecule) sequence by means of further methods known to a person skilled in the art. Suitable methods are, for example, synthesis methods using (automatic or semiautomatic) oligonucleotide synthesis devices, biochemical methods, such as, for example, in vitro transcription methods, etc.
  • the present invention also provides a pharmaceutical composition, comprising the at least one "complexed precomplexed nucleic acid" as defined according to the present invention and optionally a pharmaceutically acceptable carrier and/or vehicle.
  • the inventive pharmaceutical composition comprises the at least one "complexed precomplexed nucleic acid" as defined according to the present invention, i.e. at least one complexed precomplexed nucleic acid (molecule), e.g. an mRNA, which has been precomplexed in a first step with (a high-molecular) PEI in an N/P ratio between 0.05 and 2, preferably in an N/P ratio between 0.1 and 1 and have been further complexed in a second step with a cationic or polycationic compound.
  • molecule e.g. an mRNA
  • inventive pharmaceutical composition may comprise a pharmaceutically acceptable carrier and/or vehicle.
  • a pharmaceutically acceptable carrier typically includes the liquid or non-liquid basis of the inventive inventive pharmaceutical composition. If the inventive pharmaceutical composition is provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions.
  • water or preferably a buffer preferably an aqueous buffer
  • a sodium salt preferably at least 50 mM of a sodium salt
  • a calcium salt preferably at least 0.01 mM of a calcium salt
  • optionally a potassium salt preferably at least 3 mM of a potassium salt.
  • the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.
  • examples of sodium salts include e.g.
  • examples of the optional potassium salts include e.g. KCI, Kl, KBr, K 2 CO 3 , KHCO 3 , K 2 SO 4
  • examples of calcium salts include e.g. CaCl 2 , CaI 2 , CaBr 2 , CaCO 3 , CaSO 4 , Ca(OH) 2 .
  • organic anions of the aforementioned cations may be contained in the buffer.
  • the buffer suitable for injection purposes as defined above may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCl 2 ) and optionally potassium chloride (KCI), wherein further anions may be present additional to the chlorides.
  • CaCI 2 can also be replaced by another salt like KCI.
  • the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI) and at least 0,01 mM calcium chloride (CaCl 2 ).
  • the injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e.
  • the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
  • Reference media are e.g. liquids occurring in "in vivd' methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Ringer-Lactate solution is particularly preferred as a liquid basis.
  • one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well for the inventive pharmaceutical composition, which are suitable for administration to a patient to be treated.
  • the term "compatible” as used here means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the at least one "complexed precomplexed nucleic acid" as defined according to the present invention in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions.
  • Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated.
  • Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
  • sugars such as, for example, lactose, glucose and sucrose
  • starches such as, for example, corn star
  • the inventive pharmaceutical composition may comprise an adjuvant.
  • an adjuvant may be understood as any compound, which is suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response.
  • the inventive pharmaceutical composition when administered, typically elicits an innate immune response due to the adjuvant, optionally contained therein.
  • Such an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal, except of cationic or polycationic compounds as defined above in order to prevent complexation of the at least one free mRNA.
  • the adjuvant may be selected from the group consisting of, without being limited thereto, including chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMERTM (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAY R1005TM ((N-(2-deoxy-2-L-leucylamino-b-D- glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate);
  • inventive pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques.
  • the inventive pharmaceutical composition may be administered by parenteral injection, more preferably by subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or via infusion techniques.
  • Sterile injectable forms of the inventive pharmaceutical compositions may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenteral ly-acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol.
  • a non-toxic parenteral ly-acceptable diluent or solvent for example as a solution in 1 ,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di- glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutical ly-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation of the inventive pharmaceutical composition.
  • inventive pharmaceutical composition as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • the active ingredient i.e. the at least one "complexed precomplexed nucleic acid" as defined according to the present invention, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • the inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the inventive immunostimulatory composition, particularly its components as defined above, suspended or dissolved in one or more carriers.
  • Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the inventive pharmaceutical composition can be formulated in a suitable lotion or cream.
  • suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the inventive pharmaceutical composition typically comprises a "safe and effective amount" of the components of the inventive pharmaceutical composition, particularly of the at least one "complexed precomplexed nucleic acid” as defined according to the present invention.
  • a "safe and effective amount” means an amount of the at least one "complexed precomplexed nucleic acid” as defined according to the present invention that is sufficient to significantly induce a positive modification of a disease or disorder as defined herein.
  • a "safe and effective amount” is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.
  • a "safe and effective amount" of the components of the inventive pharmaceutical composition, particularly of the the at least one "complexed precomplexed nucleic acid” as defined according to the present invention will furthermore vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the activity of the specific "complexed precomplexed nucleic acid" as defined herein employed, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying doctor.
  • the inventive pharmaceutical composition may be used for human and also for veterinary medical purposes, preferably for human medical purposes, as a pharmaceutical composition in general or as a vaccine.
  • the inventive pharmaceutical composition may be provided as a vaccine.
  • Such an inventive vaccine is typically composed like the inventive pharmaceutical composition and preferably supports or elicits an immune response of the immune system of a patient to be treated, e.g. an innate immune response, if an RNA or mRNA is used as the nucleic acid molecule of the at least one "complexed precomplexed nucleic acid" as defined according to the present invention.
  • the inventive vaccine may elicit an adaptive immune response, preferably, if the the at least one "complexed precomplexed nucleic acid" as defined according to the present invention encodes any of the above mentioned antigens or proteins, which elicit an adaptive immune response.
  • the inventive vaccine may also comprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicle as defined above for the inventive pharmaceutical composition.
  • a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive vaccine is administered.
  • the inventive vaccine can be administered, for example, systemically or locally. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes.
  • Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections. More preferably, vaccines may be administered by an intradermal, subcutaneous, or intramuscular route. Inventive vaccines are therefore preferably formulated in liquid (or sometimes in solid) form. The suitable amount of the inventive vaccine to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4.
  • Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices.
  • Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the inventive vaccine is to be administered orally, tablets, capsules and the like are the preferred unit dose form.
  • the pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.
  • the inventive vaccine can additionally contain one or more auxiliary substances in order to further increase its immunogenicity.
  • auxiliary substances various mechanisms can come into consideration in this respect. For example, compounds that permit the maturation of dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable auxiliary substances.
  • DCs dendritic cells
  • TNF-alpha or CD40 ligand form a first class of suitable auxiliary substances.
  • auxiliary substance any agent that influences the immune system in the manner of a "danger signal" (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow an immune response produced by the immune-stimulating adjuvant according to the invention to be enhanced and/or influenced in a targeted manner.
  • a "danger signal” LPS, GP96, etc.
  • cytokines such as GM-CFS
  • auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-I , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-I 7, IL- 18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, INF-alpha, IFN-beta, INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
  • cytokines such as monokines, lymphokines,
  • emulsifiers such as, for example, Tween ® ; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.
  • the inventive vaccine can also additionally or alternatively contain any further compound, which is known to be immune-stimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13.
  • any further compound which is known to be immune-stimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR13.
  • CpG nucleic acids in particular CpG-RNA or CpG-DNA.
  • a CpG-RNA or CpG- DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA).
  • the CpG nucleic acid is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA).
  • the CpG nucleic acid preferably contains at least one or more (mitogenic) cytosine/guanine dinucleotide sequence(s) (CpG motif(s)).
  • CpG motif(s) cytosine/guanine dinucleotide sequence(s)
  • at least one CpG motif contained in these sequences that is to say the C (cytosine) and the G (guanine) of the CpG motif, is unmethylated. All further cytosines or guanines optionally contained in these sequences can be either methylated or unmethylated.
  • the C (cytosine) and the G (guanine) of the CpG motif can also be present in methylated form.
  • the inventive vaccine can also additionally or alternatively contain an immunostimulatory RNA, i.e. an RNA derived from an immunostimulatory RNA, which triggers or increases an (innate) immune response.
  • an immunostimulatory RNA i.e. an RNA derived from an immunostimulatory RNA, which triggers or increases an (innate) immune response.
  • an RNA may be in general be as defined above for RNAs.
  • those classes of RNA molecules, which can induce an innate immune response may be selected e.g.
  • TLRs Toll-like receptors
  • RNA sequences representing and/or encoding ligands of TLRs preferably selected from human family members TLR1 - TLRI O or murine family members TLR1 - TLR13, more preferably from TLR7 and TLR8, ligands for intracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. MoI. Cell 22, 561 -569), or any other immunostimulatory RNA sequence.
  • Such an immunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides.
  • the inventive vaccine may contain immunostimulatory RNA sequences of formula (I):
  • G is guanosine, uracil or an analogue of guanosine or uracil;
  • X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above- mentioned nucleotides
  • the inventive vaccine may contain immunostimulatory RNA sequences of formula (II):
  • C is cytosine, uracil or an analogue of cytosine or uracil;
  • X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue of the above- mentioned nucleotides
  • nucleic acids of formula (I) or (II), which may be used for the inventive vaccine are typically relatively short nucleic acid molecules and typically have a length of approximately from 5 to 100 (but may also be longer than 100 nucleotides for specific embodiments, e.g. up to 200 nucleotides), from 5 to 90 or from 5 to 80 nucleotides, preferably a length of approximately from 5 to 70, more preferably a length of approximately from 8 to 60 and, more preferably a length of approximately from 15 to 60 nucleotides, more preferably from 20 to 60, most preferably from 30 to 60 nucleotides. If the nucleic acid of the invention has a maximum length of e.g.
  • nucleotides G in the nucleic acid of formula (I) is determined by I or n.
  • a nucleotide adjacent to X m in the nucleic acid of formula (I) according to the invention is preferably not a uracil.
  • the number of nucleotides C in the nucleic acid of formula (II) according to the invention is determined by I or n.
  • a nucleotide adjacent to X n , in the nucleic acid of formula (II) according to the invention is preferably not a uracil.
  • I or n > 1 at least 60%, 70%, 80%, 90% or even 100% of the nucleotides are guanosine or an analogue thereof, as defined above.
  • the remaining nucleotides to 100% (when guanosine constitutes less than 100% of the nucleotides) in the flanking sequences G 1 and/or G n are uracil or an analogue thereof, as defined hereinbefore.
  • I and n independently of one another, are each an integer from 2 to 30, more preferably an integer from 2 to 20 and yet more preferably an integer from 2 to 15.
  • the lower limit of I or n can be varied if necessary and is at least 1 , preferably at least 2, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10. This definition applies correspondingly to formula (II).
  • the inventive vaccine may contain immunostimulatory RNA sequences selected from a sequence consisting or comprising any of the fol lowi ng sequences:
  • the inventive vaccine may contain immunostimulatory RNA sequences of formula (I):
  • the at least one "complexed precomplexed nucleic acid” as defined according to the present invention i.e. at least one complexed precomplexed nucleic acid (molecule), e.g.
  • an mRNA which has been precomplexed in a first step with (a high-molecular) PEI in an N/P ratio between 0.05 and 2, preferably in an N/P ratio between 0.1 and 1 and have been further complexed in a second step with a cationic or polycationic compound as defined above, may be used for the preparation of a pharmaceutical composition or a vaccine, preferably all as defined herein, for the prophylaxis, treatment and/or amelioration of any of the diseases and disorders as defined herein, e.g. cancer or tumor diseases, infectious diseases, preferably (viral, bacterial or protozoological) infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, cardiovascular diseases and/or neural diseases.
  • infectious diseases preferably (viral, bacterial or protozoological) infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, cardiovascular diseases
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention i.e. at least one complexed precomplexed nucleic acid (molecule), e.g. an mRNA, which has been precomplexed in a first step with (a high-molecular) PEI in an N/P ratio between 0.05 and 2, preferably in an N/P ratio between 0.1 and 1 and have been further complexed in a second step with a cationic or polycationic compound as defined above, may be used (for the preparation of a medicament, e.g. a pharmaceutical composition or a vaccine as defined above) for the prophylaxis, treatment and/or amelioration of e.g.
  • a medicament e.g. a pharmaceutical composition or a vaccine as defined above
  • cervical carcinoma cervical cancer
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention may be used (for the preparation of a medicament, e.g. a pharmaceutical composition or a vaccine as defined above) for the prophylaxis, treatment, and/or amelioration of e.g. infectious diseases, preferably (viral, bacterial or protozoological) infectious diseases.
  • infectious diseases preferably (viral, bacterial or protozoological
  • infectious diseases are typically selected from influenza, malaria, SARS, yellow fever, AIDS, Lyme borreliosis, Leishmaniasis, anthrax, meningitis, viral infectious diseases such as AIDS, Condyloma acuminata, hollow warts, Dengue fever, three-day fever, Ebola virus, cold, early summer meningoencephalitis (FSME), flu, shingles, hepatitis, herpes simplex type I, herpes simplex type II, Herpes zoster, influenza, Japanese encephalitis, Lassa fever, Marburg virus, measles, foot-and-mouth disease, mononucleosis, mumps, Norwalk virus infection, Pfeiffer's glandular fever, smallpox, polio (childhood lameness), pseudo- croup, fifth disease, rabies, warts, West Nile fever, chickenpox, cytomegalic
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention may be used (for the preparation of a medicament, e.g. a pharmaceutical composition or a vaccine as defined above) for the prophylaxis, treatment, and/or amelioration of e.g. autoimmune diseases.
  • a medicament e.g. a pharmaceutical composition or a vaccine as defined above
  • autoimmune diseases can be broadly divided into systemic and organ-specific or localised autoimmune disorders, depending on the principal clinico-pathologic features of each disease.
  • Autoimmune diseases may be divided into the categories of systemic syndromes, including systemic lupus erythematosus (SLE), Sjogren's syndrome, Scleroderma, Rheumatoid Arthritis and polymyositis or local syndromes which may be endocrinologic (type I diabetes (Diabetes mellitus Type 1 ), Hashimoto's thyroiditis, Addison's disease etc.), dermatologic (pemphigus vulgaris), haematologic (autoimmune haemolytic anaemia), neural (multiple sclerosis) or can involve virtually any circumscribed mass of body tissue.
  • SLE systemic lupus erythematosus
  • Sjogren's syndrome Sjogren's syndrome
  • Scleroderma Scleroderma
  • Rheumatoid Arthritis and polymyositis or local syndromes
  • endocrinologic type I diabetes (Diabetes mellitus Type 1 ), Hashimoto's thyroiditis, Add
  • the autoimmune diseases to be treated may be selected from the group consisting of type I autoimmune diseases or type Il autoimmune diseases or type III autoimmune diseases or type IV autoimmune diseases, such as, for example, multiple sclerosis (MS), rheumatoid arthritis, diabetes, type I diabetes (Diabetes mellitus Type 1 ), chronic polyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis, colitis ulcerosa, type I allergy diseases, type Il allergy diseases, type III allergy diseases, type IV allergy diseases, fibromyalgia, hair loss, Bechterew's disease, Crohn's disease, Myasthenia gravis, neurodermitis, Polymyalgia rheumatica, progressive systemic sclerosis (PSS), Reiter's syndrome, rheumatic arthritis, psoriasis, vasculitis, etc, or type Il diabetes.
  • MS multiple sclerosis
  • rheumatoid arthritis diabetes
  • type I diabetes Diabetes mell
  • the autoreaction may be due to a T-CeII bypass.
  • a normal immune system requires the activation of B-cells by T-cells before the former can produce antibodies in large quantities.
  • This requirement of a T-cell can be by-passed in rare instances, such as infection by organisms producing super-antigens, which are capable of initiating polyclonal activation of B-cells, or even of T-cells, by directly binding to the ⁇ - subunit of T-cell receptors in a non-specific fashion.
  • autoimmune diseases from a Molecular Mimicry.
  • An exogenous antigen may share structural similarities with certain host antigens; thus, any antibody produced against this antigen (which mimics the self-antigens) can also, in theory, bind to the host antigens and amplify the immune response.
  • the most striking form of molecular mimicry is observed in Group A beta-haemolytic streptococci, which shares antigens with human myocardium, and is responsible for the cardiac manifestations of rheumatic fever.
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention may be used (for the preparation of a medicament, e.g. a pharmaceutical composition or a vaccine as defined above) for the prophylaxis, treatment, and/or amelioration of allergies or allergic diseases, i.e. diseases related to allergies.
  • Allergy is a condition that typically involves an abnormal, acquired immunological hypersensitivity to certain foreign antigens or allergens, such as the allergy antigens as defined above.
  • allergy antigens or allergens may be selected from allergy antigens as defined above antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g.
  • allergies include, without being limited thereto, allergic asthma (leading to swelling of the nasal mucosa), allergic conjunctivitis (leading to redness and itching of the conjunctiva), allergic rhinitis ("hay fever"), anaphylaxis, angiodema, atopic dermatitis (eczema), urticaria (hives), eosinophilia, respiratory, allergies to insect stings, skin allergies (leading to and including various rashes, such as eczema, hives (urticaria) and (contact) dermatitis), food allergies, allergies to medicine, etc.
  • the inventive immunostimulatory composition, the inventive pharmaceutical composition or the inventive vaccine, or the at least one (m)RNA of the adjuvant component of the inventive immunostimulatory composition complexed with a cationic or polycationic compound, together with the at least one free mRNA of the inventive immunostimulatory composition may be used for the treatment of such allergic disorders or diseases, preferably by desensitizing the immune reaction which triggers a specific immune response.
  • desensitizing may be carried out by administering an effective amount of the allergen or allergic antigen encoded by a RNA of the inventive immunostimulatory composition, preferably the at least one free mRNA, to induce a slight immune reaction.
  • the amount of the allergen or allergic antigen may then be raised step by step in subsequent administrations until the immune system of the patient to be treated tolerates a specific amount of allergen or allergic antigen.
  • diseases to be treated in the context of the present invention likewise include (hereditary) diseases, or genetic diseases in general monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general.
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention may be used (for the preparation of a medicament, e.g. a pharmaceutical composition or a vaccine as defined above) for the prophylaxis, treatment, and/or amelioration of genetic diseases.
  • Such (mono-)genetic diseases, (hereditary) diseases, or genetic diseases in general are typically caused by genetic defects, e.g. due to gene mutations resulting in loss of protein activity or regulatory mutations which do not allow transcription or translation of the protein.
  • the present invention allows to treat these diseases by providing the complexed RNA as defined herein.
  • the following diseases may be treated: 3-beta- hydroxysteroid dehydrogenase deficiency (type II); 3-ketothiolase deficiency; 6- mercaptopurine sensitivity; Aarskog-Scott syndrome; Abetalipoproteinemia; Acatalasemia; Achondrogenesis; Achondrogenesis-hypochondrogenesis; Achondroplasia; Achromatopsia; Acromesomelic dysplasia (Hunter-Thompson type); ACTH deficiency; Acyl-CoA dehydrogenase deficiency (short-chain, medium chain, long chain); Adenomatous polyposis coli; Adenosin-deaminase deficiency; Adenylosuccinase deficiency; Adhalinopathy
  • Preferred diseases to be treated which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws are preferably selected from the group consisting of autosomal-recessive inherited diseases, such as, for example, adenosine deaminase deficiency, familial hypercholesterolemia, Canavan's syndrome, Gaucher's disease, Fanconi anaemia, neuronal ceroid lipofuscinoses, mucoviscidosis (cystic fibrosis), sickle cell anaemia, phenylketonuria, alcaptonuria, albinism, hypothyreosis, galactosaemia, alpha-1 -anti-trypsin deficiency, Xeroderma pigmentosum, Ribbing's syndrome, mucopolysaccharidoses, cleft lip, jaw, palate, Laurence Moon Biedl Bardet sydrome, short rib polydactylia syndrome,
  • X syndrome muscular dystrophy (Duchenne and Becker-Kiener type), haemophilia A and B, G6PD deficiency, Fabry's disease, mucopolysaccharidosis, Norrie's syndrome, Retinitis pigmentosa, septic granulomatosis, X-SCID, ornithine transcarbamylase deficiency, Lesch- Nyhan syndrome, or from autosomal-dominant inherited diseases, such as, for example, hereditary angiooedema, Marfan syndrome, neurofibromatosis, type I progeria, Osteogenesis imperfecta, Klippel-Trenaurnay syndrome, Sturge-Weber syndrome, Hippel- Lindau syndrome and tuberosis sclerosis.
  • hereditary angiooedema Marfan syndrome
  • neurofibromatosis type I progeria
  • Osteogenesis imperfecta Klippel-Trenaurnay syndrome
  • Sturge-Weber syndrome
  • the present invention also allows treatment of diseases, which have not been inherited, or which may not be summarized under the above categories.
  • Such dieseases may include e.g. the treatment of patients, which are in need of a specific protein factor, e.g. a specific therapeutically active protein as mentioned above.
  • This may e.g. include dialysis patients, e.g. patients which undergo a (regular) a kidney or renal dialysis, and which may be in need of specific therapeutically active proteins as defined above, e.g. erythropoietin (EPO), etc.
  • EPO erythropoietin
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention may be used for (the preparation of a medicament, e.g. a pharmaceutical composition or a vaccine as defined above for) the prophylaxis, treatment, and/or amelioration of cardiovascular diseases chosen from, without being limited thereto, coronary heart disease, arteriosclerosis, apoplexy and hypertension, etc.
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention may be used for (the preparation of a medicament, e.g. a pharmaceutical composition or a vaccine as defined above for) the prophylaxis, treatment, and/or amelioration of neuronal diseases chosen from Alzheimer's disease, amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's disease etc.
  • the invention furthermore relates also to the use of the inventive pharmaceutical composition or the inventive vaccine, or the components thereof, i.e. the at least one "complexed precomplexed nucleic acid" as defined according to the present invention, preferably the at least one complexed precomplexed nucleic acid (molecule), e.g.
  • an mRNA which has been precomplexed in a first step with (a high-molecular) PEI in an N/P ratio between 0.05 and 2, preferably in an N/P ratio between 0.1 and 1 and have been further complexed in a second step with a cationic or polycationic compound as defined above, for the prophylaxis, treatment, and/or amelioration of diseases or disorders as mentioned herein.
  • a high-molecular PEI in an N/P ratio between 0.05 and 2
  • a cationic or polycationic compound as defined above
  • such a method for prophylaxis, treatment, and/or amelioration of the above-mentioned diseases or disorders, or an inoculation method for preventing the above-mentioned diseases typically comprises administering the described pharmaceutical composition or vaccine or components thereof to a patient in need thereof (e.g. suffering from any of the above diseases or showing symptoms thereof), in particular to a human being, preferably in a "safe and effective amount" and in one of the above formulations as described above for inventive pharmaceutical compositions.
  • the administration mode also may be as described above for inventive pharmaceutical compositions or vaccines.
  • the present patent application also provides a method of transfecting and optionally administering the inventive pharmaceutical composition or the inventive vaccine, or its components as defined above, e.g. the at least one "complexed precomplexed nucleic acid" as defined according to the present invention, comprising the following steps:
  • blood cells are preferably understood according to the invention as meaning a mixture or an enriched to substantially pure population of red blood cells, granulocytes, mononuclear cells (PBMCs) and/or blood platelets from whole blood, blood serum or another source, e.g. from the spleen or lymph nodes, only a small proportion of professional APCs being present.
  • blood cells in the present invention are typically characterized in that they contain a small proportion of well- differentiated professional antigen presenting cells (APCs), especially dendritic cells (DCs).
  • APCs professional antigen presenting cells
  • DCs dendritic cells
  • the blood cells as used according to the present invention may be preferably fresh blood cells, i.e.
  • the period between collection of the blood cells (especially blood withdrawal) and transfection being only short, e.g. less than 12 h, preferably less than 6 h, particularly preferably less than 2 h and very particularly preferably less than 1 h.
  • the blood cells as used according to the present invention may also be blood cells obtained upon withdrawal of blood prior to need, e.g. an operation or a treatment as defined herein, and which are stored subsequently until use.
  • Blood cells may be collected from an animal or human patient by standard methods, for example. Thus whole blood can easily be obtained by puncturing a suitable vessel. Serum is obtained in known manner by coagulating the solid blood constituents.
  • PBMCs may be mentioned as an example of an enriched partial population of blood cells. These are conventionally isolated by a method first described by B ⁇ yum (Nature 204, 793-794, 1964; Scan. J. Lab. Clin. Invest. Suppl. 97, 1967). This is generally done by withdrawing blood from the individual and adding it e.g. to a solution of density 1.077 g/ml (25°C), conventionally containing Ficoll and sodium diatrizoate, for density gradient centrifugation.
  • the PBMCs collect at the Ficoll/blood interface whereas the red blood cells and the remaining white blood cells are sedimented.
  • the interface with the PBMCs is recovered and conventionally washed with a suitable buffer, e.g. sterile PBS.
  • the PBMCs are preferably subjected to a short isotonic treatment with an aqueous solution of e.g. ammonium chloride.
  • the PBMCs are washed a further one or more times with a buffer such as PBS (sterile).
  • PBS sterile
  • the blood cells immediately prior to transfection are fresh blood cells, i.e. there is only a short period between the collection of blood cells (especially blood withdrawal) in step (a) and the transfection according to step (b), e.g. less than 12 h, preferably less than 6 h, particularly preferably less than 2 h and very particularly preferably less than 1 h.
  • dendritic cells are typically potent antigen presenting cells (APCs) that typically possess the ability to stimulate na ⁇ ve T cells. They comprise a system of leukocytes widely distributed in all tissues, especially in those that provide an environmental interface. DCs posses a heterogeneous haemopoietic lineage, in that subsets from different tissues have been shown to posses a differential morphology, phenotype and function. The ability to stimulate na ⁇ ve T cell proliferation appears to be shared between these various DC subsets.
  • APCs antigen presenting cells
  • DCs are derived from bone marrow progenitors and circulate in the blood as immature precursors prior to migration into peripheral tissues. Within different tissues, DCs differentiate and become active in the taking up and processing of antigens, and their subsequent presentation on the cell surface linked to major histocompatibility (MHC) molecules. Upon appropriate stimulation, DCs undergo further maturation and migrate to secondary lymphoid tissues where they present antigens to T cells and induce an immune response.
  • MHC major histocompatibility
  • DCs are receiving increasing scientific and clinical interest due to their key role in anti-cancer host responses and potential use as biological adjuvants in tumour vaccines, as well as their involvement in the immunobiology of tolerance and autoimmunity.
  • DCs Dendritic cells
  • Such dendritic cells (DCs), as defined above, may be isolated from different tissues as mentioned above or from blood, particularly from blood samples as described above.
  • the blood cells, professional antigen presenting cells (APCs), especially dendritic cells (DCs), used for the inventive method of transfection and optionally administration of the inventive pharmaceutical composition or the inventive vaccine preferably originate from the actual patient who will be treated with the pharmaceutical composition of the present invention (autologous cells).
  • the pharmaceutical composition or the vaccine according to the invention therefore preferably contains autologous blood cells, professional antigen presenting cells (APCs), especially dendritic cells (DCs). All types of autologous cells are encompassed hereby.
  • the transfection of the blood cells, professional antigen presenting cells (APCs), especially dendritic cells (DCs), is likewise carried out by common methods, e.g. by means of electroporation or chemical methods, especially lipofection, preferably only by administration of the inventive composition without further transfection reagents.
  • the at least one "complexed precomplexed nucleic acid" as defined according to the present invention i.e. at least one complexed precomplexed nucleic acid (molecule), e.g.
  • an mRNA which has been precomplexed in a first step with (a high-molecular) PEI in an N/P ratio between 0.05 and 2, preferably in an N/P ratio between 0.1 and 1 and have been further complexed in a second step with a cationic or polycationic compound as defined above, is preferably prepared by methods known to those skilled in the art, especially by chemical synthesis or particularly preferably by means of molecular biological methods which have already been mentioned above, more preferably by methods as described above
  • transfect the blood cells professional antigen presenting cells (APCs), especially dendritic cells (DCs), used for the inventive method of transfection and optionally administration, with the inventive pharmaceutical composition or vaccine or its components, e.g. the at least one "complexed precomplexed nucleic acid" as defined according to the present invention.
  • APCs professional antigen presenting cells
  • DCs dendritic cells
  • administration of such transfected cells to a patient, to living tissues and/or organisms in vivo, particularly retransplantation into the host organism in the case of autologous cells may be envisaged as well and carried out as an optional step c) of the above mentioned method.
  • an organism typically means mammals, selected from, without being restricted thereto, the group comprising humans, and animals, including e.g. pig, goat, cattle, swine, dog, cat, donkey, monkey, ape or rodents, including mouse, hamster and rabbit.
  • living tissues as mentioned above, are preferably derived from these organisms.
  • Administration of the transfected cells to those living tissues and/or organisms may occur via any suitable administration route, e.g. systemically, and include e.g. intra- or transdermal, oral, parenteral, including subcutaneous, intramuscular or intravenous injections, topical and/or intranasal routes as defined above.
  • the present invention also provides a method for the preparation of the at least one "complexed precomplexed nucleic acid" as defined according to the present invention, comprising following steps: a) providing a nucleic acid precomplexed with PEI by precomplexing a nucleic acid (molecule) as defined above with PEI in a ratio as defined above, preferably in an N/P ratio between 0.05 and 2, more preferably in an N/P ratio between 0.05 and 1 , even more preferably in an N/P ratio between 0.1 and 1 ; and subsequently b) complexing the nucleic acid precomplexed with PEI with a cationic or polycationic compound, preferably a cationic peptide, preferbly in an N/P ratio of about about 0.1 -50, preferably in a range of about 0.5-50 and most preferably in a range of about 0.75-25 or 1 -25 regarding the ratio of RNA:cationic peptide in the complex, even more preferably in the range of about
  • kits particularly kits of parts, comprising as components alone or in combination, the inventive inventive pharmaceutical composition or vaccine, and/or the at least one "complexed precomplexed nucleic acid" as defined according to the present invention, and optionally technical instructions with information on the administration and dosage of these components.
  • kits preferably kits of parts, may be applied, e.g., for any of the above mentioned applications or uses.
  • Figure 1 shows a GC enriched luciferase encoding mRNA sequence according to SEQ ID NO: 1
  • pCV19-Pp luc(GC)-muag-A70-C30 The mRNA sequence of pCV19-Pp luc(GC)-muag-A7O-C3O encodes luciferase and contains following sequence elements:
  • Figure 2 shows a wildtype luciferase encoding mRNA sequence according to SEQ ID NO: 1
  • Ppluc(wt)-muag-A70-C30 The mRNA sequence of CAP-PpI uc(wt)-muag- A70-C30 encodes wildtype luciferase and contains following sequence elements:
  • the ORF is indicated in italic letters, muag (mutated alpha-globin-3'-UTR is indicated with a dotted line, the poly-A-tail is underlined with a single line and the poly-C-tail is underlined with a double line.
  • Figure 3 shows a GC enriched luciferase encoding mRNA sequence according to SEQ
  • CAP- Ppluc(GC)-muag-A70-C30 which exhibits a length of 1857 nuclotides and was termed "CAP- Ppluc(GC)-muag-A70-C30".
  • the mRNA sequence of CAP-Ppluc(wt)-muag- A70-C30 encodes luciferase and contains following sequence elements: • GC-optimized sequence for a better codon usage and stabilization;
  • the ORF is indicated in italic letters, muag (mutated alpha-globin-3'-UTR is indicated with a dotted line, the poly-A-tail is underlined with a single line and the poly-C-tail is underlined with a double line.
  • Figure 4 shows the coding sequence of CAP-Ppluc(wt)-muag-A70-C30 (SEQ ID NO:
  • Figure 5 depicts the coding sequence of CAP-Ppluc(GC)-muag-A70-C30 (SEQ ID NO:
  • Figure 6 shows that precomplexation of the luciferase encoding mRNA, which has been precomplexed with PEI (800 kDa) in an N/P ratio of 0.04 to 0.67 and further complexed with the cationic peptide R 9 H 3 ( Figure 6, N/P PEI (RNA:peptide 1 :5000) leads to a significantly increased luciferase expression in HeLa-cells, whereas a complexation with PEI alone ( Figure 6, N/P PEI w/o peptide) has no influence on luciferase expression (see Figure 6).
  • the control was carried out with RNA alone (4 ⁇ g RNA) or without RNA (w/o RNA).
  • Figure 7 reflects that precomplexation of the luciferase encoding mRNA, which has been precomplexed with PEI (800 kDa) in an N/P ratio of 0.04 to 0.67 and further complexed with the cationic peptide R 9 H 3 (Figure 7, N/P PEI (molar ratio of RNA:peptide 1 :5000, corresponding to an N/P ratio of about 25)) leads to a significantly increased luciferase expression in CHO-cells.
  • the control was carried out with RNA alone (4 ⁇ g RNA), Peptide alone without
  • RNA w/o RNA
  • medium without RNA w/o RNA
  • Figure 8 shows that precomplexation of the luciferase encoding mRNA, which has been precomplexed with PEI (800 kDa) in an N/P ratio of 0.1 7 to 0.67 and further complexed with the cationic peptide R 9 H 3 ( Figure 8, N/P PEI (molar ratio of RNA.peptide 1 :5000, corresponding to an N/P ratio of about 25)) leads to a significantly increased luciferase expression in L929-cells, whereas a complexation with PEI alone ( Figure 8, N/P PEI w/o peptide) has no influence on luciferase expression (see Figure 8).
  • the control was carried out with RNA alone (4 ⁇ g RNA).
  • Figure 9 shows that precomplexation of the luciferase encoding mRNA, which has been precomplexed with PEI (800 kDa) in an N/P ratio of 0.04 to 0.1 7 and further complexed with the cationic peptide protamin in a mass ratio of 8:1 (corresponding to a molar ratio of 1 :35 and an N/P ratio of 0.12) leads to a significantly increased luciferase expression in HeLa-cells, whereas a complexation with PEI alone ( Figure 9, w/o peptide) has no influence on luciferase expression.
  • PEI 800 kDa
  • Figure 10 depicts that precomplexation of the luciferase encoding mRNA, which has been precomplexed either high-molecular PEI (800 kDa) in an N/P ratio of 0.04 to 0.67 or low-molecular PEI (25 kDa) in an N/P ratio of 0.04, respectively, and further complexed with the cationic peptide R 9 H 3 (Figure 10, N/P PEI 800 kDa (molar ratio of RNA:peptide 1 :5000, corresponding to an N/P ratio of about 25) or N/P PEI 25 kDa (molar ratio of RNA:peptide 1 :5000, corresponding to an N/P ratio of about 25)) leads to a significantly increased luciferase expression in HeLa-cells, whereas a complexation with
  • PEI alone ( Figure 10, N/P PEI w/o peptide) has no influence on luciferase expression (see Figure 10).
  • the control was carried out with RNA alone (4 ⁇ g RNA), with medium without RNA (w/o RNA) or with peptide without RNA (w/o RNA).
  • Figure 1 1 illustrates that precomplexation of the luciferase encoding mRNA, which has been precomplexed with PEI (800 kDa) in an N/P ratio of 0.04 to 0.67 and further complexed with the cationic peptide R 9 ( Figure 1 1 , N/P PEI (molar ratio of RNA:peptide 1 :5000, corresponding to an N/P ratio of about 25)) leads to a significantly increased luciferase expression in HeLa-cells, whereas a complexation with PEI alone ( Figure 1 1 , N/P PEI w/o) has no influence on luciferase expression (see Figure 1 1 ).
  • the control was carried out with RNA alone (luc-RNActive 4 ⁇ g RNA) or with medium without RNA (medium w/o
  • Figure 12 shows that precomplexation of the luciferase encoding mRNA, which has been precomplexed with PEI (800 kDa) in an N/P ratio of 0.04 to 0.1 7 and further complexed with the cationic polymer Lipofectamine (not
  • Lipofectamine 2000 in an RNA:Lipofectamine ratio of 1 ⁇ g:1 ⁇ l (see Figure 12) leads to a significantly increased luciferase expression in HeLa-cells, whereas a complexation with PEI alone has no influence on luciferase expression (see Figure 12).
  • the control was carried out with RNA alone (luc- RNActive 4 ⁇ g RNA) or with medium without RNA (w/o RNA).
  • DNA sequences encoding Pp luciferase ⁇ Photinus pyralis
  • corresponding mRNA sequences encoding Pp luciferase sequences were prepared and used for subsequent in vitro transcription reactions, transfection and vaccination experiments.
  • the DNA sequence and corresponding mRNA sequences termed pCV19-Ppluc(GC)-muag-A70-C30 sequence were prepared, which corresponds to the luciferase coding sequence.
  • the constructs were prepared by modifying the wildtype luciferase encoding DNA sequence by introducing a GC- optimized sequence for a better codon usage and stabilization, stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR)), a stretch of 70 x adenosine at the 3'-terminal end (poly-A-tail) and a stretch of 30 x cytosine at the 3'- terminal end (poly-C-tail), leading to SEQ ID NO: 1 (see Figure 1 ).
  • sequence of the final DNA construct had a length of 1863 nucleotides and was termed "pCV19-Ppluc(GC)-muag-A70-C30".
  • SEQ ID NO: 1 see Figure 1 . the sequence of the corresponding mRNA is shown.
  • CAP-Ppluc(GC)-muag-A70-C30 DNA sequence were prepared, which also correspond to the luciferase coding sequence. Therefore, a basic DNA construct was prepared termed CAP-Ppluc(wt)-muag-A70-C30 by introducing into the underlying wildtype sequence construct stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR)), a stretch of 70 x adenosine at the 3'-terminal end (poly-A-tail) and a stretch of 30 x cytosine at the 3'- terminal end (poly-C-tail), leading to a sequence according to SEQ ID NO: 2 (see Figure 2).
  • SEQ ID NO: 2 shows the corresponding mRNA sequence of the entire wt construct.
  • the mRNA coding sequence (CDS) of the basic construct termed CAP-PpIuc(wt)-muag-A70-C30 is shown in Figure 4 (SEQ ID NO: 4).
  • This construct was further modified by introducing a GC-optimized sequence for a better codon usage and stabilization, leading to SEQ ID NO: 3 (see Figure 3).
  • SEQ ID NO: 3 shows the corresponding mRNA sequence of the GC modified construct.
  • the final GC modified construct had a length of 1857 nucleotides and was termed "CAP-Ppluc(GC)-muag-A70-C30".
  • the mRNA coding sequence (CDS) of the final construct termed CAP-Ppluc(GC)-muag-A70-C30 is shown in Figure 5 (SEQ ID NO: 5).
  • the stabilized luciferase mRNA according to SEQ ID NOs: 1 or 3 were transcribed in vitro using T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tubingen, Germany) starting from the above DNA constructs following the manufactures instructions.
  • All mRNA-transkripts contained a GC-optimized sequence for a better codon usage and stabilization, stabilizing sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR)), a stretch of 70 x adenosine at the 3'-terminal end (poly-A-tail), a stretch of 30 x cytosine at the 3'- terminal end (poly-C-tail), and a 5'- Cap-structure.
  • the 5 '-Cap-structure was obtained by adding an excess of N7-
  • RNA stabilized luciferase mRNA according to SEQ ID NOs: 1 or 2 were precomplexed by mixing the mRNA in different N/P ratios with PEI (high molecular
  • PEI 800 kDa or low molecular PEI (25 kDa)
  • precomplexed mRNA mRNA-PEI precomplexes
  • a cationic transfection reagent in the indicated molar ratio and diluted with water to a final volume of 40 ⁇ l, leading to "complexed precomplexed mRNA".
  • the cationic transfection reagent used in the specific example R 9 H 3 , R 9 , protamine or lipofectamine (not lipofectamine 2000)
  • Transfection and expression of stabilized luciferase mRNA constructs 4.1 Hela-cells
  • HeLa-cells (1 x10Vwell) were seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out.
  • a precomplexed complexed mRNA e.g. a precomplexed complexed mRNA according to SEQ ID NO: 1 or 3
  • the transfection solution (300 ⁇ l per well) was added to the cells and the cells were incubated for 4 h at 37°C. Subsequently 300 ⁇ l RPMI-medium (Camprex) containing 10% FCS was added per well and the cells were incubated for additional 20 h at 37°C. The transfection solution was sucked off 24 h after transfection and the cells were lysed in 300 ⁇ l lysis buffer (25 mM THs-PO 4 , 2 mM EDTA, 10% glycerol, 1 % Triton-X 100, 2 mM DTT).
  • luciferin buffer 25 mM Glycylglycin, 15 mM MgSO 4 , 5 mM ATP, 62,5 ⁇ M luciferin
  • luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).
  • CHO-cells (1 x10 5 /well) were seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out.
  • precomplexed complexed mRNA construct as disclosed above, e.g. a precomplexed complexed mRNA according to SEQ ID NO: 1 or 3, were mixed with 260 ⁇ l serum free medium and added to the cells (final RNA concentration of each preparation was 13 ⁇ g/ml).
  • the transfection solution Prior to addition of the transfection solution the CHO- cells were washed gently and carefully 2 times with 2 ml Optimen (Invitrogen) per well. Then, the transfection solution (300 ⁇ l per well) was added to the cells and the cells were incubated for 4 h at 37°C. Subsequently 300 ⁇ l Ham's F12-medium was added per well and the cells were incubated for additional 20 h at 37°C.
  • the transfection solution was sucked off 24 h after transfection and the cells were lysed in 300 ⁇ l lysis buffer (25 mM THs-PO 4 , 2 mM EDTA, 10% glycerol, 1 % Triton-X 100, 2 mM DTT).
  • the supernatants were then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO 4 , 5 mM ATP, 62,5 ⁇ M luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad
  • L929-cells (1 x10 5 /well) were seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out.
  • the L929- cells Prior to addition of the transfection solution the L929- cells were washed gently and carefully 2 times with 2 ml Optimen (Invitrogen) per well. Then, the transfection solution (300 ⁇ l per well) was added to the cells and the cells were incubated for 4 h at 37°C. Subsequently 300 ⁇ l RPMI-medium (Camprex) containing 10% FCS was added per well and the cells were incubated for additional 20 h at 37°C.
  • the transfection solution was sucked off 24 h after transfection and the cells were lysed in 300 ⁇ l lysis buffer (25 mM THs-PO 4 , 2 mM EDTA, 10% glycerol, 1 % Triton-X 100, 2 mM DTT).
  • the supernatants were then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO 4 , 5 mM ATP, 62,5 ⁇ M luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).
  • HeLa-cells were transfected with a precomplexed complexed mRNA according to SEQ ID NO: 3 (mRNA concentration: 4 ⁇ g CAP-Ppluc(GC)-muag-A70- C30 construct encoding luciferase).
  • Precomplexation of the mRNA was carried out with PEI (800 kDa) in an N/P ratio of RNA:PEI of 0.04, 0.1 7, 0.67, or 1 .08.
  • Preparing the precomplexed complexed mRNA constructs and the transfection into HeLa-cells was generally carried out as described above under sections 3. and 4.2. Particularly, CHO-cells were transfected with a precomplexed complexed mRNA according to SEQ ID NO: 3 (mRNA concentration: 4 ⁇ g CAP-Ppluc(GC)-muag-A70-
  • L929-cells were transfected with a precomplexed complexed mRNA according to SEQ ID NO: 3 (mRNA concentration: 4 ⁇ g CAP-Ppluc(GC)-muag-A70- C30 construct encoding luciferase).
  • Precomplexation of the mRNA was carried out with PEI (800 kDa) in an N/P ratio of RNA:PEI of 0.04, 0.1 7, 0.67, 1.08 or 2.1 7.
  • HeLa-cells was generally carried out as described above under sections 3. and 4.1 . Particularly, HeLa-cells were transfected with a precomplexed complexed mRNA according to SEQ ID NO: 3 (mRNA concentration: 4 ⁇ g CAP-Ppluc(GC)-muag-A70- C30 construct encoding luciferase). Precomplexation of the mRNA was carried out with PEI (800 kDa) in an N/P ratio of RNA:PEI of 0.04, 0.17, 0.67, or 1 .08.
  • SEQ ID NO: 3 mRNA concentration: 4 ⁇ g CAP-Ppluc(GC)-muag-A70- C30 construct encoding luciferase.
  • Precomplexation of the mRNA was carried out with PEI (800 kDa) in an N/P ratio of RNA:PEI of 0.04, 0.17, 0.67, or 1 .08.
  • precomplexation of the luciferase encoding mRNA which has been precomplexed with PEI (800 kDa) in an N/P ratio of 0.04 to 0.17 and further complexed with the cationic peptide protamine in a mass ratio of 8:1 (corresponding to a molar ratio of 1 :35 and an N/P ratio of 0.12) leads to a significantly increased luciferase expression in HeLa-cells, whereas a complexation with PEI alone ( Figure 9, w/o peptide) has no influence on luciferase expression.
  • Preparing the precomplexed complexed mRNA constructs and the transfection into HeLa-cells was generally carried out as described above under sections 3. and 4.1 .
  • HeLa-cells were transfected with a precomplexed complexed mRNA according to SEQ ID NO: 3 (mRNA concentration: 4 ⁇ g CAP-Ppluc(GC)-muag-A70-
  • Precomplexation of the mRNA was carried out with either high-molecular PEI (800 kDa) or low-molecular PEI (25 kDa) in an N/P ratio of RNA:PEI of 0.04, 0.17, 0.67, or 1.08.
  • RNA alone (4 ⁇ g RNA), with medium without RNA medium (w/o RNA) or with peptide without RNA (peptide w/o RNA).
  • RNA alone (4 ⁇ g RNA), with medium without RNA medium (w/o RNA) or with peptide without RNA (peptide w/o RNA).
  • peptide w/o RNA peptide without RNA
  • Preparing the precomplexed complexed mRNA constructs and the transfection into HeLa-cells was generally carried out as described above under sections 3. and 4.1 .
  • HeLa-cells were transfected with a precomplexed complexed mRNA according to SEQ ID NO: 3 (mRNA concentration: 4 ⁇ g CAP-PpI uc(GC)-muag-A70- C30 construct encoding luciferase).
  • Precomplexation of the mRNA was carried out with PEI (800 kDa) in an N/P ratio of RNA:PEI of 0.04, 0.1 7, 0.67, 1 .08 or 2.1 7.
  • HeLa-cells were transfected with a precomplexed complexed mRNA according to SEQ ID NO: 3 (mRNA concentration: 4 ⁇ g CAP-Ppluc(GC)-muag-A70- C30 construct encoding luciferase).
  • Precomplexation of the mRNA was carried out with PEI (800 kDa) in an N/P ratio of RNA:PEI of 0.04, 0.17, 0.67 or 1.08.
  • Complexation of the precomplexed mRNA was carried out with the cationic polymer Lipofectamine (not Lipofectamine 2000) in an RNA:Lipofectamine ratio of 1 ⁇ g:1 ⁇ l.
  • LipofectamineTM Reagent is suitable for the transfection of DNA into eukaryotic cells (1 ), and is a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl- 1 -propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water.
  • DOSPA polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl- 1 -propanaminium trifluoroacetate
  • DOPE neutral lipid dioleoyl phosphatidylethanolamine

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Abstract

La présente invention porte sur des acides nucléiques, de préférence des ARNm, précomplexés complexés, qui ont été précomplexés dans une première étape avec du PEI en un rapport N/P compris entre 0,05 et 2, de préférence en un rapport N/P compris entre 0,1 et 1, et qui ont été complexés encore davantage dans une seconde étape avec un composé cationique. La présente invention porte en outre sur l'utilisation de tels acides nucléiques précomplexés complexés pour l'amélioration de la libération endosomale d'acides nucléiques et facultativement l'amélioration de l'expression d'une protéine ou d'un peptide codé. L'invention porte également sur des compositions comportant de tels acides nucléiques précomplexés complexés et sur l'utilisation de tels acides nucléiques précomplexés complexés ou de compositions de ceux-ci pour la thérapie génique et/ou le traitement de diverses maladies telles que mentionnées par les présentes, par exemple par vaccination. La présente invention porte également sur des procédés de préparation et d'administration de ces acides nucléiques précomplexés complexés ou de compositions de ceux-ci et sur des kits comportant ces acides nucléiques précomplexés complexés ou des compositions de ceux-ci.
PCT/EP2009/000886 2009-02-09 2009-02-09 Utilisation de pei pour l'amélioration de la libération endosomale et de l'expression d'acides nucléiques transfectés, complexés par des composés cationiques ou polycationiques Ceased WO2010088927A1 (fr)

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